MIT Students Turn Internet Into a Sixth Human Sense -- Video
By Kim Zetter EmailFebruary 05, 2009 | 3:59:35 PMCategories: TED Conference
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LONG BEACH, California -- Students at the MIT Media Lab have developed a wearable computing system that turns any surface into an interactive display screen. The wearer can summon virtual gadgets and internet data at will, then dispel them like smoke when they're done.
Pattie Maes of the lab's Fluid Interfaces group said the research is aimed at creating a new digital "sixth sense" for humans.
In the tactile world, we use our five senses to take in information about our environment and respond to it, Maes explained. But a lot of the information that helps us understand and respond to the world doesn't come from these senses. Instead, it comes from computers and the internet. Maes' goal is to harness computers to feed us information in an organic fashion, like our existing senses.
The prototype was built from an ordinary webcam and a battery-powered 3M projector, with an attached mirror -- all connected to an internet-enabled mobile phone. The setup, which costs less than $350, allows the user to project information from the phone onto any surface -- walls, the body of another person or even your hand.
Maes showed a video of her student Pranav Mistry who she describes as the brains behind the project. Mistry wore the device on a lanyard around his neck, and colored Magic Marker caps on four fingers (red, blue, green and yellow) helped the camera distinguish the four fingers and recognize his hand gestures with software that Mistry created.
The gestures can be as simple as using his fingers and thumbs to create a picture frame that tells the camera to snap a photo, which is saved to his mobile phone. When he gets back to an office, he projects the images onto a wall and begins to size them.
When he encounters someone at a party, the system projects a cloud of words on the person's body to provide more information about him -- his blog URL, the name of his company, his likes and interests. "This is a more controversial [feature]," Maes said over the audience's laughter.
In another frame, Mistry picks up a boarding pass while he's sitting in a car. He projects the current status of his flight and gate number he's retrieved from the flight-status page of the airline onto the card.
"If you need to know what time it is, it's as simple as drawing a watch on your arm," Maes said, while Mistry used his right finger to draw a circle on his left wrist. The face of a watch popped up on his hand, which the audience liked.
When Mistry folds his hands in "namaste" fashion, the system opens a menu to allow him to choose an application. If he wants to read e-mail on his phone, he draws an @ symbol in the air with his finger. He can project a phone pad onto his palm and dial a number without removing the phone from his pocket. As he reads the newspaper on the subway he can project a video onto the page that provides more information about the topic he's reading.
Maes and Mistry told Wired they've been working on the project for four months, day and night, and have filed a patent for it.
Maes' MIT group, which includes seven graduate students, were thinking about how a person could be more integrated into the world around them and access information without having to do something like take out a phone. They initially produced a wristband that would read an RFID tag to know, for example, which book a user is holding in a store.
They also had a ring that used infrared to communicate by beacon to supermarket smart shelves to give you information about products. As you grab a package of macaroni, the ring would glow red or green to tell you if the product was organic or free of peanut traces -- whatever criteria you program into the system.
"We wanted to make information more useful to people in real time with minimal effort in a way that doesn't require any behavior changes," Maes said. "The wristband was getting close, but you still had to take out your cell phone to look at the information."
That's when they struck on the idea of accessing information from the internet and projecting it. So someone wearing the wristband could pick up a paperback in the bookstore and immediately call up reviews about the book, projecting them onto a surface in the store or doing a keyword search through the book by accessing digitized pages on Amazon or Google books.
They started with a larger projector that was mounted on a helmet. But that proved cumbersome if someone was projecting data onto a wall then turned to speak to friend -- the data would project on the friend's face. Last month, they switched to a smaller projector and created the pendant prototype to be worn around the neck.
The TED demo was the first time they've shown it in public, though they're far from making a commercial product or forming a company around their invention. "But we're really excited about the potential," Maes said.
They learned recently that cellphone makers soon plan to release cellphones with projectors integrated in them, which will simplify their system even more.
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Wow, so we're getting pretty close to the full gargoyle setup [see Snow Crash, Neal Stephenson]. I'll buy this in a heartbeat once it's something more than a vaporware tech demo.
Posted by: AJ | Feb 5, 2009 1:32:46 PM
Now someone needs to invent self-cleaning glass to handle the fingerprints people are going to make all over glass surfaces using this thing.
Posted by: James A Woods | Feb 5, 2009 3:53:52 PM
http://www.turkokey.org
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http://www.turkokey.gen.tr
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http://www.chatyeli.gen.tr
http://www.mirckiz.com
http://www.chatmerkezi.org
Posted by: sohbet odaları | Feb 5, 2009 4:19:06 PM
http://www.chatkeyfim.com
http://www.chatyeli.com
http://www.sohbetant.com
Posted by: sohbet | Feb 5, 2009 4:20:00 PM
We've been using the same damn keyboard, mouse, monitor setup forever now. It's been more than 10 years and I don't even hear about voice recognition anymore. Seems like I read a ways back that computers won't be powerful enough until around 10 GHZ, but that was before multicores, so not sure where they are with that. This seems like it would be beyond that and we won't see it working in forever.
Posted by: capcom | Feb 5, 2009 4:20:47 PM
It looks like this project has some great potential here, but it seems that this may be the start of a world in the future as portrayed in M.T. Anderson's book, "Feed". I hope that this project isn't the start of an apocalypse.
Posted by: Mitch | Feb 5, 2009 4:23:23 PM
it will be cool when i dont have to were marker caps on my finger tips all the time.
Posted by: mike | Feb 5, 2009 4:46:23 PM
full-time projection just seems intrusive and lame.
I'd want lightweight glasses that can overlay info on my environment without others seeing the overlay.
Being able to route output to a pendant projector, or other display would be a nice addition.
Posted by: Jeff | Feb 5, 2009 4:48:42 PM
I don't want to, but have to be naysayer on this.
How brain-dead you have to be to outsource your taste for books, preference of grocery, and memory of friends to some central A.I.
Nay!
Posted by: nay | Feb 5, 2009 4:52:02 PM
Maybe the privacy concerns of public projection could be mitigated by linking the projector to glasses which use the same modulating polarization filter, although I've no idea whether that is technically feasible.
Posted by: Carsten | Feb 5, 2009 5:13:58 PM
I'd prefer the majority of the data on a HUD, but I still want one.
Posted by: Tisi | Feb 5, 2009 5:14:40 PM
Now I can project porn onto anyone I don't find at least mildly amusing!
"What's that? Of course I want to hear about the specifics of your 401k." :)
Posted by: hell yeah! | Feb 5, 2009 5:16:49 PM
I didnt see anything in the article that stated where the information came from. So projecting information on someone would be no more of a privacy violation then writing the info down in your PDA. Its not like there's some giant database thats memorized everyones book faves. (Other then amazon. :P )
I like it.. there should be a HUD glasses version, with switchable preferences. Similuate the same effects as actual projection (the watch on hand) but just in the glassees, that would be coolest.
heh..so all those computer wearing geeks that have been around since the 90s have finally come out with something thats doable. Nice.
..oh yes about the voice recognition. Yes the hype around that has died down quite a bit. Though the technology seems like its around to handle it.
I think most people just dont like the idea of being in a room of people talking to their gadget and giving it commands. That and literally no one Ive ever met likes to take the time it takes to train the software about their voice.
Posted by: david b | Feb 5, 2009 5:27:52 PM
@Mitch: "start of an apocalypse" ? Aren't we in the midst of one now? It's the end days, my friend! Singularity will save some but the world and the rest? Down in flames! Fire and brimstone!
Posted by: Dale | Feb 5, 2009 5:42:11 PM
I'm with Nay. If people keep using this stuff long term they will become morons with no memory or decision making capacity - oh wait, that's the goal isn't it? Complete Corporate Control.
Sigh. I guess it could be useful for education . . . depends on who does the programming.
Posted by: Naytoo | Feb 5, 2009 5:54:52 PM
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Thursday, February 5, 2009
Wednesday, February 4, 2009
the cooption of pink
at the supermarket checkout line rows of people with veggies, that take a while to look up and weigh as specialty items, then you have the older women with the potential checkbookwriting skills, and you wonder what is next as you put the hokey pokey, the one foot in the one foot out, and you are doing this rocking back movement, and finally you see that you are this cia dude totally watching out to see if you have calcculated the fastest line checkout, should i go to the self checkout or go to the express even though the express line is long
Bubble brightness
Cats on bubble but not interested in scenarios; 'we just want to win'
By Bruce Pascoe
Arizona Daily Star
Tucson, Arizona | Published: 02.03.2009
advertisement
Quantcast
Anybody with an Arizona Wildcat basketball schedule can come up with a plan, and Russ Pennell has heard plenty of them.
You know, something like this: To get to the NCAA tournament from their current 14-8 record, the Wildcats should … sweep this week at Oregon State and Oregon … beat everyone, except maybe UCLA, at home … see if they can pull out a surprise at ASU, or at Washington.
But the UA interim head coach, and his players, don't want to hear it.
"I've got friends who say, 'Hey, we figured it all out: Win the rest of the home games, and do this and this and this,'" Pennell said. "That's not the way I want to think. If it was that simple none of us would worry about it."
Instead, point guard Nic Wise says the Wildcats look at every game like they should — and can — win.
"We don't look at it like we have to win this one if we lose this one," Wise said. "I don't know who thinks like that."
Well, we do.
It's a safe bet the Wildcats need to win at least five of their final nine regular-season games — barring a very successful Pac-10 tournament run — to make a 25th straight NCAA tournament appearance.
As of Monday, the Wildcats had a bubble-bursting No. 63 RPI that isn't likely to go up much this weekend even with a sweep in Oregon. They are not even listed in the eight schools just outside of ESPN's Bracketology projections Monday (though they were at least among eight others considered).
So maybe sweeping this week in Oregon is a good idea. Then returning home to beat at least one of the Los Angeles schools and …
"We just want to win," Wise said. "We don't want to be left out of the NCAA. That's what we came here for, to get a chance to go to the tournament and play in big games like that. If we don't make it, we'll be devastated. So we're doing everything we can right now to make it there."
Especially this season, with their third coaching staff in three years, making it could be special.
With "all the stuff we've been through, we need something to keep us happy," forward Jordan Hill said. "The freshmen, sophomores and juniors would just have something to look at and wonder, 'Man — all the things we been through and we still made it? This is a good team right here.'"
Pac-10 honors Budinger
Junior forward Chase Budinger was named the Pac-10 Player of the Week on Monday for the second time in his career after leading the Wildcats to a sweep of the Washington schools last week.
Budinger averaged 22 points, 9.5 rebounds, 5.5 assists and no turnovers in the two games, while getting his second double-double of the year (19 points, 11 rebounds) against Washington State. Budinger shot .387 from the field, .333 from three-point range and .833 from the free-throw line, though he missed 11 of his first 12 shots against Washington State.
Budinger is the third Arizona player to receive the weekly league honor this season, joining Hill on Dec. 15 and Jamelle Horne on Dec. 29.
Wise on roll
Had the Pac-10 award been considered for the Wildcats' previous three games, Wise might have had a shot at it.
Wise has scored 58 points, with 14 assists and 11 turnovers, in the three games beginning with the the Wildcats' Jan. 24 win over Houston. He shot just 2 of 8 from the field against Washington State but had four assists and three steals, with only one turnover.
"He's been more aggressive offensively, looking for his shot more, which we want him to do," Pennell said. "Nic's got a real good feel for when to shoot and when not to. He rarely takes a bad shot. He may overpenetrate sometimes but he rarely takes a bad shot."
Pennell said Wise has moved past a brief midseason slump despite playing on a "bad wheel" — the knee injury that forced him to miss seven games last season.
"His knee gets sore, and he never says anything," Pennell said. "He just plays through it. So you just never know. He's a gutty little player, and you'd probably have to pry his brain open to have him tell you exactly how he feels."
Comment on this story
By Bruce Pascoe
Arizona Daily Star
Tucson, Arizona | Published: 02.03.2009
advertisement
Quantcast
Anybody with an Arizona Wildcat basketball schedule can come up with a plan, and Russ Pennell has heard plenty of them.
You know, something like this: To get to the NCAA tournament from their current 14-8 record, the Wildcats should … sweep this week at Oregon State and Oregon … beat everyone, except maybe UCLA, at home … see if they can pull out a surprise at ASU, or at Washington.
But the UA interim head coach, and his players, don't want to hear it.
"I've got friends who say, 'Hey, we figured it all out: Win the rest of the home games, and do this and this and this,'" Pennell said. "That's not the way I want to think. If it was that simple none of us would worry about it."
Instead, point guard Nic Wise says the Wildcats look at every game like they should — and can — win.
"We don't look at it like we have to win this one if we lose this one," Wise said. "I don't know who thinks like that."
Well, we do.
It's a safe bet the Wildcats need to win at least five of their final nine regular-season games — barring a very successful Pac-10 tournament run — to make a 25th straight NCAA tournament appearance.
As of Monday, the Wildcats had a bubble-bursting No. 63 RPI that isn't likely to go up much this weekend even with a sweep in Oregon. They are not even listed in the eight schools just outside of ESPN's Bracketology projections Monday (though they were at least among eight others considered).
So maybe sweeping this week in Oregon is a good idea. Then returning home to beat at least one of the Los Angeles schools and …
"We just want to win," Wise said. "We don't want to be left out of the NCAA. That's what we came here for, to get a chance to go to the tournament and play in big games like that. If we don't make it, we'll be devastated. So we're doing everything we can right now to make it there."
Especially this season, with their third coaching staff in three years, making it could be special.
With "all the stuff we've been through, we need something to keep us happy," forward Jordan Hill said. "The freshmen, sophomores and juniors would just have something to look at and wonder, 'Man — all the things we been through and we still made it? This is a good team right here.'"
Pac-10 honors Budinger
Junior forward Chase Budinger was named the Pac-10 Player of the Week on Monday for the second time in his career after leading the Wildcats to a sweep of the Washington schools last week.
Budinger averaged 22 points, 9.5 rebounds, 5.5 assists and no turnovers in the two games, while getting his second double-double of the year (19 points, 11 rebounds) against Washington State. Budinger shot .387 from the field, .333 from three-point range and .833 from the free-throw line, though he missed 11 of his first 12 shots against Washington State.
Budinger is the third Arizona player to receive the weekly league honor this season, joining Hill on Dec. 15 and Jamelle Horne on Dec. 29.
Wise on roll
Had the Pac-10 award been considered for the Wildcats' previous three games, Wise might have had a shot at it.
Wise has scored 58 points, with 14 assists and 11 turnovers, in the three games beginning with the the Wildcats' Jan. 24 win over Houston. He shot just 2 of 8 from the field against Washington State but had four assists and three steals, with only one turnover.
"He's been more aggressive offensively, looking for his shot more, which we want him to do," Pennell said. "Nic's got a real good feel for when to shoot and when not to. He rarely takes a bad shot. He may overpenetrate sometimes but he rarely takes a bad shot."
Pennell said Wise has moved past a brief midseason slump despite playing on a "bad wheel" — the knee injury that forced him to miss seven games last season.
"His knee gets sore, and he never says anything," Pennell said. "He just plays through it. So you just never know. He's a gutty little player, and you'd probably have to pry his brain open to have him tell you exactly how he feels."
Comment on this story
Washington State Arizona
MCKALE center was electric infrequently today. There is much to say about their win over the washington state cougars, and they are a terrific outside shooting team; a group of men with amazing hearts, smarts, and they are completely angry at themselves for handing over turnovers today as the wildcats trapped to claw, and where chase played like a veteran. Before we discuss a casual nonchalant double double by first team all american jordan hill, there is a moment when you see freshman no longer play like freshman, they are simply just more comfortable, and these boys are in their eighteenth year of age, and they are completely ready to sprout forth into the colllege basketball world. one example is arizona's kyle fogg, who has sleek, and elegant, lengthy arms: appendages so impressive they lead to countless strips of the ball, a defensive specialty of his and of jordan hill. chase was tough, relentlessly driving to the whole, and absolutely aware of the fact that cbs had broadcast his 1 for ten, or one for alot of shots stat in the first half. ending up with his own double double, the man of the hour was zane johnson, who hit a big bucket when it was needed to bring the cats back from a seven point down defecit. clearly we had a problem on our hands, and washington state plays some of the slowest, most obnoxious, style of play of any team in the country they know when to slow it down, and they are defensively ready to stop transition buckets. but they are white guys. and white guys can not make athletic plays, and white boys from pullman can not finish alley oops very well, and the college game has been watered down, and there is a very difficult decision one makes when playing the rain up there. You have to be in the cold, in the country, stuck with butt ugly chicks.
but anyways the game was long, especially cause of commercials, there was plenty to say about the free bags that alltell gave away. we never should have put their logo on the court. uccla just says john wooden court. ours is lute's and bobby's
anyways, bob saget is in hell, because his rerunds drive him insane.
the game from the bleachers was best, and no one looked at me, or what i was wearing, though the red and blue i was bleeding today was for a pivotal, mid-season win. russ and dunlap are doing a great job extending the bench, and there is very little that has not already been said about our range of play; going from fifty pooints today and over one hundred two days ago against the best team in the pac ten (as far as standings went, at least). There was plenty of time to sit back \\
there were different angles at which to observe the game, differing people to avoid and to seek ouot, hands to shake, smiles to share.
the lemonade lady who peddles lemon drink for five american dollars is not wealthy. and there is not much wealth in the places where the fans are the best and most involved. the people who stay outside and guard the gate in the late moments of the second half when no one is coming in. the ticket takers are always nice and yet angry they are not in the game watching, with the people who pay a ton for parking and bad concession food.
there are those who direct traffic outside in good and bad weather and under the protective umbrella of the parking garage as well.
there are tons of people who sit in wheelchairs and would make good color analysts. at halftime, when taking a smoke break and not smoking and i observed that there were people with marlboros, who said we were lucky at half to not have wsu make a three that went in and out at the end of the half, which would have put them up eight.
no : say no to mammograms, and pink commerce
Dr. Morrow ended her lecture by saying that “the routine use of MRI in cancer patients requires some evidence of clinical benefit. To date, this does not exist.” In her last slide, she itemized concepts in breast cancer that were intuitively obvious, but that proved incorrect:
* high dose chemo + bone marrow transplant is superior to conventional chemotherapy;
* Endocrine therapy is inferior to chemotherapy and will not result in a survival advantage;
* Treatment of breast cancer with less than mastectomy is dangerous;
* local therapy does not influence survival.
And she added to that list: MRI finds cancer not found by other modalities. It must be useful.
Dr. Morrow’s presentation was followed by a report on the first and only prospective study of MRI, the COMICE trial, which was sponsored by the research arm of the British National Health Service. (England and Canada sponsor significant research on actual effectiveness as a means of cost containment). COMICE randomized 1623 women into two groups: those who received MRI prior to BCS, and those who did not. The primary endpoint was the rate of re-operation (those who had to have further excision, and those who had a subsequent mastectomy). There were not significant differences between the two study groups in re-operation rates, which substantiated Morrow’s perspective.
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ABC Slams Companies for Donating to Breast Cancer Research
Reporter David Muir cited liberal critic of corporations who also advises women to forego regular mammograms.
By Ken Shepherd
Business & Media Institute
10/13/2006 2:14:17 PM
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Companies can’t win – even when they literally give away money. ABC attacked businesses for helping “the corporate bottom line” by funding breast cancer research. In the process, the network relied on a critic of routine mammograms to make its attack.
On the October 12 “World News with Charles Gibson,” reporter David Muir turned a critical eye to businesses such as the makers of Campbell’s Soup or Yoplait yogurt that engage in breast cancer fundraising efforts.
“These pink ribbon campaigns often mean much more to the corporate bottom line than they do to people living with or at risk for breast cancer,” scoffed Barbara Brenner, the executive director of Breast Cancer Action.
Nowhere in his story did Muir mention that corporate fundraising efforts such as Yoplait’s “Save Lids to Save Lives” are often made in direct partnership with the Susan G. Komen Breast Cancer Foundation, perhaps the leading national advocate for breast cancer research and treatment.
Brennan was the only breast cancer research advocate featured in his story, but Muir suggested to viewers those views were shared by research proponents broadly.
“Advocates say” despite raising money for research, “they’re raising their profits a whole lot more,” the ABC reporter closed his story.
What Muir left out was how unorthodox Brenner’s group is and how some argue those views are a distraction in the fight against breast cancer.
A look at Brennan’s Web site reveals her organization is skeptical of, if not downright hostile to corporate sources of research financing. For example, her group refuses any contribution from health insurance or pharmaceutical companies.
But perhaps more disturbing is that her group disputes the wisdom of regular mammogram screenings for women.
“The benefit of routine mammograms for healthy premenopausal women is unproven,” Brenner’s group asserts in a Web page entitled “What You Should Know About Mammograms.”
“Healthy premenopausal women should not have mammograms as a routine matter. The risk of radiation, combined with the high incidence of both false negatives and positives, means that routine mammography for premenopausal women may well do more harm than good,” Breast Cancer Action asserts.
Dr. Elizabeth Whelan, an epidemiologist and Business & Media Institute (BMI) adviser, told BMI that such a claim was “wholly irresponsible.”
“I’m really shocked that they would say that. The benefits of early screening are overwhelmingly important,” Whelan countered, adding that “none of this recognizes what’s happened in the last eight years. It’s become largely a curable disease” when the cancer is caught early, thanks in part to drugs like Tamoxifen, Whelan argued.
Whelan added that it wasn’t surprising corporate donors would hope to get good PR and a boost in sales from their philanthropy, but added that media scrutiny would be better served finding where money is spent by breast cancer advocates, including Brenner’s group.
Being diagnosed with breast cancer is often frightening and confusing. It is easy to feel lost in a world of unfamiliar medical terms and procedures. Questions about diagnosis, treatment, money, relationships, and quality of life can cause you worry and stress. Sometimes you don’t even know where to begin or what questions to ask. You are not alone in this journey. (This document was written by women who have gone through this experience.) By reading this, you are already taking the first steps toward recovery. The following pages contain ideas and tips that will hopefully make dealing with health care providers, hospitals, and your health easier and less intimidating for you and your loved ones.
The best thing that you can do for yourself now is to give yourself time to think about your options. You need not feel rushed into making decisions you are not comfortable with or do not fully understand. You have this time to gather information and make informed decisions. A few weeks most likely will not influence your treatment outcome, but it may make a huge difference in your state of mind. Your diagnosis will be presented to you with a series of choices; what matters is that you make the choices that are right for you. The process may seem overwhelming and leave you with a lot of uncertainty. You can ask people you trust to help you make the choices that lie ahead. You and your family or friends can become advocates by doing research and talking to experts. Breast Cancer Action (BCA) is one resource for you during this time.
#9-Tetrahydrocannabinol Inhibits Cell Cycle Progression in Human
Breast Cancer Cells through Cdc2 Regulation
Marı´a M. Caffarel,1 David Sarrio´,2 Jose´ Palacios,2 Manuel Guzma´n,1 and Cristina Sa´nchez1
1Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University and 2Breast and Gynecological Cancer
Group, Molecular Pathology Programme, Centro Nacional de Investigaciones Oncolo´gicas, Madrid, Spain
Abstract
It has been proposed that cannabinoids are involved in the
control of cell fate. Thus, these compounds can modulate
proliferation, differentiation, and survival in different manners
depending on the cell type and its physiopathologic
context. However, little is known about the effect of
cannabinoids on the cell cycle, the main process controlling
cell fate. Here, we show that #9-tetrahydrocannabinol (THC),
through activation of CB2 cannabinoid receptors, reduces
human breast cancer cell proliferation by blocking the
progression of the cell cycle and by inducing apoptosis. In
particular, THCarres ts cells in G2-M via down-regulation of
Cdc2, as suggested by the decreased sensitivity to THC
acquired by Cdc2-overexpressing cells. Of interest, the
proliferation pattern of normal human mammary epithelial
cells was much less affected by THC. We also analyzed by realtime
quantitative PCR the expression of CB1 and CB2
cannabinoid receptors in a series of human breast tumor
and nontumor samples. We found a correlation between CB2
expression and histologic grade of the tumors. There was also
an association between CB2 expression and other markers of
prognostic and predictive value, such as estrogen receptor,
progesterone receptor, and ERBB2/HER-2 oncogene. Importantly,
no significant CB2 expression was detected in nontumor
breast tissue. Taken together, these data might set the
bases for a cannabinoid therapy for the management of breast
cancer. (Cancer Res 2006; 66(13): 6615-21)
Introduction
There are very few critical decisions that cells must take during
their lifetime. Basically, these are whether to proliferate, differentiate,
or die. A tight regulation of the cell cycle is crucial to control
all these decisions, and its deregulation has devastating consequences,
such as cancer (1). It has been proposed that
cannabinoids, the active components of Cannabis sativa, play a
role in the control of the aforementioned decisions. For example,
they can modulate survival, proliferation, and differentiation
depending on the cell type and its physiopathologic context
(2, 3). Among the f70 cannabinoids synthesized by C. sativa,
D9-tetrahydrocannabinol (THC) is the most important in terms of
potency and abundance (4). THC exerts a wide variety of biological
effects by mimicking endogenous compounds, the endocannabinoids
anandamide and 2-arachidonoylglycerol, which activate
specific cannabinoid receptors. Thus far, two G protein–coupled
cannabinoid-specific receptors have been cloned from mammalian
tissues: CB1, abundantly expressed in the brain and at many
peripheral sites, and CB2, almost exclusively expressed in the
immune system (5). Engagement of these receptors by THC or
endocannabinoids affects several signaling pathways, some of them
directly involved in the control of cell fate. For instance,
cannabinoids modulate mitogen-activated protein kinases and
the phosphatidylinositol 3-kinase/Akt survival pathway, which have
a prominent role in the control of cell growth and differentiation
(6). Due to the growing evidence that cannabinoids participate in
the control of cell fate and to the fact that the cell cycle is a key
process underlying the regulation of survival/proliferation/differentiation
decisions, we decided to study the effect of THC on the
cell cycle and the mechanism of cannabinoid action on this
process. Because breast tumors are one of the most common
human neoplasias and one of the leading causes of death among
Western women (7), we decided to focus our studies on this
particular type of cancer.
Materials and Methods
Cell culture and viability. EVSA-T, MDA-MB-231, MDA-MB-468, and
SKBr3 cells were kindly given by Dr. Lo´pez-Rivas [Centro Andaluz de
Biologı´a del Desarrollo, Consejo Superior de Investigaciones Cientı´ficas
(CSIC), Sevilla, Spain], and MCF-7 and T-47D cells were obtained from the
American Type Culture Collection (ATCC; Manassas, VA). Cells were
maintained in RPMI 1640 (EVSA-T, MDA-MB-231, MCF-7, and T-47D) or
DMEM (MDA-MB-468 and SKBr3) supplemented with 10% fetal bovine
serum (FBS), 5 units/mL penicillin, and 5 mg/mL streptomycin. Human
mammary epithelial cells (HMEC) were kindly given by Dr. Lacal (Instituto
de Investigaciones Biome´dicas, CSIC, Madrid, Spain) and grown in
mammary epithelial growth medium (Cambrex, East Rutherford, NJ)
according to the manufacturer’s instructions. Cannabinoid ligands were
prepared in DMSO. Control incubations had the corresponding DMSO
content. No significant influence of DMSO was observed on cell viability
at the final concentration used (0.1-0.2%, v/v). Cell viability was determined
by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test
(Sigma, St. Louis, MO) according to the manufacturer’s instructions.
Western blot analysis. Samples were subjected to SDS-PAGE, and
proteins were transferred onto polyvinylidene fluoride membranes. Blots
were incubated with the following antibodies: anti-phosphorylated Cdc2
(Tyr15), anti-cyclin B1, anti-Cdc25C, anti-p27, anti-caspase-3, and antipoly(
ADP-ribose) polymerase (PARP) (Cell Signaling, Beverly, MA); anti-
Cdc2 and anti-p21 (Santa Cruz, Santa Cruz, CA); anti-Wee1 (BioVision,
Mountain View, CA); anti-survivin (R&D Systems, Minneapolis, MN); and
anti-a-tubulin as loading control (Sigma). Luminograms were obtained with
an enhanced chemiluminescence detection kit (Amersham Life Sciences,
Arlington Heights, IL), and densitometric analysis was done with Multianalyst
software (Bio-Rad, Hercules, CA).
Cell cycle analysis. Cells were permeabilized and fixed in 1% (w/v)
bovine serum albumin and 30% ethanol-PBS and labeled with 5 Ag/mL
Hoechst 33342 (Molecular Probes, Leiden, the Netherlands). Fluorescence
intensity was analyzed using a LSR flow cytometer (Becton Dickinson, San
Jose, CA). Ten thousand cells per analysis were recorded.
Requests for reprints: Cristina Sa´nchez, Department of Biochemistry and
Molecular Biology I, School of Biology, Complutense University, 28040 Madrid,
Spain. Phone: 34-913944668; Fax: 34-913944672; E-mail: csg@bbm1.ucm.es.
I2006 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-05-4566
www.aacrjournals.org 6615 Cancer Res 2006; 66: (13). July 1, 2006
Research Article
Apoptosis. Cells were incubated in binding buffer [10 mmol/L HEPES
(pH 7.4), 2.5 mmol/L CaCl2, 140 mmol/L NaCl] supplemented with Annexin
V-FITC (Molecular Probes). Propidium iodide (PI; Sigma) was added
1 minute before sample analysis. Fluorescence intensity was analyzed using
a FACS Scalibur flow cytometer (Becton Dickinson). For triple staining
experiments, cells were labeled with Hoechst 33342, and apoptosis was
analyzed as described above. Ten thousand cells per analysis were
recorded.
Caspase-3 activity. Caspase-3/7 activity was determined with a
luminogenic substrate (Caspase-Glo, Promega, Madison, WI) according to
the manufacturer’s instructions. Luminescence was determined in a
Microplate Fluorescence Reader (BMG Labtech, Offenburg, Germany).
Tissue samples. Samples were obtained from the Centro Nacional de
Investigaciones Oncolo´gicas Tumor Bank (Madrid, Spain). Histologic
grade was assessed according to Elston and Ellis criteria. Immunohistochemical
staining for prognostic and predictive factors was done by
the EnVision method with a heat-induced antigen retrieval step.
Monoclonal antibodies for estrogen receptor (ER), progesterone receptor
(PR), p53 (Novocastra, Newcastle, United Kingdom), and Ki67 (DAKO,
Glostrup, Denmark) were used. ERBB2/HER-2 expression was evaluated
using Herceptest (DAKO). The percentage of cells with unequivocal
nuclear staining for ER, PR, Ki67, and p53 was scored, and a cutoff of
5% was used for positivity for ER and PR and 15% for Ki67 and p53. For
ERBB2/HER-2, only cases with 3+ membranous staining were scored as
positive.
Confocal microscopy analysis of cannabinoid receptors. Human
breast cancer and normal breast 5-Am paraffin-embedded tissue sections
were analyzed. Primary antibodies against CB1 and CB2 receptors (Affinity
Bioreagents, Golden, CO) were used. Secondary anti-rabbit antibody Alexa
Fluor 594 was from Molecular Probes. Cell nuclei were stained with YOYO-1
(Molecular Probes). Confocal fluorescence images were acquired using
Laser Sharp 2000 software (Bio-Rad).
Reverse transcription-PCR analysis. Total RNA was isolated using the
RNeasy Protect kit (Qiagen, Hilden, Germany). cDNA was obtained using
Figure 1. Effect of cannabinoids on
human breast cell proliferation. Cells were
incubated in low-FBS medium (2.5% for
MCF-7 and 0.5% for the rest of the tumor
cell lines) for 72 hours in the presence of
5 Amol/L THC (A), different concentrations
of THC ranging from 1 to 12 Amol/L (B),
or the specified THC concentration (C).
When antagonists were used (1 Amol/L
rimonabant and 2 Amol/L SR144528), they
were added 1 hour before THC. ER and
p53 status was obtained from ATCC and
German National Resource Centre for
Biological Material (DSMZ, Braunschweig,
Germany). IC50s were defined as
concentrations of THC required to
decrease cell viability to 50%. A, columns,
mean for each cell line (n z 3); bars, SE.
B, significant differences from MCF-7
(*, P < 0.05; **, P < 0.01) or T-47D cells
(b, P < 0 .0 5; bb, P < 0.01). For real-time
quantitative PCR experiments, an arbitrary
value of 1 was assigned to cannabinoid
receptor expression in MDA-MB 468 cells
(reference). Representative experiment
(n = 3). A and C, columns, means cell
viability in THC-treated cells versus their
respective vehicle-treated cells (set at
100%; n z 3); bars, SE. Significant
differences (*, P < 0.05; **, cc, P < 0 .0 1)
from control (*) or 5 Amol/L THC alone (c).
D, CB1 and CB2 mRNA expression in
the cell lines was determined by RT-PCR
(representative gels, n z 3). U373 MG
astrocytoma cells and Jurkat leukemia
cells were used as positive controls for CB1
and CB2, respectively.
Cancer Research
Cancer Res 2006; 66: (13). July 1, 2006 6616 www.aacrjournals.org
Transcriptor Reverse Transcriptase (Roche, Applied Science, Penzberg,
Germany). Primer sequences were CB1 (sense), 5¶-CGTGGGCAGCCTGTTCCTCA-
3¶; CB1 (antisense), 5¶-CATGCGGGCTTGGTCTGG-3¶; CB2 (sense),
5¶-TGGGACAGGGTCAGTACAAGT-3¶; CB2
(antisense), 5¶-CTTTGGCTCCTGGTGGTCT-
3¶; glyceraldehyde-3-phosphate dehydrogenase (GAPDH;
sense), 5¶-GGGAAGCTCACTGGCATGGCCTTCC-3¶; and GAPDH (antisense),
5¶-CATGTGGGCCATGAGGTCCACCAC-3¶.
Real-time quantitative PCR. Total RNA and cDNA were obtained from
frozen breast tumors or cell lines as described above. Taqman probes for
human CB1, CB2, and 18S RNA (used as internal reference) were from
Applied Biosystems (Foster City, CA).
Plasmids and transfections. pIRESpuro2 and pIRESpuro2-Cdc2 were
kindly given by Dr. Huang ( Johns Hopkins University, Baltimore, MD).
Transfections were carried out with Fugene 6 (Roche Applied Science,
Indianapolis, IN) according to the manufacturer’s protocol. Transfected
cells were selected with puromycin.
Statistical analysis. ANOVA with a post hoc analysis by the Student‘s-
Newman-Keuls’ test was routinely used. For cannabinoid receptor
expression in human samples, data were log transformed to achieve
normality in the distribution. An F-test was subsequently done to compare
equality of variances in each group, and a classic t test or a t test with
different variances was applied using the Welch modification.
Results
THCinhibits proliferation of human breast cancer cells.
Several human breast cell lines were incubated with THC, and
viable cell numbers were estimated. THC decreased proliferation in
all the tumor cells tested (Fig. 1A). Among the tumor cells, those
with more aggressive phenotype (ER) were more sensitive to THC
(Fig. 1B). Remarkably, nontumor HMEC cells were the most
resistant to cannabinoid treatment (IC50 >12 Amol/L; Fig. 1A).
Rimonabant, a selective CB1 receptor antagonist, did not block
THC effect in EVSA-T cells (Fig. 1C) or any of the other cell lines
studied (data not shown). In contrast, SR144528, a selective CB2
receptor antagonist, partially prevented the THC-induced decrease
of EVSA-T cell proliferation (Fig. 1C). Both reverse transcription-
PCR (RT-PCR; Fig. 1D) and real-time quantitative PCR experiments
(Fig. 1B) confirmed the expression of CB2 mRNA in this cell line,
whereas CB1 mRNA was undetectable (Fig. 1B and D).
THC-induced decrease of cell proliferation is due to the
blockade of the G2-M transition. We next sought to examine
whether an alteration of EVSA-T cell cycle underlies THC
antiproliferative effect. The cannabinoid increased the number of
cells in the G0-G1 compartment and, in parallel, decreased the
number of cells in S phase (Fig. 2A and C). At the highest
concentration tested (5 Amol/L), THC also produced the following:
(a) an increase in the number of cells in G2-M phases and (b) the
appearance of a population of hypodiploid cells (Fig. 2A and C).
The latter two effects were prevented by SR144528 (Fig. 2C).
Importantly, THC did not alter the cell cycle profile of HMEC cells
(Fig. 2B).
To analyze the precise mechanism of THC action, we first
studied the expression of several proteins involved in the G2-M
transition. THC decreased the total levels of Cdc2 [p34, cyclindependent
kinase (CDK) 1; Fig. 3A], the major CDK controlling the
entrance of cells in mitosis after completing G2 events (8), and
SR144528 (2 Amol/L) completely prevented this effect [relative
optical density (OD) after 16 hours of treatment relative to vehicle:
SR144528+THC, 105 F 4]. The expression of cyclin B1, the positive
regulatory subunit of Cdc2 (9), did not significantly change on
cannabinoid challenge (data not shown). The levels of p21, a CDK
inhibitor known to prevent Cdc2-cyclin B activation (8), were
enhanced by THC (Fig. 3A). It has been recently proposed that p27,
a CDK inhibitor traditionally associated to the regulation of G1-S
transition, can also inhibit Cdc2 at G2-M (10). In our system,
however, THC did not modify p27 levels (data not shown).
To be active, Cdc2 has to be dephosphorylated in the Tyr15
residue (11). THC treatment decreased the amount of phosphorylated
Tyr15-Cdc2 to a lower extent than total Cdc2 levels (Fig. 3A),
indicating that the ratio inactive/active Cdc2 was augmented by
THC. Phosphorylation of Cdc2 in Tyr15 is controlled by the Wee1/
Mik1 family of protein kinases and by the phosphatase Cdc25C
(11). Our results show that THC enhances Wee1 and reduces
Cdc25C protein levels (Fig. 3A).
In view of the aforementioned results, it is conceivable that
THC exposure prevents EVSA-T cells to reach the required levels
of active Cdc2 to enter mitosis. To test whether Cdc2 downregulation
is important in the growth-inhibiting effect of THC,
we heterologously expressed this kinase. As shown in Fig. 3B,
cells overexpressing Cdc2 became significantly more resistant
to THC.
Figure 2. Effect of THC on cell cycle
dynamics. Cell cycle profiles of EVSA-T
(A) and HMEC cells (B) incubated with
5 Amol/L THC or the corresponding vehicle
for 48 hours. Representative experiments
(n z 3). C, percentage of EVSA-T cells
(mean F SE) in every phase of the cell
cycle after 48 hours of incubation with the
indicated compounds. Where indicated,
rimonabant (SR1; 1 Amol/L) or SR144528
(SR2; 2 Amol/L) was added 1 hour
before THC. Significant differences from
control (*, P < 0.05; **, P < 0.01) or the
corresponding concentration of THC alone
(b, P < 0.05).
Control of Cell Cycle by Cannabinoids
www.aacrjournals.org 6617 Cancer Res 2006; 66: (13). July 1, 2006
THC-induced cell cycle arrest is associated with apoptosis.
We next tried to elucidate whether THC-induced inhibition of
proliferation was associated with cell death. The cannabinoid
induced apoptosis, a process that was prevented by SR144528
(Fig. 4A). THC also induced a two-fold increase in caspase-3
activity, an effect that was prevented by SR144528 (Fig. 4B).
Likewise, we observed reduced levels of both pro-caspase-3 (the
inactive precursor of caspase-3) and PARP (a caspase-3 substrate)
in cannabinoid-treated cells (Fig. 4C). We subsequently addressed
the question of whether apoptotic cells were those arrested in G2-M
by THC. We conducted triple staining experiments to analyze the
percentage of apoptotic cells in every phase of the cell cycle. As
shown in Fig. 5A, THC induced apoptosis in all the cell cycle
phases, but the majority of apoptotic cells were in the G2-M
compartment.
It is known that survivin, a member of the inhibitor of apoptosis
family, can be phosphorylated in Thr34 by Cdc2. This phosphorylation
results in enhanced stability of survivin and the
consequent inhibition of caspase activity (12). Survivin was highly
expressed in all the breast cancer cell lines tested but was hardly
detectable in HMEC (Fig. 5B), in agreement with previous reports
showing a sharp differential expression in cancer (high levels)
versus normal (undetectable levels) tissues (12). Moreover, THC
decreased survivin levels in EVSA-T cells (Fig. 5C), which may
explain why THC-induced Cdc2 inactivation results in apoptosis.
In fact, when Cdc2 levels were enhanced by overexpression,
survivin decrease on cannabinoid treatment was attenuated
(relative optical density after 16 hours of treatment for
pIRESpuro2-transfected cells: vehicle, 100 F 6; 5 Amol/L THC,
31 F 4. For pIRESpuro2-Cdc2-transfected cells: vehicle, 100 F 5;
5 Amol/L THC, 74 F 3; Fig. 5D).
Cannabinoid receptors are expressed in human breast
tumors. The presence of CB1 and CB2 receptors in human breast
tumors was evaluated by real-time quantitative PCR and confocal
microscopy. Lower levels of CB1 mRNA were detected in tumors
of low-medium (grade 1-2) and high (grade 3) histologic grade
compared with normal, noncancerous breast tissue (grade 1-2
versus noncancerous breast tissue (P = 0.008); grade 3 versus
noncancerous breast tissue (P = 0.0007); Fig. 6A). CB2 expression
was higher than CB1 expression in all the tumors analyzed (P =
0.00002) and seemed to correlate with their histologic grade
(grade 1-2 versus grade 3; P = 0.04; Fig. 6A). Of interest, CB2
transcripts were hardly detectable in normal breast tissue
(Fig. 6A). Moreover, the expression of CB2 showed an association
with molecular markers of prognostic value. Thus, ER tumors
expressed more CB2 mRNA than ER+ tumors (Fig. 6B). CB2
expression was also higher in PR than in PR+ samples. ER/
PR tumors (response rate <10% to conventional therapies)
Figure 3. Effect of THC on the expression
of proteins involved in the control of the
G2-M transition. A, Western blot analysis
of the indicated proteins was done at least
twice. Representative luminograms. T3,
3 Amol/L THC and T5, 5 Amol/L THC.
Optical density (arbitrary units, mean F
SE) of the different proteins relative to their
respective time-point control incubations
(set at 100). B, EVSA-T cell viability was
assayed after 72 hours of incubation
with 5 Amol/L THC or vehicle. Columns,
mean cell viability in THC-treated cells
versus their respective vehicle-treated
cells (set at 100%); bars, SE. Inset,
Western blot of total Cdc2 levels in
EVSA-T cells (lane 1), EVSA-T cells
transfected with pIRESpuro2 (lane 2),
and EVSA-T cells transfected with
pIRESpuro2-Cdc2 (lane 3). A and B,
significant differences from the
corresponding vehicle-treated cells
(*, P < 0.05; **, P < 0.01) or between
THC-treated cells transfected with
either pIRESpuro2 or pIRESpuro2-Cdc2
(b, P < 0.05).
Cancer Research
Cancer Res 2006; 66: (13). July 1, 2006 6618 www.aacrjournals.org
expressed more CB2 mRNA than ER+/PR+ tumors (60-70%
response rate; ref. 7; Fig. 6B). ERBB2/HER-2-positive tumors
(with worse survival prediction at least in node-positive patients;
ref. 7) expressed higher levels of CB2 mRNA than ERBB2/HER-2-
negative tumors (Fig. 6B). Confocal microscopy experiments
confirmed the presence of CB1 and CB2 proteins in tumor cells
(Fig. 6C). None of the receptor proteins was detected in normal
ducts.
Figure 4. Analysis of THC-induced cell
death. Fluorescence-activated cell sorting
analysis (A) and caspase-3 activity (B)
of EVSA-T cells after incubation with the
indicated compounds for 24 hours.
Antagonists (1 Amol/L rimonabant or
2 Amol/L SR144528) were added 1 hour
before THC (5 Amol/L). A, percentage
of apoptotic cells (FITC positive/PI
negative) within the total cell population.
B, columns, means of at least five or
more experiments; bars, SE. Significant
differences from control (*, P < 0 .0 5;
**, P < 0.01) or THC alone (b, P < 0 .0 5;
bb, P < 0.01). C, Western blot analysis of
pro-caspase-3 and PARP. T3, 3 Amol/L
THC and T5, 5 Amol/L THC.
Representative luminograms (n z 3).
Figure 5. Connection between cell
cycle arrest and apoptosis. A, percentage
of apoptotic cells in different phases
of the cell cycle as assessed by triple
staining with Annexin V-FITC, PI, and
Hoechst 33342. Significant differences
from the corresponding vehicle-treated
cells (*, P < 0.05; **, P < 0.01) or
between THC-treated cells in G2-M
and THC-treated cells in G0-G1 or S
(b, P < 0.05). B-D, Western blot of survivin
in different human breast cell lines (B),
EVSA-T cells incubated with THC (T3, 3
Amol/L THC; T5, 5 Amol/L THC) or the
corresponding vehicle (C), and EVSA-T
cells transfected with pIRESpuro2 or
pIRESpuro-Cdc2 and incubated for 16
hours with the indicated compounds (D).
Representative luminograms (n z 2).
Control of Cell Cycle by Cannabinoids
www.aacrjournals.org 6619 Cancer Res 2006; 66: (13). July 1, 2006
Discussion
There is scant information on the effect of cannabinoids on the
cell cycle. It has been shown previously that the endogenous
cannabinoid anandamide arrests the cycle of hepatoma HepG2
cells (13), epidermal growth factor–stimulated PC3 prostate cancer
cells (14), and the breast cancer cell line EFM-19 (15) at the G1-S
transition. The same effect was exerted by a metabolically stable
analogue of anandamide, met-F-anandamide, on KiMol thyroid
carcinoma cells (16). Results presented herein (a) show that the
plant-derived cannabinoid THC is able to block the progression of
breast cancer cell cycle and (b) provide a mechanism for this
action. Of interest, nontumor mammary epithelial cells were rather
insensitive to THC, suggesting that cannabinoids would fulfill one
of the demanded requirements of any compound intended to be
used in cancer therapy: the selectivity for tumor cells. In our
experimental model, THC induces a CB2-mediated cell cycle arrest
at the G2-M transition via Cdc2 down-regulation. We are
nonetheless aware that our observation that THC effects are
significantly but not completely prevented by the CB2 selective
antagonist also points to the existence of CB2-independent
Figure 6. Cannabinoid receptor
expression in human breast tumors.
A and B, mRNA expression (medians F
SE) of CB1 and CB2 receptors as
determined by real-time quantitative PCR.
An arbitrary value of 1 unit was assigned to
CB2 expression in one of the tumors
(reference), and the relative expression of
the rest of the samples was calculated
by comparison with the reference. Note the
different scale in the y axes of (A).
Significantly different values (*, P < 0 .0 5;
**, P < 0.01) from ‘‘+ variable’’ or ‘‘+/+
variable.’’ ER; PR; S1, <2 cm; S2, 2 to
5 cm; and S3, >5 cm; metastasis was
determined as presence/absence of
positive axillary lymph nodes. C, confocal
microscopy analysis of cannabinoid
receptors in a grade 1 (left ) and a grade
3 tumor (right ). Green, cell nuclei; red,
cannabinoid receptors. Arrows, different
areas within the tumor samples.
Cancer Research
Cancer Res 2006; 66: (13). July 1, 2006 6620 www.aacrjournals.org
processes in cannabinoid antiproliferative action. Most cancer cells
evade antigrowth signals because they have defective G1 checkpoints,
which makes the G2 checkpoint an attractive target for
cancer therapy (11). Since its discovery in 1986 (17), it has been
clear that Cdc2 is essential for cell cycle progression (18). In fact,
Cdc2-deficient mice die at very early stages of embryonic
development (18). The pivotal importance of this particular protein
could explain why, in our system, a somewhat moderate decrease
of total Cdc2 levels (f40%) results in large changes in cell viability.
In addition, our data indicate that THC increases the inactive/
active Cdc2 ratio, supporting that the cannabinoid decreases not
only total Cdc2 levels but also enzyme-specific activity.
It is important to point out that, although the antiproliferative
effect of cannabinoids on different tumor cells has been extensively
confirmed both in vitro and in vivo (6), a recent report indicates
that THC may enhance breast cancer cell growth under certain
circumstances. In that study, the authors showed a direct
association between the degree of sensitivity of a tumor to THC
and the level of CB1 and CB2 expression. Thus, THC has
antiproliferative effect in tumors expressing cannabinoid receptors,
whereas those with low to no expression suffer increased growth
and metastasis due to THC-induced suppression of the antitumor
immune response (19). Results presented herein are not in
disagreement with that report, as EVSA-T cells, which are very
sensitive to THC, express high levels of CB2.
By real-time quantitative PCR experiments, we have observed a
correlation between CB2 expression and the histologic grade of
breast tumors. Moreover, CB2 expression is higher in tumors with
predicted low response to conventional therapies, for instance
ER/PR tumors, which are weakly responsive to adjuvant
tamoxifen (7). In addition, hardly detectable levels of CB2 were
found in normal breast tissue. Because both cell cycle arrest and
apoptosis induced by THC are CB2-mediated effects, it is tempting
to speculate that the tumors with poor prognosis (i.e., those
reluctant to conventional therapies and, according to our data,
expressing the highest levels of CB2 receptor), may be the most
responsive to cannabinoids. In addition, the psychotropic effects of
cannabinoids are mediated by CB1 but not CB2 receptors, and,
therefore, a cannabinoid-based therapy selectively targeting CB2
receptors would be devoid of the side effects associated to
cannabis consumption (20).
Breast cancer is the most common malignant disease among
Western women. Although the rates of mortality of breast cancer
patients have decreased as a result of early diagnosis by
mammograms, certain breast tumors remain reluctant to conventional
therapies, and current treatments have side effects that
substantially affect the patient’s quality of life (21). Our findings
might set the basis for new strategies for the management of
breast cancer.
Acknowledgments
Received 12/22/2005; revised 4/6/2006; accepted 5/2/2006.
Grant support: Fondo de Investigaciones Sanitarias FIS 04/1029 (C. Sa´nchez) and
BEFI 01/9132 (D. Sarrio´); Complutense University PR1/05 (C. Sa´nchez); Ministerio de
Educacio´n y Ciencia SAF2003-00745 (M. Guzma´n), SAF2004-08258-C02-01
( J. Palacios), and AP2003-0063 (M.M. Caffarel); and Fundacio´n Cientı´fica de la
Asociacio´n Espan˜ola Contra el Ca´ncer (M. Guzma´n).
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank the staff at Centro de Microscopı´a y Citometrı´a (Complutense University,
Madrid, Spain) and A. A´lvarez-Barrientos for expert advice on flow cytometry
experiments; O. Rueda for expert advice on statistical analysis; and the members of our
laboratory for technical support, especially to K. Blazej, C. Bla´zquez, D. Callegarin,
A. Egia, B. Herrera, and M. Lorente.
References
1. Malumbres M, Barbacid M. To cycle or not to cycle: a
critical decision in cancer. Nat Rev Cancer 2001;1:222–31.
2. Guzman M, Sanchez C, Galve-Roperh I. Cannabinoids
and cell fate. Pharmacol Ther 2002;95:175–84.
3. Velasco G, Galve-Roperh I, Sanchez C, Blazquez C,
Guzman M.Hypothesis: cannabinoid therapy for the treatment
of gliomas? Neuropharmacology 2004;47:315–23.
4. Gaoni Y, Mechoulam R. Isolation, structure, and
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transition. Biochim Biophys Acta 2001;1519:1–12.
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Skp2-mediated degradation of p27 regulates progression
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induced hepatocyte apoptosis. Hepatology 2003;
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and ceramide production. Prostate 2003;56:1–12.
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breast cancer cell proliferation. Proc Natl Acad Sci U S A
1998;95:8375–80.
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Di Marzo V, Bifulco M. Inhibitory effects of cannabinoid
CB1 receptor stimulation on tumor growth
and metastatic spreading: actions on signals involved
in angiogenesis and metastasis. FASEB J 2003;17:
1771–3.
17. Russell P, Nurse P. Schizosaccharomyces pombe and
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18. Malumbres M, Barbacid M. Mammalian cyclindependent
kinases. Trends Biochem Sci 2005;30:630–41.
19. McKallip RJ, Nagarkatti M, Nagarkatti PS. D-9-
tetrahydrocannabinol enhances breast cancer growth
and metastasis by suppression of the antitumor
immune response. J Immunol 2005;174:3281–9.
20. Sanchez C, de Ceballos ML, del Pulgar TG, et al.
Inhibition of glioma growth in vivo by selective
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Control of Cell Cycle by Cannabinoids
www.aacrjournals.org 6621 Cancer Res 2006; 66: (13). July 1, 2006The give and take for charity
Matthew Putney / For The Times
For-profit fundraisers collect huge sums on behalf of worthy causes, but nonprofits see only a fraction of it.
By Doug Smith and Charles Piller
July 06, 2008
For 24 years, Citizens Against Government Waste has exposed pork-barrel spenders and rallied tax critics.
Its “Pig Book” and “porker” awards, meant to shame congressional leaders who exploit the public purse, have made the group a media darling and a political force.
But when it comes to policing its own fundraising practices, America’s self-proclaimed “#1 taxpayer watchdog” seems to have lost its bite.
Records filed with the California attorney general’s office show that over the last decade, for-profit fundraisers for the nonprofit kept more than 94 cents of every donated dollar. Yet donors could write off the full contribution on their tax returns.
“It’s a rip-off of the taxpayer,” said Pablo Eisenberg, senior fellow at Georgetown Public Policy Institute and a philanthropy scholar.
A Times investigation found hundreds of other examples of charities that pocketed just a sliver of what commercial fundraisers collected in their names. Some didn’t get a dime or even lost money.
According to a comprehensive review of state records filed over a decade, the problem of paltry returns extends well beyond what has been reported in recent years among benevolent societies for police, firefighters and veterans. It affects charities large and small, well-known and obscure. It spans a range of causes, including child and animal welfare, health research and opposition to drunk driving.
In more than 5,800 campaigns on behalf of charities that were registered with the state attorney general from 1997 to 2006, the fundraisers reported taking in $2.6 billion. They kept nearly $1.4 billion – about 54 cents of every dollar raised.
These numbers reflect only part of the problem. Though commercial fundraisers are required to file detailed fundraising reports with the state, many do not, and the law is not aggressively enforced because of limited staffing.
For-profit campaigns, which often employ telemarketing, mass mailings or one-time events, account for a small fraction of $223 billion in charitable giving each year in the United States. But they collect significant sums and help shape public perceptions of charities. Pairing computer-controlled dialing systems with low-wage workers, such firms can reach a large number of people in a short time.
“If I could forget about what percentage was going where,” said Dan Halfeldt, former sales manager at one Phoenix-based telemarketer for nonprofits, “I could really say, ‘Wow, I’m raising money for something good.’ ”
The firm, Midwest Publishing Inc., consistently offered among the lowest returns by large fundraisers. It did not respond to written questions about its operations.
The fundraising business is growing. More than 300 fundraisers have registered in California. Since 2000, the number of campaigns and amounts raised by for-profit firms has risen by about two-thirds.
Among the charities that netted little from such campaigns were the Humane Society of the United States, the American Breast Cancer Foundation, the Christian social-action group Concerned Women for America, the National Right to Life Committee and Students Against Destructive Decisions.
Among The Times’ findings:
* More than 100 charities raised $1 million or more from commercial appeals but netted less than 25 cents per dollar. Fundraisers got the rest.
* In 430 campaigns, charities got nothing: All $44 million donated went to fundraisers. In 337 of those cases, charities actually lost money, paying fees to fundraisers that exceeded the amount raised.
* In hundreds of other campaigns, charities apparently entered into contracts that limited their share of donations to 20% or less, no matter how successful the campaign.
* Groups with strong emotional or patriotic appeal – those supporting animals, children, veterans and public safety workers, for instance – often fared worst. Missing-children charities received less than 15% of more than $28 million raised on their behalf.
Many rely heavily on set-rate contracts and aim their campaigns toward a mass audience, which is less efficient than targeting a defined set of donors.
In general, charities argue that it takes money to make money and that the benefits of commercially run campaigns may not show in state filings.
For instance, Citizens Against Government Waste said that its telemarketing was meant to find donors who would give regularly, not necessarily to raise a large sum in a single campaign.
To critics, that argument often excuses wastefulness or profiteering – and every charity pays for that.
Some charities “take advantage of American generosity,” said Bennett Weiner, an executive with the Better Business Bureau research program Wise Giving Alliance. They “accomplish very little, siphon off good money from the community and tarnish the well for more legitimate nonprofits.”
By donating to inefficient charities, said Daniel Borochoff, president of the American Institute of Philanthropy, “you are taking money out of the mouth of a hungry kid.”
Urgent accounts
“Where are the children? Where did they go?” a child’s mournful voice sings on the website of Operation Lookout – National Center for Missing Youth. “Are they held captive or do they run free? … Oh, where can they be?”
The group’s online, telemarketing and door-to-door outreach materials offer urgent accounts of runaways or kidnapping victims – and equally urgent pleas for cash.
Commercial firms reported having raised more than $6 million for Operation Lookout, based in Everett, Wash., since 1999. Less than $1 million reached the charity.
A current contract between the fundraiser and the charity sets a minimum 15% return on donations. In practice, over the years, 15% has been the maximum. In each year from 2000 to 2006, it received exactly 15% – not a penny more or less – of funds collected in its name by commercial fundraiser Midwest Publishing.
Operation Lookout did not respond to requests for comment.
The charity has posted a defense of its fundraising on its website, blaming accounting requirements for distorting its results: “In some instances, accounting rules require that we combine volunteer recruitment, calls to action and public education making them appear as a ‘fundraising’ expense.”
Neither of the nation’s two major accounting standards bodies has ruled that such costs must be combined.
Based on tax returns and other sources, the American Institute of Philanthropy ferrets out hidden costs of fundraising by charities – including but not limited to commercial campaigns. It estimated that Operation Lookout spent 84 cents to raise each dollar in its 2006 budget of $2.1 million, a worse record than all but 18 of more than 500 rated charities.
The philanthropy institute normally considers up to 35 cents to raise a dollar as a reasonable cost.
The Times analysis of commercial campaigns in California found inefficiency to be typical among missing-child groups: On average more than 85 cents per dollar went to the fundraiser.
However, some charities of this kind eschew commercial fundraisers, including the National Center for Missing and Exploited Children. Ernie Allen, its chief executive, said groups that rely on minuscule returns taint the entire field.
Patty Wetterling co-founded the Jacob Wetterling Foundation in 1990, after her 11-year-old son was kidnapped at gunpoint. Jacob has not been found.
“Missing children’s organizations spring up out of crisis,” she said. “Fundraisers exploit these devastating situations.”
But groups shouldn’t allow themselves to be overcharged by fundraisers over and over, she said. They “need to learn.”
6% for the charity
Vickie Bouska, a retiree in Hiawatha, Iowa, became a donor to Citizens Against Government Waste after she heard leaders’ arguments on television and received a fundraising letter. The group addressed her fears about the future.
“I’m scared to death of what we’re leaving our children,” she said. “The Chinese or the Canadians are going to own us, because no one in Washington can close the pocketbooks.”
But she was stunned to learn from a reporter how much money the group ceded to its commercial fundraisers. Of $879,000 raised in the decade examined, $49,000 – 6% – reached the charity.
“They aren’t really getting the money,” Bouska said, “so why am I sending it?”
Thomas Schatz, president of the Washington, D.C.-based group founded by industrialist J. Peter Grace and muckraking journalist Jack Anderson, defended its fundraising.
“The purpose of telemarketing is not always to ‘come out ahead,’ though that’s always the goal, but to reinforce [donors] for the future,” he said.
The Los Angeles-based fundraiser Facter Direct, which conducted the group’s telemarketing campaigns, declined to comment on its work for Citizens Against Government Waste. On average, state data show, it returns 39% of what it raises to its clients, slightly less than the industry norm.
“The numbers on the surface don’t always tell the whole story, said the firm’s president, Tom Siegel. “Organizations are not stupid. They recognize the cost of telemarketing and the annoyance of it. But … they recognize that it’s one of the most effective ways to raise money” over the long run.
Schatz noted that the telemarketing campaigns reported to the state reflect a small portion of his organization’s overall fundraising. Direct mail by commercial solicitors – an approach used by the group for two decades – provides most of its approximately $5.4 million in annual revenue and is more efficient, he said.
It proved impossible to verify that claim, because only one direct-mail campaign of behalf of Citizens Against Government Waste was registered with the state, as required, in the 10-year period. That one showed no revenue.
Citizens Against Government Waste suggested that its overall fundraising was highly efficient – costing just 19 cents per dollar raised, according to its 2006 tax return.
But the group came up with that figure by designating most direct-mail and telemarketing costs as “services.” The practice is legal if information, such as criticism of pork-barrel projects, is included in fundraising pitches. The American Institute of Philanthropy estimated the actual cost of the group’s overall fundraising at 69 cents per dollar.
Some other economic-policy nonprofits steer clear of commercial fundraisers, emphasizing foundation grants and direct contact with donors.
Telemarketing and mass mailings can raise awareness, but “there’s always something about it that rubs me the wrong way,” said Ryan Alexander, president of Taxpayers for Common Sense in Washington, D.C. “It’s important that people know that their dollars are going to the mission.”
Joseph Bast, president of the Heartland Institute, a Chicago nonprofit that promotes free-market approaches to environmental and social problems and accepts corporate donations, said he didn’t trust telemarketers to safeguard his group’s image, especially when returns can be low.
“It’s not a very good risk,” he said.
Costs and credibility
American generosity has its limits. The charitable pie in the U.S. has accounted for about 2% of the gross domestic product since 1970.
Meanwhile, the number of charities has risen dramatically. More than 1.9 million nonprofits – one for every 150 U.S. citizens – fill gaps in government services, give voice to diverse views and compete fiercely for this limited share of donor dollars.
Tax laws, designed to encourage giving, don’t take into account how much money reaches the charitable cause. Donors to commercially run drives can write off the entire donation, even if most of it goes to the fundraiser.
Lawmakers periodically have considered reining in the fundraising industry. But the U.S. Supreme Court has limited their options by upholding the free-speech rights of fundraisers and charities.
Recently, public confidence has been shaken by news of inefficient for-profit fundraisers used by police and firefighter charities. And congressional hearings depicted some veterans’ charities as little more than shells that enrich fundraisers and executives.
In a national survey conducted in March, 70% of those polled said charities waste “a great deal” or “a fair amount” of their funds.
Charities “may be losing their most prized possession: their reputation for caring,” said Paul C. Light, a professor of public service at New York University who wrote the survey and has studied nonprofit governance for more than two decades.
Many charities hire for-profit fundraisers precisely because they need help selling themselves to a skeptical public. They can be “a godsend and a lifesaver,” particularly for charities without staff fundraisers, said Diana Aviv, chief executive of Independent Sector, a trade group for nonprofits.
Limited information is available about such firms; most are privately held and many shun the media.
Those that specialize in nonprofits with long-standing patron networks tend to offer better returns. New York-based telemarketer DCM Inc. works exclusively for arts groups, including the Los Angeles Philharmonic, targeting ticket buyers and former donors. It enjoys one of the best records in the business, returning, on average, 72 cents per dollar raised.
“I don’t think that telemarketing is a great contribution to Western civilization, but art is,” said Phil Miller, DCM’s president. “We provide a good service for great organizations.”
Even so, many charities get stuck with unfavorable terms.
A contract between Michigan-based TeleService USA and the veterans charity VietNow makes donor lists “the exclusive property” of the fundraiser. If the charity switches fundraisers, it would lose access to past supporters – the most likely future donors.
TeleService USA, which delivered to its clients, on average, 11 cents per dollar raised, did not respond to requests for an interview.
Hundreds of charities follow the practice used by Operation Lookout, many accepting set-rate contracts that deliver a sliver of gross donations. For small charities, the contracts seem to minimize risk: Fundraisers pay upfront costs, and charities get guaranteed – if often small – returns.
But major charities are the dominant users of for-profit fundraising, whether to raise money for basic costs or to expand a donor base. Among 1,614 charities with commercial campaigns registered in California, 100 accounted for 73% of gross donations. Just two, Paralyzed Veterans of America and the American Diabetes Assn., together took almost 14%. In theory, large charities use for-profit firms more efficiently because they can tap long-standing donor networks. In practice, they do little better than small ones.
Fundraisers for these major groups, meanwhile, reaped a windfall. Those for the 100 top-grossing charities received nearly $977 million, and firms for just the top two pocketed $171 million combined.
“Often the most popular causes … solicit everyone under the sun to get a few dollars from almost everyone,” said Borochoff, of the American Institute of Philanthropy. “That’s a really expensive way to raise money.”
Donors often have no idea where their money goes.
Disillusioned donor
Pamela Kay Weeks lost both breasts to cancer. After she recovered, she received letters and calls from the American Breast Cancer Foundation, a Baltimore charity that listed research as a priority. Hoping to spare others what she suffered, Weeks wrote a check – the first of many.
“I’m not a wealthy person,” said Weeks, 51, an executive secretary who lives in Sneads, Fla. “I’ve given what I can.”
Weeks thought her donations were headed for a philanthropic powerhouse. But the foundation is one of the nation’s least efficient charities, according to the American Institute of Philanthropy and Charity Navigator, another watchdog group.
In 2006 just 2.5% of the American Breast Cancer Foundation budget went to research and 10.5% to mammograms or other services unrelated to fundraising.
In reports filed with the California attorney general from 2003 through 2006, the foundation said it raised $5.8 million from fundraisers, netting just $700,000, or 12%.
The foundation had a particularly close link to one fundraiser, although there was no sign of it in state files.
Phyllis Wolf, executive director of the foundation, created the charity in 1997 with her son Joseph Wolf and two friends. The son worked for the foundation in its early years, then began a for-profit fundraising firm called Non Profit Promotions.
From 2002 through 2006, the foundation paid Non Profit Promotions an average of almost $3 million annually, according to foundation tax returns. That’s not illegal, but it violated conflict-of-interest policies used by several large charities and a model policy by the Better Business Bureau.
And despite state laws requiring fundraisers to submit results, no reports by Non Profit Promotions were on record in California or other states whose files were checked by The Times.
Neither Phyllis Wolf nor Joseph Wolf replied to written questions about their fundraising.
Weeks was surprised – and saddened – to hear where most of the donors’ money went.
“I figured that a lot of it goes to paying people to research cancer,” she said. “It’s unreal, hard to believe.”
charles.piller@latimes.com
doug.smith@latimes.com
Researchers Maloy Moore and Scott Wilson contributed to this report.
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Copyright 2008 Los Angeles Times
Records filed with the California attorney general’s office show that over the last decade, for-profit fundraisers for the nonprofit kept more than 94 cents of every donated dollar. Yet donors could write off the full contribution on their tax returns.
“It’s a rip-off of the taxpayer,” said Pablo Eisenberg, senior fellow at Georgetown Public Policy Institute and a philanthropy scholar.
A Times investigation found hundreds of other examples of charities that pocketed just a sliver of what commercial fundraisers collected in their names. Some didn’t get a dime or even lost money
The Sin in Doing Good Deeds?
December 30, 2008
If a businessman rakes in a hefty profit while doing good works, is that charity or greed? Do we applaud or hiss?
A new book, “Uncharitable,” seethes with indignation at public expectations that charities be prudent, nonprofit and saintly. The author, Dan Pallotta, argues that those expectations make them less effective, and he has a point.
Mr. Pallotta’s frustration is intertwined with his own history as the inventor of fund-raisers like AIDSRides and Breast Cancer 3-Days - events that, he says, netted $305 million over nine years for unrestricted use by charities. In the aid world, that’s a breathtaking sum.
But Mr. Pallotta’s company wasn’t a charity, but rather a for-profit company that created charitable events. Critics railed at his $394,500 salary - low for a corporate chief executive, but stratospheric in the aid world - and at the millions of dollars spent on advertising and marketing and other expenses.
Taken from New York Times, thanks to Karen for sending on
“Shame on Pallotta,” declared one critic at the time, accusing him of “greed and unabashed profiteering.” In the aftermath of a wave of criticism, his company collapsed.
One breast cancer charity that parted ways with Mr. Pallotta began producing its own fund-raising walks, but the net sum raised by those walks for breast cancer research plummeted from $71 million to $11 million, he says.
Mr. Pallotta argues powerfully that the aid world is stunted because groups are discouraged from using such standard business tools as advertising, risk-taking, competitive salaries and profits to lure capital.
“We allow people to make huge profits doing any number of things that will hurt the poor, but we want to crucify anyone who wants to make money helping them,” Mr. Pallotta says. “Want to make a million selling violent video games to kids? Go for it. Want to make a million helping cure kids of cancer? You’re labeled a parasite.”
I confess to ambivalence. I deeply admire the other kind of aid workers, those whose passion for their work is evident by the fact that they’ve gone broke doing it. I’m filled with awe when I go to a place like Darfur and see unpaid or underpaid aid workers in groups like Doctors Without Borders, risking their lives to patch up the victims of genocide.
I also worry that if aid groups paid executives as lavishly as Citigroup, they would be managed as badly as Citigroup.
Yet there’s a broad recognition in much of the aid community that a major rethink is necessary, that groups would be more effective if they borrowed more tools from the business world, and that there is too much “gotcha” scrutiny on overhead rather than on what they actually accomplish. It’s notable that leaders of Oxfam and Save the Children have publicly endorsed the book, and it’s certainly becoming more socially acceptable to note that businesses can also play a powerful role in fighting poverty.
“Howard Schultz has done more for coffee-growing regions of Africa than anybody I can think of,” Michael Fairbanks, a development expert, said of the chief executive of Starbucks. By helping countries improve their coffee-growing practices and brand their coffees, Starbucks has probably helped impoverished African coffee farmers more than any aid group has.
Mr. Fairbanks himself demonstrates that a businessman can do good even as he does well. Rwanda’s president, Paul Kagame, hired Mr. Fairbanks’s consulting company and paid it millions of dollars between 2000 and 2007.
In turn, Mr. Fairbanks helped Rwanda market its coffee, tea and gorillas. Rwandan coffee now retails for up to $55 a pound in Manhattan, wages in the Rwandan coffee sector have soared up to eight-fold, and zillionaires stumble through the Rwandan jungle to admire the wildlife. President Kagame thanked Mr. Fairbanks by granting him Rwandan citizenship.
There are lots of saintly aid workers in Rwanda, including the heroic Dr. Paul Farmer of Partners in Health, and they do extraordinary work. But sometimes, so do the suits. Isaac Durojaiye, a Nigerian businessman, is an example of the way the line is beginning to blur between businesses and charities. He runs a for-profit franchise business that provides fee-for-use public toilets in Nigeria. When he started, there was one public toilet in Nigeria for every 200,000 people, but by charging, he has been able to provide basic sanitation to far more people than any aid group.
In the war on poverty, there is room for all kinds of organizations. Mr. Pallotta may be right that by frowning on aid groups that pay high salaries, advertise extensively and even turn a profit, we end up hurting the world’s neediest.
“People continue to die as a result,” he says bluntly. “This we call morality.”
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An Iranian rocket carrying the satellite "Omid," or "Hope" in English, in this photo released by the Fars News Agency at an undisclosed location on Monday. (Fars News Agency via The Associated Press)
Iranian satellite launch prompts concern
By Alan Cowell and William J. Broad
Published: February 4, 2009
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Iran said Tuesday that it had launched its first domestically produced satellite, a move that has prompted concerns in the United States and other nations about Iran's nuclear ambitions and its ability to deploy long-range ballistic missiles.
The launch on Monday, coinciding with celebrations marking the 30th anniversary of the Islamic revolution, also creates an early challenge for President Barack Obama, who has sought to strike a conciliatory tone toward Iran by conditionally offering dialogue after years of tensions. The United States and other nations believe Tehran wants to develop nuclear weapons, a charge that Iran's leaders deny.
Iran said it had also used a domestically produced rocket to launch the satellite. That would make it part of the exclusive club of states that can loft objects into orbit, which now numbers at least nine. Weapons experts say the same technology used to put satellites into orbit can also be used for launching weapons.
In Washington, State Department spokesman Robert Wood called the reports a matter of "great concern" and potentially in violation of United Nations agreements limiting Iran from missile activity.
"Developing a space launch vehicle that could be put a satellite into orbit could possibly lead to the development of a ballistic missile system," he said.
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The head of Israel's Space Agency, Zvi Kaplan, said initial reports showed that a satellite had been launched.
"From what I have been investigating it is true," he said, according to The Associated Press. "We are not surprised because in this day and age of information and technology and with Iranian scientists studying abroad they can obtain the knowledge."
The official Iranian news agency, IRNA, said the satellite was launched using a Safir-2 rocket and was "successfully sent into orbit."
The satellite, which weighed about 60 pounds, is named Omid, or Hope, IRNA said, and was sent into space as a "data-processing satellite project" that began in March 2005 as "the first practical step toward acquiring national space technology."
"The project's experts focused on manufacturing the equipment and helping develop the potential of domestic companies to carry out such projects," the IRNA report said.
David Albright, the president of the Institute for Science and International Security, a private group in Washington that tracks nuclear proliferation, said he felt the technology used by Iran to launch the satellite remained rudimentary by international standards.
"It's not a very capable missile. The payload and diameter aren't that great," he said. "It doesn't say much, if anything, about their ability to deliver a nuclear weapon. But part of the concern here is that Iran is continuing its steady drip-drip-drip toward a nuclear weapons capability."
Aerospace experts said that while Tehran was still not capable of launching an intercontinental ballistic missile, the satellite still took many years of preparation and required the mastery of staging — the art of creating a multistage rocket that drops stages (and thus weight) as it races ever higher.
Last August, Iran test-fired a new rocket capable of carrying a satellite into orbit. Western experts said at the time that the launching represented a potentially significant, if much-delayed, step in Iran's efforts to join the international space club. And in 2005, Tehran launched a Russian-made satellite with a Russian-made rocket.
"They've been at this game for about five years, working on a rocket big enough to put a satellite into space," said Charles Vick, an expert on Iranian rockets at GlobalSecurity.org, a private research group in Alexandria, Virginia.
He added that the Iranian rocket had two stages. If carrying a warhead, he said, the Iranian missile could fire the weapon about 1,550 miles.
For Iran to achieve the technical step of launching an intercontinental ballistic missile, Vick said, it would have to develop a more powerful basic rocket or more upper stages — goals it is pursuing.
Since Obama's inauguration, outside powers have been looking for clues as to whether Tehran is prepared to make new concessions in the dispute over Iran's alleged efforts to develop nuclear weapons. They have also been waiting to see how the new American president will pursue his overtures of dialogue, which are designed to halt Iran's uranium enrichment program.
Nations dealing with the Iran issue — the United States, Russia, Britain, China, France and Germany — will meet Wednesday in Frankfurt, Germany, for their first talks since Obama took office.
Iranian state television on Tuesday showed footage of a rocket blasting off from a firing platform in a huge blast of smoke flame as it clambered into the night sky.
Breast Cancer Month Spurs Pink-Product Debate
Run Date: 10/21/04
By Rebecca Vesely
WeNews correspondent
October is Breast Cancer Awareness Month and many companies have been peddling pink products to help the cause. Critics, however, say it's a dubious way to raise funds and urge shoppers to know how businesses handle the proceeds.
Save Lids to Save Lives
SAN FRANCISCO (WOMENSENEWS)--Seeing pink these days?
You're not the only one. From Estee Lauder's pink lipstick to pink Keds to Conair pink travel hair dryers to Jimmy Choo hot pink high heels, numerous companies are peddling pink products.
That's because October is National Breast Cancer Awareness Month, which each year raises millions of dollars for breast cancer charities and reminds women that early detection works.
So what's wrong with that?
A lot, says Breast Cancer Action, a San Francisco nonprofit that this month launched "Think Before You Pink," an awareness campaign that casts a critical eye on social marketing campaigns for breast cancer.
"Companies have figured out that if you slap a pink ribbon on a product, people will buy it," says Barbara Brenner, executive director of Breast Cancer Action. "It's time to look past the ribbon and ask, 'Where is the money going?'"
Some manufacturers clearly state on their packaging how much money is going to a specific organization and who will benefit. Others aren't so clear. They say only that a "portion of proceeds" or, more commonly, a "generous portion of proceeds" will go to breast cancer research.
In a partnership with journalist and activist Barbara Ehrenreich, Breast Cancer Action is urging consumers to ask where their money goes after they buy a pink product.
Consumers, Brenner says, should educate themselves on how much their purchases will really help breast cancer organizations financially and how those groups, in turn, use the money. If a retailer can't tell them, they should walk away.
Breast Cancer Action is also calling for more coordination within breast cancer research funding to speed work toward a cure. Although millions of dollars pour into breast cancer research, no one knows exactly how much money is being raised and spent every year or where all the money goes, Brenner says.
The group is suggesting a worldwide effort, along the lines of polio eradication, to find a cure for breast cancer. Since 1994, California has been coordinating its efforts on a state level under the California Breast Cancer Research Program, and Brenner says it's a good model. Funded by a tobacco tax and a tax check-off on state income tax forms and administered by the University of California, the program has awarded nearly $165 million to breast cancer research and education in the state.
Yoplait Lid Drive
Breast Cancer Action is particularly critical of the "Save Lids to Save Lives" by the Yoplait. General Mills, which owns Yoplait, donates 10 cents to breast cancer research for every lid consumers send back to the company in September and October.
At that rate, each consumer would have to eat three Yoplait yogurts a day for the month of October to raise just $36, excluding postage, Breast Cancer Action points out.
Pam Becker, spokeswoman for Minneapolis, Minn.-based General Mills, says the company is proud of the campaign, which has been running for seven years, and will raise $2.1 million for the Susan G. Komen Breast Cancer Foundation, in Dallas, this year. About $1.1 million will come from the lid campaign and another $900,000 will be from a company donation.
"That contribution is not insignificant," Becker says. "And pink lids help raise awareness."
Beyond Awareness
Brenner says that at this point, awareness isn't enough.
"At this point aren't we all aware of breast cancer?" she retorts. "With 40,000 American women dying from breast cancer each year, shouldn't we focus on solving the problem?"
Breast Cancer Action is also critical of Yoplait because some of its yogurts may contain recombinant bovine growth hormone, a drug used to increase milk production in dairy cows.
Some companies have moved to eliminate rBGH from their products and Canada and the European Union have both banned rBGH due to concerns about links between a number of illnesses and growth hormones.
The U.S. Food and Drug Administration approved rBGH in 1993 after conducting a safety review.
General Mills' Becker says more awareness is needed to encourage women to do self-breast exams and mammograms.
"One thing that is helping people these days is early detection and we need to encourage that," she says. She also points out that no direct link between rBGH and breast cancer has been established.
Environmental Links
A study released this month by the Breast Cancer Fund, a San Francisco environmental group, and Breast Cancer Action indicates that as many as half of all breast cancer cases remain unexplained by personal characteristics, such as genetics. The report points out that numerous researchers believe many cases are linked to environmental factors. About 216,000 women will be diagnosed with breast cancer in the United States this year.
Shelley Alpern, vice president and manager of social research at Trillium Assets Management Corp., a Boston-based socially responsible investment firm, has looked closely at social marketing and breast cancer. She says the Breast Cancer Action is on the right track.
"There's the whole question of where the proceeds go and how much of it goes directly to the campaign," Alpern says. "The answer is, 'however much the company decides.'"
Alpern says consumers who are collecting yogurt-container lids to raise money for breast cancer would be helping more by giving directly to the Komen Foundation. Consumers should also be wary of any campaign, she adds, with an "inherent conflict of interest," such as a car dealership running a breast cancer campaign.
"The product pumps out carcinogens, and yet they are trying to pinkwash their own image."
Corporate 'Pinkwashing'
"Pinkwashing," or aligning a product with the fight against breast cancer, helps companies associate themselves with a good cause, Alpern says.
But companies that produce pink products say their contribution has been significant.
"The progress in the last 15 years, since the cosmetic companies have been involved, has been tremendous," says Irene Malbin, vice president of the Cosmetic, Toiletry and Fragrance Association, a trade group in Washington, D.C. "Awareness programs have not only helped educate women about the benefits of early detection, but the media attention that the programs have generated has also mobilized the clinical and medical field to push for more advancements in finding a cure."
And it's unlikely that everyone who buys a pink product would otherwise write a check directly to a breast cancer organization, Malbin points out.
Recipients of the cosmetic industry's fundraising include hospitals, cancer centers and nonprofit breast health programs.
One large beneficiary is the Susan G. Komen Breast Cancer Foundation, which over its 18-year history has raised $740 million to fight breast cancer. A peer-review committee at the foundation evaluates applications for funding for research, as well as education, screening and treatment for underserved populations.
Rebecca Vesely is a health care reporter at the Oakland Tribune.
For more information:
The Susan G. Komen Breast Cancer Foundation--
http://www.komen.org/
Yoplait--Save Lids to Save Lives:
http://www.yoplaitusa.com/breastcancer_lids.aspx
say it's a dubious way to raise funds and urge shoppers to know how businesses handle the proceeds.
You're not the only one. From Estee Lauder's pink lipstick to pink Keds to Conair pink travel hair dryers to Jimmy Choo hot pink high heels, numerous companies are peddling pink products.
That's because October is National Breast Cancer Awareness Month, which each year raises millions of dollars for breast cancer charities and reminds women that early detection works.
So what's wrong with that?
A lot, says Breast Cancer Action, a San Francisco nonprofit that this month launched "Think Before You Pink," an awareness campaign that casts a critical eye on social marketing campaigns for breast cancer.
#9-Tetrahydrocannabinol Inhibits Cell Cycle Progression in Human
Breast Cancer Cells through Cdc2 Regulation
Marı´a M. Caffarel,1 David Sarrio´,2 Jose´ Palacios,2 Manuel Guzma´n,1 and Cristina Sa´nchez1
1Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University and 2Breast and Gynecological Cancer
Group, Molecular Pathology Programme, Centro Nacional de Investigaciones Oncolo´gicas, Madrid, Spain
Abstract
It has been proposed that cannabinoids are involved in the
control of cell fate. Thus, these compounds can modulate
proliferation, differentiation, and survival in different manners
depending on the cell type and its physiopathologic
context. However, little is known about the effect of
cannabinoids on the cell cycle, the main process controlling
cell fate. Here, we show that #9-tetrahydrocannabinol (THC),
through activation of CB2 cannabinoid receptors, reduces
human breast cancer cell proliferation by blocking the
progression of the cell cycle and by inducing apoptosis. In
particular, THCarres ts cells in G2-M via down-regulation of
Cdc2, as suggested by the decreased sensitivity to THC
acquired by Cdc2-overexpressing cells. Of interest, the
proliferation pattern of normal human mammary epithelial
cells was much less affected by THC. We also analyzed by realtime
quantitative PCR the expression of CB1 and CB2
cannabinoid receptors in a series of human breast tumor
and nontumor samples. We found a correlation between CB2
expression and histologic grade of the tumors. There was also
an association between CB2 expression and other markers of
prognostic and predictive value, such as estrogen receptor,
progesterone receptor, and ERBB2/HER-2 oncogene. Importantly,
no significant CB2 expression was detected in nontumor
breast tissue. Taken together, these data might set the
bases for a cannabinoid therapy for the management of breast
cancer. (Cancer Res 2006; 66(13): 6615-21)
Introduction
There are very few critical decisions that cells must take during
their lifetime. Basically, these are whether to proliferate, differentiate,
or die. A tight regulation of the cell cycle is crucial to control
all these decisions, and its deregulation has devastating consequences,
such as cancer (1). It has been proposed that
cannabinoids, the active components of Cannabis sativa, play a
role in the control of the aforementioned decisions. For example,
they can modulate survival, proliferation, and differentiation
depending on the cell type and its physiopathologic context
(2, 3). Among the f70 cannabinoids synthesized by C. sativa,
D9-tetrahydrocannabinol (THC) is the most important in terms of
potency and abundance (4). THC exerts a wide variety of biological
effects by mimicking endogenous compounds, the endocannabinoids
anandamide and 2-arachidonoylglycerol, which activate
specific cannabinoid receptors. Thus far, two G protein–coupled
cannabinoid-specific receptors have been cloned from mammalian
tissues: CB1, abundantly expressed in the brain and at many
peripheral sites, and CB2, almost exclusively expressed in the
immune system (5). Engagement of these receptors by THC or
endocannabinoids affects several signaling pathways, some of them
directly involved in the control of cell fate. For instance,
cannabinoids modulate mitogen-activated protein kinases and
the phosphatidylinositol 3-kinase/Akt survival pathway, which have
a prominent role in the control of cell growth and differentiation
(6). Due to the growing evidence that cannabinoids participate in
the control of cell fate and to the fact that the cell cycle is a key
process underlying the regulation of survival/proliferation/differentiation
decisions, we decided to study the effect of THC on the
cell cycle and the mechanism of cannabinoid action on this
process. Because breast tumors are one of the most common
human neoplasias and one of the leading causes of death among
Western women (7), we decided to focus our studies on this
particular type of cancer.
Materials and Methods
Cell culture and viability. EVSA-T, MDA-MB-231, MDA-MB-468, and
SKBr3 cells were kindly given by Dr. Lo´pez-Rivas [Centro Andaluz de
Biologı´a del Desarrollo, Consejo Superior de Investigaciones Cientı´ficas
(CSIC), Sevilla, Spain], and MCF-7 and T-47D cells were obtained from the
American Type Culture Collection (ATCC; Manassas, VA). Cells were
maintained in RPMI 1640 (EVSA-T, MDA-MB-231, MCF-7, and T-47D) or
DMEM (MDA-MB-468 and SKBr3) supplemented with 10% fetal bovine
serum (FBS), 5 units/mL penicillin, and 5 mg/mL streptomycin. Human
mammary epithelial cells (HMEC) were kindly given by Dr. Lacal (Instituto
de Investigaciones Biome´dicas, CSIC, Madrid, Spain) and grown in
mammary epithelial growth medium (Cambrex, East Rutherford, NJ)
according to the manufacturer’s instructions. Cannabinoid ligands were
prepared in DMSO. Control incubations had the corresponding DMSO
content. No significant influence of DMSO was observed on cell viability
at the final concentration used (0.1-0.2%, v/v). Cell viability was determined
by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test
(Sigma, St. Louis, MO) according to the manufacturer’s instructions.
Western blot analysis. Samples were subjected to SDS-PAGE, and
proteins were transferred onto polyvinylidene fluoride membranes. Blots
were incubated with the following antibodies: anti-phosphorylated Cdc2
(Tyr15), anti-cyclin B1, anti-Cdc25C, anti-p27, anti-caspase-3, and antipoly(
ADP-ribose) polymerase (PARP) (Cell Signaling, Beverly, MA); anti-
Cdc2 and anti-p21 (Santa Cruz, Santa Cruz, CA); anti-Wee1 (BioVision,
Mountain View, CA); anti-survivin (R&D Systems, Minneapolis, MN); and
anti-a-tubulin as loading control (Sigma). Luminograms were obtained with
an enhanced chemiluminescence detection kit (Amersham Life Sciences,
Arlington Heights, IL), and densitometric analysis was done with Multianalyst
software (Bio-Rad, Hercules, CA).
Cell cycle analysis. Cells were permeabilized and fixed in 1% (w/v)
bovine serum albumin and 30% ethanol-PBS and labeled with 5 Ag/mL
Hoechst 33342 (Molecular Probes, Leiden, the Netherlands). Fluorescence
intensity was analyzed using a LSR flow cytometer (Becton Dickinson, San
Jose, CA). Ten thousand cells per analysis were recorded.
Requests for reprints: Cristina Sa´nchez, Department of Biochemistry and
Molecular Biology I, School of Biology, Complutense University, 28040 Madrid,
Spain. Phone: 34-913944668; Fax: 34-913944672; E-mail: csg@bbm1.ucm.es.
I2006 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-05-4566
www.aacrjournals.org 6615 Cancer Res 2006; 66: (13). July 1, 2006
Research Article
Apoptosis. Cells were incubated in binding buffer [10 mmol/L HEPES
(pH 7.4), 2.5 mmol/L CaCl2, 140 mmol/L NaCl] supplemented with Annexin
V-FITC (Molecular Probes). Propidium iodide (PI; Sigma) was added
1 minute before sample analysis. Fluorescence intensity was analyzed using
a FACS Scalibur flow cytometer (Becton Dickinson). For triple staining
experiments, cells were labeled with Hoechst 33342, and apoptosis was
analyzed as described above. Ten thousand cells per analysis were
recorded.
Caspase-3 activity. Caspase-3/7 activity was determined with a
luminogenic substrate (Caspase-Glo, Promega, Madison, WI) according to
the manufacturer’s instructions. Luminescence was determined in a
Microplate Fluorescence Reader (BMG Labtech, Offenburg, Germany).
Tissue samples. Samples were obtained from the Centro Nacional de
Investigaciones Oncolo´gicas Tumor Bank (Madrid, Spain). Histologic
grade was assessed according to Elston and Ellis criteria. Immunohistochemical
staining for prognostic and predictive factors was done by
the EnVision method with a heat-induced antigen retrieval step.
Monoclonal antibodies for estrogen receptor (ER), progesterone receptor
(PR), p53 (Novocastra, Newcastle, United Kingdom), and Ki67 (DAKO,
Glostrup, Denmark) were used. ERBB2/HER-2 expression was evaluated
using Herceptest (DAKO). The percentage of cells with unequivocal
nuclear staining for ER, PR, Ki67, and p53 was scored, and a cutoff of
5% was used for positivity for ER and PR and 15% for Ki67 and p53. For
ERBB2/HER-2, only cases with 3+ membranous staining were scored as
positive.
Confocal microscopy analysis of cannabinoid receptors. Human
breast cancer and normal breast 5-Am paraffin-embedded tissue sections
were analyzed. Primary antibodies against CB1 and CB2 receptors (Affinity
Bioreagents, Golden, CO) were used. Secondary anti-rabbit antibody Alexa
Fluor 594 was from Molecular Probes. Cell nuclei were stained with YOYO-1
(Molecular Probes). Confocal fluorescence images were acquired using
Laser Sharp 2000 software (Bio-Rad).
Reverse transcription-PCR analysis. Total RNA was isolated using the
RNeasy Protect kit (Qiagen, Hilden, Germany). cDNA was obtained using
Figure 1. Effect of cannabinoids on
human breast cell proliferation. Cells were
incubated in low-FBS medium (2.5% for
MCF-7 and 0.5% for the rest of the tumor
cell lines) for 72 hours in the presence of
5 Amol/L THC (A), different concentrations
of THC ranging from 1 to 12 Amol/L (B),
or the specified THC concentration (C).
When antagonists were used (1 Amol/L
rimonabant and 2 Amol/L SR144528), they
were added 1 hour before THC. ER and
p53 status was obtained from ATCC and
German National Resource Centre for
Biological Material (DSMZ, Braunschweig,
Germany). IC50s were defined as
concentrations of THC required to
decrease cell viability to 50%. A, columns,
mean for each cell line (n z 3); bars, SE.
B, significant differences from MCF-7
(*, P < 0.05; **, P < 0.01) or T-47D cells
(b, P < 0 .0 5; bb, P < 0.01). For real-time
quantitative PCR experiments, an arbitrary
value of 1 was assigned to cannabinoid
receptor expression in MDA-MB 468 cells
(reference). Representative experiment
(n = 3). A and C, columns, means cell
viability in THC-treated cells versus their
respective vehicle-treated cells (set at
100%; n z 3); bars, SE. Significant
differences (*, P < 0.05; **, cc, P < 0 .0 1)
from control (*) or 5 Amol/L THC alone (c).
D, CB1 and CB2 mRNA expression in
the cell lines was determined by RT-PCR
(representative gels, n z 3). U373 MG
astrocytoma cells and Jurkat leukemia
cells were used as positive controls for CB1
and CB2, respectively.
Cancer Research
Cancer Res 2006; 66: (13). July 1, 2006 6616 www.aacrjournals.org
Transcriptor Reverse Transcriptase (Roche, Applied Science, Penzberg,
Germany). Primer sequences were CB1 (sense), 5¶-CGTGGGCAGCCTGTTCCTCA-
3¶; CB1 (antisense), 5¶-CATGCGGGCTTGGTCTGG-3¶; CB2 (sense),
5¶-TGGGACAGGGTCAGTACAAGT-3¶; CB2
(antisense), 5¶-CTTTGGCTCCTGGTGGTCT-
3¶; glyceraldehyde-3-phosphate dehydrogenase (GAPDH;
sense), 5¶-GGGAAGCTCACTGGCATGGCCTTCC-3¶; and GAPDH (antisense),
5¶-CATGTGGGCCATGAGGTCCACCAC-3¶.
Real-time quantitative PCR. Total RNA and cDNA were obtained from
frozen breast tumors or cell lines as described above. Taqman probes for
human CB1, CB2, and 18S RNA (used as internal reference) were from
Applied Biosystems (Foster City, CA).
Plasmids and transfections. pIRESpuro2 and pIRESpuro2-Cdc2 were
kindly given by Dr. Huang ( Johns Hopkins University, Baltimore, MD).
Transfections were carried out with Fugene 6 (Roche Applied Science,
Indianapolis, IN) according to the manufacturer’s protocol. Transfected
cells were selected with puromycin.
Statistical analysis. ANOVA with a post hoc analysis by the Student‘s-
Newman-Keuls’ test was routinely used. For cannabinoid receptor
expression in human samples, data were log transformed to achieve
normality in the distribution. An F-test was subsequently done to compare
equality of variances in each group, and a classic t test or a t test with
different variances was applied using the Welch modification.
Results
THCinhibits proliferation of human breast cancer cells.
Several human breast cell lines were incubated with THC, and
viable cell numbers were estimated. THC decreased proliferation in
all the tumor cells tested (Fig. 1A). Among the tumor cells, those
with more aggressive phenotype (ER) were more sensitive to THC
(Fig. 1B). Remarkably, nontumor HMEC cells were the most
resistant to cannabinoid treatment (IC50 >12 Amol/L; Fig. 1A).
Rimonabant, a selective CB1 receptor antagonist, did not block
THC effect in EVSA-T cells (Fig. 1C) or any of the other cell lines
studied (data not shown). In contrast, SR144528, a selective CB2
receptor antagonist, partially prevented the THC-induced decrease
of EVSA-T cell proliferation (Fig. 1C). Both reverse transcription-
PCR (RT-PCR; Fig. 1D) and real-time quantitative PCR experiments
(Fig. 1B) confirmed the expression of CB2 mRNA in this cell line,
whereas CB1 mRNA was undetectable (Fig. 1B and D).
THC-induced decrease of cell proliferation is due to the
blockade of the G2-M transition. We next sought to examine
whether an alteration of EVSA-T cell cycle underlies THC
antiproliferative effect. The cannabinoid increased the number of
cells in the G0-G1 compartment and, in parallel, decreased the
number of cells in S phase (Fig. 2A and C). At the highest
concentration tested (5 Amol/L), THC also produced the following:
(a) an increase in the number of cells in G2-M phases and (b) the
appearance of a population of hypodiploid cells (Fig. 2A and C).
The latter two effects were prevented by SR144528 (Fig. 2C).
Importantly, THC did not alter the cell cycle profile of HMEC cells
(Fig. 2B).
To analyze the precise mechanism of THC action, we first
studied the expression of several proteins involved in the G2-M
transition. THC decreased the total levels of Cdc2 [p34, cyclindependent
kinase (CDK) 1; Fig. 3A], the major CDK controlling the
entrance of cells in mitosis after completing G2 events (8), and
SR144528 (2 Amol/L) completely prevented this effect [relative
optical density (OD) after 16 hours of treatment relative to vehicle:
SR144528+THC, 105 F 4]. The expression of cyclin B1, the positive
regulatory subunit of Cdc2 (9), did not significantly change on
cannabinoid challenge (data not shown). The levels of p21, a CDK
inhibitor known to prevent Cdc2-cyclin B activation (8), were
enhanced by THC (Fig. 3A). It has been recently proposed that p27,
a CDK inhibitor traditionally associated to the regulation of G1-S
transition, can also inhibit Cdc2 at G2-M (10). In our system,
however, THC did not modify p27 levels (data not shown).
To be active, Cdc2 has to be dephosphorylated in the Tyr15
residue (11). THC treatment decreased the amount of phosphorylated
Tyr15-Cdc2 to a lower extent than total Cdc2 levels (Fig. 3A),
indicating that the ratio inactive/active Cdc2 was augmented by
THC. Phosphorylation of Cdc2 in Tyr15 is controlled by the Wee1/
Mik1 family of protein kinases and by the phosphatase Cdc25C
(11). Our results show that THC enhances Wee1 and reduces
Cdc25C protein levels (Fig. 3A).
In view of the aforementioned results, it is conceivable that
THC exposure prevents EVSA-T cells to reach the required levels
of active Cdc2 to enter mitosis. To test whether Cdc2 downregulation
is important in the growth-inhibiting effect of THC,
we heterologously expressed this kinase. As shown in Fig. 3B,
cells overexpressing Cdc2 became significantly more resistant
to THC.
Figure 2. Effect of THC on cell cycle
dynamics. Cell cycle profiles of EVSA-T
(A) and HMEC cells (B) incubated with
5 Amol/L THC or the corresponding vehicle
for 48 hours. Representative experiments
(n z 3). C, percentage of EVSA-T cells
(mean F SE) in every phase of the cell
cycle after 48 hours of incubation with the
indicated compounds. Where indicated,
rimonabant (SR1; 1 Amol/L) or SR144528
(SR2; 2 Amol/L) was added 1 hour
before THC. Significant differences from
control (*, P < 0.05; **, P < 0.01) or the
corresponding concentration of THC alone
(b, P < 0.05).
Control of Cell Cycle by Cannabinoids
www.aacrjournals.org 6617 Cancer Res 2006; 66: (13). July 1, 2006
THC-induced cell cycle arrest is associated with apoptosis.
We next tried to elucidate whether THC-induced inhibition of
proliferation was associated with cell death. The cannabinoid
induced apoptosis, a process that was prevented by SR144528
(Fig. 4A). THC also induced a two-fold increase in caspase-3
activity, an effect that was prevented by SR144528 (Fig. 4B).
Likewise, we observed reduced levels of both pro-caspase-3 (the
inactive precursor of caspase-3) and PARP (a caspase-3 substrate)
in cannabinoid-treated cells (Fig. 4C). We subsequently addressed
the question of whether apoptotic cells were those arrested in G2-M
by THC. We conducted triple staining experiments to analyze the
percentage of apoptotic cells in every phase of the cell cycle. As
shown in Fig. 5A, THC induced apoptosis in all the cell cycle
phases, but the majority of apoptotic cells were in the G2-M
compartment.
It is known that survivin, a member of the inhibitor of apoptosis
family, can be phosphorylated in Thr34 by Cdc2. This phosphorylation
results in enhanced stability of survivin and the
consequent inhibition of caspase activity (12). Survivin was highly
expressed in all the breast cancer cell lines tested but was hardly
detectable in HMEC (Fig. 5B), in agreement with previous reports
showing a sharp differential expression in cancer (high levels)
versus normal (undetectable levels) tissues (12). Moreover, THC
decreased survivin levels in EVSA-T cells (Fig. 5C), which may
explain why THC-induced Cdc2 inactivation results in apoptosis.
In fact, when Cdc2 levels were enhanced by overexpression,
survivin decrease on cannabinoid treatment was attenuated
(relative optical density after 16 hours of treatment for
pIRESpuro2-transfected cells: vehicle, 100 F 6; 5 Amol/L THC,
31 F 4. For pIRESpuro2-Cdc2-transfected cells: vehicle, 100 F 5;
5 Amol/L THC, 74 F 3; Fig. 5D).
Cannabinoid receptors are expressed in human breast
tumors. The presence of CB1 and CB2 receptors in human breast
tumors was evaluated by real-time quantitative PCR and confocal
microscopy. Lower levels of CB1 mRNA were detected in tumors
of low-medium (grade 1-2) and high (grade 3) histologic grade
compared with normal, noncancerous breast tissue (grade 1-2
versus noncancerous breast tissue (P = 0.008); grade 3 versus
noncancerous breast tissue (P = 0.0007); Fig. 6A). CB2 expression
was higher than CB1 expression in all the tumors analyzed (P =
0.00002) and seemed to correlate with their histologic grade
(grade 1-2 versus grade 3; P = 0.04; Fig. 6A). Of interest, CB2
transcripts were hardly detectable in normal breast tissue
(Fig. 6A). Moreover, the expression of CB2 showed an association
with molecular markers of prognostic value. Thus, ER tumors
expressed more CB2 mRNA than ER+ tumors (Fig. 6B). CB2
expression was also higher in PR than in PR+ samples. ER/
PR tumors (response rate <10% to conventional therapies)
Figure 3. Effect of THC on the expression
of proteins involved in the control of the
G2-M transition. A, Western blot analysis
of the indicated proteins was done at least
twice. Representative luminograms. T3,
3 Amol/L THC and T5, 5 Amol/L THC.
Optical density (arbitrary units, mean F
SE) of the different proteins relative to their
respective time-point control incubations
(set at 100). B, EVSA-T cell viability was
assayed after 72 hours of incubation
with 5 Amol/L THC or vehicle. Columns,
mean cell viability in THC-treated cells
versus their respective vehicle-treated
cells (set at 100%); bars, SE. Inset,
Western blot of total Cdc2 levels in
EVSA-T cells (lane 1), EVSA-T cells
transfected with pIRESpuro2 (lane 2),
and EVSA-T cells transfected with
pIRESpuro2-Cdc2 (lane 3). A and B,
significant differences from the
corresponding vehicle-treated cells
(*, P < 0.05; **, P < 0.01) or between
THC-treated cells transfected with
either pIRESpuro2 or pIRESpuro2-Cdc2
(b, P < 0.05).
Cancer Research
Cancer Res 2006; 66: (13). July 1, 2006 6618 www.aacrjournals.org
expressed more CB2 mRNA than ER+/PR+ tumors (60-70%
response rate; ref. 7; Fig. 6B). ERBB2/HER-2-positive tumors
(with worse survival prediction at least in node-positive patients;
ref. 7) expressed higher levels of CB2 mRNA than ERBB2/HER-2-
negative tumors (Fig. 6B). Confocal microscopy experiments
confirmed the presence of CB1 and CB2 proteins in tumor cells
(Fig. 6C). None of the receptor proteins was detected in normal
ducts.
Figure 4. Analysis of THC-induced cell
death. Fluorescence-activated cell sorting
analysis (A) and caspase-3 activity (B)
of EVSA-T cells after incubation with the
indicated compounds for 24 hours.
Antagonists (1 Amol/L rimonabant or
2 Amol/L SR144528) were added 1 hour
before THC (5 Amol/L). A, percentage
of apoptotic cells (FITC positive/PI
negative) within the total cell population.
B, columns, means of at least five or
more experiments; bars, SE. Significant
differences from control (*, P < 0 .0 5;
**, P < 0.01) or THC alone (b, P < 0 .0 5;
bb, P < 0.01). C, Western blot analysis of
pro-caspase-3 and PARP. T3, 3 Amol/L
THC and T5, 5 Amol/L THC.
Representative luminograms (n z 3).
Figure 5. Connection between cell
cycle arrest and apoptosis. A, percentage
of apoptotic cells in different phases
of the cell cycle as assessed by triple
staining with Annexin V-FITC, PI, and
Hoechst 33342. Significant differences
from the corresponding vehicle-treated
cells (*, P < 0.05; **, P < 0.01) or
between THC-treated cells in G2-M
and THC-treated cells in G0-G1 or S
(b, P < 0.05). B-D, Western blot of survivin
in different human breast cell lines (B),
EVSA-T cells incubated with THC (T3, 3
Amol/L THC; T5, 5 Amol/L THC) or the
corresponding vehicle (C), and EVSA-T
cells transfected with pIRESpuro2 or
pIRESpuro-Cdc2 and incubated for 16
hours with the indicated compounds (D).
Representative luminograms (n z 2).
Control of Cell Cycle by Cannabinoids
www.aacrjournals.org 6619 Cancer Res 2006; 66: (13). July 1, 2006
Discussion
There is scant information on the effect of cannabinoids on the
cell cycle. It has been shown previously that the endogenous
cannabinoid anandamide arrests the cycle of hepatoma HepG2
cells (13), epidermal growth factor–stimulated PC3 prostate cancer
cells (14), and the breast cancer cell line EFM-19 (15) at the G1-S
transition. The same effect was exerted by a metabolically stable
analogue of anandamide, met-F-anandamide, on KiMol thyroid
carcinoma cells (16). Results presented herein (a) show that the
plant-derived cannabinoid THC is able to block the progression of
breast cancer cell cycle and (b) provide a mechanism for this
action. Of interest, nontumor mammary epithelial cells were rather
insensitive to THC, suggesting that cannabinoids would fulfill one
of the demanded requirements of any compound intended to be
used in cancer therapy: the selectivity for tumor cells. In our
experimental model, THC induces a CB2-mediated cell cycle arrest
at the G2-M transition via Cdc2 down-regulation. We are
nonetheless aware that our observation that THC effects are
significantly but not completely prevented by the CB2 selective
antagonist also points to the existence of CB2-independent
Figure 6. Cannabinoid receptor
expression in human breast tumors.
A and B, mRNA expression (medians F
SE) of CB1 and CB2 receptors as
determined by real-time quantitative PCR.
An arbitrary value of 1 unit was assigned to
CB2 expression in one of the tumors
(reference), and the relative expression of
the rest of the samples was calculated
by comparison with the reference. Note the
different scale in the y axes of (A).
Significantly different values (*, P < 0 .0 5;
**, P < 0.01) from ‘‘+ variable’’ or ‘‘+/+
variable.’’ ER; PR; S1, <2 cm; S2, 2 to
5 cm; and S3, >5 cm; metastasis was
determined as presence/absence of
positive axillary lymph nodes. C, confocal
microscopy analysis of cannabinoid
receptors in a grade 1 (left ) and a grade
3 tumor (right ). Green, cell nuclei; red,
cannabinoid receptors. Arrows, different
areas within the tumor samples.
Cancer Research
Cancer Res 2006; 66: (13). July 1, 2006 6620 www.aacrjournals.org
processes in cannabinoid antiproliferative action. Most cancer cells
evade antigrowth signals because they have defective G1 checkpoints,
which makes the G2 checkpoint an attractive target for
cancer therapy (11). Since its discovery in 1986 (17), it has been
clear that Cdc2 is essential for cell cycle progression (18). In fact,
Cdc2-deficient mice die at very early stages of embryonic
development (18). The pivotal importance of this particular protein
could explain why, in our system, a somewhat moderate decrease
of total Cdc2 levels (f40%) results in large changes in cell viability.
In addition, our data indicate that THC increases the inactive/
active Cdc2 ratio, supporting that the cannabinoid decreases not
only total Cdc2 levels but also enzyme-specific activity.
It is important to point out that, although the antiproliferative
effect of cannabinoids on different tumor cells has been extensively
confirmed both in vitro and in vivo (6), a recent report indicates
that THC may enhance breast cancer cell growth under certain
circumstances. In that study, the authors showed a direct
association between the degree of sensitivity of a tumor to THC
and the level of CB1 and CB2 expression. Thus, THC has
antiproliferative effect in tumors expressing cannabinoid receptors,
whereas those with low to no expression suffer increased growth
and metastasis due to THC-induced suppression of the antitumor
immune response (19). Results presented herein are not in
disagreement with that report, as EVSA-T cells, which are very
sensitive to THC, express high levels of CB2.
By real-time quantitative PCR experiments, we have observed a
correlation between CB2 expression and the histologic grade of
breast tumors. Moreover, CB2 expression is higher in tumors with
predicted low response to conventional therapies, for instance
ER/PR tumors, which are weakly responsive to adjuvant
tamoxifen (7). In addition, hardly detectable levels of CB2 were
found in normal breast tissue. Because both cell cycle arrest and
apoptosis induced by THC are CB2-mediated effects, it is tempting
to speculate that the tumors with poor prognosis (i.e., those
reluctant to conventional therapies and, according to our data,
expressing the highest levels of CB2 receptor), may be the most
responsive to cannabinoids. In addition, the psychotropic effects of
cannabinoids are mediated by CB1 but not CB2 receptors, and,
therefore, a cannabinoid-based therapy selectively targeting CB2
receptors would be devoid of the side effects associated to
cannabis consumption (20).
Breast cancer is the most common malignant disease among
Western women. Although the rates of mortality of breast cancer
patients have decreased as a result of early diagnosis by
mammograms, certain breast tumors remain reluctant to conventional
therapies, and current treatments have side effects that
substantially affect the patient’s quality of life (21). Our findings
might set the basis for new strategies for the management of
breast cancer.
Acknowledgments
Received 12/22/2005; revised 4/6/2006; accepted 5/2/2006.
Grant support: Fondo de Investigaciones Sanitarias FIS 04/1029 (C. Sa´nchez) and
BEFI 01/9132 (D. Sarrio´); Complutense University PR1/05 (C. Sa´nchez); Ministerio de
Educacio´n y Ciencia SAF2003-00745 (M. Guzma´n), SAF2004-08258-C02-01
( J. Palacios), and AP2003-0063 (M.M. Caffarel); and Fundacio´n Cientı´fica de la
Asociacio´n Espan˜ola Contra el Ca´ncer (M. Guzma´n).
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank the staff at Centro de Microscopı´a y Citometrı´a (Complutense University,
Madrid, Spain) and A. A´lvarez-Barrientos for expert advice on flow cytometry
experiments; O. Rueda for expert advice on statistical analysis; and the members of our
laboratory for technical support, especially to K. Blazej, C. Bla´zquez, D. Callegarin,
A. Egia, B. Herrera, and M. Lorente.
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Control of Cell Cycle by Cannabinoids
www.aacrjournals.org 6621 Cancer Res 2006; 66: (13). July 1, 2006
* high dose chemo + bone marrow transplant is superior to conventional chemotherapy;
* Endocrine therapy is inferior to chemotherapy and will not result in a survival advantage;
* Treatment of breast cancer with less than mastectomy is dangerous;
* local therapy does not influence survival.
And she added to that list: MRI finds cancer not found by other modalities. It must be useful.
Dr. Morrow’s presentation was followed by a report on the first and only prospective study of MRI, the COMICE trial, which was sponsored by the research arm of the British National Health Service. (England and Canada sponsor significant research on actual effectiveness as a means of cost containment). COMICE randomized 1623 women into two groups: those who received MRI prior to BCS, and those who did not. The primary endpoint was the rate of re-operation (those who had to have further excision, and those who had a subsequent mastectomy). There were not significant differences between the two study groups in re-operation rates, which substantiated Morrow’s perspective.
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ABC Slams Companies for Donating to Breast Cancer Research
Reporter David Muir cited liberal critic of corporations who also advises women to forego regular mammograms.
By Ken Shepherd
Business & Media Institute
10/13/2006 2:14:17 PM
Send this page to a friend! (click here)
Companies can’t win – even when they literally give away money. ABC attacked businesses for helping “the corporate bottom line” by funding breast cancer research. In the process, the network relied on a critic of routine mammograms to make its attack.
On the October 12 “World News with Charles Gibson,” reporter David Muir turned a critical eye to businesses such as the makers of Campbell’s Soup or Yoplait yogurt that engage in breast cancer fundraising efforts.
“These pink ribbon campaigns often mean much more to the corporate bottom line than they do to people living with or at risk for breast cancer,” scoffed Barbara Brenner, the executive director of Breast Cancer Action.
Nowhere in his story did Muir mention that corporate fundraising efforts such as Yoplait’s “Save Lids to Save Lives” are often made in direct partnership with the Susan G. Komen Breast Cancer Foundation, perhaps the leading national advocate for breast cancer research and treatment.
Brennan was the only breast cancer research advocate featured in his story, but Muir suggested to viewers those views were shared by research proponents broadly.
“Advocates say” despite raising money for research, “they’re raising their profits a whole lot more,” the ABC reporter closed his story.
What Muir left out was how unorthodox Brenner’s group is and how some argue those views are a distraction in the fight against breast cancer.
A look at Brennan’s Web site reveals her organization is skeptical of, if not downright hostile to corporate sources of research financing. For example, her group refuses any contribution from health insurance or pharmaceutical companies.
But perhaps more disturbing is that her group disputes the wisdom of regular mammogram screenings for women.
“The benefit of routine mammograms for healthy premenopausal women is unproven,” Brenner’s group asserts in a Web page entitled “What You Should Know About Mammograms.”
“Healthy premenopausal women should not have mammograms as a routine matter. The risk of radiation, combined with the high incidence of both false negatives and positives, means that routine mammography for premenopausal women may well do more harm than good,” Breast Cancer Action asserts.
Dr. Elizabeth Whelan, an epidemiologist and Business & Media Institute (BMI) adviser, told BMI that such a claim was “wholly irresponsible.”
“I’m really shocked that they would say that. The benefits of early screening are overwhelmingly important,” Whelan countered, adding that “none of this recognizes what’s happened in the last eight years. It’s become largely a curable disease” when the cancer is caught early, thanks in part to drugs like Tamoxifen, Whelan argued.
Whelan added that it wasn’t surprising corporate donors would hope to get good PR and a boost in sales from their philanthropy, but added that media scrutiny would be better served finding where money is spent by breast cancer advocates, including Brenner’s group.
Being diagnosed with breast cancer is often frightening and confusing. It is easy to feel lost in a world of unfamiliar medical terms and procedures. Questions about diagnosis, treatment, money, relationships, and quality of life can cause you worry and stress. Sometimes you don’t even know where to begin or what questions to ask. You are not alone in this journey. (This document was written by women who have gone through this experience.) By reading this, you are already taking the first steps toward recovery. The following pages contain ideas and tips that will hopefully make dealing with health care providers, hospitals, and your health easier and less intimidating for you and your loved ones.
The best thing that you can do for yourself now is to give yourself time to think about your options. You need not feel rushed into making decisions you are not comfortable with or do not fully understand. You have this time to gather information and make informed decisions. A few weeks most likely will not influence your treatment outcome, but it may make a huge difference in your state of mind. Your diagnosis will be presented to you with a series of choices; what matters is that you make the choices that are right for you. The process may seem overwhelming and leave you with a lot of uncertainty. You can ask people you trust to help you make the choices that lie ahead. You and your family or friends can become advocates by doing research and talking to experts. Breast Cancer Action (BCA) is one resource for you during this time.
#9-Tetrahydrocannabinol Inhibits Cell Cycle Progression in Human
Breast Cancer Cells through Cdc2 Regulation
Marı´a M. Caffarel,1 David Sarrio´,2 Jose´ Palacios,2 Manuel Guzma´n,1 and Cristina Sa´nchez1
1Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University and 2Breast and Gynecological Cancer
Group, Molecular Pathology Programme, Centro Nacional de Investigaciones Oncolo´gicas, Madrid, Spain
Abstract
It has been proposed that cannabinoids are involved in the
control of cell fate. Thus, these compounds can modulate
proliferation, differentiation, and survival in different manners
depending on the cell type and its physiopathologic
context. However, little is known about the effect of
cannabinoids on the cell cycle, the main process controlling
cell fate. Here, we show that #9-tetrahydrocannabinol (THC),
through activation of CB2 cannabinoid receptors, reduces
human breast cancer cell proliferation by blocking the
progression of the cell cycle and by inducing apoptosis. In
particular, THCarres ts cells in G2-M via down-regulation of
Cdc2, as suggested by the decreased sensitivity to THC
acquired by Cdc2-overexpressing cells. Of interest, the
proliferation pattern of normal human mammary epithelial
cells was much less affected by THC. We also analyzed by realtime
quantitative PCR the expression of CB1 and CB2
cannabinoid receptors in a series of human breast tumor
and nontumor samples. We found a correlation between CB2
expression and histologic grade of the tumors. There was also
an association between CB2 expression and other markers of
prognostic and predictive value, such as estrogen receptor,
progesterone receptor, and ERBB2/HER-2 oncogene. Importantly,
no significant CB2 expression was detected in nontumor
breast tissue. Taken together, these data might set the
bases for a cannabinoid therapy for the management of breast
cancer. (Cancer Res 2006; 66(13): 6615-21)
Introduction
There are very few critical decisions that cells must take during
their lifetime. Basically, these are whether to proliferate, differentiate,
or die. A tight regulation of the cell cycle is crucial to control
all these decisions, and its deregulation has devastating consequences,
such as cancer (1). It has been proposed that
cannabinoids, the active components of Cannabis sativa, play a
role in the control of the aforementioned decisions. For example,
they can modulate survival, proliferation, and differentiation
depending on the cell type and its physiopathologic context
(2, 3). Among the f70 cannabinoids synthesized by C. sativa,
D9-tetrahydrocannabinol (THC) is the most important in terms of
potency and abundance (4). THC exerts a wide variety of biological
effects by mimicking endogenous compounds, the endocannabinoids
anandamide and 2-arachidonoylglycerol, which activate
specific cannabinoid receptors. Thus far, two G protein–coupled
cannabinoid-specific receptors have been cloned from mammalian
tissues: CB1, abundantly expressed in the brain and at many
peripheral sites, and CB2, almost exclusively expressed in the
immune system (5). Engagement of these receptors by THC or
endocannabinoids affects several signaling pathways, some of them
directly involved in the control of cell fate. For instance,
cannabinoids modulate mitogen-activated protein kinases and
the phosphatidylinositol 3-kinase/Akt survival pathway, which have
a prominent role in the control of cell growth and differentiation
(6). Due to the growing evidence that cannabinoids participate in
the control of cell fate and to the fact that the cell cycle is a key
process underlying the regulation of survival/proliferation/differentiation
decisions, we decided to study the effect of THC on the
cell cycle and the mechanism of cannabinoid action on this
process. Because breast tumors are one of the most common
human neoplasias and one of the leading causes of death among
Western women (7), we decided to focus our studies on this
particular type of cancer.
Materials and Methods
Cell culture and viability. EVSA-T, MDA-MB-231, MDA-MB-468, and
SKBr3 cells were kindly given by Dr. Lo´pez-Rivas [Centro Andaluz de
Biologı´a del Desarrollo, Consejo Superior de Investigaciones Cientı´ficas
(CSIC), Sevilla, Spain], and MCF-7 and T-47D cells were obtained from the
American Type Culture Collection (ATCC; Manassas, VA). Cells were
maintained in RPMI 1640 (EVSA-T, MDA-MB-231, MCF-7, and T-47D) or
DMEM (MDA-MB-468 and SKBr3) supplemented with 10% fetal bovine
serum (FBS), 5 units/mL penicillin, and 5 mg/mL streptomycin. Human
mammary epithelial cells (HMEC) were kindly given by Dr. Lacal (Instituto
de Investigaciones Biome´dicas, CSIC, Madrid, Spain) and grown in
mammary epithelial growth medium (Cambrex, East Rutherford, NJ)
according to the manufacturer’s instructions. Cannabinoid ligands were
prepared in DMSO. Control incubations had the corresponding DMSO
content. No significant influence of DMSO was observed on cell viability
at the final concentration used (0.1-0.2%, v/v). Cell viability was determined
by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test
(Sigma, St. Louis, MO) according to the manufacturer’s instructions.
Western blot analysis. Samples were subjected to SDS-PAGE, and
proteins were transferred onto polyvinylidene fluoride membranes. Blots
were incubated with the following antibodies: anti-phosphorylated Cdc2
(Tyr15), anti-cyclin B1, anti-Cdc25C, anti-p27, anti-caspase-3, and antipoly(
ADP-ribose) polymerase (PARP) (Cell Signaling, Beverly, MA); anti-
Cdc2 and anti-p21 (Santa Cruz, Santa Cruz, CA); anti-Wee1 (BioVision,
Mountain View, CA); anti-survivin (R&D Systems, Minneapolis, MN); and
anti-a-tubulin as loading control (Sigma). Luminograms were obtained with
an enhanced chemiluminescence detection kit (Amersham Life Sciences,
Arlington Heights, IL), and densitometric analysis was done with Multianalyst
software (Bio-Rad, Hercules, CA).
Cell cycle analysis. Cells were permeabilized and fixed in 1% (w/v)
bovine serum albumin and 30% ethanol-PBS and labeled with 5 Ag/mL
Hoechst 33342 (Molecular Probes, Leiden, the Netherlands). Fluorescence
intensity was analyzed using a LSR flow cytometer (Becton Dickinson, San
Jose, CA). Ten thousand cells per analysis were recorded.
Requests for reprints: Cristina Sa´nchez, Department of Biochemistry and
Molecular Biology I, School of Biology, Complutense University, 28040 Madrid,
Spain. Phone: 34-913944668; Fax: 34-913944672; E-mail: csg@bbm1.ucm.es.
I2006 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-05-4566
www.aacrjournals.org 6615 Cancer Res 2006; 66: (13). July 1, 2006
Research Article
Apoptosis. Cells were incubated in binding buffer [10 mmol/L HEPES
(pH 7.4), 2.5 mmol/L CaCl2, 140 mmol/L NaCl] supplemented with Annexin
V-FITC (Molecular Probes). Propidium iodide (PI; Sigma) was added
1 minute before sample analysis. Fluorescence intensity was analyzed using
a FACS Scalibur flow cytometer (Becton Dickinson). For triple staining
experiments, cells were labeled with Hoechst 33342, and apoptosis was
analyzed as described above. Ten thousand cells per analysis were
recorded.
Caspase-3 activity. Caspase-3/7 activity was determined with a
luminogenic substrate (Caspase-Glo, Promega, Madison, WI) according to
the manufacturer’s instructions. Luminescence was determined in a
Microplate Fluorescence Reader (BMG Labtech, Offenburg, Germany).
Tissue samples. Samples were obtained from the Centro Nacional de
Investigaciones Oncolo´gicas Tumor Bank (Madrid, Spain). Histologic
grade was assessed according to Elston and Ellis criteria. Immunohistochemical
staining for prognostic and predictive factors was done by
the EnVision method with a heat-induced antigen retrieval step.
Monoclonal antibodies for estrogen receptor (ER), progesterone receptor
(PR), p53 (Novocastra, Newcastle, United Kingdom), and Ki67 (DAKO,
Glostrup, Denmark) were used. ERBB2/HER-2 expression was evaluated
using Herceptest (DAKO). The percentage of cells with unequivocal
nuclear staining for ER, PR, Ki67, and p53 was scored, and a cutoff of
5% was used for positivity for ER and PR and 15% for Ki67 and p53. For
ERBB2/HER-2, only cases with 3+ membranous staining were scored as
positive.
Confocal microscopy analysis of cannabinoid receptors. Human
breast cancer and normal breast 5-Am paraffin-embedded tissue sections
were analyzed. Primary antibodies against CB1 and CB2 receptors (Affinity
Bioreagents, Golden, CO) were used. Secondary anti-rabbit antibody Alexa
Fluor 594 was from Molecular Probes. Cell nuclei were stained with YOYO-1
(Molecular Probes). Confocal fluorescence images were acquired using
Laser Sharp 2000 software (Bio-Rad).
Reverse transcription-PCR analysis. Total RNA was isolated using the
RNeasy Protect kit (Qiagen, Hilden, Germany). cDNA was obtained using
Figure 1. Effect of cannabinoids on
human breast cell proliferation. Cells were
incubated in low-FBS medium (2.5% for
MCF-7 and 0.5% for the rest of the tumor
cell lines) for 72 hours in the presence of
5 Amol/L THC (A), different concentrations
of THC ranging from 1 to 12 Amol/L (B),
or the specified THC concentration (C).
When antagonists were used (1 Amol/L
rimonabant and 2 Amol/L SR144528), they
were added 1 hour before THC. ER and
p53 status was obtained from ATCC and
German National Resource Centre for
Biological Material (DSMZ, Braunschweig,
Germany). IC50s were defined as
concentrations of THC required to
decrease cell viability to 50%. A, columns,
mean for each cell line (n z 3); bars, SE.
B, significant differences from MCF-7
(*, P < 0.05; **, P < 0.01) or T-47D cells
(b, P < 0 .0 5; bb, P < 0.01). For real-time
quantitative PCR experiments, an arbitrary
value of 1 was assigned to cannabinoid
receptor expression in MDA-MB 468 cells
(reference). Representative experiment
(n = 3). A and C, columns, means cell
viability in THC-treated cells versus their
respective vehicle-treated cells (set at
100%; n z 3); bars, SE. Significant
differences (*, P < 0.05; **, cc, P < 0 .0 1)
from control (*) or 5 Amol/L THC alone (c).
D, CB1 and CB2 mRNA expression in
the cell lines was determined by RT-PCR
(representative gels, n z 3). U373 MG
astrocytoma cells and Jurkat leukemia
cells were used as positive controls for CB1
and CB2, respectively.
Cancer Research
Cancer Res 2006; 66: (13). July 1, 2006 6616 www.aacrjournals.org
Transcriptor Reverse Transcriptase (Roche, Applied Science, Penzberg,
Germany). Primer sequences were CB1 (sense), 5¶-CGTGGGCAGCCTGTTCCTCA-
3¶; CB1 (antisense), 5¶-CATGCGGGCTTGGTCTGG-3¶; CB2 (sense),
5¶-TGGGACAGGGTCAGTACAAGT-3¶; CB2
(antisense), 5¶-CTTTGGCTCCTGGTGGTCT-
3¶; glyceraldehyde-3-phosphate dehydrogenase (GAPDH;
sense), 5¶-GGGAAGCTCACTGGCATGGCCTTCC-3¶; and GAPDH (antisense),
5¶-CATGTGGGCCATGAGGTCCACCAC-3¶.
Real-time quantitative PCR. Total RNA and cDNA were obtained from
frozen breast tumors or cell lines as described above. Taqman probes for
human CB1, CB2, and 18S RNA (used as internal reference) were from
Applied Biosystems (Foster City, CA).
Plasmids and transfections. pIRESpuro2 and pIRESpuro2-Cdc2 were
kindly given by Dr. Huang ( Johns Hopkins University, Baltimore, MD).
Transfections were carried out with Fugene 6 (Roche Applied Science,
Indianapolis, IN) according to the manufacturer’s protocol. Transfected
cells were selected with puromycin.
Statistical analysis. ANOVA with a post hoc analysis by the Student‘s-
Newman-Keuls’ test was routinely used. For cannabinoid receptor
expression in human samples, data were log transformed to achieve
normality in the distribution. An F-test was subsequently done to compare
equality of variances in each group, and a classic t test or a t test with
different variances was applied using the Welch modification.
Results
THCinhibits proliferation of human breast cancer cells.
Several human breast cell lines were incubated with THC, and
viable cell numbers were estimated. THC decreased proliferation in
all the tumor cells tested (Fig. 1A). Among the tumor cells, those
with more aggressive phenotype (ER) were more sensitive to THC
(Fig. 1B). Remarkably, nontumor HMEC cells were the most
resistant to cannabinoid treatment (IC50 >12 Amol/L; Fig. 1A).
Rimonabant, a selective CB1 receptor antagonist, did not block
THC effect in EVSA-T cells (Fig. 1C) or any of the other cell lines
studied (data not shown). In contrast, SR144528, a selective CB2
receptor antagonist, partially prevented the THC-induced decrease
of EVSA-T cell proliferation (Fig. 1C). Both reverse transcription-
PCR (RT-PCR; Fig. 1D) and real-time quantitative PCR experiments
(Fig. 1B) confirmed the expression of CB2 mRNA in this cell line,
whereas CB1 mRNA was undetectable (Fig. 1B and D).
THC-induced decrease of cell proliferation is due to the
blockade of the G2-M transition. We next sought to examine
whether an alteration of EVSA-T cell cycle underlies THC
antiproliferative effect. The cannabinoid increased the number of
cells in the G0-G1 compartment and, in parallel, decreased the
number of cells in S phase (Fig. 2A and C). At the highest
concentration tested (5 Amol/L), THC also produced the following:
(a) an increase in the number of cells in G2-M phases and (b) the
appearance of a population of hypodiploid cells (Fig. 2A and C).
The latter two effects were prevented by SR144528 (Fig. 2C).
Importantly, THC did not alter the cell cycle profile of HMEC cells
(Fig. 2B).
To analyze the precise mechanism of THC action, we first
studied the expression of several proteins involved in the G2-M
transition. THC decreased the total levels of Cdc2 [p34, cyclindependent
kinase (CDK) 1; Fig. 3A], the major CDK controlling the
entrance of cells in mitosis after completing G2 events (8), and
SR144528 (2 Amol/L) completely prevented this effect [relative
optical density (OD) after 16 hours of treatment relative to vehicle:
SR144528+THC, 105 F 4]. The expression of cyclin B1, the positive
regulatory subunit of Cdc2 (9), did not significantly change on
cannabinoid challenge (data not shown). The levels of p21, a CDK
inhibitor known to prevent Cdc2-cyclin B activation (8), were
enhanced by THC (Fig. 3A). It has been recently proposed that p27,
a CDK inhibitor traditionally associated to the regulation of G1-S
transition, can also inhibit Cdc2 at G2-M (10). In our system,
however, THC did not modify p27 levels (data not shown).
To be active, Cdc2 has to be dephosphorylated in the Tyr15
residue (11). THC treatment decreased the amount of phosphorylated
Tyr15-Cdc2 to a lower extent than total Cdc2 levels (Fig. 3A),
indicating that the ratio inactive/active Cdc2 was augmented by
THC. Phosphorylation of Cdc2 in Tyr15 is controlled by the Wee1/
Mik1 family of protein kinases and by the phosphatase Cdc25C
(11). Our results show that THC enhances Wee1 and reduces
Cdc25C protein levels (Fig. 3A).
In view of the aforementioned results, it is conceivable that
THC exposure prevents EVSA-T cells to reach the required levels
of active Cdc2 to enter mitosis. To test whether Cdc2 downregulation
is important in the growth-inhibiting effect of THC,
we heterologously expressed this kinase. As shown in Fig. 3B,
cells overexpressing Cdc2 became significantly more resistant
to THC.
Figure 2. Effect of THC on cell cycle
dynamics. Cell cycle profiles of EVSA-T
(A) and HMEC cells (B) incubated with
5 Amol/L THC or the corresponding vehicle
for 48 hours. Representative experiments
(n z 3). C, percentage of EVSA-T cells
(mean F SE) in every phase of the cell
cycle after 48 hours of incubation with the
indicated compounds. Where indicated,
rimonabant (SR1; 1 Amol/L) or SR144528
(SR2; 2 Amol/L) was added 1 hour
before THC. Significant differences from
control (*, P < 0.05; **, P < 0.01) or the
corresponding concentration of THC alone
(b, P < 0.05).
Control of Cell Cycle by Cannabinoids
www.aacrjournals.org 6617 Cancer Res 2006; 66: (13). July 1, 2006
THC-induced cell cycle arrest is associated with apoptosis.
We next tried to elucidate whether THC-induced inhibition of
proliferation was associated with cell death. The cannabinoid
induced apoptosis, a process that was prevented by SR144528
(Fig. 4A). THC also induced a two-fold increase in caspase-3
activity, an effect that was prevented by SR144528 (Fig. 4B).
Likewise, we observed reduced levels of both pro-caspase-3 (the
inactive precursor of caspase-3) and PARP (a caspase-3 substrate)
in cannabinoid-treated cells (Fig. 4C). We subsequently addressed
the question of whether apoptotic cells were those arrested in G2-M
by THC. We conducted triple staining experiments to analyze the
percentage of apoptotic cells in every phase of the cell cycle. As
shown in Fig. 5A, THC induced apoptosis in all the cell cycle
phases, but the majority of apoptotic cells were in the G2-M
compartment.
It is known that survivin, a member of the inhibitor of apoptosis
family, can be phosphorylated in Thr34 by Cdc2. This phosphorylation
results in enhanced stability of survivin and the
consequent inhibition of caspase activity (12). Survivin was highly
expressed in all the breast cancer cell lines tested but was hardly
detectable in HMEC (Fig. 5B), in agreement with previous reports
showing a sharp differential expression in cancer (high levels)
versus normal (undetectable levels) tissues (12). Moreover, THC
decreased survivin levels in EVSA-T cells (Fig. 5C), which may
explain why THC-induced Cdc2 inactivation results in apoptosis.
In fact, when Cdc2 levels were enhanced by overexpression,
survivin decrease on cannabinoid treatment was attenuated
(relative optical density after 16 hours of treatment for
pIRESpuro2-transfected cells: vehicle, 100 F 6; 5 Amol/L THC,
31 F 4. For pIRESpuro2-Cdc2-transfected cells: vehicle, 100 F 5;
5 Amol/L THC, 74 F 3; Fig. 5D).
Cannabinoid receptors are expressed in human breast
tumors. The presence of CB1 and CB2 receptors in human breast
tumors was evaluated by real-time quantitative PCR and confocal
microscopy. Lower levels of CB1 mRNA were detected in tumors
of low-medium (grade 1-2) and high (grade 3) histologic grade
compared with normal, noncancerous breast tissue (grade 1-2
versus noncancerous breast tissue (P = 0.008); grade 3 versus
noncancerous breast tissue (P = 0.0007); Fig. 6A). CB2 expression
was higher than CB1 expression in all the tumors analyzed (P =
0.00002) and seemed to correlate with their histologic grade
(grade 1-2 versus grade 3; P = 0.04; Fig. 6A). Of interest, CB2
transcripts were hardly detectable in normal breast tissue
(Fig. 6A). Moreover, the expression of CB2 showed an association
with molecular markers of prognostic value. Thus, ER tumors
expressed more CB2 mRNA than ER+ tumors (Fig. 6B). CB2
expression was also higher in PR than in PR+ samples. ER/
PR tumors (response rate <10% to conventional therapies)
Figure 3. Effect of THC on the expression
of proteins involved in the control of the
G2-M transition. A, Western blot analysis
of the indicated proteins was done at least
twice. Representative luminograms. T3,
3 Amol/L THC and T5, 5 Amol/L THC.
Optical density (arbitrary units, mean F
SE) of the different proteins relative to their
respective time-point control incubations
(set at 100). B, EVSA-T cell viability was
assayed after 72 hours of incubation
with 5 Amol/L THC or vehicle. Columns,
mean cell viability in THC-treated cells
versus their respective vehicle-treated
cells (set at 100%); bars, SE. Inset,
Western blot of total Cdc2 levels in
EVSA-T cells (lane 1), EVSA-T cells
transfected with pIRESpuro2 (lane 2),
and EVSA-T cells transfected with
pIRESpuro2-Cdc2 (lane 3). A and B,
significant differences from the
corresponding vehicle-treated cells
(*, P < 0.05; **, P < 0.01) or between
THC-treated cells transfected with
either pIRESpuro2 or pIRESpuro2-Cdc2
(b, P < 0.05).
Cancer Research
Cancer Res 2006; 66: (13). July 1, 2006 6618 www.aacrjournals.org
expressed more CB2 mRNA than ER+/PR+ tumors (60-70%
response rate; ref. 7; Fig. 6B). ERBB2/HER-2-positive tumors
(with worse survival prediction at least in node-positive patients;
ref. 7) expressed higher levels of CB2 mRNA than ERBB2/HER-2-
negative tumors (Fig. 6B). Confocal microscopy experiments
confirmed the presence of CB1 and CB2 proteins in tumor cells
(Fig. 6C). None of the receptor proteins was detected in normal
ducts.
Figure 4. Analysis of THC-induced cell
death. Fluorescence-activated cell sorting
analysis (A) and caspase-3 activity (B)
of EVSA-T cells after incubation with the
indicated compounds for 24 hours.
Antagonists (1 Amol/L rimonabant or
2 Amol/L SR144528) were added 1 hour
before THC (5 Amol/L). A, percentage
of apoptotic cells (FITC positive/PI
negative) within the total cell population.
B, columns, means of at least five or
more experiments; bars, SE. Significant
differences from control (*, P < 0 .0 5;
**, P < 0.01) or THC alone (b, P < 0 .0 5;
bb, P < 0.01). C, Western blot analysis of
pro-caspase-3 and PARP. T3, 3 Amol/L
THC and T5, 5 Amol/L THC.
Representative luminograms (n z 3).
Figure 5. Connection between cell
cycle arrest and apoptosis. A, percentage
of apoptotic cells in different phases
of the cell cycle as assessed by triple
staining with Annexin V-FITC, PI, and
Hoechst 33342. Significant differences
from the corresponding vehicle-treated
cells (*, P < 0.05; **, P < 0.01) or
between THC-treated cells in G2-M
and THC-treated cells in G0-G1 or S
(b, P < 0.05). B-D, Western blot of survivin
in different human breast cell lines (B),
EVSA-T cells incubated with THC (T3, 3
Amol/L THC; T5, 5 Amol/L THC) or the
corresponding vehicle (C), and EVSA-T
cells transfected with pIRESpuro2 or
pIRESpuro-Cdc2 and incubated for 16
hours with the indicated compounds (D).
Representative luminograms (n z 2).
Control of Cell Cycle by Cannabinoids
www.aacrjournals.org 6619 Cancer Res 2006; 66: (13). July 1, 2006
Discussion
There is scant information on the effect of cannabinoids on the
cell cycle. It has been shown previously that the endogenous
cannabinoid anandamide arrests the cycle of hepatoma HepG2
cells (13), epidermal growth factor–stimulated PC3 prostate cancer
cells (14), and the breast cancer cell line EFM-19 (15) at the G1-S
transition. The same effect was exerted by a metabolically stable
analogue of anandamide, met-F-anandamide, on KiMol thyroid
carcinoma cells (16). Results presented herein (a) show that the
plant-derived cannabinoid THC is able to block the progression of
breast cancer cell cycle and (b) provide a mechanism for this
action. Of interest, nontumor mammary epithelial cells were rather
insensitive to THC, suggesting that cannabinoids would fulfill one
of the demanded requirements of any compound intended to be
used in cancer therapy: the selectivity for tumor cells. In our
experimental model, THC induces a CB2-mediated cell cycle arrest
at the G2-M transition via Cdc2 down-regulation. We are
nonetheless aware that our observation that THC effects are
significantly but not completely prevented by the CB2 selective
antagonist also points to the existence of CB2-independent
Figure 6. Cannabinoid receptor
expression in human breast tumors.
A and B, mRNA expression (medians F
SE) of CB1 and CB2 receptors as
determined by real-time quantitative PCR.
An arbitrary value of 1 unit was assigned to
CB2 expression in one of the tumors
(reference), and the relative expression of
the rest of the samples was calculated
by comparison with the reference. Note the
different scale in the y axes of (A).
Significantly different values (*, P < 0 .0 5;
**, P < 0.01) from ‘‘+ variable’’ or ‘‘+/+
variable.’’ ER; PR; S1, <2 cm; S2, 2 to
5 cm; and S3, >5 cm; metastasis was
determined as presence/absence of
positive axillary lymph nodes. C, confocal
microscopy analysis of cannabinoid
receptors in a grade 1 (left ) and a grade
3 tumor (right ). Green, cell nuclei; red,
cannabinoid receptors. Arrows, different
areas within the tumor samples.
Cancer Research
Cancer Res 2006; 66: (13). July 1, 2006 6620 www.aacrjournals.org
processes in cannabinoid antiproliferative action. Most cancer cells
evade antigrowth signals because they have defective G1 checkpoints,
which makes the G2 checkpoint an attractive target for
cancer therapy (11). Since its discovery in 1986 (17), it has been
clear that Cdc2 is essential for cell cycle progression (18). In fact,
Cdc2-deficient mice die at very early stages of embryonic
development (18). The pivotal importance of this particular protein
could explain why, in our system, a somewhat moderate decrease
of total Cdc2 levels (f40%) results in large changes in cell viability.
In addition, our data indicate that THC increases the inactive/
active Cdc2 ratio, supporting that the cannabinoid decreases not
only total Cdc2 levels but also enzyme-specific activity.
It is important to point out that, although the antiproliferative
effect of cannabinoids on different tumor cells has been extensively
confirmed both in vitro and in vivo (6), a recent report indicates
that THC may enhance breast cancer cell growth under certain
circumstances. In that study, the authors showed a direct
association between the degree of sensitivity of a tumor to THC
and the level of CB1 and CB2 expression. Thus, THC has
antiproliferative effect in tumors expressing cannabinoid receptors,
whereas those with low to no expression suffer increased growth
and metastasis due to THC-induced suppression of the antitumor
immune response (19). Results presented herein are not in
disagreement with that report, as EVSA-T cells, which are very
sensitive to THC, express high levels of CB2.
By real-time quantitative PCR experiments, we have observed a
correlation between CB2 expression and the histologic grade of
breast tumors. Moreover, CB2 expression is higher in tumors with
predicted low response to conventional therapies, for instance
ER/PR tumors, which are weakly responsive to adjuvant
tamoxifen (7). In addition, hardly detectable levels of CB2 were
found in normal breast tissue. Because both cell cycle arrest and
apoptosis induced by THC are CB2-mediated effects, it is tempting
to speculate that the tumors with poor prognosis (i.e., those
reluctant to conventional therapies and, according to our data,
expressing the highest levels of CB2 receptor), may be the most
responsive to cannabinoids. In addition, the psychotropic effects of
cannabinoids are mediated by CB1 but not CB2 receptors, and,
therefore, a cannabinoid-based therapy selectively targeting CB2
receptors would be devoid of the side effects associated to
cannabis consumption (20).
Breast cancer is the most common malignant disease among
Western women. Although the rates of mortality of breast cancer
patients have decreased as a result of early diagnosis by
mammograms, certain breast tumors remain reluctant to conventional
therapies, and current treatments have side effects that
substantially affect the patient’s quality of life (21). Our findings
might set the basis for new strategies for the management of
breast cancer.
Acknowledgments
Received 12/22/2005; revised 4/6/2006; accepted 5/2/2006.
Grant support: Fondo de Investigaciones Sanitarias FIS 04/1029 (C. Sa´nchez) and
BEFI 01/9132 (D. Sarrio´); Complutense University PR1/05 (C. Sa´nchez); Ministerio de
Educacio´n y Ciencia SAF2003-00745 (M. Guzma´n), SAF2004-08258-C02-01
( J. Palacios), and AP2003-0063 (M.M. Caffarel); and Fundacio´n Cientı´fica de la
Asociacio´n Espan˜ola Contra el Ca´ncer (M. Guzma´n).
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank the staff at Centro de Microscopı´a y Citometrı´a (Complutense University,
Madrid, Spain) and A. A´lvarez-Barrientos for expert advice on flow cytometry
experiments; O. Rueda for expert advice on statistical analysis; and the members of our
laboratory for technical support, especially to K. Blazej, C. Bla´zquez, D. Callegarin,
A. Egia, B. Herrera, and M. Lorente.
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Control of Cell Cycle by Cannabinoids
www.aacrjournals.org 6621 Cancer Res 2006; 66: (13). July 1, 2006The give and take for charity
Matthew Putney / For The Times
For-profit fundraisers collect huge sums on behalf of worthy causes, but nonprofits see only a fraction of it.
By Doug Smith and Charles Piller
July 06, 2008
For 24 years, Citizens Against Government Waste has exposed pork-barrel spenders and rallied tax critics.
Its “Pig Book” and “porker” awards, meant to shame congressional leaders who exploit the public purse, have made the group a media darling and a political force.
But when it comes to policing its own fundraising practices, America’s self-proclaimed “#1 taxpayer watchdog” seems to have lost its bite.
Records filed with the California attorney general’s office show that over the last decade, for-profit fundraisers for the nonprofit kept more than 94 cents of every donated dollar. Yet donors could write off the full contribution on their tax returns.
“It’s a rip-off of the taxpayer,” said Pablo Eisenberg, senior fellow at Georgetown Public Policy Institute and a philanthropy scholar.
A Times investigation found hundreds of other examples of charities that pocketed just a sliver of what commercial fundraisers collected in their names. Some didn’t get a dime or even lost money.
According to a comprehensive review of state records filed over a decade, the problem of paltry returns extends well beyond what has been reported in recent years among benevolent societies for police, firefighters and veterans. It affects charities large and small, well-known and obscure. It spans a range of causes, including child and animal welfare, health research and opposition to drunk driving.
In more than 5,800 campaigns on behalf of charities that were registered with the state attorney general from 1997 to 2006, the fundraisers reported taking in $2.6 billion. They kept nearly $1.4 billion – about 54 cents of every dollar raised.
These numbers reflect only part of the problem. Though commercial fundraisers are required to file detailed fundraising reports with the state, many do not, and the law is not aggressively enforced because of limited staffing.
For-profit campaigns, which often employ telemarketing, mass mailings or one-time events, account for a small fraction of $223 billion in charitable giving each year in the United States. But they collect significant sums and help shape public perceptions of charities. Pairing computer-controlled dialing systems with low-wage workers, such firms can reach a large number of people in a short time.
“If I could forget about what percentage was going where,” said Dan Halfeldt, former sales manager at one Phoenix-based telemarketer for nonprofits, “I could really say, ‘Wow, I’m raising money for something good.’ ”
The firm, Midwest Publishing Inc., consistently offered among the lowest returns by large fundraisers. It did not respond to written questions about its operations.
The fundraising business is growing. More than 300 fundraisers have registered in California. Since 2000, the number of campaigns and amounts raised by for-profit firms has risen by about two-thirds.
Among the charities that netted little from such campaigns were the Humane Society of the United States, the American Breast Cancer Foundation, the Christian social-action group Concerned Women for America, the National Right to Life Committee and Students Against Destructive Decisions.
Among The Times’ findings:
* More than 100 charities raised $1 million or more from commercial appeals but netted less than 25 cents per dollar. Fundraisers got the rest.
* In 430 campaigns, charities got nothing: All $44 million donated went to fundraisers. In 337 of those cases, charities actually lost money, paying fees to fundraisers that exceeded the amount raised.
* In hundreds of other campaigns, charities apparently entered into contracts that limited their share of donations to 20% or less, no matter how successful the campaign.
* Groups with strong emotional or patriotic appeal – those supporting animals, children, veterans and public safety workers, for instance – often fared worst. Missing-children charities received less than 15% of more than $28 million raised on their behalf.
Many rely heavily on set-rate contracts and aim their campaigns toward a mass audience, which is less efficient than targeting a defined set of donors.
In general, charities argue that it takes money to make money and that the benefits of commercially run campaigns may not show in state filings.
For instance, Citizens Against Government Waste said that its telemarketing was meant to find donors who would give regularly, not necessarily to raise a large sum in a single campaign.
To critics, that argument often excuses wastefulness or profiteering – and every charity pays for that.
Some charities “take advantage of American generosity,” said Bennett Weiner, an executive with the Better Business Bureau research program Wise Giving Alliance. They “accomplish very little, siphon off good money from the community and tarnish the well for more legitimate nonprofits.”
By donating to inefficient charities, said Daniel Borochoff, president of the American Institute of Philanthropy, “you are taking money out of the mouth of a hungry kid.”
Urgent accounts
“Where are the children? Where did they go?” a child’s mournful voice sings on the website of Operation Lookout – National Center for Missing Youth. “Are they held captive or do they run free? … Oh, where can they be?”
The group’s online, telemarketing and door-to-door outreach materials offer urgent accounts of runaways or kidnapping victims – and equally urgent pleas for cash.
Commercial firms reported having raised more than $6 million for Operation Lookout, based in Everett, Wash., since 1999. Less than $1 million reached the charity.
A current contract between the fundraiser and the charity sets a minimum 15% return on donations. In practice, over the years, 15% has been the maximum. In each year from 2000 to 2006, it received exactly 15% – not a penny more or less – of funds collected in its name by commercial fundraiser Midwest Publishing.
Operation Lookout did not respond to requests for comment.
The charity has posted a defense of its fundraising on its website, blaming accounting requirements for distorting its results: “In some instances, accounting rules require that we combine volunteer recruitment, calls to action and public education making them appear as a ‘fundraising’ expense.”
Neither of the nation’s two major accounting standards bodies has ruled that such costs must be combined.
Based on tax returns and other sources, the American Institute of Philanthropy ferrets out hidden costs of fundraising by charities – including but not limited to commercial campaigns. It estimated that Operation Lookout spent 84 cents to raise each dollar in its 2006 budget of $2.1 million, a worse record than all but 18 of more than 500 rated charities.
The philanthropy institute normally considers up to 35 cents to raise a dollar as a reasonable cost.
The Times analysis of commercial campaigns in California found inefficiency to be typical among missing-child groups: On average more than 85 cents per dollar went to the fundraiser.
However, some charities of this kind eschew commercial fundraisers, including the National Center for Missing and Exploited Children. Ernie Allen, its chief executive, said groups that rely on minuscule returns taint the entire field.
Patty Wetterling co-founded the Jacob Wetterling Foundation in 1990, after her 11-year-old son was kidnapped at gunpoint. Jacob has not been found.
“Missing children’s organizations spring up out of crisis,” she said. “Fundraisers exploit these devastating situations.”
But groups shouldn’t allow themselves to be overcharged by fundraisers over and over, she said. They “need to learn.”
6% for the charity
Vickie Bouska, a retiree in Hiawatha, Iowa, became a donor to Citizens Against Government Waste after she heard leaders’ arguments on television and received a fundraising letter. The group addressed her fears about the future.
“I’m scared to death of what we’re leaving our children,” she said. “The Chinese or the Canadians are going to own us, because no one in Washington can close the pocketbooks.”
But she was stunned to learn from a reporter how much money the group ceded to its commercial fundraisers. Of $879,000 raised in the decade examined, $49,000 – 6% – reached the charity.
“They aren’t really getting the money,” Bouska said, “so why am I sending it?”
Thomas Schatz, president of the Washington, D.C.-based group founded by industrialist J. Peter Grace and muckraking journalist Jack Anderson, defended its fundraising.
“The purpose of telemarketing is not always to ‘come out ahead,’ though that’s always the goal, but to reinforce [donors] for the future,” he said.
The Los Angeles-based fundraiser Facter Direct, which conducted the group’s telemarketing campaigns, declined to comment on its work for Citizens Against Government Waste. On average, state data show, it returns 39% of what it raises to its clients, slightly less than the industry norm.
“The numbers on the surface don’t always tell the whole story, said the firm’s president, Tom Siegel. “Organizations are not stupid. They recognize the cost of telemarketing and the annoyance of it. But … they recognize that it’s one of the most effective ways to raise money” over the long run.
Schatz noted that the telemarketing campaigns reported to the state reflect a small portion of his organization’s overall fundraising. Direct mail by commercial solicitors – an approach used by the group for two decades – provides most of its approximately $5.4 million in annual revenue and is more efficient, he said.
It proved impossible to verify that claim, because only one direct-mail campaign of behalf of Citizens Against Government Waste was registered with the state, as required, in the 10-year period. That one showed no revenue.
Citizens Against Government Waste suggested that its overall fundraising was highly efficient – costing just 19 cents per dollar raised, according to its 2006 tax return.
But the group came up with that figure by designating most direct-mail and telemarketing costs as “services.” The practice is legal if information, such as criticism of pork-barrel projects, is included in fundraising pitches. The American Institute of Philanthropy estimated the actual cost of the group’s overall fundraising at 69 cents per dollar.
Some other economic-policy nonprofits steer clear of commercial fundraisers, emphasizing foundation grants and direct contact with donors.
Telemarketing and mass mailings can raise awareness, but “there’s always something about it that rubs me the wrong way,” said Ryan Alexander, president of Taxpayers for Common Sense in Washington, D.C. “It’s important that people know that their dollars are going to the mission.”
Joseph Bast, president of the Heartland Institute, a Chicago nonprofit that promotes free-market approaches to environmental and social problems and accepts corporate donations, said he didn’t trust telemarketers to safeguard his group’s image, especially when returns can be low.
“It’s not a very good risk,” he said.
Costs and credibility
American generosity has its limits. The charitable pie in the U.S. has accounted for about 2% of the gross domestic product since 1970.
Meanwhile, the number of charities has risen dramatically. More than 1.9 million nonprofits – one for every 150 U.S. citizens – fill gaps in government services, give voice to diverse views and compete fiercely for this limited share of donor dollars.
Tax laws, designed to encourage giving, don’t take into account how much money reaches the charitable cause. Donors to commercially run drives can write off the entire donation, even if most of it goes to the fundraiser.
Lawmakers periodically have considered reining in the fundraising industry. But the U.S. Supreme Court has limited their options by upholding the free-speech rights of fundraisers and charities.
Recently, public confidence has been shaken by news of inefficient for-profit fundraisers used by police and firefighter charities. And congressional hearings depicted some veterans’ charities as little more than shells that enrich fundraisers and executives.
In a national survey conducted in March, 70% of those polled said charities waste “a great deal” or “a fair amount” of their funds.
Charities “may be losing their most prized possession: their reputation for caring,” said Paul C. Light, a professor of public service at New York University who wrote the survey and has studied nonprofit governance for more than two decades.
Many charities hire for-profit fundraisers precisely because they need help selling themselves to a skeptical public. They can be “a godsend and a lifesaver,” particularly for charities without staff fundraisers, said Diana Aviv, chief executive of Independent Sector, a trade group for nonprofits.
Limited information is available about such firms; most are privately held and many shun the media.
Those that specialize in nonprofits with long-standing patron networks tend to offer better returns. New York-based telemarketer DCM Inc. works exclusively for arts groups, including the Los Angeles Philharmonic, targeting ticket buyers and former donors. It enjoys one of the best records in the business, returning, on average, 72 cents per dollar raised.
“I don’t think that telemarketing is a great contribution to Western civilization, but art is,” said Phil Miller, DCM’s president. “We provide a good service for great organizations.”
Even so, many charities get stuck with unfavorable terms.
A contract between Michigan-based TeleService USA and the veterans charity VietNow makes donor lists “the exclusive property” of the fundraiser. If the charity switches fundraisers, it would lose access to past supporters – the most likely future donors.
TeleService USA, which delivered to its clients, on average, 11 cents per dollar raised, did not respond to requests for an interview.
Hundreds of charities follow the practice used by Operation Lookout, many accepting set-rate contracts that deliver a sliver of gross donations. For small charities, the contracts seem to minimize risk: Fundraisers pay upfront costs, and charities get guaranteed – if often small – returns.
But major charities are the dominant users of for-profit fundraising, whether to raise money for basic costs or to expand a donor base. Among 1,614 charities with commercial campaigns registered in California, 100 accounted for 73% of gross donations. Just two, Paralyzed Veterans of America and the American Diabetes Assn., together took almost 14%. In theory, large charities use for-profit firms more efficiently because they can tap long-standing donor networks. In practice, they do little better than small ones.
Fundraisers for these major groups, meanwhile, reaped a windfall. Those for the 100 top-grossing charities received nearly $977 million, and firms for just the top two pocketed $171 million combined.
“Often the most popular causes … solicit everyone under the sun to get a few dollars from almost everyone,” said Borochoff, of the American Institute of Philanthropy. “That’s a really expensive way to raise money.”
Donors often have no idea where their money goes.
Disillusioned donor
Pamela Kay Weeks lost both breasts to cancer. After she recovered, she received letters and calls from the American Breast Cancer Foundation, a Baltimore charity that listed research as a priority. Hoping to spare others what she suffered, Weeks wrote a check – the first of many.
“I’m not a wealthy person,” said Weeks, 51, an executive secretary who lives in Sneads, Fla. “I’ve given what I can.”
Weeks thought her donations were headed for a philanthropic powerhouse. But the foundation is one of the nation’s least efficient charities, according to the American Institute of Philanthropy and Charity Navigator, another watchdog group.
In 2006 just 2.5% of the American Breast Cancer Foundation budget went to research and 10.5% to mammograms or other services unrelated to fundraising.
In reports filed with the California attorney general from 2003 through 2006, the foundation said it raised $5.8 million from fundraisers, netting just $700,000, or 12%.
The foundation had a particularly close link to one fundraiser, although there was no sign of it in state files.
Phyllis Wolf, executive director of the foundation, created the charity in 1997 with her son Joseph Wolf and two friends. The son worked for the foundation in its early years, then began a for-profit fundraising firm called Non Profit Promotions.
From 2002 through 2006, the foundation paid Non Profit Promotions an average of almost $3 million annually, according to foundation tax returns. That’s not illegal, but it violated conflict-of-interest policies used by several large charities and a model policy by the Better Business Bureau.
And despite state laws requiring fundraisers to submit results, no reports by Non Profit Promotions were on record in California or other states whose files were checked by The Times.
Neither Phyllis Wolf nor Joseph Wolf replied to written questions about their fundraising.
Weeks was surprised – and saddened – to hear where most of the donors’ money went.
“I figured that a lot of it goes to paying people to research cancer,” she said. “It’s unreal, hard to believe.”
charles.piller@latimes.com
doug.smith@latimes.com
Researchers Maloy Moore and Scott Wilson contributed to this report.
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Records filed with the California attorney general’s office show that over the last decade, for-profit fundraisers for the nonprofit kept more than 94 cents of every donated dollar. Yet donors could write off the full contribution on their tax returns.
“It’s a rip-off of the taxpayer,” said Pablo Eisenberg, senior fellow at Georgetown Public Policy Institute and a philanthropy scholar.
A Times investigation found hundreds of other examples of charities that pocketed just a sliver of what commercial fundraisers collected in their names. Some didn’t get a dime or even lost money
The Sin in Doing Good Deeds?
December 30, 2008
If a businessman rakes in a hefty profit while doing good works, is that charity or greed? Do we applaud or hiss?
A new book, “Uncharitable,” seethes with indignation at public expectations that charities be prudent, nonprofit and saintly. The author, Dan Pallotta, argues that those expectations make them less effective, and he has a point.
Mr. Pallotta’s frustration is intertwined with his own history as the inventor of fund-raisers like AIDSRides and Breast Cancer 3-Days - events that, he says, netted $305 million over nine years for unrestricted use by charities. In the aid world, that’s a breathtaking sum.
But Mr. Pallotta’s company wasn’t a charity, but rather a for-profit company that created charitable events. Critics railed at his $394,500 salary - low for a corporate chief executive, but stratospheric in the aid world - and at the millions of dollars spent on advertising and marketing and other expenses.
Taken from New York Times, thanks to Karen for sending on
“Shame on Pallotta,” declared one critic at the time, accusing him of “greed and unabashed profiteering.” In the aftermath of a wave of criticism, his company collapsed.
One breast cancer charity that parted ways with Mr. Pallotta began producing its own fund-raising walks, but the net sum raised by those walks for breast cancer research plummeted from $71 million to $11 million, he says.
Mr. Pallotta argues powerfully that the aid world is stunted because groups are discouraged from using such standard business tools as advertising, risk-taking, competitive salaries and profits to lure capital.
“We allow people to make huge profits doing any number of things that will hurt the poor, but we want to crucify anyone who wants to make money helping them,” Mr. Pallotta says. “Want to make a million selling violent video games to kids? Go for it. Want to make a million helping cure kids of cancer? You’re labeled a parasite.”
I confess to ambivalence. I deeply admire the other kind of aid workers, those whose passion for their work is evident by the fact that they’ve gone broke doing it. I’m filled with awe when I go to a place like Darfur and see unpaid or underpaid aid workers in groups like Doctors Without Borders, risking their lives to patch up the victims of genocide.
I also worry that if aid groups paid executives as lavishly as Citigroup, they would be managed as badly as Citigroup.
Yet there’s a broad recognition in much of the aid community that a major rethink is necessary, that groups would be more effective if they borrowed more tools from the business world, and that there is too much “gotcha” scrutiny on overhead rather than on what they actually accomplish. It’s notable that leaders of Oxfam and Save the Children have publicly endorsed the book, and it’s certainly becoming more socially acceptable to note that businesses can also play a powerful role in fighting poverty.
“Howard Schultz has done more for coffee-growing regions of Africa than anybody I can think of,” Michael Fairbanks, a development expert, said of the chief executive of Starbucks. By helping countries improve their coffee-growing practices and brand their coffees, Starbucks has probably helped impoverished African coffee farmers more than any aid group has.
Mr. Fairbanks himself demonstrates that a businessman can do good even as he does well. Rwanda’s president, Paul Kagame, hired Mr. Fairbanks’s consulting company and paid it millions of dollars between 2000 and 2007.
In turn, Mr. Fairbanks helped Rwanda market its coffee, tea and gorillas. Rwandan coffee now retails for up to $55 a pound in Manhattan, wages in the Rwandan coffee sector have soared up to eight-fold, and zillionaires stumble through the Rwandan jungle to admire the wildlife. President Kagame thanked Mr. Fairbanks by granting him Rwandan citizenship.
There are lots of saintly aid workers in Rwanda, including the heroic Dr. Paul Farmer of Partners in Health, and they do extraordinary work. But sometimes, so do the suits. Isaac Durojaiye, a Nigerian businessman, is an example of the way the line is beginning to blur between businesses and charities. He runs a for-profit franchise business that provides fee-for-use public toilets in Nigeria. When he started, there was one public toilet in Nigeria for every 200,000 people, but by charging, he has been able to provide basic sanitation to far more people than any aid group.
In the war on poverty, there is room for all kinds of organizations. Mr. Pallotta may be right that by frowning on aid groups that pay high salaries, advertise extensively and even turn a profit, we end up hurting the world’s neediest.
“People continue to die as a result,” he says bluntly. “This we call morality.”
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An Iranian rocket carrying the satellite "Omid," or "Hope" in English, in this photo released by the Fars News Agency at an undisclosed location on Monday. (Fars News Agency via The Associated Press)
Iranian satellite launch prompts concern
By Alan Cowell and William J. Broad
Published: February 4, 2009
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Iran said Tuesday that it had launched its first domestically produced satellite, a move that has prompted concerns in the United States and other nations about Iran's nuclear ambitions and its ability to deploy long-range ballistic missiles.
The launch on Monday, coinciding with celebrations marking the 30th anniversary of the Islamic revolution, also creates an early challenge for President Barack Obama, who has sought to strike a conciliatory tone toward Iran by conditionally offering dialogue after years of tensions. The United States and other nations believe Tehran wants to develop nuclear weapons, a charge that Iran's leaders deny.
Iran said it had also used a domestically produced rocket to launch the satellite. That would make it part of the exclusive club of states that can loft objects into orbit, which now numbers at least nine. Weapons experts say the same technology used to put satellites into orbit can also be used for launching weapons.
In Washington, State Department spokesman Robert Wood called the reports a matter of "great concern" and potentially in violation of United Nations agreements limiting Iran from missile activity.
"Developing a space launch vehicle that could be put a satellite into orbit could possibly lead to the development of a ballistic missile system," he said.
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The head of Israel's Space Agency, Zvi Kaplan, said initial reports showed that a satellite had been launched.
"From what I have been investigating it is true," he said, according to The Associated Press. "We are not surprised because in this day and age of information and technology and with Iranian scientists studying abroad they can obtain the knowledge."
The official Iranian news agency, IRNA, said the satellite was launched using a Safir-2 rocket and was "successfully sent into orbit."
The satellite, which weighed about 60 pounds, is named Omid, or Hope, IRNA said, and was sent into space as a "data-processing satellite project" that began in March 2005 as "the first practical step toward acquiring national space technology."
"The project's experts focused on manufacturing the equipment and helping develop the potential of domestic companies to carry out such projects," the IRNA report said.
David Albright, the president of the Institute for Science and International Security, a private group in Washington that tracks nuclear proliferation, said he felt the technology used by Iran to launch the satellite remained rudimentary by international standards.
"It's not a very capable missile. The payload and diameter aren't that great," he said. "It doesn't say much, if anything, about their ability to deliver a nuclear weapon. But part of the concern here is that Iran is continuing its steady drip-drip-drip toward a nuclear weapons capability."
Aerospace experts said that while Tehran was still not capable of launching an intercontinental ballistic missile, the satellite still took many years of preparation and required the mastery of staging — the art of creating a multistage rocket that drops stages (and thus weight) as it races ever higher.
Last August, Iran test-fired a new rocket capable of carrying a satellite into orbit. Western experts said at the time that the launching represented a potentially significant, if much-delayed, step in Iran's efforts to join the international space club. And in 2005, Tehran launched a Russian-made satellite with a Russian-made rocket.
"They've been at this game for about five years, working on a rocket big enough to put a satellite into space," said Charles Vick, an expert on Iranian rockets at GlobalSecurity.org, a private research group in Alexandria, Virginia.
He added that the Iranian rocket had two stages. If carrying a warhead, he said, the Iranian missile could fire the weapon about 1,550 miles.
For Iran to achieve the technical step of launching an intercontinental ballistic missile, Vick said, it would have to develop a more powerful basic rocket or more upper stages — goals it is pursuing.
Since Obama's inauguration, outside powers have been looking for clues as to whether Tehran is prepared to make new concessions in the dispute over Iran's alleged efforts to develop nuclear weapons. They have also been waiting to see how the new American president will pursue his overtures of dialogue, which are designed to halt Iran's uranium enrichment program.
Nations dealing with the Iran issue — the United States, Russia, Britain, China, France and Germany — will meet Wednesday in Frankfurt, Germany, for their first talks since Obama took office.
Iranian state television on Tuesday showed footage of a rocket blasting off from a firing platform in a huge blast of smoke flame as it clambered into the night sky.
Breast Cancer Month Spurs Pink-Product Debate
Run Date: 10/21/04
By Rebecca Vesely
WeNews correspondent
October is Breast Cancer Awareness Month and many companies have been peddling pink products to help the cause. Critics, however, say it's a dubious way to raise funds and urge shoppers to know how businesses handle the proceeds.
Save Lids to Save Lives
SAN FRANCISCO (WOMENSENEWS)--Seeing pink these days?
You're not the only one. From Estee Lauder's pink lipstick to pink Keds to Conair pink travel hair dryers to Jimmy Choo hot pink high heels, numerous companies are peddling pink products.
That's because October is National Breast Cancer Awareness Month, which each year raises millions of dollars for breast cancer charities and reminds women that early detection works.
So what's wrong with that?
A lot, says Breast Cancer Action, a San Francisco nonprofit that this month launched "Think Before You Pink," an awareness campaign that casts a critical eye on social marketing campaigns for breast cancer.
"Companies have figured out that if you slap a pink ribbon on a product, people will buy it," says Barbara Brenner, executive director of Breast Cancer Action. "It's time to look past the ribbon and ask, 'Where is the money going?'"
Some manufacturers clearly state on their packaging how much money is going to a specific organization and who will benefit. Others aren't so clear. They say only that a "portion of proceeds" or, more commonly, a "generous portion of proceeds" will go to breast cancer research.
In a partnership with journalist and activist Barbara Ehrenreich, Breast Cancer Action is urging consumers to ask where their money goes after they buy a pink product.
Consumers, Brenner says, should educate themselves on how much their purchases will really help breast cancer organizations financially and how those groups, in turn, use the money. If a retailer can't tell them, they should walk away.
Breast Cancer Action is also calling for more coordination within breast cancer research funding to speed work toward a cure. Although millions of dollars pour into breast cancer research, no one knows exactly how much money is being raised and spent every year or where all the money goes, Brenner says.
The group is suggesting a worldwide effort, along the lines of polio eradication, to find a cure for breast cancer. Since 1994, California has been coordinating its efforts on a state level under the California Breast Cancer Research Program, and Brenner says it's a good model. Funded by a tobacco tax and a tax check-off on state income tax forms and administered by the University of California, the program has awarded nearly $165 million to breast cancer research and education in the state.
Yoplait Lid Drive
Breast Cancer Action is particularly critical of the "Save Lids to Save Lives" by the Yoplait. General Mills, which owns Yoplait, donates 10 cents to breast cancer research for every lid consumers send back to the company in September and October.
At that rate, each consumer would have to eat three Yoplait yogurts a day for the month of October to raise just $36, excluding postage, Breast Cancer Action points out.
Pam Becker, spokeswoman for Minneapolis, Minn.-based General Mills, says the company is proud of the campaign, which has been running for seven years, and will raise $2.1 million for the Susan G. Komen Breast Cancer Foundation, in Dallas, this year. About $1.1 million will come from the lid campaign and another $900,000 will be from a company donation.
"That contribution is not insignificant," Becker says. "And pink lids help raise awareness."
Beyond Awareness
Brenner says that at this point, awareness isn't enough.
"At this point aren't we all aware of breast cancer?" she retorts. "With 40,000 American women dying from breast cancer each year, shouldn't we focus on solving the problem?"
Breast Cancer Action is also critical of Yoplait because some of its yogurts may contain recombinant bovine growth hormone, a drug used to increase milk production in dairy cows.
Some companies have moved to eliminate rBGH from their products and Canada and the European Union have both banned rBGH due to concerns about links between a number of illnesses and growth hormones.
The U.S. Food and Drug Administration approved rBGH in 1993 after conducting a safety review.
General Mills' Becker says more awareness is needed to encourage women to do self-breast exams and mammograms.
"One thing that is helping people these days is early detection and we need to encourage that," she says. She also points out that no direct link between rBGH and breast cancer has been established.
Environmental Links
A study released this month by the Breast Cancer Fund, a San Francisco environmental group, and Breast Cancer Action indicates that as many as half of all breast cancer cases remain unexplained by personal characteristics, such as genetics. The report points out that numerous researchers believe many cases are linked to environmental factors. About 216,000 women will be diagnosed with breast cancer in the United States this year.
Shelley Alpern, vice president and manager of social research at Trillium Assets Management Corp., a Boston-based socially responsible investment firm, has looked closely at social marketing and breast cancer. She says the Breast Cancer Action is on the right track.
"There's the whole question of where the proceeds go and how much of it goes directly to the campaign," Alpern says. "The answer is, 'however much the company decides.'"
Alpern says consumers who are collecting yogurt-container lids to raise money for breast cancer would be helping more by giving directly to the Komen Foundation. Consumers should also be wary of any campaign, she adds, with an "inherent conflict of interest," such as a car dealership running a breast cancer campaign.
"The product pumps out carcinogens, and yet they are trying to pinkwash their own image."
Corporate 'Pinkwashing'
"Pinkwashing," or aligning a product with the fight against breast cancer, helps companies associate themselves with a good cause, Alpern says.
But companies that produce pink products say their contribution has been significant.
"The progress in the last 15 years, since the cosmetic companies have been involved, has been tremendous," says Irene Malbin, vice president of the Cosmetic, Toiletry and Fragrance Association, a trade group in Washington, D.C. "Awareness programs have not only helped educate women about the benefits of early detection, but the media attention that the programs have generated has also mobilized the clinical and medical field to push for more advancements in finding a cure."
And it's unlikely that everyone who buys a pink product would otherwise write a check directly to a breast cancer organization, Malbin points out.
Recipients of the cosmetic industry's fundraising include hospitals, cancer centers and nonprofit breast health programs.
One large beneficiary is the Susan G. Komen Breast Cancer Foundation, which over its 18-year history has raised $740 million to fight breast cancer. A peer-review committee at the foundation evaluates applications for funding for research, as well as education, screening and treatment for underserved populations.
Rebecca Vesely is a health care reporter at the Oakland Tribune.
For more information:
The Susan G. Komen Breast Cancer Foundation--
http://www.komen.org/
Yoplait--Save Lids to Save Lives:
http://www.yoplaitusa.com/breastcancer_lids.aspx
say it's a dubious way to raise funds and urge shoppers to know how businesses handle the proceeds.
You're not the only one. From Estee Lauder's pink lipstick to pink Keds to Conair pink travel hair dryers to Jimmy Choo hot pink high heels, numerous companies are peddling pink products.
That's because October is National Breast Cancer Awareness Month, which each year raises millions of dollars for breast cancer charities and reminds women that early detection works.
So what's wrong with that?
A lot, says Breast Cancer Action, a San Francisco nonprofit that this month launched "Think Before You Pink," an awareness campaign that casts a critical eye on social marketing campaigns for breast cancer.
#9-Tetrahydrocannabinol Inhibits Cell Cycle Progression in Human
Breast Cancer Cells through Cdc2 Regulation
Marı´a M. Caffarel,1 David Sarrio´,2 Jose´ Palacios,2 Manuel Guzma´n,1 and Cristina Sa´nchez1
1Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University and 2Breast and Gynecological Cancer
Group, Molecular Pathology Programme, Centro Nacional de Investigaciones Oncolo´gicas, Madrid, Spain
Abstract
It has been proposed that cannabinoids are involved in the
control of cell fate. Thus, these compounds can modulate
proliferation, differentiation, and survival in different manners
depending on the cell type and its physiopathologic
context. However, little is known about the effect of
cannabinoids on the cell cycle, the main process controlling
cell fate. Here, we show that #9-tetrahydrocannabinol (THC),
through activation of CB2 cannabinoid receptors, reduces
human breast cancer cell proliferation by blocking the
progression of the cell cycle and by inducing apoptosis. In
particular, THCarres ts cells in G2-M via down-regulation of
Cdc2, as suggested by the decreased sensitivity to THC
acquired by Cdc2-overexpressing cells. Of interest, the
proliferation pattern of normal human mammary epithelial
cells was much less affected by THC. We also analyzed by realtime
quantitative PCR the expression of CB1 and CB2
cannabinoid receptors in a series of human breast tumor
and nontumor samples. We found a correlation between CB2
expression and histologic grade of the tumors. There was also
an association between CB2 expression and other markers of
prognostic and predictive value, such as estrogen receptor,
progesterone receptor, and ERBB2/HER-2 oncogene. Importantly,
no significant CB2 expression was detected in nontumor
breast tissue. Taken together, these data might set the
bases for a cannabinoid therapy for the management of breast
cancer. (Cancer Res 2006; 66(13): 6615-21)
Introduction
There are very few critical decisions that cells must take during
their lifetime. Basically, these are whether to proliferate, differentiate,
or die. A tight regulation of the cell cycle is crucial to control
all these decisions, and its deregulation has devastating consequences,
such as cancer (1). It has been proposed that
cannabinoids, the active components of Cannabis sativa, play a
role in the control of the aforementioned decisions. For example,
they can modulate survival, proliferation, and differentiation
depending on the cell type and its physiopathologic context
(2, 3). Among the f70 cannabinoids synthesized by C. sativa,
D9-tetrahydrocannabinol (THC) is the most important in terms of
potency and abundance (4). THC exerts a wide variety of biological
effects by mimicking endogenous compounds, the endocannabinoids
anandamide and 2-arachidonoylglycerol, which activate
specific cannabinoid receptors. Thus far, two G protein–coupled
cannabinoid-specific receptors have been cloned from mammalian
tissues: CB1, abundantly expressed in the brain and at many
peripheral sites, and CB2, almost exclusively expressed in the
immune system (5). Engagement of these receptors by THC or
endocannabinoids affects several signaling pathways, some of them
directly involved in the control of cell fate. For instance,
cannabinoids modulate mitogen-activated protein kinases and
the phosphatidylinositol 3-kinase/Akt survival pathway, which have
a prominent role in the control of cell growth and differentiation
(6). Due to the growing evidence that cannabinoids participate in
the control of cell fate and to the fact that the cell cycle is a key
process underlying the regulation of survival/proliferation/differentiation
decisions, we decided to study the effect of THC on the
cell cycle and the mechanism of cannabinoid action on this
process. Because breast tumors are one of the most common
human neoplasias and one of the leading causes of death among
Western women (7), we decided to focus our studies on this
particular type of cancer.
Materials and Methods
Cell culture and viability. EVSA-T, MDA-MB-231, MDA-MB-468, and
SKBr3 cells were kindly given by Dr. Lo´pez-Rivas [Centro Andaluz de
Biologı´a del Desarrollo, Consejo Superior de Investigaciones Cientı´ficas
(CSIC), Sevilla, Spain], and MCF-7 and T-47D cells were obtained from the
American Type Culture Collection (ATCC; Manassas, VA). Cells were
maintained in RPMI 1640 (EVSA-T, MDA-MB-231, MCF-7, and T-47D) or
DMEM (MDA-MB-468 and SKBr3) supplemented with 10% fetal bovine
serum (FBS), 5 units/mL penicillin, and 5 mg/mL streptomycin. Human
mammary epithelial cells (HMEC) were kindly given by Dr. Lacal (Instituto
de Investigaciones Biome´dicas, CSIC, Madrid, Spain) and grown in
mammary epithelial growth medium (Cambrex, East Rutherford, NJ)
according to the manufacturer’s instructions. Cannabinoid ligands were
prepared in DMSO. Control incubations had the corresponding DMSO
content. No significant influence of DMSO was observed on cell viability
at the final concentration used (0.1-0.2%, v/v). Cell viability was determined
by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test
(Sigma, St. Louis, MO) according to the manufacturer’s instructions.
Western blot analysis. Samples were subjected to SDS-PAGE, and
proteins were transferred onto polyvinylidene fluoride membranes. Blots
were incubated with the following antibodies: anti-phosphorylated Cdc2
(Tyr15), anti-cyclin B1, anti-Cdc25C, anti-p27, anti-caspase-3, and antipoly(
ADP-ribose) polymerase (PARP) (Cell Signaling, Beverly, MA); anti-
Cdc2 and anti-p21 (Santa Cruz, Santa Cruz, CA); anti-Wee1 (BioVision,
Mountain View, CA); anti-survivin (R&D Systems, Minneapolis, MN); and
anti-a-tubulin as loading control (Sigma). Luminograms were obtained with
an enhanced chemiluminescence detection kit (Amersham Life Sciences,
Arlington Heights, IL), and densitometric analysis was done with Multianalyst
software (Bio-Rad, Hercules, CA).
Cell cycle analysis. Cells were permeabilized and fixed in 1% (w/v)
bovine serum albumin and 30% ethanol-PBS and labeled with 5 Ag/mL
Hoechst 33342 (Molecular Probes, Leiden, the Netherlands). Fluorescence
intensity was analyzed using a LSR flow cytometer (Becton Dickinson, San
Jose, CA). Ten thousand cells per analysis were recorded.
Requests for reprints: Cristina Sa´nchez, Department of Biochemistry and
Molecular Biology I, School of Biology, Complutense University, 28040 Madrid,
Spain. Phone: 34-913944668; Fax: 34-913944672; E-mail: csg@bbm1.ucm.es.
I2006 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-05-4566
www.aacrjournals.org 6615 Cancer Res 2006; 66: (13). July 1, 2006
Research Article
Apoptosis. Cells were incubated in binding buffer [10 mmol/L HEPES
(pH 7.4), 2.5 mmol/L CaCl2, 140 mmol/L NaCl] supplemented with Annexin
V-FITC (Molecular Probes). Propidium iodide (PI; Sigma) was added
1 minute before sample analysis. Fluorescence intensity was analyzed using
a FACS Scalibur flow cytometer (Becton Dickinson). For triple staining
experiments, cells were labeled with Hoechst 33342, and apoptosis was
analyzed as described above. Ten thousand cells per analysis were
recorded.
Caspase-3 activity. Caspase-3/7 activity was determined with a
luminogenic substrate (Caspase-Glo, Promega, Madison, WI) according to
the manufacturer’s instructions. Luminescence was determined in a
Microplate Fluorescence Reader (BMG Labtech, Offenburg, Germany).
Tissue samples. Samples were obtained from the Centro Nacional de
Investigaciones Oncolo´gicas Tumor Bank (Madrid, Spain). Histologic
grade was assessed according to Elston and Ellis criteria. Immunohistochemical
staining for prognostic and predictive factors was done by
the EnVision method with a heat-induced antigen retrieval step.
Monoclonal antibodies for estrogen receptor (ER), progesterone receptor
(PR), p53 (Novocastra, Newcastle, United Kingdom), and Ki67 (DAKO,
Glostrup, Denmark) were used. ERBB2/HER-2 expression was evaluated
using Herceptest (DAKO). The percentage of cells with unequivocal
nuclear staining for ER, PR, Ki67, and p53 was scored, and a cutoff of
5% was used for positivity for ER and PR and 15% for Ki67 and p53. For
ERBB2/HER-2, only cases with 3+ membranous staining were scored as
positive.
Confocal microscopy analysis of cannabinoid receptors. Human
breast cancer and normal breast 5-Am paraffin-embedded tissue sections
were analyzed. Primary antibodies against CB1 and CB2 receptors (Affinity
Bioreagents, Golden, CO) were used. Secondary anti-rabbit antibody Alexa
Fluor 594 was from Molecular Probes. Cell nuclei were stained with YOYO-1
(Molecular Probes). Confocal fluorescence images were acquired using
Laser Sharp 2000 software (Bio-Rad).
Reverse transcription-PCR analysis. Total RNA was isolated using the
RNeasy Protect kit (Qiagen, Hilden, Germany). cDNA was obtained using
Figure 1. Effect of cannabinoids on
human breast cell proliferation. Cells were
incubated in low-FBS medium (2.5% for
MCF-7 and 0.5% for the rest of the tumor
cell lines) for 72 hours in the presence of
5 Amol/L THC (A), different concentrations
of THC ranging from 1 to 12 Amol/L (B),
or the specified THC concentration (C).
When antagonists were used (1 Amol/L
rimonabant and 2 Amol/L SR144528), they
were added 1 hour before THC. ER and
p53 status was obtained from ATCC and
German National Resource Centre for
Biological Material (DSMZ, Braunschweig,
Germany). IC50s were defined as
concentrations of THC required to
decrease cell viability to 50%. A, columns,
mean for each cell line (n z 3); bars, SE.
B, significant differences from MCF-7
(*, P < 0.05; **, P < 0.01) or T-47D cells
(b, P < 0 .0 5; bb, P < 0.01). For real-time
quantitative PCR experiments, an arbitrary
value of 1 was assigned to cannabinoid
receptor expression in MDA-MB 468 cells
(reference). Representative experiment
(n = 3). A and C, columns, means cell
viability in THC-treated cells versus their
respective vehicle-treated cells (set at
100%; n z 3); bars, SE. Significant
differences (*, P < 0.05; **, cc, P < 0 .0 1)
from control (*) or 5 Amol/L THC alone (c).
D, CB1 and CB2 mRNA expression in
the cell lines was determined by RT-PCR
(representative gels, n z 3). U373 MG
astrocytoma cells and Jurkat leukemia
cells were used as positive controls for CB1
and CB2, respectively.
Cancer Research
Cancer Res 2006; 66: (13). July 1, 2006 6616 www.aacrjournals.org
Transcriptor Reverse Transcriptase (Roche, Applied Science, Penzberg,
Germany). Primer sequences were CB1 (sense), 5¶-CGTGGGCAGCCTGTTCCTCA-
3¶; CB1 (antisense), 5¶-CATGCGGGCTTGGTCTGG-3¶; CB2 (sense),
5¶-TGGGACAGGGTCAGTACAAGT-3¶; CB2
(antisense), 5¶-CTTTGGCTCCTGGTGGTCT-
3¶; glyceraldehyde-3-phosphate dehydrogenase (GAPDH;
sense), 5¶-GGGAAGCTCACTGGCATGGCCTTCC-3¶; and GAPDH (antisense),
5¶-CATGTGGGCCATGAGGTCCACCAC-3¶.
Real-time quantitative PCR. Total RNA and cDNA were obtained from
frozen breast tumors or cell lines as described above. Taqman probes for
human CB1, CB2, and 18S RNA (used as internal reference) were from
Applied Biosystems (Foster City, CA).
Plasmids and transfections. pIRESpuro2 and pIRESpuro2-Cdc2 were
kindly given by Dr. Huang ( Johns Hopkins University, Baltimore, MD).
Transfections were carried out with Fugene 6 (Roche Applied Science,
Indianapolis, IN) according to the manufacturer’s protocol. Transfected
cells were selected with puromycin.
Statistical analysis. ANOVA with a post hoc analysis by the Student‘s-
Newman-Keuls’ test was routinely used. For cannabinoid receptor
expression in human samples, data were log transformed to achieve
normality in the distribution. An F-test was subsequently done to compare
equality of variances in each group, and a classic t test or a t test with
different variances was applied using the Welch modification.
Results
THCinhibits proliferation of human breast cancer cells.
Several human breast cell lines were incubated with THC, and
viable cell numbers were estimated. THC decreased proliferation in
all the tumor cells tested (Fig. 1A). Among the tumor cells, those
with more aggressive phenotype (ER) were more sensitive to THC
(Fig. 1B). Remarkably, nontumor HMEC cells were the most
resistant to cannabinoid treatment (IC50 >12 Amol/L; Fig. 1A).
Rimonabant, a selective CB1 receptor antagonist, did not block
THC effect in EVSA-T cells (Fig. 1C) or any of the other cell lines
studied (data not shown). In contrast, SR144528, a selective CB2
receptor antagonist, partially prevented the THC-induced decrease
of EVSA-T cell proliferation (Fig. 1C). Both reverse transcription-
PCR (RT-PCR; Fig. 1D) and real-time quantitative PCR experiments
(Fig. 1B) confirmed the expression of CB2 mRNA in this cell line,
whereas CB1 mRNA was undetectable (Fig. 1B and D).
THC-induced decrease of cell proliferation is due to the
blockade of the G2-M transition. We next sought to examine
whether an alteration of EVSA-T cell cycle underlies THC
antiproliferative effect. The cannabinoid increased the number of
cells in the G0-G1 compartment and, in parallel, decreased the
number of cells in S phase (Fig. 2A and C). At the highest
concentration tested (5 Amol/L), THC also produced the following:
(a) an increase in the number of cells in G2-M phases and (b) the
appearance of a population of hypodiploid cells (Fig. 2A and C).
The latter two effects were prevented by SR144528 (Fig. 2C).
Importantly, THC did not alter the cell cycle profile of HMEC cells
(Fig. 2B).
To analyze the precise mechanism of THC action, we first
studied the expression of several proteins involved in the G2-M
transition. THC decreased the total levels of Cdc2 [p34, cyclindependent
kinase (CDK) 1; Fig. 3A], the major CDK controlling the
entrance of cells in mitosis after completing G2 events (8), and
SR144528 (2 Amol/L) completely prevented this effect [relative
optical density (OD) after 16 hours of treatment relative to vehicle:
SR144528+THC, 105 F 4]. The expression of cyclin B1, the positive
regulatory subunit of Cdc2 (9), did not significantly change on
cannabinoid challenge (data not shown). The levels of p21, a CDK
inhibitor known to prevent Cdc2-cyclin B activation (8), were
enhanced by THC (Fig. 3A). It has been recently proposed that p27,
a CDK inhibitor traditionally associated to the regulation of G1-S
transition, can also inhibit Cdc2 at G2-M (10). In our system,
however, THC did not modify p27 levels (data not shown).
To be active, Cdc2 has to be dephosphorylated in the Tyr15
residue (11). THC treatment decreased the amount of phosphorylated
Tyr15-Cdc2 to a lower extent than total Cdc2 levels (Fig. 3A),
indicating that the ratio inactive/active Cdc2 was augmented by
THC. Phosphorylation of Cdc2 in Tyr15 is controlled by the Wee1/
Mik1 family of protein kinases and by the phosphatase Cdc25C
(11). Our results show that THC enhances Wee1 and reduces
Cdc25C protein levels (Fig. 3A).
In view of the aforementioned results, it is conceivable that
THC exposure prevents EVSA-T cells to reach the required levels
of active Cdc2 to enter mitosis. To test whether Cdc2 downregulation
is important in the growth-inhibiting effect of THC,
we heterologously expressed this kinase. As shown in Fig. 3B,
cells overexpressing Cdc2 became significantly more resistant
to THC.
Figure 2. Effect of THC on cell cycle
dynamics. Cell cycle profiles of EVSA-T
(A) and HMEC cells (B) incubated with
5 Amol/L THC or the corresponding vehicle
for 48 hours. Representative experiments
(n z 3). C, percentage of EVSA-T cells
(mean F SE) in every phase of the cell
cycle after 48 hours of incubation with the
indicated compounds. Where indicated,
rimonabant (SR1; 1 Amol/L) or SR144528
(SR2; 2 Amol/L) was added 1 hour
before THC. Significant differences from
control (*, P < 0.05; **, P < 0.01) or the
corresponding concentration of THC alone
(b, P < 0.05).
Control of Cell Cycle by Cannabinoids
www.aacrjournals.org 6617 Cancer Res 2006; 66: (13). July 1, 2006
THC-induced cell cycle arrest is associated with apoptosis.
We next tried to elucidate whether THC-induced inhibition of
proliferation was associated with cell death. The cannabinoid
induced apoptosis, a process that was prevented by SR144528
(Fig. 4A). THC also induced a two-fold increase in caspase-3
activity, an effect that was prevented by SR144528 (Fig. 4B).
Likewise, we observed reduced levels of both pro-caspase-3 (the
inactive precursor of caspase-3) and PARP (a caspase-3 substrate)
in cannabinoid-treated cells (Fig. 4C). We subsequently addressed
the question of whether apoptotic cells were those arrested in G2-M
by THC. We conducted triple staining experiments to analyze the
percentage of apoptotic cells in every phase of the cell cycle. As
shown in Fig. 5A, THC induced apoptosis in all the cell cycle
phases, but the majority of apoptotic cells were in the G2-M
compartment.
It is known that survivin, a member of the inhibitor of apoptosis
family, can be phosphorylated in Thr34 by Cdc2. This phosphorylation
results in enhanced stability of survivin and the
consequent inhibition of caspase activity (12). Survivin was highly
expressed in all the breast cancer cell lines tested but was hardly
detectable in HMEC (Fig. 5B), in agreement with previous reports
showing a sharp differential expression in cancer (high levels)
versus normal (undetectable levels) tissues (12). Moreover, THC
decreased survivin levels in EVSA-T cells (Fig. 5C), which may
explain why THC-induced Cdc2 inactivation results in apoptosis.
In fact, when Cdc2 levels were enhanced by overexpression,
survivin decrease on cannabinoid treatment was attenuated
(relative optical density after 16 hours of treatment for
pIRESpuro2-transfected cells: vehicle, 100 F 6; 5 Amol/L THC,
31 F 4. For pIRESpuro2-Cdc2-transfected cells: vehicle, 100 F 5;
5 Amol/L THC, 74 F 3; Fig. 5D).
Cannabinoid receptors are expressed in human breast
tumors. The presence of CB1 and CB2 receptors in human breast
tumors was evaluated by real-time quantitative PCR and confocal
microscopy. Lower levels of CB1 mRNA were detected in tumors
of low-medium (grade 1-2) and high (grade 3) histologic grade
compared with normal, noncancerous breast tissue (grade 1-2
versus noncancerous breast tissue (P = 0.008); grade 3 versus
noncancerous breast tissue (P = 0.0007); Fig. 6A). CB2 expression
was higher than CB1 expression in all the tumors analyzed (P =
0.00002) and seemed to correlate with their histologic grade
(grade 1-2 versus grade 3; P = 0.04; Fig. 6A). Of interest, CB2
transcripts were hardly detectable in normal breast tissue
(Fig. 6A). Moreover, the expression of CB2 showed an association
with molecular markers of prognostic value. Thus, ER tumors
expressed more CB2 mRNA than ER+ tumors (Fig. 6B). CB2
expression was also higher in PR than in PR+ samples. ER/
PR tumors (response rate <10% to conventional therapies)
Figure 3. Effect of THC on the expression
of proteins involved in the control of the
G2-M transition. A, Western blot analysis
of the indicated proteins was done at least
twice. Representative luminograms. T3,
3 Amol/L THC and T5, 5 Amol/L THC.
Optical density (arbitrary units, mean F
SE) of the different proteins relative to their
respective time-point control incubations
(set at 100). B, EVSA-T cell viability was
assayed after 72 hours of incubation
with 5 Amol/L THC or vehicle. Columns,
mean cell viability in THC-treated cells
versus their respective vehicle-treated
cells (set at 100%); bars, SE. Inset,
Western blot of total Cdc2 levels in
EVSA-T cells (lane 1), EVSA-T cells
transfected with pIRESpuro2 (lane 2),
and EVSA-T cells transfected with
pIRESpuro2-Cdc2 (lane 3). A and B,
significant differences from the
corresponding vehicle-treated cells
(*, P < 0.05; **, P < 0.01) or between
THC-treated cells transfected with
either pIRESpuro2 or pIRESpuro2-Cdc2
(b, P < 0.05).
Cancer Research
Cancer Res 2006; 66: (13). July 1, 2006 6618 www.aacrjournals.org
expressed more CB2 mRNA than ER+/PR+ tumors (60-70%
response rate; ref. 7; Fig. 6B). ERBB2/HER-2-positive tumors
(with worse survival prediction at least in node-positive patients;
ref. 7) expressed higher levels of CB2 mRNA than ERBB2/HER-2-
negative tumors (Fig. 6B). Confocal microscopy experiments
confirmed the presence of CB1 and CB2 proteins in tumor cells
(Fig. 6C). None of the receptor proteins was detected in normal
ducts.
Figure 4. Analysis of THC-induced cell
death. Fluorescence-activated cell sorting
analysis (A) and caspase-3 activity (B)
of EVSA-T cells after incubation with the
indicated compounds for 24 hours.
Antagonists (1 Amol/L rimonabant or
2 Amol/L SR144528) were added 1 hour
before THC (5 Amol/L). A, percentage
of apoptotic cells (FITC positive/PI
negative) within the total cell population.
B, columns, means of at least five or
more experiments; bars, SE. Significant
differences from control (*, P < 0 .0 5;
**, P < 0.01) or THC alone (b, P < 0 .0 5;
bb, P < 0.01). C, Western blot analysis of
pro-caspase-3 and PARP. T3, 3 Amol/L
THC and T5, 5 Amol/L THC.
Representative luminograms (n z 3).
Figure 5. Connection between cell
cycle arrest and apoptosis. A, percentage
of apoptotic cells in different phases
of the cell cycle as assessed by triple
staining with Annexin V-FITC, PI, and
Hoechst 33342. Significant differences
from the corresponding vehicle-treated
cells (*, P < 0.05; **, P < 0.01) or
between THC-treated cells in G2-M
and THC-treated cells in G0-G1 or S
(b, P < 0.05). B-D, Western blot of survivin
in different human breast cell lines (B),
EVSA-T cells incubated with THC (T3, 3
Amol/L THC; T5, 5 Amol/L THC) or the
corresponding vehicle (C), and EVSA-T
cells transfected with pIRESpuro2 or
pIRESpuro-Cdc2 and incubated for 16
hours with the indicated compounds (D).
Representative luminograms (n z 2).
Control of Cell Cycle by Cannabinoids
www.aacrjournals.org 6619 Cancer Res 2006; 66: (13). July 1, 2006
Discussion
There is scant information on the effect of cannabinoids on the
cell cycle. It has been shown previously that the endogenous
cannabinoid anandamide arrests the cycle of hepatoma HepG2
cells (13), epidermal growth factor–stimulated PC3 prostate cancer
cells (14), and the breast cancer cell line EFM-19 (15) at the G1-S
transition. The same effect was exerted by a metabolically stable
analogue of anandamide, met-F-anandamide, on KiMol thyroid
carcinoma cells (16). Results presented herein (a) show that the
plant-derived cannabinoid THC is able to block the progression of
breast cancer cell cycle and (b) provide a mechanism for this
action. Of interest, nontumor mammary epithelial cells were rather
insensitive to THC, suggesting that cannabinoids would fulfill one
of the demanded requirements of any compound intended to be
used in cancer therapy: the selectivity for tumor cells. In our
experimental model, THC induces a CB2-mediated cell cycle arrest
at the G2-M transition via Cdc2 down-regulation. We are
nonetheless aware that our observation that THC effects are
significantly but not completely prevented by the CB2 selective
antagonist also points to the existence of CB2-independent
Figure 6. Cannabinoid receptor
expression in human breast tumors.
A and B, mRNA expression (medians F
SE) of CB1 and CB2 receptors as
determined by real-time quantitative PCR.
An arbitrary value of 1 unit was assigned to
CB2 expression in one of the tumors
(reference), and the relative expression of
the rest of the samples was calculated
by comparison with the reference. Note the
different scale in the y axes of (A).
Significantly different values (*, P < 0 .0 5;
**, P < 0.01) from ‘‘+ variable’’ or ‘‘+/+
variable.’’ ER; PR; S1, <2 cm; S2, 2 to
5 cm; and S3, >5 cm; metastasis was
determined as presence/absence of
positive axillary lymph nodes. C, confocal
microscopy analysis of cannabinoid
receptors in a grade 1 (left ) and a grade
3 tumor (right ). Green, cell nuclei; red,
cannabinoid receptors. Arrows, different
areas within the tumor samples.
Cancer Research
Cancer Res 2006; 66: (13). July 1, 2006 6620 www.aacrjournals.org
processes in cannabinoid antiproliferative action. Most cancer cells
evade antigrowth signals because they have defective G1 checkpoints,
which makes the G2 checkpoint an attractive target for
cancer therapy (11). Since its discovery in 1986 (17), it has been
clear that Cdc2 is essential for cell cycle progression (18). In fact,
Cdc2-deficient mice die at very early stages of embryonic
development (18). The pivotal importance of this particular protein
could explain why, in our system, a somewhat moderate decrease
of total Cdc2 levels (f40%) results in large changes in cell viability.
In addition, our data indicate that THC increases the inactive/
active Cdc2 ratio, supporting that the cannabinoid decreases not
only total Cdc2 levels but also enzyme-specific activity.
It is important to point out that, although the antiproliferative
effect of cannabinoids on different tumor cells has been extensively
confirmed both in vitro and in vivo (6), a recent report indicates
that THC may enhance breast cancer cell growth under certain
circumstances. In that study, the authors showed a direct
association between the degree of sensitivity of a tumor to THC
and the level of CB1 and CB2 expression. Thus, THC has
antiproliferative effect in tumors expressing cannabinoid receptors,
whereas those with low to no expression suffer increased growth
and metastasis due to THC-induced suppression of the antitumor
immune response (19). Results presented herein are not in
disagreement with that report, as EVSA-T cells, which are very
sensitive to THC, express high levels of CB2.
By real-time quantitative PCR experiments, we have observed a
correlation between CB2 expression and the histologic grade of
breast tumors. Moreover, CB2 expression is higher in tumors with
predicted low response to conventional therapies, for instance
ER/PR tumors, which are weakly responsive to adjuvant
tamoxifen (7). In addition, hardly detectable levels of CB2 were
found in normal breast tissue. Because both cell cycle arrest and
apoptosis induced by THC are CB2-mediated effects, it is tempting
to speculate that the tumors with poor prognosis (i.e., those
reluctant to conventional therapies and, according to our data,
expressing the highest levels of CB2 receptor), may be the most
responsive to cannabinoids. In addition, the psychotropic effects of
cannabinoids are mediated by CB1 but not CB2 receptors, and,
therefore, a cannabinoid-based therapy selectively targeting CB2
receptors would be devoid of the side effects associated to
cannabis consumption (20).
Breast cancer is the most common malignant disease among
Western women. Although the rates of mortality of breast cancer
patients have decreased as a result of early diagnosis by
mammograms, certain breast tumors remain reluctant to conventional
therapies, and current treatments have side effects that
substantially affect the patient’s quality of life (21). Our findings
might set the basis for new strategies for the management of
breast cancer.
Acknowledgments
Received 12/22/2005; revised 4/6/2006; accepted 5/2/2006.
Grant support: Fondo de Investigaciones Sanitarias FIS 04/1029 (C. Sa´nchez) and
BEFI 01/9132 (D. Sarrio´); Complutense University PR1/05 (C. Sa´nchez); Ministerio de
Educacio´n y Ciencia SAF2003-00745 (M. Guzma´n), SAF2004-08258-C02-01
( J. Palacios), and AP2003-0063 (M.M. Caffarel); and Fundacio´n Cientı´fica de la
Asociacio´n Espan˜ola Contra el Ca´ncer (M. Guzma´n).
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank the staff at Centro de Microscopı´a y Citometrı´a (Complutense University,
Madrid, Spain) and A. A´lvarez-Barrientos for expert advice on flow cytometry
experiments; O. Rueda for expert advice on statistical analysis; and the members of our
laboratory for technical support, especially to K. Blazej, C. Bla´zquez, D. Callegarin,
A. Egia, B. Herrera, and M. Lorente.
References
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3. Velasco G, Galve-Roperh I, Sanchez C, Blazquez C,
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(IARC/World Health Organization Classification of
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Skp2-mediated degradation of p27 regulates progression
into mitosis. Dev Cell 2004;6:661–72.
11. Kawabe T. G2 checkpoint abrogators as anticancer
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division and apoptosis in cancer. Oncogene 2003;22:
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induced hepatocyte apoptosis. Hepatology 2003;
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14. Mimeault M, Pommery N, Wattez N, Bailly C,
Henichart JP. Anti-proliferative and apoptotic effects of
anandamide in human prostatic cancer cell lines: implication
of epidermal growth factor receptor down-regulation
and ceramide production. Prostate 2003;56:1–12.
15. De Petrocellis L, Melck D, Palmisano A, et al. The
endogenous cannabinoid anandamide inhibits human
breast cancer cell proliferation. Proc Natl Acad Sci U S A
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16. Portella G, Laezza C, Laccetti P, De Petrocellis L,
Di Marzo V, Bifulco M. Inhibitory effects of cannabinoid
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and metastatic spreading: actions on signals involved
in angiogenesis and metastasis. FASEB J 2003;17:
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18. Malumbres M, Barbacid M. Mammalian cyclindependent
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19. McKallip RJ, Nagarkatti M, Nagarkatti PS. D-9-
tetrahydrocannabinol enhances breast cancer growth
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20. Sanchez C, de Ceballos ML, del Pulgar TG, et al.
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Control of Cell Cycle by Cannabinoids
www.aacrjournals.org 6621 Cancer Res 2006; 66: (13). July 1, 2006
pink commerce
In one trial, investigators at Complutense University in Spain and the Institut National de la Sante et de la Recherche Medicale (INSERM) in France assessed the anti-cancer activity of cannabinoids in pancreatic cancer cell lines and in animals. Cannabinoid administration selectively increased apoptosis (programmed cell death) in pancreatic tumor cells while ignoring healthy cells, researchers found. In addition, "cannabinoid treatment inhibited the spreading of pancreatic tumor cells ... and reduced the growth of tumor cells" in animals.
"These findings may contribute to ... a new therapeutic approach for the treatment of pancreatic cancer," authors concluded.
In the second trial, investigators at Spain's Complutense University reported that THC administration "reduces human breast cancer cell proliferation [in vitro] by blocking the progression of the cell cycle and by inducing apoptosis." Authors concluded that their findings "may set the bases for a cannabinoid therapy for the management of breast cancer."
Previous preclinical data published in May in the Journal of Pharmacological and Experimental Therapeutics reported that non-psychoactive cannabinoids, particularly cannabidiol (CBD), dramatically halt the spread of breast cancer cells and recommended their use in cancer therapy.
Separate trials have also shown cannabinoids to reduce the size and halt the spread of glioma (brain tumor) cells in animals and humans in a dose dependent manner. Additional preclinical studies have demonstrated cannabinoids to inhibit cancer cell growth and selectively trigger malignant cell death in skin cancer cells, leukemic cells, lung cancer cells, and prostate carcinoma cells, among other cancerous cell lines.
For more information, please contact Paul Armentano, NORML Senior Policy Analyst, at (202) 483-5500. Full text of both studies, "Cannabinoids induce apoptosis of pancreatic tumor cells via endoplasmic reticulum stress-related genes" and "Delta-9-tetrahydrocannabinol inhibits cell cycle progression in human breast cancer cells through Cdc2 regulation" are available in the July 1, 2006 issue of Cancer Research, available online at: http://cancerres.aacrjournals.org/. Additional information on cannabinoids' anti-cancer properties is available in NORML's report, "Cannabinoids as Cancer Hope," online at: http://www.norml.org/index.cfm?Group_ID=6814
updated: Jul 06, 2006
dimes are assholes and they have inferiority complexes, nickels are like the unattractive but strong older brother who is on the high school wrestling team and gets and in state scholarship but is really not much of a contributor to human history nickels are the type of coint to own a dog,
"These findings may contribute to ... a new therapeutic approach for the treatment of pancreatic cancer," authors concluded.
In the second trial, investigators at Spain's Complutense University reported that THC administration "reduces human breast cancer cell proliferation [in vitro] by blocking the progression of the cell cycle and by inducing apoptosis." Authors concluded that their findings "may set the bases for a cannabinoid therapy for the management of breast cancer."
Previous preclinical data published in May in the Journal of Pharmacological and Experimental Therapeutics reported that non-psychoactive cannabinoids, particularly cannabidiol (CBD), dramatically halt the spread of breast cancer cells and recommended their use in cancer therapy.
Separate trials have also shown cannabinoids to reduce the size and halt the spread of glioma (brain tumor) cells in animals and humans in a dose dependent manner. Additional preclinical studies have demonstrated cannabinoids to inhibit cancer cell growth and selectively trigger malignant cell death in skin cancer cells, leukemic cells, lung cancer cells, and prostate carcinoma cells, among other cancerous cell lines.
For more information, please contact Paul Armentano, NORML Senior Policy Analyst, at (202) 483-5500. Full text of both studies, "Cannabinoids induce apoptosis of pancreatic tumor cells via endoplasmic reticulum stress-related genes" and "Delta-9-tetrahydrocannabinol inhibits cell cycle progression in human breast cancer cells through Cdc2 regulation" are available in the July 1, 2006 issue of Cancer Research, available online at: http://cancerres.aacrjournals.org/. Additional information on cannabinoids' anti-cancer properties is available in NORML's report, "Cannabinoids as Cancer Hope," online at: http://www.norml.org/index.cfm?Group_ID=6814
updated: Jul 06, 2006
dimes are assholes and they have inferiority complexes, nickels are like the unattractive but strong older brother who is on the high school wrestling team and gets and in state scholarship but is really not much of a contributor to human history nickels are the type of coint to own a dog,
the "we are usually ripping you off but not today" sale, or the "were gonna stop pricefucking u for a day" sale, or the cable is now ten bucks a month for six months,
we can play golf on the moon but pot will never be legal because then prison wouldnt really be prison, people wouldnt mind going, they would pass blunts all day, and they would just chill and not get lung cancer, and
tiger denzel chris rock eminem and then you have ichael jordan betting islands in a black skins game
espn in yiddish, the kurt warner pastrami trophy, the college bowls we're live at the Grand Knish in Knoxville, new york,
its a football stadium where jeremiah wright stars at running back as a converted ethipiona jew going to zion like his islamic brethren, then you have
we can play golf on the moon but pot will never be legal because then prison wouldnt really be prison, people wouldnt mind going, they would pass blunts all day, and they would just chill and not get lung cancer, and
tiger denzel chris rock eminem and then you have ichael jordan betting islands in a black skins game
espn in yiddish, the kurt warner pastrami trophy, the college bowls we're live at the Grand Knish in Knoxville, new york,
its a football stadium where jeremiah wright stars at running back as a converted ethipiona jew going to zion like his islamic brethren, then you have
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