Thursday, April 10, 2014

Forbidden Cancer Treatment They Don’t Want You To Know About

German-born biochemist Johanna Budwig (1908-2003) was acknowledged as perhaps the world’s leading expert on lipid biochemistry – the field of study dealing with fats (lipids that are solid at room temperature) and oils (lipids that are liquid at room temperature), and their activity within biological systems.

She held a Ph.D. in Natural Science, with emphasis in chemistry and physics, and was also formally trained as a physician, botanist, and biologist.  On seven different occasions she was nominated by her peers to receive a Nobel Prize.
Dr. Budwig was familiar with the work of 1931 Nobel laureate Otto Warburg who demonstrated that, unlike normal cells which receive their energy from oxygen gas, the energy that maintains cancer cells is derived principally from the fermentation of glucose.
Warburg stated:
“Cancer, above all other diseases, has countless secondary causes.  But, even for cancer, there is only one prime cause.  Summarized in a few words, the prime cause of cancer is the replacement of the respiration of oxygen in normal body cells by a fermentation of sugar [glucose] … all cancer cells without exception must ferment, and no normal growing cell ought to exist that ferments in the body.”
Warburg’s work never fully postulated the cause(s) of this reversion of cancer cells to the more primitive anaerobic state. He did, however, theorize that he could raise the oxygen levels in anaerobic cells through the consumption of saturated fats. Pursuing this reasoning, he unsuccessfully attempted to increase oxygen transfer into cancer cells using the saturated fat butyric acid (which Budwig later determined was not sufficiently energetic to effect oxygen transport). Building on Warburg’s work, 1937 Nobel laureate Albert Szent-Györgyi demonstrated that essential fatty acids (EFAs), combined with sulphur-rich proteins, are able to increase cellular oxygenation.
Budwig understood that cancer cells revert to the more primitive fermentation of glucose because they are deprived of oxygen to the extent they must find another source of energy in order to survive.  One of her greatest contributions is the discovery of the reason this oxygen deprivation and subsequent reversion occurs. Simply stated, the anaerobic environment that spawns the proliferation of cancer is caused by the lack of sufficient omega-3 and omega-6 essential fatty acids in the diet, as well as the overconsumption of modern foods containing unhealthy forms of fats and oils, according to Budwig.
Essential fatty acids are so labeled because they are essential for life and cannot be manufactured by the body. Consequently, they must be supplied through the diet.  If these health-providing fatty acids aren’t consumed in sufficient amounts, physical processes within the cells degrade to the more primitive form of energy production – fermentation of glucose within a poorly oxygenated environment.  Also, and equally important, is the fact that the modern-day overconsumption of unhealthy forms of fats and oils so prevalent in today’s processed foods, competes with the uptake and use of healthy EFAs, further limiting their availability.
Based on her own extensive research over many decades, as well as probable knowledge of Szent-Györgyi’s findings regarding the use of essential fatty acids combined with sulphur-rich proteins to raise cellular energy levels, Dr. Budwig pioneered a protocol for cancer prevention and treatment based on the use of small amounts of flaxseed (linseed) oil combined with a rich supply of sulphur-based proteins.  She recommended using organic cottage cheese as the best sources of sulphur-based protein.
Flax oil is one of the richest sources of the EFAs omega-3s and -6s, and cottage cheese is perhaps the most convenient, richest source of sulphur-based protein.
Forbidden Cancer Treatment They Dont Want You To Know AboutBudwig found that neither ingredient alone is effective in either the prevention or treatment of disease.  The flax oil must be “activated” by thoroughly mixing it with the cottage cheese in an electric blender at a ratio ranging from 1 tablespoon flaxseed oil per ¼ cup of organic low fat cottage cheese, to 3-4 tablespoons flax oil per ½ cup cottage cheese, depending on the severity of illness – taken on a daily basis.  Other ingredients such as fruit and honey may be added to taste.
For those who are lactose intolerant or simply want to avoid animal-derived foods, alternatives to cottage cheese can be found in the references below.  One possibility is the product by Nature’s Distributors, an Arizona-based company that claims one capsule of the dried, sulphurated protein in Companion Nutrients can activate the EFAs in one tablespoon of flaxseed oil.  Because the precise cancer treatment is so critical, Companion Nutrients might best be used in addition to but not as a substitute for cottage cheese.
As amazing as it may sound to some – especially to oncologists who practice traditional forms of cancer therapy, not to mention the unsuspecting and naive public who have observed for decades the many twists and turns of the War On Cancer – the use of these two inexpensive (and unpatentable) food substances provides a powerful and effective means of treating even the most advanced cancers.
According to Dr. Budwig, “… 99% of the sick that come to see me … are cancer patients who have had operations and radiation sessions, and were diagnosed as being far too advanced for another operation to be of any help.  Even in these cases health can be restored, usually within a few months, I would say in 90% of cases.”
Forbidden Cancer Treatment They Dont Want You To Know AboutThe preceding special report is an excerpt from the popular, doctor-recommended The Encyclopedia of Medical Breakthroughs and Forbidden Treatments
Dr. Johanna Budwig’s flax oil & sulphur-based protein therapy is just one of 12 scientifically validated cures for cancer featured in The Encyclopedia of Medical Breakthroughs and Forbidden Treatments.
The Encyclopedia of Medical Breakthroughs and Forbidden Treatments is a 380-page book (also available as an e-book) containing a definitive, never-before-published compilation of alternative medicine discoveries.  It features cutting-edge, state-of-the-art health information, alternative treatment options and effective solutions for virtually any disease or health problem you or your loved ones might have.
1. Warburg, Otto. “The Prime Cause and Prevention of Cancer” (Revised Lindau Lecture),
3. Budwig, J., “Der Tod des Tumors, Band II” (“The Death of Tumors, Vol. II”), Transcribed interview, broadcast Sept. 11, 1967 by Süddeutscher Rundfunk Stuttgart (Germany).

Turmeric: Doctors Say This Spice Is a Brain Health Miracle

Dear Health Conscious Reader,

Are you concerned about maintaining the health of your brain as you age? You're not alone. Losing one's memory and mental abilities to cognitive decline is something we all fear, and the current statistics on the prevalence of cognitive decline in this country are not pretty. By age 65, sadly 1 in 8 Americans will suffer from severe cognitive decline, and by age 80, an astonishing 1 in 2 will.[1]

Researchers have been working for years to develop an effective treatment. But one "miracle" drug after another has failed to live up to its promises, and it's becoming more and more apparent that pharmaceuticals are not the solution. Unfortunately, the medical establishment is so narrowly focused on finding an elusive "cure" that scientifically proven prevention strategies are often ignored.

I'm Joshua Corn, Editor-in-Chief of Live in the Now, one of the fastest growing natural health publications in the nation. My passion for natural health drives me to seek the truth about the causes of health problems and to educate people on alternative solutions that are both safe and effective.

Please keep reading, because I'll tell you about an amazingly effective way to protect your brain from the ravages of cognitive decline and boost your thinking and memory abilities quickly, naturally and safely.

Nature's Brain-Protecting Miracle

There are a number of natural brain protectors out there, but did you know that one herb has shown more promise when it comes to supporting your neurological system than any other medical finding to date? It's a scientific fact that curcumin, an antioxidant compound found in the root of the turmeric plant, is one of the most powerful natural brain protecting substances on the planet! It continues to amaze scientists with its remarkable cognitive health benefits.

You may be familiar with turmeric as the bright yellow spice that is commonly found in curry powder. Turmeric has a long history as a healing herb and culinary spice in India. Interestingly, India has the highest per capita consumption of turmeric AND the lowest incidence of cognitive decline worldwide![2]

Joshua Corn, Editor-in-Chief of the Live in the Now newsletter, is a health freedom advocate who's been involved in the natural health movement for over 15 years. He's always been dedicated to promoting health, vitality, longevity and natural living. Josh is currently writing a book on natural remedies and is gearing up to launch the Live in the Now radio show. In addition to his work in the natural health field, Josh is an avid outdoorsman, organic gardener, animal lover and enjoys "living in the now" with his wife and two sons.

Your Brain on Curcumin

The hallmark process associated with certain types of cognitive decline is the formation in the brain of abnormal protein structures. Normally when malformed proteins are formed within the brain, the immune system sends out cells known as macrophages, which engulf and destroy the proteins. If this ordinary function fails, defective proteins accumulate in the brain and cognitive decline can follow.

That's why I was excited to read that recent research is showing that curcumin encourages the immune system to send macrophages to the brain. A landmark clinical trial involving people with severe cognitive decline that measured the effects of curcumin. Amazingly, the participants taking curcumin had significantly higher levels of dissolved abnormal proteins in their blood compared to those in the placebo group.

This study showed that curcumin has the ability to effectively pass into the brain, bind to beta-amyloid plaques and assist the body in their breakdown.[3] Curcumin is one of the only substances known to have such a profound protective effect on the brain.

The Incredible Health Benefits of Curcumin
Protects brain cells from aging Increases memory retention and clarity
Boosts overall cognitive function Supports joint and muscle health
Promotes healthy cardiovascular function Supports a healthy inflammatory response
Supports healthy mood balance Promotes a healthy digestive system
Boosts detoxification and liver health Supports natural weight loss

Increases memory retention and clarity
Supports joint and muscle health
Supports a healthy inflammatory response
Promotes a healthy digestive system
Supports natural weight loss
Curcumin Combats Dangerous Inflammation

You may have heard about the dangers of "silent" inflammation. It's been discussed by Dr. Oz and has made headlines in publications such as Newsweek and Time. It's important for you to know that low-grade inflammation is rapidly becoming recognized as the root cause of the development of cognitive decline, as well as a wide range of serious health problems.[4]

Unfortunately, most people don't do anything until the initial symptoms, such as muscle aches and joint discomfort, appear. When this happens, their gut reaction often is to go see a doctor, who is unlikely to correctly diagnose the problem, and more than likely will prescribe drugs that are far from safe and only provide short-term benefits.

But the initial symptoms of pain are just the beginning. If left unchecked, inflammation can damage your body in many ways. One of the worst examples of this is cognitive decline, which can ravage your memory and severely impact your quality of life in what should be your golden years.

Here are 7 ways inflammation can damage your body:

  1. Memory loss and cognitive decline
  2. Increased cardiovascular risk
  3. Joint pain and loss of mobility
  4. Allergies and breathing discomfort
  5. Accelerated skin aging and wrinkles
  6. Compromised digestive function
  7. Weight gain and loss of muscle tone
Cutting-edge science is showing that curcumin is one of the most powerful, natural anti-inflammatories ever studied. Curcumin is so effective because it is a potent inhibitor of the body's most powerful inflammation-causing chemical, called Nf-kappa beta. Miraculously, curcumin has been shown in numerous studies to tame joint pain and muscle soreness, protect the brain, support cardiovascular health, bolster immunity and more.[5,6,7,8,9] If you have a lingering health problem that no doctor can resolve, you may be suffering from inflammation and curcumin may be just the natural solution you've been looking for.

How to Get the Most Out of Curcumin

Although you may be able to find high quality turmeric powder at your local market, it's very difficult to verify its purity and potency. Plus, it's almost impossible to incorporate therapeutic amounts of curcumin into your diet on a daily basis as a spice. So, to put it simply, a high-quality curcumin supplement is your best bet!

However, not all curcumin supplements are created equal. Unfortunately, most supplements out there don't have the necessary doses and components to get the job done. If you take the wrong supplement, you'll end up not only wasting money, but missing out on an incredible opportunity to protect your brain and keep harmful inflammation at bay.

There are 3 major factors to consider when deciding on a curcumin supplement:

  1. Standardization and Dose: Standardization is the process by which the active ingredients in a plant are concentrated and brought to a consistent level. The active ingredients of turmeric are called curcuminoids, and there are actually three components: curcumin, demethoxycurcumin and bisdemethoxycurcumin. Any good curcumin supplement should include all 3 of these curcuminoids and be standardized to at least 75%. Also, the minimum recommended daily dose is 1,000 mg per day.
  2. Absorption: One problem with curcumin is that it's not easily absorbed. However, it was discovered that an extract of black pepper, called piperine, significantly enhances absorption of curcumin. In fact, one study found that the addition of a piperine increased bioavailablity by 2,000%![10] So make sure that any curcumin supplement you take contains this important extract.
  3. Price: Some people mistakenly believe if you pay more you get better quality. My advice is that you don't need to pay more than $30 for a one month supply. Many companies are taking advantage of the hype surrounding curcumin and charging more than double this.

The Curcumin Supplement I Personally Recommend

Since cognitive decline and inflammation are so widespread, I consider curcumin a "must take" supplement. However, when I was looking at the different options out there, I was shocked by how many poor quality curcumin supplements there were. The ones that had "all the right stuff" were absurdly expensive, making this lifesaving nutrient off-limits for too many people!

That's why I took it upon myself to develop an effective AND affordable curcumin supplement called Curcumin2K™. In addition to my work with Live in the Now, I'm also the CEO of Stop Aging Now, a company that has been making premium grade dietary supplements for over 15 years. I developed Curcumin2K working with my Scientific Advisory Board, and it's simply the most effective and most affordable curcumin supplement on the market.

I invite you to do your own research to make sure I'm not just shamelessly promoting my own product, but I think you'll find no other curcumin product like Curcumin2K. Here are just a few reasons why:

  • Curcumin2K is made with 1,330 mg of turmeric extract standardized to contain a minimum of 95% the full spectrum of curcuminoids, which includes curcumin, demethoxycurcumin and bisdemethoxycurcumin.
  • Curcumin2K is enhanced with 20 mg of BioPerine® black pepper extract standardized to contain 95% piperine. BioPerine is the brand used in all the research that has shown piperine increases curcumin's absorption by up to 2,000%!
  • Curcumin2K is made in the USA in an FDA inspected facility to meet or exceed stringent USP stringent standards for quality, purity and potency.
  • Curcumin2K ships to you for free, is tax-free and comes with an amazing 365-day "any reason" guarantee. This way you risk nothing!
  • Curcumin2K is available for as little as $19.95 per bottle (which lasts a full month). This is up to 3 times less than similar high quality brands.

Our Curcumin2K Customers Say It Best...

We already have tens of thousands of satisfied Curcumin2K customers. One of the best parts of my job is reading some of their remarkable success stories. Here are just a few that I've recently received:

"I only use Stop Aging Now supplements because they're made in the USA and I've had great results. I've noticed since taking Curcumin2K that my memory is quicker. A great company with great products."
- Terry Bart from Jackson, WY
"Curcumin2K is far superior to the curcumin I had been taking. The potency is much higher, plus it contains black pepper extract. I always find your products to be superior even to the higher-priced brands."
- Kathleen Roper from Fennville, MI
"I noticed shortly after taking Curcumin2K that my joint discomfort started to decrease and I was able to become more active again!!!! This product has really been a lifesaver for me! I HIGHLY RECOMMEND it!"
- Peter Dialfonso from Somerset, NJ
"I am 66 years old and recently decided to try Curcumin2K. Within 5 days I noticed the difference and after 1 week 90% of all my "old age" symptoms had TOTALLY disappeared! I now enjoy life without pain."
- Ruth Rich from Possum Creek, AU

I Want to Do More Than Just Sell You a Bottle

I truly believe that Curcumin2K will improve your health in more ways than you dreamed possible. I take it daily, and so do my wife, parents and many of my friends. But I certainly don't expect you take my word for it!

You can try Curcumin2K risk-free. Not only is it extremely affordable, will ship to you for free and is tax free, but if you don't get results, your entire order is free! Curcumin2K also comes with an amazing 365-day "any reason" guarantee. So even if you've taken every last capsule, and even if one full year has passed, if you're not satisfied, you'll get every penny back. I personally guarantee the quality.

Order Now And Feel Better Within 7 Days!

The best thing about Curcumin2K is how rapidly it begins to work. When I began taking it, I noticed the benefits within a few days, and I personally guarantee that you'll have a similar experience. Here's what you can expect:
Protection for your brain and cognitive function
Improved mental clarity and memory retention
Better mood balance and more energy
A healthier heart and more balanced cholesterol
Fewer aches and pains and less stiffness
Smoother, more youthful-looking skin
Support for liver health and detoxification
Antioxidant protection against free radicals
An overall increased sense of vitality

Whether you decide to use Curcumin2K or take another approach, I hope I've convinced you to take protecting your cognitive health seriously. I consider Curcumin2K one of the most important supplements I currently take. I think you'll find the same is true for you, so I hope that you'll give it a try!

To Your Good Health,

Joshua Corn
CEO of Stop Aging Now
Editor-in-Chief of Live in the Now

Scientific References:
2. Neurology. 1998; 51(4): 1000-1008.
3. Arthr Res Ther, 2005:8(1): doi:10.1186/ar1846.
4. Dr. David Graham, Testimony to the Senate Finance Committee, November 18, 2004.
5. Int J Biochem Cell Biol. 2009; 41(1): 40?59.
6. J Neurosci Res. 2004 Mar 15; 75(6): 742-50.
7. Nutrition. Sept 2009, 25(9): 964-972.)
8. J Pharmacol Exp Ther. 2007 May; 321(2): 616-25.
9. Biochem Pharmacol. 2005 Sep 1; 70(5): 700-13.
10. Planta Med. 1998; 64(4): 353-356.

Wednesday, March 12, 2014

How To Remove Skin Tags With Apple cider vinegar

I am known to my friends a bit of a fiddler.  I make my own scrubs and tonics and now I have turned the fiddling with natural skin tag removal.  I know the doctor or even myself can lance them off… but they bleed heavily and can leave a scar so I thought I would try a different approach, not to mention the money saved for a doctor’s appointment.  Here is what I did and what worked and what didn’t work.  I only have 2 skin tags (one on my arm and one on my neck), so I don’t have that much to experiment on.  This goes without saying I am not a medical professional and this is only my experience, so make sure you check with your doctor that the skin tag/mole is not of concern before you attempt to remove it yourself.

First attempt:  Used clear nail polish.  Painted over the skin tag (in my inner crook of the arm).  Skin tag blew up in size — almost blister like and little scary — and my skin reacted very poorly to the nail polish and I developed quite a rash.  Opinion:  I would NOT do this again.  And it did not finish the tag off.  I had to use Apple cider vinegar to finish the job.

Second attempt:  Apple cider vinegar.  I cut a tiny square of cotton pad to cover the tag and soaked it in organic apple cider vinegar and then used a band aid to secure and left over night.  Repeat if necessary.  Results:  Skin tag turned black and fell off in about 3-5 days with no bleeding and no scarring.  Once it is at the black/dark brown stage it is dead and you can stop using the vinegar and just wait for the tag to fall off.  Do not pick!  I do have slightly red skin around the area from the nail polish but that is fading over time and my skin is smooth.  I can’t believe a tag used to be there!  Opinion & Mistakes:  I would use this method again if I had more skin tags!  Apple cider vinegar is strong acid and I did not protect the surround skin so I got a bit of an acid burn around and near the skin tag.  I am using Rose Hip Oil to reduce the redness and heal the skin.  If I did this again what I would do differently:  1. protect surrounding skin with Vaseline and 2. maybe start by just holding a q tip on the tag 3 times a day to see if that works before leaving it on over night.

Good luck and let me know how you get on.

Thursday, March 6, 2014

Kidney Stone Remedy

***Kidney Stone Remedy***

Healthy lifestyle along with proper diet can help in the elimination of kidney stones and kidney diseases.

1. Lemon Juice and Olive Oil

The combination of lemon juice and olive oil is traditionally used as a home remedy to expel gallbladder stones but it can also be used to treat kidney stones. The citric acid present in lemons helps break down calcium-based kidney stones and stops further growth.

1. Take four tablespoons or a quarter cup of fresh lemon juice.
2. Add an equal amount of olive oil.
3. Drink this mixture followed by plenty of water.
4. Do this two to three times a day, up to three days. You need not continue this remedy if you pass the stones in a single dose.

NOTE: Replace olive oil with coconut oil making it more effective and healthier!

2. Apple Cider Vinegar-

Apple cider vinegar helps dissolve kidney stones. It also has an alkalinizing effect on blood and urine.

Mix two tablespoons of organic apple cider vinegar and one teaspoon of honey in one cup of warm water.
Drink this a few times a day.

3. Pomegranate-

Both the seeds and juice of pomegranates have astringent properties that can help in the treatment of kidney stones.

Try to eat one whole pomegranate or drink one glass of freshly squeezed pomegranate juice daily. You can mix pomegranate in a fruit salad also.

Warning: This remedy may not be suitable for passing large kidney stones.

Saturday, February 22, 2014

Medical Studies that Prove Cannabis Can Cure Breast Cancer (11) :Pathways mediating the effects of cannabidiol on the reduction of breast cancer cell proliferation, invasion, and metastasis.

Pathways mediating the effects of cannabidiol on the reduction of breast cancer cell proliferation, invasion, and metastasis.

Medical Studies that Prove Cannabis Can Cure Breast Cancer (11)


Invasion and metastasis of aggressive breast cancer cells are the final and fatal steps during cancer progression. Clinically, there are still limited therapeutic interventions for aggressive and metastatic breast cancers available. Therefore, effective, targeted, and non-toxic therapies are urgently required. Id-1, an inhibitor of basic helix-loop-helix transcription factors, has recently been shown to be a key regulator of the metastatic potential of breast and additional cancers. We previously reported that cannabidiol (CBD), a cannabinoid with a low toxicity profile, down-regulated Id-1 gene expression in aggressive human breast cancer cells in culture. Using cell proliferation and invasion assays, cell flow cytometry to examine cell cycle and the formation of reactive oxygen species, and Western analysis, we determined pathways leading to the down-regulation of Id-1 expression by CBD and consequently to the inhibition of the proliferative and invasive phenotype of human breast cancer cells. Then, using the mouse 4T1 mammary tumor cell line and the ranksum test, two different syngeneic models of tumor metastasis to the lungs were chosen to determine whether treatment with CBD would reduce metastasis in vivo. We show that CBD inhibits human breast cancer cell proliferation and invasion through differential modulation of the extracellular signal-regulated kinase (ERK) and reactive oxygen species (ROS) pathways, and that both pathways lead to down-regulation of Id-1 expression. Moreover, we demonstrate that CBD up-regulates the pro-differentiation factor, Id-2. Using immune competent mice, we then show that treatment with CBD significantly reduces primary tumor mass as well as the size and number of lung metastatic foci in two models of metastasis. Our data demonstrate the efficacy of CBD in pre-clinical models of breast cancer. The results have the potential to lead to the development of novel non-toxic compounds for the treatment of breast cancer metastasis, and the information gained from these experiments broaden our knowledge of both Id-1 and cannabinoid biology as it pertains to cancer progression.

Results: 6

Fig. 1
CBD up-regulates ERK phosphorylation and Id-2 expression. (A) Proteins from MDA-MB231 cells treated with 1.5 μM CBD (as previously described [21]) for 1 or 2 days were extracted and analyzed for Id-1, total ERK, pERK, or p38 by Western blot analysis. (B) Proteins from MDA-MB231 cells treated with CBD for 3 days were extracted and analyzed for Id-1, Id-2, or NFkappaB by Western blot analysis
Sean D. McAllister, et al. Breast Cancer Res Treat. 2011 August;129(1):37-47.
Fig. 6
CBD reduces the number of metastatic foci in the syngeneic model of tail vein injection. Lung metastases were generated in BALB/c mice after tail vein injection of 5 × 105 4T1 cells. One day after the injection, the tumor-bearing mice were injected i.p. once a day with vehicle or 1 mg/kg CBD for 15 days. Visible lung metastases were counted and measured by using a dissecting microscope (A). Lung metastases measured included those (B) <1 b="" mm="">C
) 1–2 mm, and (D) >2 mm
Sean D. McAllister, et al. Breast Cancer Res Treat. 2011 August;129(1):37-47.
Fig. 3
Production of ROS represents another factor involved in the inhibitory activity of CBD. MDA-MB231 cells were treated for 3 days with vehicle (Control) or 1.5 μM CBD in the presence and absence of 20 μM TOC. Cell proliferation (A) and invasion (B) were measured using the MTT and Boyden chamber assay, respectively. (C) Proteins from cells treated with vehicle (control) or 1.5 μM of CBD for 3 days in the absence or presence of TOC were extracted and analyzed for Id-1 by Western blot analysis. (D) The production of ROS was measured using 2′-7′Dichloro-dihydrofluorescein (Sigma-Aldrich). (*) indicates statistically significant differences from control (P < 0.05)
Sean D. McAllister, et al. Breast Cancer Res Treat. 2011 August;129(1):37-47.
Fig. 4
CBD inhibits the expression of Id-1 and corresponding breast cancer proliferation and invasion in mouse 4T1 cells. (A) 4T1 cells were treated for 3 days with 1.5 μM CBD, proteins were extracted and analyzed for Id-1 expression. (B) 4T1 cells were collected and cell cycle analyzed using a desktop FACS Calibur with Cell Quest Pro software (BD Bioscience, CA). The distribution of cells in different cell cycle stages was determined according to their DNA content. (C) Invasion assays were carried out using the Boyden chamber assay. (*) indicates statistically significant differences from control (P < 0.05)
Sean D. McAllister, et al. Breast Cancer Res Treat. 2011 August;129(1):37-47.
Fig. 2
ERK partly mediates the inhibitory activity of CBD on cell growth and invasion. MDA-MB231 cells were treated for 3 days with vehicle (Control) or 1.5 μM CBD in the presence and absence of 0.1–0.5 μM U0126. Cell proliferation (A) and invasion (B) were measured using the MTT and Boyden chamber assays, respectively. Data are presented as relative proliferation or invasiveness of the cells, where the respective controls are set as 100%. (C) Proteins from MDA-MB231 cells treated with vehicle (control) or 1.5 μM of CBD for 3 days in the absence or presence of U0126 were extracted and analyzed for Id-1 by Western blot analysis. (*) indicates statistically significant difference from control (P < 0.05). (#) indicates statistically significant difference from CBD (P < 0.05)
Sean D. McAllister, et al. Breast Cancer Res Treat. 2011 August;129(1):37-47.
Fig. 5
CBD reduces primary tumor growth and metastasis of 4T1 cancer cells in an orthotopic mouse model. Primary tumors and subsequent secondary tumors (metastases) were generated in BALB/c mice by subcutaneous injection of 1 × 105 4T1 cells under the fourth major nipple. Treatment with CBD was initiated upon detection of the first palpable tumor (approximately 7 days). (A) The primary tumor volume was calculated by measuring the perpendicular largest diameters of the tumor with a caliper. (B) The weight of the tumors was also measured. (C) The visible lung metastases were measured using a dissecting microscope. (D) The average volume per metastatic foci was calculated as described in the methods. (*) indicates statistically significant differences from vehicle (P < 0.05)
Sean D. McAllister, et al. Breast Cancer Res Treat. 2011 August;129(1):37-47.

Medical Studies that Prove Cannabis Can Cure Mouth and Throat Cancer (10) :

Cannabinoids inhibit cellular respiration of human oral cancer cells.



The primary cannabinoids, Delta(9)-tetrahydrocannabinol (Delta(9)-THC) and Delta(8)-tetrahydrocannabinol (Delta(8)-THC) are known to disturb the mitochondrial function and possess antitumor activities. These observations prompted us to investigate their effects on the mitochondrial O(2) consumption in human oral cancer cells (Tu183). This epithelial cell line overexpresses bcl-2 and is highly resistant to anticancer drugs.


A phosphorescence analyzer that measures the time-dependence of O(2) concentration in cellular or mitochondrial suspensions was used for this purpose.


A rapid decline in the rate of respiration was observed when Delta(9)-THC or Delta(8)-THC was added to the cells. The inhibition was concentration-dependent, and Delta(9)-THC was the more potent of the two compounds. Anandamide (an endocannabinoid) was ineffective; suggesting the effects of Delta(9)-THC and Delta(8)-THC were not mediated by the cannabinoidreceptors. Inhibition of O(2) consumption by cyanide confirmed the oxidations occurred in the mitochondrial respiratory chain. Delta(9)-THC inhibited the respiration of isolated mitochondria from beef heart.


These results show the cannabinoids are potent inhibitors of Tu183 cellular respiration and are toxic to this highly malignant tumor.


Author information

  • 1Department of Pediatricsy, State University of New York, Upstate Medical University, Syracuse, NY, USA.

Medical Studies that Prove Cannabis Can Cure Brain Cancer ( 9 ) : Cannabinoids Inhibit the Vascular Endothelial Growth Factor Pathway in Gliomas

  Medical Studies that Prove Cannabis Can Cure Brain Cancer ( 9 )


Cannabinoids inhibit tumor angiogenesis in mice, but the mechanism of their antiangiogenic action is still unknown. Because the vascular endothelial growth factor (VEGF) pathway plays a critical role in tumor angiogenesis, here we studied whether cannabinoids affect it. As a first approach, cDNA array analysis showed that cannabinoid administration to mice bearing s.c. gliomas lowered the expression of various VEGF pathway-related genes. The use of other methods (ELISA, Western blotting, and confocal microscopy) provided additional evidence that cannabinoids depressed the VEGF pathway by decreasing the production of VEGF and the activation of VEGF receptor (VEGFR)-2, the most prominent VEGF receptor, in cultured glioma cells and in mouse gliomas. Cannabinoid-induced inhibition of VEGF production and VEGFR-2 activation was abrogated both in vitro and in vivo by pharmacological blockade of ceramide biosynthesis. These changes in the VEGF pathway were paralleled by changes in tumor size. Moreover, intratumoral administration of the cannabinoid Δ9-tetrahydrocannabinol to two patients with glioblastoma multiforme (grade IV astrocytoma) decreased VEGF levels and VEGFR-2 activation in the tumors. Because blockade of the VEGF pathway constitutes one of the most promising antitumoral approaches currently available, the present findings provide a novel pharmacological target for cannabinoid-based therapies.


To grow beyond minimal size, tumors must generate a new vascular supply for purposes of gas exchange, cell nutrition, and waste disposal (1, 2, 3, 4) . They do so by secreting proangiogenic cytokines that promote the formation of blood vessels. Vascular endothelial growth factor (VEGF; also known as VEGF-A) is considered the most important proangiogenic molecule because it is expressed abundantly by a wide variety of animal and human tumors and because of its potency, selectivity, and ability to regulate most and perhaps all of the steps in the angiogenic cascade (1, 2, 3, 4) . The best characterized VEGF receptors are two related receptor tyrosine kinases termed VEGF receptor (VEGFR)-1 (also known as Flt-1) and VEGFR-2 (also known as kinase domain region or Flk-1). Although VEGF binds to VEGFR-1 with higher affinity, numerous studies in cultured cells and laboratory animals have provided evidence that VEGFR-2 is the major mediator of the mitogenic, antiapoptotic, angiogenic, and permeability-enhancing effects of VEGF (1, 2, 3, 4) . Because overexpression of VEGF and VEGFR-2 is causally involved in the progression of many solid tumors, several strategies to inhibit VEGF signaling have been translated into clinical trials in cancer patients, including anti-VEGF and anti-VEGFR-2 antibodies, small VEGFR-2 inhibitors, and a soluble decoy VEGFR (5, 6, 7, 8) . In addition, clinical trials are being performed with a number of promising anticancer compounds such as Iressa and Herceptin that block proteins involved in the induction of the VEGF pathway (5 , 8) .
Cannabinoids, the active components of Cannabis sativa L. (marijuana), and their derivatives exert a wide array of effects by activating their specific G protein-coupled receptors CB1 and CB2, which are normally engaged by a family of endogenous ligands–the endocannabinoids (9 , 10) . Marijuana and its derivatives have been used in medicine for many centuries, and there is currently a renaissance in the study of the therapeutic effects of cannabinoids. Today, cannabinoids are approved to palliate the wasting and emesis associated with cancer and AIDS chemotherapy (11) , and ongoing clinical trials are determining whether cannabinoids are effective agents in the treatment of pain (12) , neurodegenerative disorders such as multiple sclerosis (13) , and traumatic brain injury (14) . In addition, cannabinoid administration to mice and/or rats induces the regression of lung adenocarcinomas (15) , gliomas (16) , thyroid epitheliomas (17) , lymphomas (18) , and skin carcinomas (19) . These studies have also evidenced that cannabinoids display a fair drug safety profile and do not produce the generalized cytotoxic effects of conventional chemotherapies, making them potential antitumoral agents (20 , 21) .
Little is known, however, about the mechanism of cannabinoid antitumoral action in vivo. By modulating key cell signaling pathways, cannabinoids directly induce apoptosis or cell cycle arrest in different transformed cells in vitro (20) . However, the involvement of these events in their antitumoral action in vivo is as yet unknown. More recently, immunohistochemical and functional analyses of the vasculature of gliomas (22) and skin carcinomas (19) have shown that cannabinoid administration to mice inhibits tumor angiogenesis. These findings prompted us to explore the mechanism by which cannabinoids impair angiogenesis of gliomas and, particularly, the possible impact of cannabinoids on the VEGF pathway. Here, we report that cannabinoid administration inhibits the VEGF pathway in cultured glioma cells, in glioma-bearing mice, and in two patients with glioblastoma multiforme. In addition, this effect may be mediated by ceramide, a sphingolipid second messenger implicated previously in cannabinoid signaling in glioma cells (23) .



The Δ9-tetrahydrocannabinol was kindly given by Alfredo Dupetit (The Health Concept, Richelbach, Germany). JWH-133 was kindly given by Dr. John Huffman (Department of Chemistry, Clemson University, Clemson, SC; Ref. 24 ). WIN-55,212-2 and anandamide were from Sigma (St. Louis, MO). SR141716 and SR144528 were kindly given by Sanofi-Synthelabo (Montpellier, France). For in vitro incubations, cannabinoid agonists and antagonists were directly applied at a final DMSO concentration of 0.1–0.2% (v/v). For in vivo experiments, ligands were prepared at 1% (v/v) DMSO in 100 μl PBS supplemented with 5 mg/ml BSA. No significant influence of the vehicle was observed on any of the parameters determined.

Cell Culture.

The rat C6 glioma (25) , the human U373 MG astrocytoma (25) , the mouse PDV.C57 epidermal carcinoma (19) , and the human ECV304 bladder cancer epithelioma (22) were cultured as described previously. Human glioma cells were prepared from a glioblastoma multiforme (grade IV astrocytoma; Ref. 26 ). The biopsy was digested with collagenase (type Ia; Sigma) in DMEM at 37°C for 90 min, the supernatant was seeded in DMEM containing 15% FCS and 1 mm glutamine, cells were grown for 2 passages, and 24 h before the experiments, cells were transferred to 0.5%-serum DMEM. Cell viability was determined by trypan blue exclusion. Rat recombinant VEGF and N-acetylsphingosine (C2-ceramide) were from Sigma.

Tumor Induction in Mice.

Tumors were induced in mice deficient in recombination activating gene 2 by s.c. flank inoculation of 5 × 106 C6 glioma cells in 100 μl PBS supplemented with 0.1% glucose (16) . When tumors had reached a volume of 350–450 mm3, animals were assigned randomly to the various groups and injected intratumorally for up to 8 days with 50 μg/day JWH-133 and/or 60 μg/day fumonisin B1 (Alexis, San Diego, CA). Control animals were injected with vehicle. Tumors were measured with external caliper, and volume was calculated as (4π/3) × (width/2)2 × (length/2).

Human Tumor Samples.

Tumor biopsies were obtained from two of the patients enrolled in an ongoing Phase I/II clinical trial (at the Neurosurgery Department of Tenerife University Hospital, Spain) aimed at investigating the effect of Δ9-tetrahydrocannabinol administration on the growth of recurrent glioblastoma multiforme. The patients had failed standard therapy, which included surgery, radiotherapy (60 Gy), and temozolomide chemotherapy (4 cycles). Patients had clear evidence of tumor progression on sequential magnetic resonance scanning before enrollment in the study, had received no anticancer therapy for ∼1 year, and had a fair health status (Karnofski performance score = 90). The patients provided written informed consent. The protocol was approved by the Clinical Trials Committee of Tenerife University Hospital and by the Spanish Ministry of Health.
Patient 1 (a 48-year-old man) had a right-occipital-lobe tumor (7.5 × 6 cm maximum diameters), and Patient 2 (a 57-year-old-man) had a right-temporal-lobe tumor (6 × 5 cm maximum diameters). Both tumors were diagnosed by the Pathology Department of Tenerife University Hospital as glioblastoma multiforme and showed the hallmarks of this type of tumor (high vascularization, necrotic areas, abundant palisading and mitotic cells, and so on). The tumors were removed extensively by surgery, biopsies were taken, and the tip (∼5 cm) of a silastic infusion cathether (9.6 French; 3.2 mm diameter) was placed into the resection cavity. The infusion cathether was connected to a Nuport subclavicular s.c. reservoir. Each day 0.5–1.5 (median 1.0) mg of Δ9-tetrahydrocannabinol (100 μg/μl in ethanol solution) were dissolved in 30 ml of physiological saline solution supplemented with 0.5% (w/v) human serum albumin, and the resulting solution was filtered and subsequently administered at a rate of 0.3 ml/min with a syringe pump connected to the s.c. reservoir. Patient 1 started the treatment 4 days after the surgery and received a total amount of 24.5 mg of Δ9-tetrahydrocannabinol for 19 days. The posttreatment biopsy was taken 19 days after the cessation of Δ9-tetrahydrocannabinol administration. Patient 2 started the treatment 4 days after the surgery and received a total amount of 13.5 mg of Δ9-tetrahydrocannabinol for 16 days. The posttreatment biopsy was taken 43 days after the cessation of Δ9-tetrahydrocannabinol administration. Samples were either transferred to DMEM containing 15% FCS and 1 mm glutamine (for tumor-cell isolation, see above; Fig. 2B ) and frozen (for VEGF determination, Patients 1 and 2; and for VEGFR-2 Western blotting, Patient 1; Fig. 6, A and C ) or fixed in formalin and embedded in paraffin (for VEGFR-2 confocal microscopy, Patients 1 and 2; Fig. 6B ).

The cDNA Arrays.

Total RNA was extracted (27) from tumors of vehicle- or JWH-133-treated mice (see above), and poly(A)+ RNA was isolated with oligotex resin (Qiagen Inc., Valencia, CA) and reverse-transcribed with Moloney murine leukemia virus reverse transcriptase in the presence of 50 μCi [α-33P]dATP for the generation of radiolabeled cDNA probes. Purified radiolabeled probes were hybridized to angiogenesis, hypoxia, and metastasis gene array membranes (GEArray Q Series; Superarray Bioscience Corporation, Frederick, MD) according to the manufacturer’s instructions. 5 Hybridization signals were detected by phosphorimager and analyzed by Phoretix housekeeping genes in the blots as internal controls for normalization. The selection criteria were set conservatively throughout the process, and the genes selected were required to exhibit at least a 2-fold change of expression and a P < 0.01.


VEGF levels were determined in cell culture media and in tumor extracts, obtained by homogenization as described previously (16) , by solid-phase ELISA using the Quantikine mouse VEGF Immunoassay (R&D Systems, Abingdon, United Kingdom; 70% cross-reactivity with rat VEGF) for rat and mouse samples and the Quantikine human VEGF Immunoassay (R&D Systems) for human samples.

Western Blot.

Particulate cell or tissue fractions were subjected to SDS-PAGE, and proteins were transferred from the gels onto polyvinylidene fluoride membranes. Blots were incubated with antibodies against total VEGFR-2 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), VEGFR-2 phosphotyrosine 996 (1:250; Cell Signaling, Beverly, MA), VEGFR-2 phosphotyrosine 1214 (1:250; kindly given by Dr. Francesco Pezzella, Nuffield Department of Clinical Laboratory Science, University of Oxford, United Kingdom), and α-tubulin (1:4000, Sigma). The latter was used as a loading control. In all of the cases, samples were subjected to luminography with an enhanced chemiluminescence detection kit (Amersham Life Sciences, Arlington Heights, IL). Densitometric analysis of the blots was performed with the Multianalyst software (Bio-Rad Laboratories, Hercules, CA).

Confocal Microscopy.

Glioma cells were cultured in coverslips and fixed in acetone for 10 min. Mouse tumors were dissected and frozen, and 5-μm sections were fixed in acetone for 10 min. Human tumors were fixed in 10% buffered formalin and then paraffin-embedded, 5-μm sections were deparaffinized and rehydrated, and antigen retrieval was carried out by immersing the slides in 10 mm citrate (pH 6.0) and boiling for 3 min. All of the samples were incubated with 10% goat serum in PBS for 30 min at room temperature to block nonspecific binding. Slices were incubated for 1.5 h with the aforementioned primary antibodies against total VEGFR-2 (1:50) and VEGFR-2 phosphotyrosine 1214 (1:20). After washing with PBS, slices were additionally incubated (1 h, room temperature, darkness) with a mixture of the secondary goat antimouse antibodies Alexa Fluor 488 and Alexa Fluor 546 (both at 1:400; Molecular Probes, Leyden, The Netherlands). After washing with PBS, sections were fixed in 1% paraformaldehyde for 10 min and mounted with DAKO fluorescence mounting medium containing TOTO-3 iodide (1:1000; Molecular Probes) to stain cell nuclei. Confocal fluorescence images were acquired using a Laser Sharp 2000 software (Bio-Rad) and a Confocal Radiance 2000 coupled to Axiovert S100 TV microscope (Carl Zeiss, Oberkochen, Germany). Pixel quantification and colocalization were determined with Metamorph-Offline software (Universal Imaging, Downingtown, PA).

Ceramide Synthesis.

C6 glioma cells were cultured for 48 h in serum-free medium with the additions indicated together with 1 μCi of l-U-[14C]serine/well, lipids were extracted, and ceramide resolved by thin-layer chromatography as described previously (28) .


Results shown represent mean ± SD. Statistical analysis was performed by ANOVA with a post hoc analysis by the Student-Neuman-Keuls test or by unpaired Student’s t test.


Changes in Gene Expression Profile in Mouse Gliomas.

The cDNA array analysis was used as a first approach to test whether cannabinoid administration affects the VEGF pathway in mouse gliomas. Because cannabinoid-based therapeutic strategies should be as devoid as possible of psychotropic side effects and glioma cells express functional CB2 receptors, which do not mediate psychoactivity (16 , 26) , mice bearing s.c. gliomas were injected with the selective CB2 agonist JWH-133 (26) .
A total of 267 genes related to angiogenesis, hypoxia (perhaps the most potent stimulus for the onset of tumor angiogenesis), and metastasis (a characteristic of actively growing tumors related closely to angiogenesis) were analyzed, of which 126 were considered to be expressed in reliable amounts. JWH-133 administration altered the expression of 10 genes, all of which are directly or indirectly related to the VEGF pathway (Fig. 1) . Thus, cannabinoid treatment lowered the expression of the following: (a) VEGF-A [confirming our previous Northern blot data (22)] and its relative VEGF-B (3 , 4) ; (b) hypoxia-inducible factor-1α [one of the subunits of hypoxia-inducible factor-1, the major transcription factor involved in VEGF gene expression (29)] ; (c) two genes known to be under the control of VEGF, namely those encoding connective tissue growth factor [a mitogen involved in extracellular matrix production and angiogenesis (30)] , and heme oxygenase-1 [an enzyme highly expressed during hypoxia and inflammation (31)] ; and (d) four genes known to encode proteins functionally related to VEGF, namely Id3 [a transcription factor inhibitor involved in angiogenesis and tumor progression (32)] , midkine [a proangiogenic and tumorigenic growth factor (33)] , angiopoietin-2 [a prominent proangiogenic factor that cooperates with VEGF (3 , 19 , 22)] , and Tie-1 [an angiopoietin receptor (34)] . In addition, cannabinoid treatment increased the expression of the gene encoding type I procollagen α1 chain (a metalloproteinase substrate related to matrix remodeling during angiogenesis; Ref. 35 ).
Fig. 1.
Changes in gene expression profile in mouse gliomas after cannabinoid treatment. Animals bearing gliomas were treated with either vehicle (Control) or JWH-133 (JWH) for 8 days as described in “Materials and Methods.” Equal amounts of poly(A)+ RNA from tumors of 2 animals/group were pooled and hybridized to angiogenesis, hypoxia, and metastasis cDNA array membranes. Genes affected by cannabinoid treatment are listed. Examples of affected genes are pointed with arrows. Angiogenesis membrane, angiopoietin-2 (top), midkine (middle), and VEGF-A (bottom); Hypoxia membrane, procollagen Iα1 (top), heme oxygenase-1 (middle), and VEGF-A (bottom); and Metastasis membrane, VEGF-A.

Inhibition of VEGF Production in Cultured Glioma Cells and in Mouse Gliomas.

We focused next on the two main components of the VEGF pathway, namely VEGF and VEGFR-2, in both cultured glioma cells and gliomas in vivo. Incubation of C6 glioma cells with the synthetic cannabinoid WIN-55,212-2 (100 nm), a mixed CB1/CB2 receptor agonist, inhibited VEGF release into the medium in a time-dependent manner (Fig. 2A) . The cannabinoid did not affect cell viability throughout the time interval in which VEGF determinations were performed (up to 48 h; data not shown). Cannabinoid-induced attenuation of VEGF production was evident in another glioma cell line (the human astrocytoma U373 MG) and, more importantly, in tumor cells obtained directly from a human glioblastoma multiforme biopsy (Fig. 2B) . The cannabinoid effect was also observed in the mouse skin carcinoma PDV.C57 and in the human bladder cancer epithelioma ECV304 (Fig. 2B) .
Fig. 2.
Inhibition of VEGF production by cannabinoids in cultured glioma cells and in mouse gliomas. A, C6 glioma cells were cultured for the times indicated with vehicle (□) or 100 nm WIN-55,212-2 (▪), and VEGF levels in the medium were determined (n = 4). B, U373 MG astrocytoma cells, tumor cells obtained from a patient with glioblastoma multiforme (GBM), PDV.C57 epidermal carcinoma cells, and ECV304 bladder cancer epithelioma cells were cultured for 48 h with vehicle (□) or 100 nm WIN-55,212-2 (▪), and VEGF levels in the medium were determined. Data represent the percentage of VEGF in cannabinoid incubations versus the respective controls (n = 3–4). C, C6 glioma cells were cultured for 48 h with vehicle (Control), 100 nm WIN-55,212-2 (WIN), 100 nm JWH-133 (JWH), 2 μm anandamide (AEA), 0.5 μm SR141716 (SR1), and/or 0.5 μm SR144528 (SR2), and VEGF levels in the medium were determined (n = 4–6). D, C6 glioma cells were cultured for 48 h with vehicle (Control), 100 μm WIN-55, 212-2 (WIN), 1 μm C2-ceramide (CER), and/or 0.5 μm fumonisin B1 (FB1), and VEGF levels in the medium were determined (n = 4). E, animals bearing gliomas were treated with either vehicle (Control), JWH-133 (JWH), fumonisin B1 (FB1), or JWH-133 plus fumonisin B1 for 8 days as described in “Materials and Methods,” and VEGF levels in the tumors were determined (n = 4–6 for each experimental group). Significantly different (∗, P < 0.01; ∗ ∗, P < 0.05) from control incubations or control animals. Bars, ±SD.
To prove the specificity of WIN-55,212-2 action on VEGF release, we used other cannabinoid receptor agonists as well as selective cannabinoid receptor antagonists (Fig. 2C) . The inhibitory effect of WIN-55,212-2 was mimicked by the endocannabinoid anandamide (2 μm), another mixed CB1/CB2 agonist, and by the synthetic cannabinoid JWH-133 (100 nm), a selective CB2 agonist. In addition, the CB1 antagonist SR141716 (0.5 μm) and the CB2 antagonist SR144528 (0.5 μm) prevented WIN-55,212-2 action, pointing to the involvement of CB receptors in cannabinoid-induced inhibition of VEGF production.
The sphingolipid messenger ceramide has been implicated in the regulation of tumor cell function by cannabinoids (16 , 23 , 36) . The involvement of ceramide in cannabinoid-induced inhibition of VEGF production was tested by the use of N-acetylsphingosine (C2-ceramide), a cell-permeable ceramide analog, and fumonisin B1, a selective inhibitor of ceramide synthesis de novo. In line with our previous data in primary cultures of rat astrocytes (28) , fumonisin B1 was able to prevent cannabinoid-induced ceramide biosynthesis (relative values of [14C]serine incorporation into ceramide, n = 3: vehicle, 100; 100 nm WIN-55,212-2, 140 ± 1; 100 nm WIN-55,212-2 plus 0.5 μm fumonisin B1, 86 ± 9). C2-ceramide (1 μm) depressed VEGF production, whereas pharmacological blockade of ceramide synthesis de novo with fumonisin B1 (0.5 μm) prevented the inhibitory effect of WIN-55,212-2 (Fig. 2D) . We subsequently evaluated whether fumonisin B1 action was also evident in vivo. The decrease in tumor VEGF levels induced by cannabinoid administration (19 , 22 , 37) was prevented by cotreatment of the animals with fumonisin B1 (Fig. 2E) .

Inhibition of VEGFR-2 in Cultured Glioma Cells and in Mouse Gliomas.

VEGFR-2 activation was determined by measuring the extent of phosphorylation of two of its essential tyrosine autophosphorylation residues, namely 996 and 1214 (3 , 4) . Western blot experiments showed that C6 glioma cells express highly phosphorylated VEGFR-2 in the absence of ligand, indicating that the receptor may be constitutively active. Incubation of C6 glioma cells with WIN-55,212-2 or JWH-133 decreased VEGFR-2 activation without affecting total VEGFR-2 levels (Fig. 3A) . Confocal microscopy experiments confirmed the decrease in VEGFR-2 immunoreactivity by cannabinoid challenge when fluorescence was expressed per cell nucleus (Fig. 3B) or per total-VEGFR-2 fluorescence (data not shown). Moreover, fumonisin B1 prevented cannabinoid inhibitory action, and C2-ceramide reduced VEGFR-2 activation (Fig. 3, A and B) . Interestingly, on cannabinoid exposure the receptor seemed to be preferentially condensed in the perinuclear region, and this relocalization was prevented by fumonisin B1 (Fig. 3B) . The functional impact of VEGF on C6 glioma cells was supported by the finding that VEGF induced a prosurvival action by preventing the loss of cell viability on prolonged (72 h) cannabinoid or C2-ceramide challenge (Fig. 3C) .
Fig. 3.
Inhibition of VEGFR-2 by cannabinoids in cultured glioma cells. A, C6 glioma cells were cultured for 4 h with vehicle (Control), 100 nm WIN-55,212-2 (WIN), 100 nm JWH-133 (JWH), 10 μm C2-ceramide (CER), and/or 0.5 μm fumonisin B1 (FB1), and VEGFR-2 activation (anti-VEGFR-2 PY996 and anti-VEGR2 PY1214 antibodies) and expression (antitotal VEGFR-2 antibody) were determined by Western blot. Absorbance values relative to those of total VEGFR-2 are given in arbitrary units. Significantly different (∗, P < 0.01) from control incubations (n = 3). B, C6 glioma cells were cultured as in panel A, and VEGFR-2 activation (anti-VEGFR-2 PY1214 antibody, green) and expression (antitotal VEGFR-2 antibody, red) were determined by confocal microscopy. Cell nuclei are stained in blue. One representative experiment of 3 is shown. Relative values of activated-VEGFR-2 pixels/cell nucleus are given in parentheses. C, C6 glioma cells were cultured for 72 h with vehicle (Control), 100 nm WIN-55,212-2 (WIN), 100 nm JWH-133 (JWH), or 1 μm C2-ceramide (CER) with (▪) or without (□) 50 ng/ml VEGF, and the number of viable cells was determined. Significantly different (∗, P < 0.01) from control incubations (n = 3–4). Bars, ±SD.
The effect of cannabinoid administration on VEGFR-2 activation was subsequently tested in tumor-bearing mice. The ceramide-dependent cannabinoid-induced inhibition of VEGFR-2 activation found in cultured cells was also observed by Western blot (Fig. 4A) and confocal microscopy (Fig. 4B) in mouse gliomas. Like in the cultured-cell experiments and in line with the cDNA array experiments (data not shown), total VEGFR-2 expression in the tumors was unaffected by cannabinoid treatment (Fig. 4, A and B) .
Fig. 4.
Inhibition of VEGFR-2 by cannabinoids in mouse gliomas. A, animals bearing gliomas were treated with either vehicle (Control), JWH-133 (JWH), fumonisin B1 (FB1), or JWH-133 plus fumonisin B1 for 8 days as described in “Materials and Methods,” and VEGFR-2 activation (anti-VEGFR-2 PY996 and anti-VEGR2 PY1214 antibodies) and expression (antitotal VEGFR-2 antibody) were determined by Western blot. Absorbance values relative to those of total VEGFR-2 (phosphorylated VEGFR-2 blots) or of α-tubulin (total VEGFR-2 blots) are given in arbitrary units. Significantly different (∗, P < 0.01) from control animals (n = 3–4 for each experimental group). B, animals bearing gliomas were treated as in panel A, and VEGFR-2 activation (anti-VEGFR-2 1214 antibody, green) and expression (antitotal VEGFR-2 antibody, red) were determined by confocal microscopy. Cell nuclei are stained in blue. Low- and high-magnification pictures are shown. One representative tumor of 3–4 for each experimental group is shown. Relative values of activated-VEGFR-2 pixels/cell nucleus are given in parentheses.
Phosphorylated VEGFR-2 has been found previously in the cell nucleus, and it has been postulated that this translocation process might play a role in VEGFR-2 signaling (38, 39, 40) . However, by confocal microscopy, we found a rather variable fraction of phosphorylated VEGFR-2 in the nuclei of C6 glioma cells in culture and on inoculation in mice, and this fraction of nuclear VEGFR-2 was unaltered after treatment with cannabinoids and/or fumonisin B1 in vitro and in vivo (data not shown).

Changes in the Size of Mouse Gliomas.

To test whether the aforementioned ceramide-dependent changes in the VEGF pathway are functionally relevant, we measured tumor size along cannabinoid and fumonisin B1 treatment. In agreement with previous observations (26) , JWH-133 administration blocked the growth of s.c. gliomas in mice. Of importance, cotreatment of the animals with fumonisin B1 prevented cannabinoid antitumoral action (Fig. 5) .
Fig. 5.
Changes in the size of mouse gliomas after cannabinoid and fumonisin B1 treatment. Animals bearing gliomas (n = 4–6 for each experimental group) were treated with either vehicle (Control, ○), JWH-133 (JWH, •), fumonisin B1 (FB1, □), or JWH-133 plus fumonisin B1 (▪) for up to 8 days as described in “Materials and Methods.” Examples of formaldehyde-fixed dissected tumors after 8 days of treatment are shown. Bars, ±SD.

Inhibition of the VEGF Pathway in Two Patients with Glioblastoma Multiforme.

To obtain additional support for the potential therapeutic implication of cannabinoid-induced inhibition of the VEGF pathway, we analyzed the tumors of two patients enrolled in a clinical trial aimed at investigating the effect of Δ9-tetrahydrocannabinol, a mixed CB1/CB2 agonist, on recurrent glioblastoma multiforme. The patients were subjected to local Δ9-tetrahydrocannabinol administration, and biopsies were taken before and after the treatment. In both patients, VEGF levels in tumor extracts were lower after cannabinoid inoculation (Fig. 6A) . The Δ9-tetrahydrocannabinol also lowered the expression of phosphorylated VEGFR-2 in the tumors of the two patients, and this was accompanied (in contrast to the mouse glioma experiments shown above) by a decrease in total VEGFR-2 levels (Fig. 6B) . This was confirmed by Western blot analysis in Patient 1 (Fig. 6C) . Unfortunately, we were unable to obtain appropriate samples for Western blot from Patient 2.
Fig. 6.
Inhibition of the VEGF pathway in two patients with glioblastoma multiforme after cannabinoid treatment. The patients were subjected to Δ9-tetrahydrocannabinol (THC) administration as described in “Materials and Methods.” A, VEGF levels in the tumors before (□) and after (▪) THC treatment. B, VEGFR-2 activation (anti-VEGFR-2 PY1214 antibody, green) and expression (antitotal VEGFR-2 antibody, red) in the tumors before and after THC treatment as determined by confocal microscopy. Cell nuclei are stained in blue. Relative values of activated-VEGFR-2 pixels (parentheses) and of total-VEGFR-2 pixels (square brackets) per cell nucleus are given for the two patients. C, VEGFR-2 activation (anti-VEGFR-2 PY996 and anti-VEGR2 PY1214 antibodies) and expression (antitotal VEGFR-2 antibody) in the tumor of Patient 1 before and after THC treatment, as determined by Western blot. Absorbance values relative to those of loading controls (α-tubulin) are given in arbitrary units.


Angiogenesis is a prerequisite for the progression of most solid tumors. In particular, gliomas first acquire their blood supply by co-opting existing normal brain vessels to form a well-vascularized tumor mass without the necessity to initiate angiogenesis (41, 42, 43) . When gliomas progress, they become hypoxic as the co-opted vasculature regresses and malignant cells rapidly proliferate. These hypoxic conditions, in turn, induce robust angiogenesis via the VEGF pathway and angiopoietin-2, and in fact, this angiogenic sprouting distinguishes a grade IV astrocytoma (glioblastoma multiforme) from lower-grade astrocytomas (41, 42, 43) . Here, we show that cannabinoid treatment impairs the VEGF pathway in mouse gliomas by blunting VEGF production and signaling. Cannabinoid-induced inhibition of VEGF expression and VEGFR-2 activation also occurred in cultured glioma cells, indicating that the changes observed in vivo may reflect the direct impact of cannabinoids on tumor cells. Moreover, a depression of the VEGF pathway was also evident in two patients with glioblastoma multiforme. Although the changes in VEGFR-2 expression observed in these two patients do not fully mirror the cultured-cell and mouse data, they clearly follow the same direction. The molecular basis of this discrepancy is, however, unknown.
Our observations do not exclude that cannabinoids may also blunt tumor VEGF signaling indirectly by targeting other receptor-mediated processes that stimulate the VEGF pathway. For example, it is known that engagement of epidermal growh factor (44) and nerve growth factor (45) receptors induces the VEGF pathway, and cannabinoids have been reported to inhibit the epidermal growth factor receptor in skin carcinoma (19) and prostate carcinoma cells (46) as well as the TrkA neurotrophin receptor in breast carcinoma (47) and pheochromocytoma cells (20) . However, the molecular mechanisms by which cannabinoid receptor activation impact these growth factor receptors remain obscure.
Recent work has shown that cannabinoids can modulate sphingolipid-metabolizing pathways by increasing the intracellular levels of ceramide (23) , a lipid second messenger that controls cell fate in different systems (48 , 49) . After cannabinoid receptor activation, two peaks of ceramide generation are observed in glioma cells that have different mechanistic origin: (a) the first peak comes from sphingomyelin hydrolysis (50) ; and (b) the second peak originates from ceramide synthesis de novo (36) . The findings reported here expand the role of de novo-synthesized ceramide in cannabinoid action. Moreover, as far as we know, this is also the first report showing that ceramide depresses the VEGF pathway by interfering with VEGF production and VEGFR-2 activation, a notion that is in line with the observation that ceramide analogs prevent VEGF-induced cell survival (51 , 52) . In the context of the “sphingolipid rheostat” theory (48 , 49) , the mitogenic sphingolipid sphingosine 1-phosphate would shift the balance toward angiogenesis and tumorigenesis (5 , 53) , whereas the antiproliferative sphingolipid ceramide would blunt angiogenesis and tumorigenesis (present study).
The use of cannabinoids in medicine is limited by their psychoactive effects mediated by neuronal CB1 receptors (9 , 10) . Although these adverse effects are within the range of those accepted for other medications, especially in cancer treatment, and tend to disappear with tolerance on continuous use (20) , it is obvious that cannabinoid-based therapies devoid of side-effects would be desirable. As glioma cells express functional CB2 receptors (26) , we used a selective CB2 ligand to target the VEGF pathway. Selective CB2 receptor activation in mice also inhibits the growth and angiogenesis of skin carcinomas (19) . Unfortunately, very little is known about the pharmacokinetics and toxicology of the selective CB2 ligands synthesized to date, making them as yet unavailable for clinical trials.
Gliomas are one of the most malignant forms of cancer, resulting in the death of affected patients within 1–2 two years after diagnosis. Current therapies for glioma treatment are usually ineffective or just palliative. Therefore, it is essential to develop new therapeutic strategies for the management of glioblastoma multiforme, which will most likely require a combination of therapies to obtain significant clinical results. In line with the idea that anti-VEGF treatments constitute one of the most promising antitumoral approaches currently available (5, 6, 7) , the present laboratory and clinical findings provide a novel pharmacological target for cannabinoid-based therapies.


We are indebted to M. A. Muñoz and C. Sánchez for expert technical assistance in the confocal microscopy experiments, Dr. L. García for personal support, and Drs. G. Velasco and I. Galve-Roperh for discussion and advice.


  • Grant support: Fundación Científica de la Asociación Española Contra el Cáncer and Ministerio de Ciencia y Tecnología Grant SAF2003-00745.
  • 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.
  • Requests for reprints: Manuel Guzmán, Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain. Phone: 34-913944668; Fax: 34-913944672; E-mail:
  • 5 See Internet address for a detailed list of the genes analyzed.
  • Received December 16, 2003.
  • Revision received April 1, 2004.
  • Accepted June 10, 2004.