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LUNG CANCER and cannabis studies completed

Overview

Lung cancer, also known as carcinoma of the lung or pulmonary carcinoma, is a malignant lung tumor characterized by uncontrolled cell growth in tissues of the lung.
 
The medical science is strongly in favour of THC laden hemp oil as a primary cancer therapy, not just in a supportive role to control the side effects of chemotherapy.

There are three main types of lung cancer. Knowing which type you have is important because it affects your treatment options and your outlook (prognosis). If you aren’t sure which type of lung cancer you have, ask your doctor so you can get the right information.

Medical Cannabis:
THC has been found to reduce tumor growth in common lung cancer by 50 percent and to significantly reduce the ability of the cancer to spread, say researchers at Harvard University, who tested the chemical in both lab and mouse studies.

Science & Research

Video -Does Smoking Weed Cause Cancer?

1975 - Study - Anticancer activity of cannabinoids.

1975 - Study - Antineoplastic activity of cannabinoids.

1977 - StudyIn vivo effects of cannabinoids on macromolecular biosynthesis in Lewis lung carcinomas.

1983 - Study ~ Anti-emetic efficacy and toxicity of nabilone, a synthetic cannabinoid, in lung cancer chemotherapy.

1992 - StudyNo increase in carcinogen-DNA adducts in the lungs of monkeys exposed chronically to marijuana smoke.

1994 - News ~ Marijuana Less Harmful to Lungs than Cigarettes.

1999 - NewsSo, you thought it was the tar that caused cancer.

2000 - Report - Anti-Tumor Effects.

2004 - Study - Cannabinoids Induce Cancer Cell Proliferation via Tumor Necrosis Factor {alpha}-Converting Enzyme (TACE/ADAM17)-Mediated Transactivation of the Epidermal Growth Factor Receptor.

2005 - Study ~ Cannabis and tobacco smoke are not equally carcinogenic.

2005 - News - Smoking Cannabis Does Not Cause Cancer Of Lung or Upper Airways.

2005 - News - No association between lung cancer and cannabis smoking in large study.

2006 - News ~ Study Finds No Link Between Marijuana Use And Lung Cancer.

2006 - News ~ Study Finds No Cancer-Marijuana Connection.

2006 - News ~ No association between lung cancer and cannabis smoking in large study.

2006 - News ~ Pot Smoking Not Linked to Lung Cancer.

2006 - News ~ Large Study Finds No Link between Marijuana and Lung Cancer.

2006 - News ~ There Seems to Be No Link between Marijuana Use and Lung Cancer.

2007 - Study ~ {Delta}-9 Tetrahydrocannabinol inhibits growth and metastasis of lung cancer.

2007 - Study - Delta(9)-Tetrahydrocannabinol inhibits epithelial growth factor-induced lung cancer cell migration in vitro as well as its growth and metastasis in vivo.

2007 - News - Marijuana Ingredients Slow Invasion by Cervical and Lung Cancer Cells.

2007 - News - Marijuana May Fight Lung Tumors.

2007 - News ~ Marijuana Cuts Lung Cancer Tumor Growth In Half, Study Shows.

2007 - News ~ Pot's Active Ingredient Halts Lung Cancer Growth, Study Says.

2007 - News ~ Marijuana Helps to Combat Lung Cancer.

2007 - News ~ Cannabis as a possible treatment for lung cancer.

2007 - News ~ Marijuana Beneficial in Fighting Lung Tumors, Study.

2008 - Letter ~ Doubts about the role of cannabis in causing lung cancer.

2008 - Study ~ Inhibition of Cancer Cell Invasion by Cannabinoids via Increased Expression of Tissue Inhibitor of Matrix Metalloproteinases-1.

2008 - Study ~ Delta9-Tetrahydrocannabinol inhibits epithelial growth factor-induced lung cancer cell migration in vitro as well as its growth and metastasis in vivo.

2010 - Study ~ Decrease of plasminogen activator inhibitor-1 may contribute to the anti-invasive action of cannabidiol on human lung cancer cells.

2010 - Study ~ Cannabidiol inhibits cancer cell invasion via upregulation of tissue inhibitor of matrix metalloproteinases-1.

2011 - Study ~ Effects of smoking cannabis on lung function.

2011 - Study ~ Cannabinoid receptors, CB1 and CB2, as novel targets for inhibition of non-small cell lung cancer growth and metastasis.

2011 - Study ~ Cannabidiol inhibits lung cancer cell invasion and metastasis via intercellular adhesion molecule-1.

2012 - Study ~ Anti-proliferative and Anti-angiogenic Effects of CB2R Agonist (JWH-133) in Non-small Lung Cancer Cells (A549) and Human Umbilical Vein Endothelial Cells: an in Vitro Investigation.

2012 - Study ~ Association Between Marijuana Exposure and Pulmonary Function Over 20 Years

2012 - News ~ Marijuana compound could stop aggressive cancer metastasis

2012 - News ~ Marijuana And Cancer: Scientists Find Cannabis Compound Stops Metastasis In
Aggressive Cancers

2012 - News ~ Can marijuana stop cancer?

2012 - News ~ Media Ignored Expert's Shocking Findings That Marijuana Helps Prevent Lung Cancer: Now It's Med-School Material

2012 - News ~ Is Marijuana the Cancer Cure We’ve Waited For?

2012 - News ~ Study: Smoking Marijuana Not Linked with Lung Damage

2013 - Editorial ~ Cannabis and the Lung: No More Smoking Gun?

2013 - Study ~ COX-2 and PPAR-γ Confer Cannabidiol-Induced Apoptosis of Human Lung Cancer Cells.

2013 - Study ~ Effects of marijuana smoking on the lung.

2013 - Study ~ Cannabis smoking and lung cancer risk: pooled analysis in the International Lung Cancer Consortium

2013 - Study ~ Magnolol induces apoptosis via caspase-independent pathways in non-small cell lung cancer cells.

2013 - News ~ "Miracle" Cannabis Oil: May Treat Cancer, But Money and the Law Stand in the Way of Finding Out

2013 - News ~ Federal Government Reports Marijuana Effective in Combatting Certain Cancers Reports ADSI

2013 - News ~ Marijuana habit not linked to lung cancer

Delta(9)-Tetrahydrocannabinol inhibits epithelial growth factor-induced lung cancer cell migration in vitro as well as its growth and metastasis in vivo

Preet A, Ganju RK, Groopman JE 
Delta(9)-Tetrahydrocannabinol inhibits epithelial growth factor-induced lung cancer cell migration in vitro as well as its growth and metastasis in vivo. [JOURNAL ARTICLE]
Oncogene 2007 Jul 9.

Delta(9)-Tetrahydrocannabinol (THC) is the primary cannabinoid of marijuana and has been shown to either potentiate or inhibit tumor growth, depending on the type of cancer and its pathogenesis.

Little is known about the activity of cannabinoids like THC on epidermal growth factor receptor-overexpressing lung cancers, which are often highly aggressive and resistant to chemotherapy.

In this study, we characterized the effects of THC on the EGF-induced growth and metastasis of human non-small cell lung cancer using the cell lines A549 and SW-1573 as in vitro models.

We found that these cells express the cannabinoid receptors CB(1) and CB(2), known targets for THC action, and that THC inhibited EGF-induced growth, chemotaxis and chemoinvasion. Moreover, signaling studies indicated that THC may act by inhibiting the EGF-induced phosphorylation of ERK1/2, JNK1/2 and AKT.

THC also induced the phosphorylation of focal adhesion kinase at tyrosine 397. Additionally, in in vivo studies in severe combined immunodeficient mice, there was significant inhibition of the subcutaneous tumor growth and lung metastasis of A549 cells in THC-treated animals as compared to vehicle-treated controls. Tumor samples from THC-treated animals revealed antiproliferative and antiangiogenic effects of THC.

Our study suggests that cannabinoids like THC should be explored as novel therapeutic molecules in controlling the growth and metastasis of certain lung cancers.Oncogene advance online publication, 9 July 2007; doi:10.1038/sj.onc.1210641

 

 

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Smoking Cannabis Does Not Cause Cancer Of Lung or Upper Airways

By Fred Gardner

Marijuana smoking —“even heavy longterm use”— does not cause cancer of the lung, upper airways, or esophagus, Donald Tashkin, MD, reported at this year’s meeting of the International Cannabinoid Research Society.

Coming from Tashkin, this conclusion had extra significance for the assembled drug-company and university-based scientists (most of whom get funding from the U.S. National Institute on Drug Abuse). Over the years, Tashkin’s lab at UCLA has produced irrefutable evidence of the damage that marijuana smoke wreaks on bronchial tissue.

With NIDA’s support, Tashkin and colleagues have identified the potent carcinogens in marijuana smoke, biopsied and made photomicrographs of pre-malignant cells, and studied the molecular changes occurring within them.

It is Tashkin’s research that the Drug Czar’s office cites in ads linking marijuana to lung cancer. Tashkin himself has long believed in a causal relationship, despite a study in which Stephen Sidney, MD, examined the files of some 64,000 Kaiser patients and found that marijuana users did not develop lung cancer at a higher rate or die earlier than non-users.

Of five smaller studies on the question, only two —involving a total of about 300 patients— concluded that marijuana smoking causes lung cancer.

Tashkin decided to settle the question by conducting a large, population-based, case-controlled study. “Our major hypothesis,” he told the ICRS, “was that heavy, longterm use of marijuana will increase the risk of lung and upper-airways cancers.”

The Los Angeles County Cancer Surveillance program provided Tashkin’s team with the names of 1,209 L.A. residents aged 59 or younger with cancer (611 lung, 403 oral/pharyngeal, 90 laryngeal, 108 esophageal).

Interviewers collected extensive lifetime histories of marijuana, tobacco, alcohol and other drug use, and data on diet, occupational exposures, family history of cancer, and various “socio-demographic factors.”

Exposure to marijuana was measured in “joint years” —average number of joints per day x years that number smoked. Thus if a person had smoked two joints a day for 15 years they’d have consumed for 30 j-yrs.

Controls were found based on age, gender and neighborhood. Among them, 46% had never used marijuana, 31% had used for less than one joint year, 12% had used for 1-10 j-yrs, 5% had used 10-30 j-yrs, 2% had used for 30-60 j-yrs, and 3% had used for more than 60 j-yrs.

Tashkin controlled for tobacco use and calculated the relative risk of marijuana use resulting in lung and upper airways cancers. A relative risk ratio of .72 means that for every 100 non-users who get lung cancer, only 72 people who smoke get lung cancer. All the odds ratios in Tashkin’s study turned out to be less than one!

Compared with subjects who had used less than one joint year, the estimated odds ratios for lung cancer were .78 for 1-10 j-yrs [according to the abstract book and .66 according to notes from the talk]; .74 for 10-30 j-yrs; .85 for 30-60 j-yrs; and 0.81 for more than 60 j-yrs.

The estimated odds ratios for oral/pharyngeal cancers were 0.92 for 1-10 j-yrs; 0.89 for 10-30 j-yrs; 0.81 for 30-60 j-yrs; and 1.0 for more than 60 j-yrs. “Similar, though less precise results were obtained for the other cancer sites,” Tashkin reported. “We found absolutely no suggestion of a dose response.”

The data on tobacco use, as expected, revealed “a very potent effect and a clear dose-response relationship —a 21-fold greater risk of developing lung cancer if you smoke more than two packs a day.” Similarly high odds obtained for oral/pharyngeal cancer, laryngeal cancer and esophageal cancer. “So, in summary” Tashkin concluded, “we failed to observe a positive association of marijuana use and other potential confounders.”

There was time for only one question, said the moderator, and San Francisco oncologist Donald Abrams, M.D., was already at the microphone: “You don’t see any positive correlation, but in at least one category, it almost looked like there was a negative correlation, i.e., a protective effect. Could you comment on that?” (Abrams was referring to Tash-kin’s lung-cancer data for marijuana-only smokers, 1-10 j-yrs.)

“ Yes,” said Tashkin. “The odds ratios are less than one almost consistently, and in one category that relationship was significant, but I think that it would be difficult to extract from these data the conclusion that marijuana is protective against lung cancer. But that is not an unreasonable hypothesis.”

Abrams’s Favorable Results
Abrams had results of his own to report at the ICRS meeting. He and his colleagues at San Francisco General Hospital had conducted a randomized, placebo-controlled study involving 50 patients with HIV-related peripheral neuropathy. Over the course of five days, patients recorded their pain levels in a diary after smoking either NIDA-supplied marijuana cigarettes or cigarettes from which the THC had been extracted. About 25% didn’t know or guessed wrong as to whether they were smoking the placebos, which suggests that the blinding worked.

Abrams’s results show marijuana providing pain relief comparable to Gaba-pentin, the most widely used treatment for a condition that afflicts some 30% of patients with HIV.

After Abrams’s presentation, a questioner bemoaned the difficulty of “separating the high from the clinical benefits.” Abrams responded: “I’m an oncologist as well as an AIDS doctor and I don’t think that a drug that creates euphoria in patients with terminal diseases is having an adverse effect.” His study was funded by the University of California’s Center for Medicinal Cannabis Research.

Add ICRS Notes
The 15th annual meeting of the ICRS was held at the Clearwater, Florida, Hilton, June 24-27. Almost 300 scientists attended. R. Stephen Ellis, MD, of San Francisco, was the sole clinician from California. Medical student Sunil Aggarwal, Farmacy operator Mike Ommaha and therapist/cultivator Pat Humphrey audited the proceedings.

Some of the younger European scientists expressed consternation over the recent U.S. Supreme Court ruling and the vote in Congress re-enforcing the cannabis prohibition. “How can they dispute that it has medical effect?” an investigator working in Germany asked us earnestly. She had come to give a talk on “the role of different neuronal populations in the pharmacological actions of delta-9 THC.”

For most ICRS members, the holy grail is a legal synthetic drug that exerts the medicinal effects of the prohibited herb. To this end they study the mechanism of action by which the body’s own cannabinoids are assembled, function, and get broken down. A drug that encourages production or delays dissolution, they figure, might achieve the desired effect without being subject to “abuse.”

News on the scientific front included the likely identification of a third cannabinoid receptor expressed in tissues of the lung, brain, kidney, spleen and smaller branches of the mesenteric artery. Investigators from GlaxoSmith-Kline and AstraZeneca both reported finding the new receptor but had different versions of its pharmacology. It may have a role in regulating blood pressure.

Several talks and posters described the safety and efficacy of Sativex, G.W. Pharmaceuticals’ plant extract containing high levels of THC and cannabidiol (CBD) formulated to spray in the mouth. See “Dr. X’s Top Talks,” on page 11.

G.W. director Geoffrey Guy seemed upbeat despite the slide his company’s stock took this spring when UK regulators withheld permission to market Sati-vex pending another clinical trial. Canada recently granted approval for doctors to prescribe Sativex, and five sales reps from Bayer (to whom G.W. sold Canadian marketing rights) are promoting it to neurologists. Sativex was approved for treatment of neuropathic pain in multiple sclerosis, but can be prescribed for other purposes as doctors see fit.

Most of the work being done with CBD and CBN is done with materials provided by GW, and some two dozen papers and posters gave them acknowledgment. At last there is a realistic alternative to NIDA for the young researchers to look to for support (and plant cannabinoids to study). GW has contributed to a significant shift in attitude.

On numerous occasions during the meeting a NIDA-funded researcher would describe the negative effects of THC, and immediately a scientist with a British accent would be at the mike pointing out that such a high dose injected into the stomach of a rat had nothing to do with the human experience with cannabis. It must have happened five or six times. The Brits were always very diplomatic, but they functioned like a truth squad.

Roger Pertwee of the University of Aberdeen reported intriguing results from experiments using a cannabis strain bred by GW to be high in THCV (tetrohydrocannabivarin).

It turns out that THCV strongly antagonizes anandamide while hardly antagonizing THC! It’s as if the cannabis plant contains and makes available to the body a choice of drugs and the body uses those it needs to achieve a balanced state (homeostasis). If the body is producing endocannabinoids in excess, it can use the plant cannabinoid THCV to achieve homeostasis. If the endocannabinoid system needs a boost, the THC provides it (while the THCV shuts down the EC system, giving it a rest as it were). The key to relief, apparently, is not high cannabinoid levels but proper gradients.

Guy explained, “It’s as if the plant contains a first-aid kit giving the body everything it needs to get bettter, and the body decides which components to employ... The endocannibnoid system begins to kick in in abnormality, in pathology. Perhaps it kicks in whether the pathology is an increase in something or a decrease in something. What it’s trying to do is get whatever that abnormality is back to homeostasis.

“ The antagonist may be working to restore function back to the center, and the agonist might be working to restore function back to the center, and once they’ve achieved the norm, they don’t go any further. The endocannabinoid system is the supeme modulator. Its job is done once you’re back to the norm. Most endocannabinoid modulators simply won’t drive the physiology or biochemistry —whatever they’re controlling— past the norm to a detrimental effect.”

Rimonabant Comes Closer
Which might explain the apparent benignity of Rimonabant, a drug that works by blocking the CB1 receptor system. Rimonabant is being tested by Sanofi-Aventis for weight loss and smoking cessation. Originally known as SR-141716, it was developed in the early 1990s as an antagonist drug for use by researchers. At the 2004 ICRS meeting, Sanofi researchers described favorable results from clinical trials of Rimonabant as a diet drug. They informally predicted regulatory approval in Europe and the U.S. within a year. Some observers warned that blocking the CB1 receptor system could result in unforeseen longterm side effects and noted that at least one MS patient had experienced an exacerbation after taking Rimonabant.

Although regulatory approval has not yet been granted, Sanofi reported good news at this meeting regarding side-effects: no more MS cases in a smoking-cessation study study involving more than 1,000 patients worldwide. “Both the 5mg and 20mg doses continued to show efficacy in the maintenance of abstinence from smoking,” reported Gerard Le Fur. “The 20mg dose also demonstrated efficacy in the reduction of weight gain as well as significantly increasing the HDL-Cholesterol levels.”

A Sanofi team also reported favorable results from studies using Rimonabant to treat various rodent models of “metabolic syndrome” —obesity-related high blood pressure, high insulin levels, excessive triglycerides and “bad” cholesterol and other problems increasing the risk of diabetes, heart attack and stroke. There is growing acceptance of the notion that the body can adjust to even a heavy blockade of the CB1 system. Perhaps when the CB1 receptor is blocked, the endocannabinoids are redirected to other targets. At times the layman is struck by how rudimentary the biochemists’ understanding of the body’s mechanism of action really is.

“ We’re on plateau one or two and the answer is on plateau 12,” said Guy. “ We could spend the next 30 years on receptors and still not fully understand them. When we talk about receptors and agonists and antagonists we should be talking in the same breath about functionality —real functionality, not models in non-pathological situations. We need an understanding of the clinical outcome.”


Osteopathic Manipulation Boosts Endocannabinoid System
John McPartland of GW Pharmaceuticals reported that osteopathic manipulative treatment (OMT) works via the endocannabinoid system. McPartland and co-workers conducted a randomized, placebo-controlled study involving 31 patients of a New Zealand osteopath.

“ Cannabimimetic effects” were measured by patients filling out a questionnaire before and after treatment defining levels of light-headedness, hunger, alterness, etc. Anandamide levels in the blood were also measured before and after treatments.

The “sham” manipulation mimicked a new technique called “biodynamic osteopathy in the cranial field.” The sham practitioner sabotaged her own concentration and mental healing intention by silently reciting “backwards serial sevens” while she applied light manual contact to the patient’s head.

Subjects receiving OMT indeed reported feeling cannabi-mimetic effects (more creativity, less coherence, for example) and their serum anandamide levels increased 168% over pre-treatment levels. Subjects receiving sham manipulation reported no changes in the questionnaire and there was no change in their serum anandamide levels.

McPartland et al noted that patients receiving OMT often experience an improved sense of well-being, sedation and euphoria —effects similar to those brought on by cannabis consumption. Previous studies indicated these psychotropic effects are not elicited by endorphins (as once had been assumed).

A recent study by Andrea Giuffrida, who contributed to the OMT study, showed that “runner’s high” correlated with elevated anandamide and not endorphins. Patients receiving chiropractic, massage, acupuncture, and energy healing also experience parallel psychotropic effects. The authors conclude that the endocannabinoid system may be mediating a widespread but heretofore unrecognized therapeutic phenomenon.
 

 

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No association between lung cancer and cannabis smoking in large study

A large study on the association between cannabis smoking and lung cancer already presented at the 2005 Meeting of the International Cannabinoid Research Society was now also presented at The American Thoracic Society Conference in San Diego and received much media interest. The study with 611 lung cancer patients and 1,040 healthy controls as well as 601 patients with cancer in the head or neck region found no increased risk for lung cancer even after heavy long-term use of cannabis.

"We expected that we would find that a history of heavy marijuana use - more than 500 to 1,000 uses - would increase the risk of cancer from several years to decades after exposure to marijuana," lead researcher Dr. Donald Tashkin of the University of California, Los Angeles, said in the Scientific American. But the scientists found that even those who smoked more than 20,000 cannabis cigarettes in their life did not have an increased risk of lung cancer.

(Sources: Scientific American of 24 May 2006; Morgenstern H, et al. Marijuana use and cancers of the lung and upper aerodigestive tract: results of a case-control study. Presentation at the ICRS Conference on Cannabinoids, 24-27 June 2005, Clearwater, USA)

 

 
 
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Marijuana Smoking Found Non-Carcinogenic

By Neil Osterweil, Senior Associate Editor, MedPage Today
Published: May 24, 2006
Reviewed by Robert Jasmer, MD; Associate Clinical Professor of Medicine, University of California, San Francisco .

SAN DIEGO, May 24 — Smoking marijuana does not appear to increase the risk of lung cancer or head-and-neck malignancies, even among heavy users, researchers reported here.
Action Points  
  • Inform interested patients that the results of this study suggest that marijuana smoking does not appear to increase the risk of cancers of the lung, head, or neck, but there is also no evidence that it offers a protective effect.
  • Caution patients that marijuana smoke may cause or exacerbate other conditions such as chronic obstructive pulmonary disease.
  • This study was published as an abstract and presented orally at a conference. These data and conclusions should be considered to be preliminary as they have not yet been reviewed and published in a peer-reviewed publication.

"We expected that we would find that a history of heavy marijuana use, more than 500 to 1,000 uses, would increase the risk of cancer from several years to decades after exposure to marijuana, said Donald Tashkin, M.D., of the University of California in Los Angeles.

But in fact, they reported at the American Thoracic Society meeting here, marijuana use was associated with cancer risk ratios below 1.0, indicating that a history of pot smoking had no effect on the risk for respiratory cancers.

Dr. Tashkin was quick to point out, however, that marijuana does not appear to have a protective effect against cancer. "If it did, there would be a dose-dependent effect, with people who smoked more having a lower risk," he said. "We didn't see that."

Studies have shown that marijuana contains many compounds that when burned, produce about 50% higher concentrations of some carcinogenic chemicals than tobacco cigarettes.

In addition, heavy, habitual marijuana use can produce accelerated malignant change in lung explants, and evidence on bronchial biopsies of pre-malignant histopathologic and molecular changes, Dr. Tashkin said.

The investigators had also previously shown that smoking one marijuana cigarette leads to the deposition in the lungs of four times as much tar as smoking a tobacco cigarette containing the same amount of plant material. Marijuana cigarettes are not filtered and are more loosely packed than tobacco, so there's less filtration of the tar. In addition, pot smokers hold the smoke in their lungs about four times longer than tobacco smokers do, Dr. Tashkin pointed out.

He and his colleagues, led by epidemiologist Hal Morgenstern, Ph.D., of the University of Michigan in Ann Arbor, conducted a study to look at possible associations between marijuana use and the risk of respiratory cancers among middle-age adults in the Los Angeles area.

For the population-based case-control study, they identified cancer cases among people from the ages of 18 to 59, using the Los Angeles County Cancer Surveillance Program registry.

They identified 611 people with lung cancer, 601 with cancers of the head and neck, and 1,040 controls matched by age, gender and neighborhood (as a surrogate for socioeconomic status).

They conducted extensive personal interviews to determine lifetime marijuana use, measured in joint-years, with one joint-year equivalent to 365 marijuana cigarettes. The interviewers also asked participants about tobacco use, alcohol consumption, use of other drugs, socioeconomic status, diet, occupation, and family history of cancer.

The investigators also used logistic regression to estimate the effect of marijuana use on lung cancer risk, adjusting for age, gender, race/ethnicity, education, and cumulative tobacco smoking and alcohol use.

They found that the heaviest users in the study had smoked more than 60 joint years worth of marijuana, or more than 22,000 joints in their lifetime. Moderately heavy users smoked between 11,000 and 22,000 joints.

"That's an enormous amount of marijuana," Dr. Tashkin said.

Despite the heavy use, "in no category was there any increased risk, nor was there any suggestion that smoking more led to a higher odds ratio," he continued. "There was no dose-response—not even a suggestion of a dose response—and in all types of cancer except one, oral cancer, the odds ratios were less than one."

The confidence intervals around the odds ratios were wide however, and the odds ratios did not show a dose response.

In contrast, tobacco smoking was associated with increased risk for all cancers, and there was a "powerful" dose-response relationship. People who smoked more than two packs of cigarettes per day had a 21-fold risk for cancer, as opposed to a less than onefold risk for marijuana, Dr. Tashkin said.

"When we restricted the analysis to those who didn't smoke any tobacco we found the same results, and when we looked for interaction between tobacco and smoking—would marijuana increase the risk, potentiate the carcinogenic effect of tobacco—we didn't find that, nor did we find a protective effect against the effect of tobacco, which is very important, because the majority of marijuana smokers also smoke tobacco," he commented.

It's possible that tetrahydrocannabinol (THC) in marijuana smoke may encourage apoptosis, or programmed cell death, causing cells to die off before they have a chance to undergo malignant transformation, he said.

Dr. Tashkin also noted that "it's never a good idea to take anything into your lungs, including marijuana smoke."

Primary source: 2006 American Thoracic Society Annual Meeting
Source reference:
Tashkin DP et al. "Marijuana Use and Lung Cancer: Results of a Case-Control Study." Presented in a briefing May 23 and in an oral session May 24, 2006.
 
 
 
 
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CLAIM #4: MARIJUANA CAUSES LUNG DISEASE

It is frequently claimed that marijuana smoke contains such high concentrations of irritants that marijuana users' risk of developing lung disease is equal to or greater than that of tobacco users.

THE FACTS

Except for their psychoactive ingredients, marijuana and tobacco smoke are nearly identical. Because most marijuana smokers inhale more deeply and hold the smoke in their lungs, more dangerous material may be consumed per cigarette. However, it is the total volume of irritant inhalation - not the amount in each cigarette - that matters.

Most tobacco smokers consume more than 10 cigarettes per day and some consume 40 or more. Regular marijuana smokers seldom consume more than three to five cigarettes per day and most consume far fewer. Thus, the amount of irritant material inhaled almost never approaches that of tobacco users.
Frequent marijuana smokers experience adverse respiratory symptoms from smoking, including chronic cough, chronic phlegm, and wheezing. However, the only prospective clinical study shows no increased risk of crippling pulmonary disease (chronic bronchitis and emphysema).
Since 1982, UCLA researchers have evaluated pulmonary function and bronchial cell characteristics in marijuana-only smokers, tobacco-only smokers, smokers of both, and non-smokers. Although they have found changes in marijuana-only smokers, the changes are much less pronounced than those found in tobacco smokers.

The nature of the marijuana-induced changes were also different, occurring primarily in the lung's large airways - not the small peripheral airways affected by tobacco smoke. Since it is small-airway inflammation that causes chronic bronchitis and emphysema, marijuana smokers may not develop these diseases. 

In an epidemiological survey, approximately 1200 subjects gave information on smoking and pulmonary function at two-year intervals. A large percentage of the subjects underwent pulmonary function testing. Although a small group who reported previous marijuana smoking had significant pulmonary abnormalities, current marijuana smokers had no significant reduction in any pulmonary functions. 

There are no epidemiological or aggregate clinical data suggesting that marijuana-only smokers develop lung cancer. However, since some bronchial cell changes appear to be pre-cancerous, an increased risk of cancer among frequent marijuana smokers is possible. 

Since the pulmonary risks associated with marijuana are related to smoking, the danger is eliminated with other routes of administration. For committed smokers, pulmonary risk might be reduced with higher-potency products, which produce desired psychoactive effects with less inhalation of irritants. Smokers could also be encouraged to abandon deep inhalation and breath-holding, which increase drug delivery only slightly. Finally, pulmonary risk might be reduced if marijuana were smoked in water pipes rather than cigarettes. 

[Next Claim]

 

 

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Marijuana Ingredients Slow Invasion by Cervical and Lung Cancer Cells

 
By Daniel J. DeNoon
WebMD Health News
Reviewed by Louise Chang, MD

Dec. 26, 2007 -- THC and another marijuana-derived compound slow the spread of cervical and lung cancers, test-tube studies suggest.

The new findings add to the fast-growing number of animal and cell-culture studies showing different anticancer effects for cannabinoids, chemical compounds derived from marijuana.

Cannabinoids, and sometimes marijuana itself, are currently used to lessen the nausea and pain experienced by many cancer patients. The new findings -- yet to be proven in human studies -- suggest that cannabinoids may have a direct anticancer effect.

"Cannabinoids' ... potential therapeutic benefit in the treatment of highly invasive cancers should be addressed in clinical trials," conclude Robert Ramer, PhD, and Burkhard Hinz, PhD, of the University of Rostock, Germany.

Might cannabinoids keep dangerous tumors from spreading throughout the body? Ramer and Hinz set up an experiment in which invasive cervical and lung cancer cells had make their way through a tissue-like gel. Even at very low concentrations, the marijuana compounds THC and methanandamide (MA) significantly slowed the invading cancer cells.

Doses of THC that reduce pain in cancer patients yield blood concentrations much higher than the concentrations needed to inhibit cancer invasion.

"Thus the effects of THC on cell invasion occurred at therapeutically relevant concentrations," Ramer and Hinz note.

The researchers are quick to point out that much more study is needed to find out whether these test-tube results apply to tumor growth in animals and in humans.

Ramer and Hinz report the findings in the Jan. 2, 2008 issue of the Journal of the National Cancer Institute.

 

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Marijuana May Fight Lung Tumors

 
By Charlene Laino
WebMD Health News
Reviewed by Louise Chang, MD

April 17, 2007 (Los Angeles) -- Cannabis may be bad for the lungs, but the active ingredient in marijuana may help combat lung cancer, new research suggests.

In lab and mouse studies, the compound, known as THC, cut lung tumor growth in half and helped prevent the cancer from spreading, says Anju Preet, PhD, a Harvard University researcher in Boston who tested the chemical.

While a lot more work needs to be done, “the results suggest THC has therapeutic potential,” she tells WebMD.

Moreover, other early research suggests the cannabis compound could help fight brain, prostate, and skin cancers as well, Preet says.

The findings were presented at the annual meeting of the American Association for Cancer Research.

The finding builds on the recent discovery of the body’s own cannabinoid system, Preet says. Known as endocannabinoids, the natural cannabinoids stimulate appetite and control pain and inflammation.

THC seeks out, attaches to, and activates two specific endocannabinoids that are present in high amounts on lung cancer cells, Preet says. This revs up their natural anti-inflammatory properties. Inflammation can promote the growth and spread of cancer.

In the new study, the researchers first demonstrated that THC inhibited the growth and spread of cells from two different lung cancer cell lines and from patient lung tumors. Then, they injected THC into mice that had been implanted with human lung cancer cells. After three weeks, tumors shrank by about 50%, compared with tumors in untreated mice.

Preet notes that animals injected with THC seem to get “high,” showing signs of clumsiness and getting the munchies. “You would expect to see the same thing in humans, so if this work does pan out, getting the dose right is going to be all important,” she says.

Paul B. Fisher, PhD, a professor of clinical pathology at Columbia University, says that though the work is “interesting,” it’s still very early.

“The issue with using a drug of this type becomes the window of concentration that will be effective. Can you physiologically achieve what you want without causing unwanted effects?” he tells WebMD.

 

 

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Anti-Tumor Effects

by GW Pharmaceuticals

Emerging research indicates that cannabinoids may help protect against the development of certain types of tumors. Most recently, a Spanish research team reported in Nature that injections of synthetic THC eradicated malignant brain tumors - so-called gliomas - in one-third of treated rats, and prolonged life in another third by as much as six weeks.

Team leader Manuel Guzman called the results "remarkable" and speculated that they "may provide a new therapeutic approach for the treatment of malignant gliomas. An accompanying commentary remarked that this was the first convincing study to demonstrate that cannabis-based treatment may combat cancer. Other journals have also recently reported on cannabinoids' antitumoral potential.

Earlier studies also indicate that cannabinoids may successfully stave certain types of tumors. One study examined the effects of delta-9-tetrahydrocannabinol (THC), delta-8-THC, and cannabinol (CBN) on cancer cells in mice lungs.

Researchers reported that cannabinoids reduced the size of the tumors by 25 to 82 percent, depending on dose and duration of treatment, with a corresponding increase in survival time. A two-year federal study by the U.S.

National Toxicology Program found that mice and rats given high doses of THC over long periods of time appeared to have greater protection against malignancies than untreated controls.

Researchers concluded that in both mice and rats, "the incidence of benign and malignant neoplasms was decreased in a dose dependent manner." Dr. Lester Grinspoon writes in Marihuana: The Forbidden Medicine (with James Bakalar) that other animal studies also suggest that some cannabinoids have tumor-reducing properties.

An Italian research team reported in 1998 that the endocannabinoid anandamide, which binds to the same brain receptors as cannabis, "potently and selectively inhibits the proliferation of human breast cancer cells in vitro" by interfering with their DNA production cycle.Non-mammary tumor cells were not affected by anandamide. Clearly, further research is necessary and appropriate.

 

REFERENCES

  1. I. Galve-Roperph et al. "Antitumoral action of cannabinoids: involvement of sustained ceramide accumulation of ERK activation." Nature Medicine 6 (2000): 313-319.
  2. ACM Bulletin. "THC destroys brain cancer in animal research," March 5, 2000.
    http://www.acmed.org/english/2000/eb000305.html
  3. D. Piomelli. "Pot of gold for glioma therapy." Nature Medicine 6 (2000): 255-256.
  4. J. Benard. "Cannabinoids, among others, send malignant tumors to nirvana." Bull Cancer 87 (2000): 299-300.
  5. J. Molnar et al. "Membrane associated with antitumor effects of crocine-ginsenoside and cannabinoid derivatives." Anticancer Res 20 (2000): 861-867.
  6. L. Ruiz et al. "Delta-9-tetrahydrocannabinol induces apoptosis in human prostate PC-3 cells via a receptor-independent mechanism." FEBS Letter 458 (1999): 400-404.
  7. S. Baek et al. "Antitumor activity of cannabigerol against human oral epitheloid carcinoma cells." Arch Pharm Res 21 (1998): 353-356.
  8. L. Harris et al., "Anti-tumoral Properties of Cannabinoids," The Pharmacology of Marihuana, ed. M. Braude et al., 2 vols., New York: Raven Press (1976) 2: 773-776 as cited by L. Grinspoon et al., Marihuana: The Forbidden Medicine (second edition), New Haven, CT: Yale University Press (1997), 173.
  9. J. James, "Unpublished Federal Study Found THC-Treated Rats Lived Longer, Had Less Cancer," AIDS Treatment News 263 (1997).
  10. "Toxicology and Carcinogenesis Studies of 1 trans-delta-9-tetrahydrocannabinol in F344N/N Rats and BC63F1 Mice," National Institutes of Health National Toxicology Program, NIH Publication No. 97-3362 (November 1996).
  11. L. Grinspoon et al., Marihuana: "The Forbidden Medicine" (second edition), 173.
  12. L. De Petrocellis et al., The endogenous cannabinoid anandamide inhibits human breast cancer cell proliferation, Proceedings of the National Academy of Sciences 95 (1998): 8375-8380.
    http://www.pnas.org/cgi/content/abstract/95/14/8375
  13. "Pot Chemicals Might Inhibit Breast Tumors, Stroke Damage," Dallas Morning News, July 13, 1998.
    http://www.mapinc.org/drugnews

 
 
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Cannabinoids Induce Cancer Cell Proliferation via Tumor Necrosis Factor {alpha}-Converting Enzyme (TACE/ADAM17)-Mediated Transactivation of the Epidermal Growth Factor Receptor

  1. Efan Hart,
  2. Oliver M. Fischer
  3. Axel Ullrich

Author Affiliations

  1. Department of Molecular Biology, Max-Planck-Institute of Biochemistry, Martinsried, Germany

Abstract

Cannabinoids, the active components of marijuana and their endogenous counterparts were reported as useful analgetic agents to accompany primary cancer treatment by preventing nausea, vomiting, and pain and by stimulating appetite.

Moreover, they have been shown to inhibit cell growth and to induce apoptosis in tumor cells. Here, we demonstrate that anandamide, Δ9-tetrahydrocannabinol (THC), HU-210, and Win55,212-2 promote mitogenic kinase signaling in cancer cells.

Treatment of the glioblastoma cell line U373-MG and the lung carcinoma cell line NCI-H292 with nanomolar concentrations of THC led to accelerated cell proliferation that was completely dependent on metalloprotease and epidermal growth factor receptor (EGFR) activity. EGFR signal transactivation was identified as the mechanistic link between cannabinoid receptors and the activation of the mitogen-activated protein kinases extracellular signal-regulated kinase 1/2 as well as prosurvival protein kinase B (Akt/PKB) signaling.

Depending on the cellular context, signal cross-communication was mediated by shedding of proAmphiregulin (proAR) and/or proHeparin-binding epidermal growth factor-like growth factor (proHB-EGF) by tumor necrosis factor α converting enzyme (TACE/ADAM17).

Taken together, our data show that concentrations of THC comparable with those detected in the serum of patients after THC administration accelerate proliferation of cancer cells instead of apoptosis and thereby contribute to cancer progression in patients.

Introduction

Cannabinoids have been used in medicine for more than a century. Recently interest in their therapeutic value has been fuelled by suggestions to apply these drugs in cancer treatment to improve analgesia and to relieve insomnia.

Because of their neuroprotective properties, cannabinoids have also been proposed to be useful drugs for the therapy of neurodegenerative diseases like Parkinson‘s disease, Huntington disease, and multiple sclerosis. Orally applicable Δ9-tetrahydrocannabinol (THC; Dronabinol, Marinol) and its synthetic derivative Nabilone (Cesamet) have been approved by the United States Food and Drug Administration to stimulate the appetite of patients with AIDS and to reduce the nausea of cancer patients undergoing chemotherapy.

Moreover, recent investigations propose that drugs activating the endogenous cannabinoid system might be used in cancer therapy to slow down or block cancer growth. The endogenous cannabinoid anandamide (AEA) acts antiproliferatively in MCF-7, EFM-19, T47D, and DU145 cells. Interestingly, cannabinoid-induced inhibition of proliferation in breast cancer cells results from cycle arrest at the G1-S phase transition and is independent of apoptosis. Furthermore, depending on drug concentration, the timing of drug delivery, and cellular context, cannabinoids may either inhibit or stimulate the function of immune cells. Although high concentrations of cannabinoids block immune cells, Derocq et al. demonstrated proliferation in human B cells after cannabinoid stimulation at nanomolar concentrations . In addition, murine hematopoietic cells depend on AEA for normal growth in serum-free medium.

THC, the endogenous cannabinoid AEA and synthetic cannabinoids like HU-210 and Win55,212-2 interact with specific G protein-coupled receptors (GPCRs). Two subtypes of the cannabinoid receptors, CB1 and CB2, have been cloned and characterized.

The CB1 receptor, which is responsible for the well-known psychotropic effects of cannabinoids, is highly expressed in the central nervous system, but lower levels are also present in immune cells and peripheral tissues including testis, whereas the CB2 receptor is predominantly expressed in immune cells.

Both cannabinoid receptors are coupled to heterotrimeric Gi/o-proteins and activate the mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase (ERK)1/2 and p38 as well as the Akt/PKB survival pathway.

Extensive research efforts have addressed the question how cannabinoids induce MAPK activation.

Thus far, the accumulation of ceramides after cannabinoid stimulation has been implicated in the induction of the ERK/MAPK signal, whereas other reports suggested intracellular ceramide levels not to be required for cannabinoid-induced MAPK activation.

Previously we and others have shown that a wide variety of GPCR agonists leads to the activation of MAPK via transactivation of the epidermal growth factor receptor (EGFR) . This mechanistic concept involves the proteolytic processing of a membrane-spanning proEGF-like growth factor by a zinc-dependent metalloprotease of the ADAM family.

The aim of this study was to identify critical elements that link the cannabinoid receptors to activation of the ERK/MAPK and the Akt/PKB pathway. Hence, we tested whether cannabinoid receptors transactivate the EGFR in cancer cell lines, thereby activating downstream mitogenic signaling events.

Our results demonstrate that treatment of NCI-H292 (lung cancer), SCC-9 (squamous cell carcinoma), 5637 (bladder carcinoma), U373-MG (glioblastoma), 1321N1 (astrocytoma), and A498 (kidney cancer) cells with cannabinoids such as THC, AEA, HU-210, and Win55,212-2 leads to rapid EGFR tyrosine phosphorylation, phosphorylation of the adaptor protein Src homology 2 domain-containing (SHC), and downstream activation of ERK1/2 and Akt/PKB. EGFR transactivation is specifically mediated by cannabinoid-induced cleavage of proAmphiregulin (proAR) and/or proHeparin-binding epidermal growth factor-like growth factor (proHB-EGF) at the cell surface by tumor necrosis factor α-converting enzyme (TACE/ADAM17). Importantly, THC induced EGFR- and metalloprotease-dependent cancer cell proliferation.

Thus, this cross-communication of CB1/CB2 receptors and the EGFR provides a molecular explanation of how cannabinoid receptors are linked to MAPK and Akt/PKB activation in a wide variety of human cancer cell lines.

In the light of these results, the use of cannabinoids in cancer therapy has to be reconsidered, because relatively high concentrations of THC induce apoptosis in cancer cells, whereas nanomolar concentrations enhance tumor cell proliferation and may, therefore, accelerate cancer progression in patients.

Materials and Methods

Cell Culture.

All of the cell lines (American Type Culture Collection, Manassas, VA) were routinely grown according to the supplier’s instructions. Heparin (Sigma, St. Louis, MO), Crm197 (Quadratech Ltd., Epsom Surrey, United Kingdom), batimastat (BB94, British Biotech, Oxford, United Kingdom), TNF-α protease inhibitor (TAPI; Calbiochem), and AG1478 (Alexis Biochemicals) were added to serum-starved cells 20 min before the respective growth factor. Arachidonylethanolamide [also called anandamide (AEA)] and THC were obtained from Sigma, and WIN 55,212-2 mesylate and HU-210 from TOCRIS (Bristol, United Kingdom).

Protein Analysis.

Cells were lysed and proteins immunoprecipitated as described previously. After SDS-PAGE, proteins were transferred to nitrocellulose membrane. Western blots were performed according to standard methods.

The antibodies against human EGFR (108.1), SHC, and HER2/neu have been characterized before. Phosphotyrosine was detected with the 4G10 monoclonal antibody (UBI, Lake Placid, NY). Polyclonal anti-phospho-p44/p42 (Thr202/Tyr204) MAPK antibody and anti-phospho-Akt (Ser473) antibody were purchased from New England Biolabs (Beverly, MA). Polyclonal anti-Akt1/2 and anti-ERK2 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-TACE antibody was from Chemicon (Harrow, United Kingdom).

Apoptosis Assay.

NCI-H292 lung cancer cells were seeded and grown for 20 h. On serum starvation for 24 h, cells were treated with THC as indicated in Fig. 3C  for 6 h. Cells were collected in assay buffer (1% sodium citrate, 0.1% Triton X-100) containing propidium iodine and were incubated at 4°C for 3 h. Nuclear DNA staining was analyzed on a Becton Dickinson FACScalibur flow cytometer.

For statistical analysis, Student’s t test was used to compare data between two groups. Values are expressed as mean ± SD of at least triplicate samples. P < 0.05 was considered statistically significant.

RNA Interference and Reverse Transcription-PCR Analysis.

Transfection of 21-nucleotide siRNA duplexes (Dharmacon Research, Lafayette, CO) for targeting endogenous genes was carried out using OligofectAMINE (Invitrogen) and 4.2 μg small interfering RNA (siRNA) duplex per 6-well plate as described previously  . Transfected SCC-9 cells and NCI-H292 cells were serum starved and assayed 4 days and 3 days after transfection, respectively.

Highest efficiencies in silencing target genes were obtained by using mixtures of siRNA duplexes targeting different regions of the gene of interest. The sequences of siRNA used were described previously.

Specific silencing of targeted genes was confirmed by Western blot (TACE) and reverse transcription-PCR analysis. RNA, isolated using RNeasy Mini kit (Qiagen, Hilden, Germany), was reverse transcribed using AMV Reverse Transcriptase (Roche, Mannheim, Germany). PuReTaq Ready-To-Go PCR Beads (Amersham Biosciences, Piscataway, NJ) were used for PCR amplification.

Primers (Sigma Ark, Steinheim, Germany) were described previously . PCR products were subjected to electrophoresis on a 2.5% agarose gel, and DNA was visualized by ethidium bromide staining.

[3H]Thymidine Incorporation Assay.

For the [3H]thymidine incorporation assay , U373-MG cells were seeded into 12-well plates at 1,5 × 104 cells/well. On serum deprivation for 48 h, cells were subjected to preincubation and stimulation as indicated in Fig. 3A  . After 18 h, cells were pulse labeled with [3H]thymidine (1 mCi/ml) for 4 h, and thymidine incorporation was measured by trichloroacetic acid precipitation and subsequent liquid scintillation counting.

MTT Assay.

In a 96-well flat-bottomed plate (Nunc, Naperville, IL), ∼2000 cells/100 μl of cell suspension were seeded. On serum starvation for 24 h, cells were incubated with inhibitors and growth factors as indicated for another 24 h. MTT, a tetrazolium dye [3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide; thiazolyl blue, SIGMA, St. Louis, MO] was added to each well to a final concentration of 1 mg/ml MTT.

Plates were incubated in the presence of MTT for 4 h. Mitochondrial dehydrogenase activity reduces the yellow MTT dye to a purple formazan, which is solubilized (DMSO, acidic acid, SDS), and absorbance was read at 570 nm on a microplate reader.

Results

Cannabinoid-Induced EGFR Signal Transactivation in Human Carcinoma Cells Depends on Metalloprotease Activity.

To address the question whether cannabinoids lead to transactivation of the EGFR in human cancer cell lines, we treated NCI-H292 (lung cancer), SCC-9 (squamous cell carcinoma), 5637 (bladder carcinoma), U373-MG (glioblastoma), 1321N1 (astrocytoma), and A498 (kidney cancer) cells with the synthetic cannabinoids Win55,212-2 and HU210, the endogenous cannabinoid AEA, and the naturally occurring THC. Resulting EGFR tyrosine phosphorylation levels were monitored by immunoblot analysis. As shown in Fig. 1, A–E  , cannabinoids rapidly induced EGFR activation within 3 min.

Fig. 1.

EGFR signal transactivation requires EGFR tyrosine kinase activity and a metalloprotease activity. A, serum-starved NCI-H292 cells, SCC9 cells, and 5637 cells were preincubated with EGFR-specific tyrphostin AG1478 (250 nm, 20 min) or vehicle (DMSO) and were treated with Win55,212-2 (Win; 10 μm), Δ9-tetrahydrocannabinol (THC; 1 μm), HU210 (50 nm), anandamide [AEA (AN); 10 μm], and epidermal growth factor (EGF) (5 ng/ml) for 3 min. After lysis, EGFR was immunoprecipitated (IP) using anti-EGFR antibody.

Tyrosine-phosphorylated EGFR was detected by (IB) with anti-phosphotyrosine (PY) antibody, followed by reprobing of the same filter with anti-EGFR antibody. B, serum-starved NCI-H292 cells, SCC9 cells, and 5637 cells were preincubated with metalloprotease inhibitor batimastat (BB94; 5 μm, 20 min) or vehicle (DMSO), stimulated for 3 min, and analyzed as described in A. C, D, and E, Serum-starved U373-MG, 1321N1, and A498 cells were preincubated with inhibitors and were stimulated for 3 min as indicated, and were analyzed as described in A. F, serum-starved NCI-H292 cells were preincubated with BB94 (5 μm, 20 min) and were stimulated with arachidonyl-2′-chloroethylamide (ACEA) (200 nm) or BML-190 (5 μm) for 3 min, and were analyzed as described in A. G, serum-starved NCI-H292 cells were preincubated with BB94 (5 μm, 20 min), tyrphostin AG1478 (250 nm, 20 min), or vehicle (DMSO) and stimulated with anandamide (An; 10 μm) or THC (1 μm) for 3 min.

After lysis, HER2 was immunoprecipitated and assayed for HER2 tyrosine phosphorylation content.

Preincubation of the cells with the metalloprotease inhibitor batimastat (BB94) or the EGFR kinase-specific inhibitor AG1478 prevented EGFR tyrosine phosphorylation in response to cannabinoid stimulation (Fig. 1, A–E . Stimulation of NCI-H292 cells with receptor subtype-specific agonists arachidonyl-2′-chloroethylamide (ACEA) and BML-190 for CB1 and CB2 receptor, respectively, demonstrated that both cannabinoid receptors are capable of transactivating the EGFR (Fig. 1F).

Expression of both the CB1 and the CB2 receptor was detected by cDNA microarray and Northern blot analysis in all six cancer cell lines (data not shown). Interestingly, the EGFR relative HER2/neu, which serves as a prognostic marker in many different cancer types, was likewise activated in response to cannabinoid stimulation (Fig. 1G . Both AEA- and THC-induced tyrosine phosphorylation of HER2/neu in NCI-H292 cells depended on metalloprotease and EGFR activity.

Therefore, phosphorylation of Her2/neu appears to result from EGFR transphosphorylation. Taken together, these experiments demonstrate that cannabinoids rapidly induce EGFR and Her2/neu signal transactivation in a metalloprotease-dependent manner in different human cancer cell lines.

Cannabinoid-Induced Activation of ERK and the Akt/PKB Survival Pathway Depends on EGFR Function.

To assess whether the EGFR links cannabinoids to ERK1/2 activation, we first analyzed the tyrosine phosphorylation content of the adaptor protein SHC. Fig. 2A  demonstrates that treating cells with Win55,212-2, THC, HU210, and AEA resulted in EGFR-dependent tyrosine phosphorylation of SHC.

We further analyzed ERK1/2 activation by immunoblot using activation state-specific MAPK antibodies. As shown in Fig. 2, B–D  , treating NCI-H292, SCC9, and U373-MG cells with cannabinoids potently activated ERK1/2 to a similar extent as EGF (5 ng/ml) stimulation. Preincubation with AG1478 or BB94 completely abrogated cannabinoid-induced MAPK activation, indicating that, in these human carcinoma cells, cannabinoid-induced ERK1/2 activation completely depends on EGFR signal transactivation (Fig. 2, B–D .

Fig. 2.

Cannabinoid-induced SHC tyrosine phosphorylation and activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt/PKB pathways is EGFR- and metalloprotease-dependent. A, serum-starved SCC9 cells were treated as described in Fig. 1 A, precipitated SHC was immunoblotted with αPY antibody followed by reprobing of the same filters with anti-SHC antibody. B and C, serum-starved SCC9 cells and NCI-H292 cells were preincubated with AG1478 (250 nm, 20 min) or BB94 (5 μm, 20 min), respectively, and were stimulated for 7 min as indicated. Phosphorylated ERK1/2 was detected by immunoblotting with phospho-specific ERK1/2 (P-ERK) antibody.

The same filters were reprobed with anti-ERK antibody. D, serum-starved U373-MG cells were preincubated with inhibitors and stimulated for 7 min as indicated and were analyzed as described in B. E and F, serum-starved SCC9 cells and NCI-H292 cells were preincubated with AG1478 (250 nm, 20 min) or BB94 (5 μm, 20 min), respectively, and stimulated for 7 min as indicated. Activated Akt/PKB was detected by immunoblotting with phospho-specific Akt/PKB (P-Akt) antibody.

The same filters were reprobed with anti-Akt/PKB antibody. Win, Win55,212-2; THC, Δ9-tetrahydrocannabinol; AEA, anandamide; EGF, epidermal growth factor; IB, immunoblotting; PY, anti-phosphotyrosine; P-ERK, phospho-specific ERK1/2; kDa, Mr in thousands; P-Akt, phospho-specific Akt/PKB.

In addition to ERK stimulation, a variety of GPCR agonists were shown to activate the survival mediator Akt/PKB. Because cannabinoids were known to activate Akt/PKB in astrocytoma cells, we addressed the question as to whether cannabinoids activate Akt/PKB in human cancer cells depending on EGFR function. Indeed, activation of Akt/PKB by all four cannabinoids was completely blocked by the selective EGFR inhibitor AG1478 and by the metalloprotease inhibitor BB94 (Fig. 2, E and F .

Taken together, tyrosine phosphorylation of SHC and activation of the Akt/PKB and the ERK/MAPK pathway after cannabinoid treatment of different human cancer cell lines critically depend on metalloprotease-mediated EGFR signal transactivation.

EGFR Mediates Cannabinoid-Induced Proliferation.

Because ERK1/2 are generally known to mediate proliferation in a variety of cells, we addressed the question as to whether cannabinoids induce proliferation of cancer cells.

To determine the proliferation rate in response to THC stimulation, we measured DNA synthesis and the turnover of MTT. As shown in Fig. 3A, THC at concentrations as low as 100 nm was capable of increasing the DNA synthesis of U373-MG cells. To investigate the involvement of the EGFR signal transactivation pathway for cannabinoid-induced DNA synthesis, we preincubated the cells with AG1478 and the metalloprotease inhibitor BB94.

The increased DNA synthesis induced by THC was blocked in the presence of either AG1478 or BB94 (Fig. 3A). Furthermore, in the lung cancer cell line NCI-H292, THC (at 300 nm) was as potent as EGF (at 5 ng/ml) in increasing proliferation (Fig. 3B). THC-induced cell proliferation of NCI-H292 cells was completely blocked by preincubation with AG1478 and the metalloprotease inhibitor TAPI. In contrast to these observations, several earlier studies reported the induction of apoptosis on treatment of cells with higher concentrations of THC.

Therefore, we tested whether, and in particular at what concentrations, THC would be able to trigger cell death of NCI-H292 cells. As shown in Fig. 3C, micromolar concentrations of THC were capable of inducing apoptosis, whereas submicromolar concentrations did not affect cell survival.

Fig. 3.

Δ9-Tetrahydrocannabinol (THC)-induced proliferation of U373-MG and NCI-H292 cells depends on EGFR transactivation. A, U373-MG cells were treated with inhibitors as indicated and incubated in the presence or absence of ligands (THC, 100 nm/300 nm; EGF, 2 ng/ml) for 18 h. Cells were then pulse labeled with [3H]thymidine, and thymidine incorporation was measured by liquid scintillation counting.

Quantitative analysis is from four independent experiments. ∗, P < 0.01 for the difference between DMSO versus THC and EGF; ∗∗, P < 0.01 for the difference between agonists versus inhibitors + agonists. B, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Serum-starved NCI-H292 cells were treated with AG1478 (250 nm) and the metalloprotease inhibitor TAPI (5 μm) and were incubated for 24 h in the presence or absence of ligands (THC 300 nm, EGF 5 ng/ml).

Bars, ± SD of absorbance at 570 nm of three independent experiments. ∗, P < 0.01 for the difference between DMSO versus THC and EGF; ∗∗, P < 0.01 for the difference between agonists versus inhibitors + agonists. C, serum-starved NCI-H292 cells were treated with different concentrations of THC as indicated for 6 h. After collection of the cells in assay buffer, nuclei were stained with propidium iodide and were analyzed by flow cytometric analysis. Quantification of experiments was performed in quadruplicate; bars, ±SD. ∗, P < 0.001 for the difference between DMSO versus THC; FL2-H, fluorescence channel 2-height.

These results demonstrate that pharmacologically active concentrations of THC are capable of promoting the cell proliferation of human carcinoma cells and identify EGFR signal transactivation as the underlying molecular mechanism.

TACE/ADAM17 Mediates Cannabinoid-Induced EGFR Activation.

Our observations clearly showed that cannabinoid receptors transactivate the EGFR in a wide variety of human cancer cell lines involving metalloprotease activity (Fig. 1, A–E).

We have previously shown the critical involvement of the metalloprotease-disintegrin TACE in proAmphiregulin shedding after GPCR stimulation. Therefore, we raised the question as to whether TACE is required for cannabinoid-induced EGFR signal transactivation.

We blocked endogenous TACE expression using a siRNA approach. TACE mRNA and protein was effectively and specifically reduced by transfecting siRNAs into NCI-H292 cells (Fig. 4, A and B).

Inhibition of TACE expression but not of the related ADAM12 completely suppressed cannabinoid-induced EGFR tyrosine phosphorylation in NCI-H292 and SCC9 cells (Fig. 4C). Likewise, the cannabinoid-induced phosphorylation and activation of ERK1/2 and Akt/PKB was abolished in the absence of TACE but was unaffected by the down-regulation of ADAM12 (Fig. 4D). As expected, suppression of neither protease had an affect on signaling events induced by direct EGF stimulation of the EGFR.

Fig. 4.

Tumor necrosis factor α-converting enzyme (TACE) mediates cannabinoid-induced EGFR transactivation and extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt/PKB activation. A and B, NCI-H292 cells were transfected with TACE or ADAM12 siRNA. Gene expression was analyzed by reverse transcription-PCR or immunoblotting (IB) with TACE antibody.

C, NCI-H292 cells and SCC9 cells were transfected with siRNAs raised against TACE and ADAM12, were serum starved, and were stimulated for 3 min with Δ9-tetrahydrocannabinol (THC; 1 μm), anandamide (An; 10 μm), and EGF (5 ng/ml), and were assayed for EGFR tyrosine phosphorylation content. D, NCI-H292 cells and SCC9 cells were transfected with siRNAs and stimulated for 7 min with agonists as indicated.

Phosphorylated ERK1/2 and activated Akt/PKB were detected by immunoblotting with phospho-specific ERK1/2 (P-ERK) and Akt/PKB (P-Akt) antibodies, respectively. The same filters were reprobed with anti-ERK1/2 antibody and anti-Akt/PKB, respectively. P-ERK, phospho-specific ERK1/2; P-Akt, phospho-specific Akt/PKB. E, NCI-H292 cells were transfected with siRNAs raised against proAR, proHB-EGF, and proTGFα.

Gene expression was analyzed by reverse transcription-PCR (RT-PCR). F, SCC9 cells were transfected with siRNAs raised against proAR, proHB-EGF, and proTGFα; stimulated with THC (1 μm) and AEA (10 μm) for 3 min; and assayed for EGFR tyrosine phosphorylation content. G, serum-starved SCC9 cells were preincubated with heparin (100 ng/ml, 15 min), stimulated with THC (1 μm) and AEA (10 μm), and assayed for EGFR tyrosine phosphorylation. H, NCI-H292 cells were transfected with siRNAs as indicated, stimulated with THC (1 μm) and AEA (10 μm), and assayed for EGFR tyrosine phosphorylation. I, serum-starved NCI-H292 cells were preincubated with CRM197 (10 μg/ml, 20 min), stimulated with THC (1 μm) and AEA (10 μm) for 3 min, and assayed for EGFR tyrosine phosphorylation. IB, immunoblotting; PY, anti-phosphotyrosine; IP, immunoprecipitated.

Ectodomain Shedding of proHB-EGF and proAR Mediates Cannabinoid-Induced EGFR Activation.

Among the different EGF-like precursors, proAR, proHB-EGF, and proTGFα are predominantly expressed in NCI-H292 and SCC9 cells as indicated by cDNA microarray analysis (data not shown; Ref. 19 ). To investigate which ligand is involved in the EGFR signal transactivation pathway after cannabinoid stimulation, we transiently transfected siRNAs, and efficient and specific silencing of the endogenous expression of proAR, proHB-EGF, and proTGFα was monitored by reverse transcription-PCR (Fig. 4E ; Ref. 19 ). AEA- and THC-induced tyrosine phosphorylation of the EGFR in SCC9 cells required proAR as well as proHB-EGF expression (Fig. 4F) . Suppression of either ligand resulted in partial reduction of cannabinoid-induced EGFR phosphorylation, whereas proTGFα inhibition did not affect EGFR phosphorylation at all. Furthermore, preincubation with heparin, which abrogates both proAR and proHB-EGF function, also interfered with cannabinoid receptor-EGFR cross-talk (Fig. 4G) .

In contrast, in NCI-H292 cells, cannabinoid-induced transactivation of the EGFR did solely depend on proHB-EGF (Fig. 4H) .

The ability of the diphtheria toxin mutant Crm197, a specific inhibitor of proHB-EGF function, to block EGFR phosphorylation in response to THC stimulation substantiated this observation (Fig. 4I) .

Together, these results show that cannabinoid-induced EGFR signal transactivation is mediated by specific proteolytic processing of the two heparin-binding EGFR ligands, proAR and proHB-EGF, by one and the same zinc-dependent metalloprotease TACE.

Discussion

Cannabinoids were shown to induce apoptosis in cells of the neuronal system including neurons, astrocytes, human grade IV astrocytoma, glioma C6, astrocytoma U373-MG, neuroblastoma N18 TG2, and pheochromocytoma PC12 cells and to inhibit proliferation of MCF-7, EFM-19, T47D, and DU145 cells. On the basis of these findings and their analgesic properties, cannabinoids were suggested as useful drugs to support cancer therapy.

Here we show that various cannabinoids potently induce mitogenic kinase signaling in different cancer cell lines. Moreover, we demonstrate, in contrast to other studies that used cannabinoids such as THC at micromolar concentrations, that nanomolar concentrations of THC induce proliferation of cancer cells (Fig. 3, A and B) .

Importantly, the concentration of THC that was used here is more likely to reflect the therapeutically relevant situation detected in serum after drug treatment.

The binding of cannabinoids to their cognate receptors has been shown to enhance the activity of the MAPKs ERK1/2. The activation of the MAPK pathway in glioma cells on cannabinoid treatment was suggested to involve the activation of Raf1 by increased ceramide levels.

However, our results identified signal transactivation of the EGFR as the key mechanism linking cannabinoid receptors to MAPK signaling cascades in a wide variety of human cancer cell lines. Activation of ERK1/2 by four different cannabinoids coincides with the phosphorylation of the EGFR and was blocked by a specific inhibitor of the EGFR, AG1478 (Fig. 2, B–D) .

Although the ability of AG1478 to block cannabinoid-induced MAPK activation was noted before in U373-MG cells by Galve-Roperh et al , they excluded the existence of cannabinoid receptor-mediated EGFR transactivation because of the inability to detect tyrosine-phosphorylated EGFR, which was probably caused by a lack of sensitivity of detection.

Interestingly, in addition to ERK1/2 activation, Akt/PKB phosphorylation was detected after cannabinoid treatment in an EGFR-dependent manner (Fig. 2E) .

Such a parallel contiguous activation of ERK1/2 and of Akt/PKB was observed before, e.g., in glioblastoma cells treated with cannabinoid receptor agonists, and was suggested to protect astrocytes from ceramide-induced apoptosis in a dose- and time-dependent manner.

Our experimental finding that cannabinoid-induced EGFR cross-talk is established in a variety of human cancer cell lines (Fig. 1, A–E) implicates the EGFR as a central integrator of cannabinoid signaling. The cross-communication between GPCRs and the EGFR involves the proteolytic processing of different membrane-spanning proEGF-like growth factor ligands like proAR, proHB-EGF, and proTGFα by zinc-dependent metalloproteases like ADAM10, ADAM12, and TACE, depending on the cellular context .

In human cancer cell lines, we demonstrate that TACE mediates transactivation of the EGFR after cannabinoid stimulation via proteolytic processing of proHB-EGF and/or proAmphiregulin (Fig. 4, F–I) . Abrogation of either TACE or the respective proEGF-like growth-factor function completely blocked cannabinoid-induced EGFR tyrosine phosphorylation and subsequent activation of the mitogenic ERK pathway and the prosurvival Akt/PKB pathway (Fig. 4) .

We previously described the involvement of TACE in EGFR signal transactivation after lysophosphatidic acid (LPA) and carbachol stimulation. Moreover, TACE was found to mediate EGFR activation by cigarette smoke via proAR shedding in NCI-H292 cells .

However, this is the first report demonstrating a TACE- and HB-EGF-dependent EGFR signal transactivation after GPCR stimulation. Interestingly, knockout experiments by Jackson et al. show that newborn mice lacking TACE, HB-EGF, and the EGFR have similar defective valvulogenesis and suggest EGFR activation by TACE-processed proHB-EGF.

Moreover, our data substantiate the concept that, depending on the cell type and the stimulated GPCR, different ADAM proteases and proEGF-like growth factor ligands are capable of activating the EGFR.

Cannabis-based drugs are in phase three clinical trials for treating pain associated with cancer.

Furthermore, THC is currently used to treat nausea in cancer patients undergoing extensive chemotherapy. In contrast, Grand and Gandhi  recently presented a case study of acute pancreatitis induced by cannabis smoking, indicating that cannabinoids may be a risk factor for pancreatic cancer.

Smoking of THC is the most effective route of delivery, as THC is rapidly absorbed after inhalation, and the effects become fully apparent within minutes. Pharmacological activity of smoked THC depends on the depth and length of inhalation.

Maximum serum concentrations up to 267 ng/ml (850 nm) are measured after smoking THC, whereas maximum serum concentrations of oral or rectal administered THC or its derivatives as a drug are lower (35–350 nm; Refs..

Here we observed a proliferative response of glioblastoma and lung cancer cells at concentrations of 100–300 nm THC, whereas THC at micromolar concentrations induced cell death in agreement with previous observations with neuronal cell types and immune cells (Fig. 3, A–C ; Refs. 5 , 40, 41, 42 ).

These findings indicate that the biological responses to cannabinoids critically depend on drug concentration and cellular context. Taken together, these results have to be taken into account when considering therapeutic applications of cannabinoids.

The risk in the medical use of THC or cannabis for the treatment of patients with established tumors is the further acceleration of tumor growth due to the proliferative potential of cannabinoids.

Acknowledgments

We thank U. Eichelsbacher, R. Gautsch, and R. Hornberger for their help with cell culture; T. Knyazeva for cDNA; P. Knyazeva for help with cDNA arrays and Northern blot analysis; and N. Prenzel and M. Buschbeck for critically reading the manuscript.

Footnotes

  • Grant support: O. Fischer has been supported by a Boehringer Ingelheim Fonds Ph.D. scholarship.

  • 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: Axel Ullrich, Department of Molecular Biology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18A, D-82152 Martinsried, Germany. E-mail: [email protected]

  • Received November 28, 2003.
  • Revision received January 27, 2004.
  • Accepted February 4, 2004.

References

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Anticancer activity of cannabinoids

Journal of the National Cancer Institute,

Vol. 55, No. 3, September 1975, pp.597-602

By A.E. Munson, L.S. Harris, M.A. Friedman, W.L. Dewey, and R.A. Carchman

Department of Pharmacology and the MCV/VCU Cancer Center, Medical College of Virginia, Virginia Commonwealth University. Richmond, Va. 23298

Supported by Public Health Service grant DA00490 from the National Institute on Drug Abuse, Health Services & Mental Health Administration; by a grant from the Alexander and Margaret Stewart Trust Fund; and by an institutional grant from the American Cancer Society.

Summary --- Lewis lung adenocarcinoma growth was retarded by the oral administration of delta-9-tetrahydrocannabinol, delta-8-tetrahydrocannabinol, and cannabinol (CBN), but not cannabidiol (CBD). Animals treated for 10 consecutive days with delta-9-THC, beginning the day after tumor implantation, demonstrated a dose-dependent action of retarded tumor growth.

Mice treated for 20 consecutive days with delta-8-THC and CBN had reduced primary tumor size. CBD showed no inhibitory effect on tumor growth at 14, 21, or 28 days. Delta-9-THC, delta-8-THC, and CBN increased the mean survival time (36% at 100 mg/kg, 25% at 200 mg/kg, and 27% at 50 mg/kg;, respectively), whereas CBD did not. Delta-9-THC administered orally daily until death in doses of 50, 100, or 200 mg/kg did not increase the life-spans of (C57BL/6 X DBA/2) F (BDF) mice hosting the L1210 murine leukemia.

However, delta-9-THC administered daily for 10 days significantly inhibited Friend leukemia virus-induced splenomegaly by 71% at 200 mg/kg as compared to 90.2% for actinomycin D. Experiments with bone marrow and isolated Lewis lung cells incubated in vitro with delta-8-THC and delta-9-THC showed a dose-dependent (10 -4 10 -7) inhibition (80-20%, respectively) of tritiated thymidine and 14C -uridine uptake into these cells. CBD was active only in high concentrations (10 -4). ----J Natl Cancer Inst 55: 597-602, 1975.

Investigations into the physiologic processes affected by the psychoactive constitutuents of marihuana [delta-9-tetrahydrocannabinol (delta-9-THC) and delta-8-tetrahydrocannabinol (delta-8-THC)] purified from Cannabis sativa are extensive.

However, only recently have attempts been made to elucidate the biochemical basis for their cytotoxic or cytostatic activity. Leuchtenberger et al. demonstrated that human lung cultures exposed to marihuana smoke showed alterations in DNA synthesis, with the appearance of anaphase bridges. Zimmerman and McClean, studying macromolecular synthesis in Tetrahymena, indicated that very low concentrations of delta-9-THC inhibited RNA, DNA, and protein synthesis and produced cytolysis. Stenchever et al. showed an increase in the number of damaged or broken chromosomes in chronic users of marihuana.

Delta-9-THC administered iv inhibited bone marrow leukopoieses, and Kolodny et al. reported that marihuana ;may impair testosterone secretion and spermatogenesis.

Furthermore, Nahas et al. showed that in chronic marihuana users there is a decreased lymphocyte reactivity to mitogens as measured by thymidine uptake.

These and other observations suggest that marihuana (delta-9-THC) interferes with vital cell biochemical processes, though no definite mechanism has yet been established.

A preliminary report from this laboratory indicated that the ability of delta-9-THC to interfere with normal cell functions might prove efficacious against neoplasms.

This report represents an effort to test various cannabinoids in several in vivo and in vitro tumor systems to determine the kinds of tumors that are sensitive to these compounds and reveal their possible biochemical sites of action(s).

MATERIALS AND METHODS

The tumor systems used were the Lewis lung adenocarcinoma, leukemia L1210, and B-tropic Friend leukemia.

In vivo systems.---Lewis lung tumor: For the maintenance of the Lewis lung carcinoma, approximately 1-mm3 pieces of tumor were transplanted into C57BL/6 mice with a 15-gauge trocar.

In experiments involving chemotherapy, 14- to 18-day-old tumors were excised, cleared of debris and necrotic tissue, and cut into small fragments (=1mm3).

Tumor tissue was then placed in 0.25% trypsin in Dulbecco's medium with 100 U Penicillin/ml and 100 mcg streptomycin/ml. After 90 minutes' incubation at 22 Degrees C, trypsin action was stopped by the addition of complete medium containing heat-inactivated fetal calf serum (final concentration, 20%). Cells were washed two times in complete medium, enumerated in a Coulter counter (Model ZB1) or on a hemocytometer, and suspended in serum-free medium at a concentration of 5 X 10 6 cells / ml. Next 1 X 10 6 cells were injected into the right hind gluteur muscle, and drugs administered as described in "Results."

Standard regimens provided for 10 consecutive daily doses beginning 24 hours after tumor inoculation. Body weights were recorded before tumor inoculation and weekly for 2 weeks. Tumor size was measured weekly for the duration of the experiment and converted to mg tumor weight, as described by Mayo (10).

Friend leukemia: B-tropic Friend leukemia virus (FLV) was maintained in BALB / c mice, and drug evaluation performed in the same animals. Pools of virus were prepared from the plasma of mice given FLV and stored at -70 Degrees C.

In experiments with FLV, 0.2 ml of a 1/20 dilution of plasma (derived from FLV-infected mice) in medium was inoculated ip into BALB / c mice. Cannabinoids were administered orally daily for 10 consecutive days beginning 24 hours after virus inoculation. Twenty-four hours after the last drug administration, the mice were killed by cervical dislocation, and the spleens removed and weighed.

Mice not given FLV were treated as described above, to evaluate possible drug-induced splenomegaly.

L1210 leukemia: The murine leukemia L1210 was maintained in DBA/2 mice by weekly transfers of 10 (to the fifth power) cells derived from the peritoneal cavity. In these experiments, 10 (fifth power) leukemia cells were inoculated ip into (C57BL/6 X DBA/2) F 1 (BDF 1) mice, and the mice were treated daily for 10 consecutive days beginning 24 hours after tumor cell inoculation. Mean survival time was used as an index of drug activity.

In vitro cell systems. ---Lewis lung tumor: We obtained isolated Lewis lung tumor cells by subjecting 1-mm (third power) sections of tumor to 0.25% trypsin at 22 degrees C and stirring for 60-90 minutes. After trypsinization, the cells were centrifuged (1,000 rpm for 10 min) and washed twice in Dulbecco's medium containing 20% heat-inactivated fetal calf serum.

They were then reconstituted to 10 7 cells/ml of 200 mm glutamine, 5,000 U penicillin, and 5,000 mcg streptomycin. Tumor cells (3-6 ml) were dispensed into 25-ml Erlenmeyer flasks and preincubated with eithe the drug or the drug vehicle for 15 minutes in a Dubnoff metabolic shaker at 37 degrees C in an atmosphere of 5% CO2--95% )2. After preincubation, 10 ucl tritiated thymidine (3H-TDR) (10 uCi, 57 Ci/mmole; New England Nuclear Corp., Boston, Mas.) was added to each flask and incubated for various times, after which 1-ml aliquots were removed and placed in 10 X 75-mm test tubes containing 1 ml 10% trichloroacetic acid (TCA) at 4 degrees C. The TCA-precipated samples were then filtered on 0.45-u Millipore filters and washed twice with 5 ml of 10% TCA at 4 degrees C.

The filters were transferred to liquid scintillation vials and counted in a toluene cocktail containing Liquifluor (New England Nuclear Corp.) (4 liters toluene to 160 ml Liquifluor). Samples were then counted in a liquid scintillator.

Bone marrow: Bone marrow cells were derived from the tibias and fibulas of BDF 1 mice. One ml Dulbecco's medium containing 1 U heparin/ml was forced through each bone by a 1-ml syringe with a 26-gauge needle. The cells were washed three times, nucleated cells were enumerated on a hemocytometer, and cell viability was ascertained by trypan blue exclusion. Cell number was adjusted to 10 (seventh) cells/ml with heparin-free Dulbecco's medium and incubated at 4 degrees C for 15 minutes.

Bone marrow cells were then dispensed (3-5 ml) into a25-ml Erlenmeyer flasks containing the test drug or the drug vehicle. This preincubation period was followed by the addition of 10 ul 3H-TDR and the procedures done as outlined for the isolated Lewis lung cells.

L1210: L1210 cells were derived from DBA/2 mice as described above. They were obtained from DBA/2 mice and inoculated 7 days before the experiment by the peritoneal cavity being flushed with 10 ml Dulbecco's medium containing heparin (5 u/ml).

The cells were washed three times in medium, and the final medium wash did not contain heparin. The cells were resuspended at 10 (seventh) cells/ml and treated as described above. Cells were routinely counted with a hemocytometer for the determination of cell viability with trypan blue; for Lewis lung tumor and L1210 cells, a Coulter apparatus (Mode ZB1) was also used.

All other reagents were of the highest quality grade available. Actinomycin D, 5-fluorouracil (5-FU), and cytosine arabinoside (ara-C) were provided by the Drug Development Branch, National Cancer Institute (NCI).

Cannabinoids. ---The structures of the four compounds are shown in text-figure 1. All occur naturally in marihuana and were chemically synthesized. These drugs were provided by the National Institute on Drug Abuse or the Sheehan Institute for Research, Cambridge, Massachusetts.

In the preparation of the drugs, the cannabinoids were complexed to albumin or solubilized in Emulphor-alcohol. Both preparations produced similar antitumor activity. With albumin, the cannabinoids were prepared in the following manner: A stock solution of 150 mg cannabinoid per ml absolute ethanol was made. Six ml of this solution was placed in a 200-ml flask.

The ethanol was evaporated off under a stream of nitrogen and 2,100 mg lyophilized bovine serum albumin (BSA) added. After the addition of 20 ml distilled water, the substances were stirred with a glass rod in a sonicator until a good suspension was achieved.

Sufficient distilled water was the aldded to make the desired dilution. Concentrations were routinely checked with a gas chromatograph. When Emulphor-alcohol was used as the vehicle, the desired amount of cannabinoid was sonicated in a solution of equal volumes by absolute ethanol and Emulphor (El-620; GAF Corp., New York, N.Y.) and then diluted with 0.15 N NaCL for a final ratio of 1: 1: 4 (ethanol: Emulphor: NaCL).

RESULTS

Effects of Cannabinoids on Murine Tumors

Delta-9-THC, delta-8-THC, and cannabinol (CBN) all inhibited primary Lewis lung tumor growth, whereas cannabidiol (CBD) enhanced tumor growth. Oral administration of 25, 50, or 100 mg delta-9-THC/kg inhibited primary tumor growth by 48, 72, and 75% respectively, when measured 12 days post tumor inoculation (table 1).

On day 19, mice given delta-9-THC had a 34% reduction in primary tumor size. On day 30, primary tumor size was 76% that of controls and only those given 100 mg delta-9-THC/kg had a significant increase in survival time (36%).

Mice treated with a delta-9-THC showed a slight weight loss over the 2-week period (average loss, 0.3 g at 50 mg/kg and 0.1 g at 100 mg/kg). This can be compared to cyclo-ohosphamide, which caused weight loss approaching 20% (table 2).

Delta-8-THC activity was similar to that of delta-9-THC when administered orally daily until death (table 2). However, as with delta-9-THC, primary tumor growth approached control values after 3 weeks. When measured 12 days post tumor inoculation, all doses (50-400 mg/kg) of delta-8-THC inhibited primary tumor growth between 40 and 60%.

Significant inhibition was also seen on day 21, which was comparable to cyclophosphamide-treated mice. Although this was not the optimum regim;en for cyclophosphamide, it was the positive control protocol provided by the NCI.

All mice given delta-8-THC survived significantly longer than controls, except those treated with 100 mg/kg. Mice given 50, 200, and 400 mg/kg delta-8-THC had an increased life-span of 22.6, 24.6, and 27.2%, respectively, as compared to 33% for mice treated with 20 mg cyclophosphamide/kg. Pyran copolymer, an immunopotentiator when administered at 50 mg/kg, also significantly increased the survival time of the animals (39.3%).

CBN, administered by gavage daily until death, demonstrated antitumor activity against the Lewis lung carcinoma when evaluated on day 14 post tumor inoculation (table 3). Primary tumor growth was inhibited by 77%, at doses of 100 mg/kg on day 14 but only by 11% on day 24. At 50 mg/kg on day 14 but only by 11% on day 24. At 50 mg/kg, CBN inhibited primary tumor growth by only 32% when measured on day 14, and no inhibition was observed on day 24; however, these animals did survive 27% longer. CBD, administered at 25 or 200 mg/kg daily until death, showed no tumor-inhibitory properties as measured by primary Lewis lung tumor size or survival time (table 4). In this experiment, CBD-treated mice showed enhanced primary tumor growth.

However, the control tumor growth rate in this experiment was decreased as compared to the previous studies.

Survival time of BDF 1 mice hosting L1210 leukemia was not prolonged by delta-9-THC treatment (table 5). Mice treated with delta-9-THC at doses of 50, 100, and 200 mg/kg administered orally daily until death, survived 8.5, 7.8, and 8.6 days, respectively, as compared to 8.6 days for mice treated with the diluent. However, delta-9-THC inhigited FLV-induced splenomegaly by 71% at 200 mg/kg as compared to 90.2% for the positive control actinomycin D (0.25 mg/kg). Although there was a dose-related inhibition, only the high dose was statistically significant (table 6).

Effect of Cannabinoids on Isolated Cells In Vitro

Isolated cells incubated in vitro represent a simple, reliable, and, hopefully, predictive method for the monitoring of the effects of agents on several biochemical parameters at the same time. The incorporation of 3H-TDR into TCA-precipitable counts in isolated Lewis lung cells is shown in text-figure 2. Similar types of curves were seen for bone marrow and L1210 cells. In all instances, for 15-45 minutes there was a linear increase in 3H-TDR uptake into the TCA-precipitable fraction.

Qualitatively, similar data (not shown) were seen after a pulse with 14C-uridine. Actinomycin D (1 mcg/ml) preferentially inhibited 14C-uridine incorporation after uridine uptake had decreased to less than 30% that of control (data not shown).

This is indirect evidence that we were measuring RNA synthesis. Experiments (data not shown) done with 5-FU (10 -4 M) indicated that, in isolated bone marrow cells, both thymidine uptake with time by delta-9-THC (10 -5 M) on Lewis lung cells is depicted in text-figure 2. In this experiment, delta-9-THC caused a nonlinear uptake of 3H-TDR.

At 30 minutes, uptake of 3H-TDR into the acid-precipitable fraction was about 50% that of control Longer incubations (i.e., 60 min) did not significantly change the uptake pattern for control and de;ta-9-THC treated tumor cells.

The effect of several cannabinoids on the uptake of 3H-TDR into cells incubated in vitro indicated that delta-9-THC, delta-8-THC, and CBN produced a dose-dependent inhibition of radiolabel uptake in the three cell types (table 7).

These results, presented as percent inhibition of radiolabel uptake as compared to control, represented an effectof cannabinoids on one aspect of macromolecular synthesis. CBD was the least active of the cannabinoids, but showed its greatest activity in the L1210 leukemia cells. Other data (not shown) indicate that these compounds similarly effect the uptake of 14C-uridine into the acid-precipitable fraction. Ara-C markedly inhibited 3H-TDR uptake more dramatically than did the cannabinoids (table 7).

Note that delta-9-THC exhibited inhibitory properties in the isolated Lewis lung tumor and L1210 cells at concentrations that did not interfere with thymidine uptake into bone marrow cells. At certain concentrations of CBD (2,5 X 10 -6 and 2.5 X 10 -7M), radiolabel uptake was consistently stimulated in bone marrow cells and in several experiments with the isolated Lewis lung cells.

DISCUSSION

We investigated four cannabinoids for antineoplastic activity against three animal tumor models in vivo and for cytotoxic or cystostatic activity in two tumor cell lines and bone marrow cells in vitro. The cannabinoids (delta-9-THC, delta-8-THC, and CBN) active in vivo against the Lewis lung tumor cells are also active in the in vitro systems.

The differential sensitivity of delta-9-THC against Lewis lung cells versus bone marrow cells is unique in that delta-8-THC and CBN are equally active in these systems. Johnson and Wiersma (5) reported that delta-9-THC administered iv caused a reduction in bone marrow metamyelocytes and an increase in lymphocytes.

It is unclear from the data whether this is a depression of myelopoiesis or if it represents a lymphocyte infiltration into the bone marrow.

The use of isolated bone marrow cells, which represent a nonneoplastic rapidly proliferating tissue, enables the rapid evaluation and assessment of drug sensititity and specificity, and thereby may predict toxicity related to bone marrow suppression. CBD showed noninhibitory activity either against the Lewis lung cells in vivo or Lewis lung and bone marrow cells in vitro at 10 -5M an 10 -6M, respectively. Indeed, the tumor growth rate in mice treated with CBD was significantly increased over controls.

This may, in part, be the consequence of the observation made in vitro (i.e., 10 -7M CBD stimulated thymidine uptake), which may be reflected by an increased rate of tumor growth.

One problem related to the use of cannabinoids is the development of tolerance to many of its behavioral effects (13). It also appears that tolerance functions in the chemotherapy of neoplsms in that the growth of the Lewis lung tumor is initially markedly inhibited but, by 3 weeks, approaches that of vehicle-treated mice (tables 1, 3).

This, in part, may reflect drug regimens, doses used, increased drug metabolism, or conversion to metabolites with antagonistic actions to delta-9-THC. It may also represent some tumor cell modifications rendering the cell insensitive to these drugs. Of further interest was the lack of activity of delta-9-THC against the L1210 in vivo, whereas the invitro L1210 studies indicated that delta-9-THC could effectively inhibit thymidine uptake.

The apparent reason for this discrepancy may be related to the high growth fraction and the short doubling time of this tumor. The in vitro data do not indicate that the cannabinids possess that degree of activity; e.g., ara-C, which "cures"L1210 mice, is several orders of magnitude more potent on a molar basis than delta-9-THC in vitro.

Inhibition of tumor growth and increased animal survival after treatment with delta-9-THC may, in part, be due to the ability of the drug to inhibit nucleic acid synthesis. Preliminary data with Lewis lung cells grown in tissue culture indicate that 10 -5M delta-9-THC inhibits by 50% the uptake of 3H-TDR into acid-precipitable counts over a 4-hour incubation period. Simultaneous determination of acid-soluble fractions did not show any inhibitory effects on radiolabeled uptake.

Therefore, delta-9-THC may be acting at site(s) distal to the uptake of precursor. We are currently evaluating the acid-soluble pool to see if phosphorylation of precursor is involved in the action of delta-9-THC.

These results lend further support to increasing evidence that, in addition to the well-known behavioral effects of delta-9-THC, this agent modifies other cell responses that may have greater biologic significance in that they have antineoplastic activity. The high doses of delta-9-THC (i.e., 200 mg/kg) are not tolerable in humans.

On a body-surface basis, this would be about 17 mg/m(2) for mice. Extrapolation to a 60-kg man would require 1,020 mg for comparable dosage.

The highest doses administered to man have been 250-300 mg. Whether only cannabinoids active in the central nervous system (CNS) exhibit this antineoplastic property is not the question, since CBN, which lacks marihuana-like psychoactivity, is quite active in our systems.

With structure-activity investigations, more active agents may be designed and synthesized which are devoid of or have reduced CNS activity. That these compounds readily cross the blood-brain barrier and do not possess many of the toxic manifestations of presently used cytotoxic agents, makes them an appealing group of drugs to study.

REFERENCES

1. Singer AJ: Marihuana: Chemistry, pharmacology and patterns of social use. Ann NY Acad Sci 191: 3-261, 1971

2. Leuchtenberger C, Leuchtenberger R, Schneider A: Effects of marijuana and tobacco smoke on human lung physiology. Nature 241: 137-139,1973

3. Zimmerman AM, McClean DK: Action of narcotic and hallucinogenic agents on the cell cycle. In Drugs and the Cell Cycle (Zimmerman AM, Padilla GM. Cameron IL, eds.). New York, Academic Press, 1973, pp.67-94

4. Stenchever MA, Kunysz TJ, Allen MA: Chromosome breakage in users of marihuana. Am J Obstet Gynecol 118: 105-113, 1974

5. JohnsonRT, Wiersema V: Repression of bone marrow leukopoieses by delta-9-THC . Res Commun Chem Pathol Pharmacol 7: 613-616, 1974

6. Kolodny RC, Masters WH, Kolodner RM, et al: Depression of plasma testosterone levels after chronic intensive marijuana use. N Engl J Med 290: 872-874, 1974

7. Nahas GG, Suchu-Foca N, Armand JP, et al: Inhibition of cellular immunity in marihuana smokers. Science 183: 419-420, 1974

8. Levy JA, Munson AE, Haris LS, et al: Effect of delta-8 and delta-9-tetrahydrocannabinol on the immunre response in mice. The Pharmacologist 16: 259, 1974

9. Harris LS, Munson AE, Friedman MA, et al: Retardation of tumor growth by delta-9-tetradydrocannabinol. The Pharmacologist 16: 259, 1974

10. Mayo JG: Biologic characterization of the subcutaneously implanted Lewis lung tumor. Cancer Chemother Rep 3: 325-330, 1972

11. Geran RI, Greenberg NH, MacDonald MM, et al: Protocols for screening chemical agents and natural products against animal tumors and other biological systems. Cancer Chemother Rep 3: 13, 1972

12. Munson AE, Regelson W, Lawrence W: The biphasic response of the reticuloendothelial system (RES) produced by pyran copolymer and its relationship to immunologic response. J Reticuloendothel Soc 7: 375-385, 1970

13. McMillan DE, Dewey WL, Turk RF, et al: Blood levels of 3H-delta-9-tetrahydrocannabinol and its metabolites in tolerant and non-tolerant pigeons. Biochem Pharmacol 22: 383-397, 1973

14. Jones RT: The 30-day trip-clinical studies of cannabis tolerance and dependence. Proceedings of the International Conference on the Pharmacology of Cannabis. Savanah, Georgia, 1974, p.29

15. Hollister LE: Structure-activity relationships in man of cannabis constituents and homologs and metabolites of delta-9-tetrahydrocannabinol. Pharmacology 11: 3-11, 1974

 

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Antineoplastic activity of cannabinoids

A.E. Munson, L.S. Harris, M.A. Friedman, W.L. Dewey, and R.A. Carchman

Journal of the National Cancer Institute, Vol. 55, No. 3, September 1975

Supported by Public Health Service grant DA00490 from the National Institute on Drug Abuse, Health Services & Mental Health Administration; by a grant from the Alexander and Margaret Stewart Trust Fund; and by an institutional grant from the American Cancer Society.

Department of Pharmacology and the MCV/VCU Cancer Center, Medical College of Virginia, Virginia Commonwealth University. Richmond, Va. 23298

Abstract

Lewis lung adenocarcinoma growth was retarded by the oral administration of delta-9-tetrahydrocannabinol, delta-8-tetrahydrocannabinol, and cannabinol (CBN), but not cannabidiol (CBD). Animals treated for 10 consecutive days with delta-9-THC, beginning the day after tumor implantation, demonstrated a dose-dependent action of retarded tumor growth.

Mice treated for 20 consecutive days with delta-8-THC and CBN had reduced primary tumor size. CBD showed no inhibitory effect on tumor growth at 14, 21, or 28 days.

Delta-9-THC, delta-8-THC, and CBN increased the mean survival time (36% at 100 mg/kg, 25% at 200 mg/kg, and 27% at 50 mg/kg;, respectively), whereas CBD did not. Delta-9-THC administered orally daily until death in doses of 50, 100, or 200 mg/kg did not increase the life-spans of (C57BL/6 X DBA/2) F (BDF) mice hosting the L1210 murine leukemia.

However, delta-9-THC administered daily for 10 days significantly inhibited Friend leukemia virus-induced splenomegaly by 71% at 200 mg/kg as compared to 90.2% for actinomycin D. Experiments with bone marrow and isolated Lewis lung cells incubated in vitro with delta-8-THC and delta-9-THC showed a dose-dependent (10 -4 10 -7) inhibition (80-20%, respectively) of tritiated thymidine and 14C -uridine uptake into these cells. CBD was active only in high concentrations (10 -4)

Introduction

Investigations into the physiologic processes affected by the psychoactive constitutuents of marihuana [delta-9-tetrahydrocannabinol (delta-9-THC) and delta-8-tetrahydrocannabinol (delta-8-THC)] purified from Cannabis sativa are extensive.

However, only recently have attempts been made to elucidate the biochemical basis for their cytotoxic or cytostatic activity. Leuchtenberger et al. demonstrated that human lung cultures exposed to marihuana smoke showed alterations in DNA synthesis, with the appearance of anaphase bridges. Zimmerman and McClean, studying macromolecular synthesis in Tetrahymena, indicated that very low concentrations of delta-9-THC inhibited RNA, DNA, and protein synthesis and produced cytolysis. Stenchever et al. (4) showed an increase in the number of damaged or broken chromosomes in chronic users of marihuana. Delta-9-THC administered iv inhibited bone marrow leukopoieses, and Kolodny et al. reported that marihuana; may impair testosterone secretion and spermatogenesis.

Furthermore, Nahas et al. showed that in chronic marihuana users there is a decreased lymphocyte reactivity to mitogens as measured by thymidine uptake.

These and other observations suggest that marihuana (delta-9-THC) interferes with vital cell biochemical processes, though no definite mechanism has yet been established.

A preliminary report from this laboratory indicated that the ability of delta-9-THC to interfere with normal cell functions might prove efficacious against neoplasms.

This report represents an effort to test various cannabinoids in several in vivo and in vitro tumor systems to determine the kinds of tumors that are sensitive to these compounds and reveal their possible biochemical sites of action(s).

Materials and methods

The tumor systems used were the Lewis lung adenocarcinoma, leukemia L1210, and B-tropic Friend leukemia.

In vivo systems.

Lewis lung tumor: For the maintenance of the Lewis lung carcinoma, approximately 1-mm3 pieces of tumor were transplanted into C57BL/6 mice with a 15-gauge trocar. In experiments involving chemotherapy, 14- to 18-day-old tumors were excised, cleared of debris and necrotic tissue, and cut into small fragments (=1mm3).

Tumor tissue was then placed in 0.25% trypsin in Dulbecco's medium with 100 U Penicillin/ml and 100 mcg streptomycin/ml. After 90 minutes' incubation at 22 Degrees C, trypsin action was stopped by the addition of complete medium containing heat-inactivated fetal calf serum (final concentration, 20%). Cells were washed two times in complete medium, enumerated in a Coulter counter (Model ZB1) or on a hemocytometer, and suspended in serum-free medium at a concentration of 5 X 10 6 cells / ml.

Next 1 X 10 6 cells were injected into the right hind gluteur muscle, and drugs administered as described in "Results." Standard regimens provided for 10 consecutive daily doses beginning 24 hours after tumor inoculation. Body weights were recorded before tumor inoculation and weekly for 2 weeks.

Tumor size was measured weekly for the duration of the experiment and converted to mg tumor weight, as described by Mayo (10).

Friend leukemia: B-tropic Friend leukemia virus (FLV) was maintained in BALB / c mice, and drug evaluation performed in the same animals. Pools of virus were prepared from the plasma of mice given FLV and stored at -70 Degrees C. In experiments with FLV, 0.2 ml of a 1/20 dilution of plasma (derived from FLV-infected mice) in medium was inoculated ip into BALB / c mice. Cannabinoids were administered orally daily for 10 consecutive days beginning 24 hours after virus inoculation.

Twenty-four hours after the last drug administration, the mice were killed by cervical dislocation, and the spleens removed and weighed. Mice not given FLV were treated as described above, to evaluate possible drug-induced splenomegaly.

L1210 leukemia: The murine leukemia L1210 was maintained in DBA/2 mice by weekly transfers of 10 (to the fifth power) cells derived from the peritoneal cavity. In these experiments, 10 (fifth power) leukemia cells were inoculated ip into (C57BL/6 X DBA/2) F 1 (BDF 1) mice, and the mice were treated daily for 10 consecutive days beginning 24 hours after tumor cell inoculation. Mean survival time was used as an index of drug activity.

In vitro cell systems

Lewis lung tumor: We obtained isolated Lewis lung tumor cells by subjecting 1-mm (third power) sections of tumor to 0.25% trypsin at 22 degrees C and stirring for 60-90 minutes.

After trypsinization, the cells were centrifuged (1,000 rpm for 10 min) and washed twice in Dulbecco's medium containing 20% heat-inactivated fetal calf serum. They were then reconstituted to 10 7 cells/ml of 200 mm glutamine, 5,000 U penicillin, and 5,000 mcg streptomycin.

Tumor cells (3-6 ml) were dispensed into 25-ml Erlenmeyer flasks and preincubated with eithe the drug or the drug vehicle for 15 minutes in a Dubnoff metabolic shaker at 37 degrees C in an atmosphere of 5% CO2--95% )2. After preincubation, 10 ucl tritiated thymidine (3H-TDR) (10 uCi, 57 Ci/mmole;

New England Nuclear Corp., Boston, Mas.) was added to each flask and incubated for various times, after which 1-ml aliquots were removed and placed in 10 X 75-mm test tubes containing 1 ml 10% trichloroacetic acid (TCA) at 4 degrees C. The TCA-precipated samples were then filtered on 0.45-u Millipore filters and washed twice with 5 ml of 10% TCA at 4 degrees C.

The filters were transferred to liquid scintillation vials and counted in a toluene cocktail containing Liquifluor (New England Nuclear Corp.) (4 liters toluene to 160 ml Liquifluor). Samples were then counted in a liquid scintillator.

Bone marrow: Bone marrow cells were derived from the tibias and fibulas of BDF 1 mice. One ml Dulbecco's medium containing 1 U heparin/ml was forced through each bone by a 1-ml syringe with a 26-gauge needle. The cells were washed three times, nucleated cells were enumerated on a hemocytometer, and cell viability was ascertained by trypan blue exclusion.

Cell number was adjusted to 10 (seventh) cells/ml with heparin-free Dulbecco's medium and incubated at 4 degrees C for 15 minutes. Bone marrow cells were then dispensed (3-5 ml) into a25-ml Erlenmeyer flasks containing the test drug or the drug vehicle. This preincubation period was followed by the addition of 10 ul 3H-TDR and the procedures done as outlined for the isolated Lewis lung cells.

L1210: L1210 cells were derived from DBA/2 mice as described above. They were obtained from DBA/2 mice and inoculated 7 days before the experiment by the peritoneal cavity being flushed with 10 ml Dulbecco's medium containing heparin (5 u/ml).

The cells were washed three times in medium, and the final medium wash did not contain heparin. The cells were resuspended at 10 (seventh) cells/ml and treated as described above.

Cells were routinely counted with a hemocytometer for the determination of cell viability with trypan blue; for Lewis lung tumor and L1210 cells, a Coulter apparatus (Mode ZB1) was also used. All other reagents were of the highest quality grade available. Actinomycin D, 5-fluorouracil (5-FU), and cytosine arabinoside (ara-C) were provided by the Drug Development Branch, National Cancer Institute (NCI).

Cannabinoids: The structures of the four compounds are shown in text-figure 1.

All occur naturally in marihuana and were chemically synthesized. These drugs were provided by the National Institute on Drug Abuse or the Sheehan Institute for Research, Cambridge, Massachusetts.

In the preparation of the drugs, the cannabinoids were complexed to albumin or solubilized in Emulphor-alcohol. Both preparations produced similar antitumor activity. With albumin, the cannabinoids were prepared in the following manner: A stock solution of 150 mg cannabinoid per ml absolute ethanol was made. Six ml of this solution was placed in a 200-ml flask.

The ethanol was evaporated off under a stream of nitrogen and 2,100 mg lyophilized bovine serum albumin (BSA) added. After the addition of 20 ml distilled water, the substances were stirred with a glass rod in a sonicator until a good suspension was achieved. Sufficient distilled water was the aldded to make the desired dilution. Concentrations were routinely checked with a gas chromatograph.

When Emulphor-alcohol was used as the vehicle, the desired amount of cannabinoid was sonicated in a solution of equal volumes by absolute ethanol and Emulphor (El-620; GAF Corp., New York, N.Y.) and then diluted with 0.15 N NaCL for a final ratio of 1: 1: 4 (ethanol: Emulphor: NaCL).

Results

Effects of Cannabinoids on Murine Tumors

Delta-9-THC, delta-8-THC, and cannabinol (CBN) all inhibited primary Lewis lung tumor growth, whereas cannabidiol (CBD) enhanced tumor growth.

Oral administration of 25, 50, or 100 mg delta-9-THC/kg inhibited primary tumor growth by 48, 72, and 75% respectively, when measured 12 days post tumor inoculation (table 1).

On day 19, mice given delta-9-THC had a 34% reduction in primary tumor size. On day 30, primary tumor size was 76% that of controls and only those given 100 mg delta-9-THC/kg had a significant increase in survival time (36%). Mice treated with a delta-9-THC showed a slight weight loss over the 2-week period (average loss, 0.3 g at 50 mg/kg and 0.1 g at 100 mg/kg).

This can be compared to cyclo-ohosphamide, which caused weight loss approaching 20% (table 2).

Delta-8-THC activity was similar to that of delta-9-THC when administered orally daily until death (table 2). However, as with delta-9-THC, primary tumor growth approached control values after 3 weeks. When measured 12 days post tumor inoculation, all doses (50-400 mg/kg) of delta-8-THC inhibited primary tumor growth between 40 and 60%. Significant inhibition was also seen on day 21, which was comparable to cyclophosphamide-treated mice. Although this was not the optimum regime for cyclophosphamide, it was the positive control protocol provided by the NCI.

All mice given delta-8-THC survived significantly longer than controls, except those treated with 100 mg/kg. Mice given 50, 200, and 400 mg/kg delta-8-THC had an increased life-span of 22.6, 24.6, and 27.2%, respectively, as compared to 33% for mice treated with 20 mg cyclophosphamide/kg. Pyran copolymer, an immunopotentiator when administered at 50 mg/kg, also significantly increased the survival time of the animals (39.3%).

CBN, administered by gavage daily until death, demonstrated antitumor activity against the Lewis lung carcinoma when evaluated on day 14 post tumor inoculation (table 3).

Primary tumor growth was inhibited by 77%, at doses of 100 mg/kg on day 14 but only by 11% on day 24. At 50 mg/kg on day 14 but only by 11% on day 24. At 50 mg/kg, CBN inhibited primary tumor growth by only 32% when measured on day 14, and no inhibition was observed on day 24; however, these animals did survive 27% longer.

CBD, administered at 25 or 200 mg/kg daily until death, showed no tumor-inhibitory properties as measured by primary Lewis lung tumor size or survival time (table 4).

In this experiment, CBD-treated mice showed enhanced primary tumor growth. However, the control tumor growth rate in this experiment was decreased as compared to the previous studies.

Survival time of BDF 1 mice hosting L1210 leukemia was not prolonged by delta-9-THC treatment (table 5).

Mice treated with delta-9-THC at doses of 50, 100, and 200 mg/kg administered orally daily until death, survived 8.5, 7.8, and 8.6 days, respectively, as compared to 8.6 days for mice treated with the diluent. However, delta-9-THC inhigited FLV-induced splenomegaly by 71% at 200 mg/kg as compared to 90.2% for the positive control actinomycin D (0.25 mg/kg).

Although there was a dose-related inhibition, only the high dose was statistically significant (table 6).

Effect of Cannabinoids on Isolated Cells In Vitro

Isolated cells incubated in vitro represent a simple, reliable, and, hopefully, predictive method for the monitoring of the effects of agents on several biochemical parameters at the same time.

The incorporation of 3H-TDR into TCA-precipitable counts in isolated Lewis lung cells is shown in text-figure 2. Similar types of curves were seen for bone marrow and L1210 cells. In all instances, for 15-45 minutes there was a linear increase in 3H-TDR uptake into the TCA-precipitable fraction. Qualitatively, similar data (not shown) were seen after a pulse with 14C-uridine.

Actinomycin D (1 mcg/ml) preferentially inhibited 14C-uridine incorporation after uridine uptake had decreased to less than 30% that of control (data not shown). This is indirect evidence that we were measuring RNA synthesis.

Experiments (data not shown) done with 5-FU (10 -4 M) indicated that, in isolated bone marrow cells, both thymidine uptake with time by delta-9-THC (10 -5 M) on Lewis lung cells is depicted in text-figure 2.

In this experiment, delta-9-THC caused a nonlinear uptake of 3H-TDR. At 30 minutes, uptake of 3H-TDR into the acid-precipitable fraction was about 50% that of control Longer incubations (i.e., 60 min) did not significantly change the uptake pattern for control and de;ta-9-THC treated tumor cells.

The effect of several cannabinoids on the uptake of 3H-TDR into cells incubated in vitro indicated that delta-9-THC, delta-8-THC, and CBN produced a dose-dependent inhibition of radiolabel uptake in the three cell types (table 7). These results, presented as percent inhibition of radiolabel uptake as compared to control, represented an effect of cannabinoids on one aspect of macromolecular synthesis.

CBD was the least active of the cannabinoids, but showed its greatest activity in the L1210 leukemia cells. Other data (not shown) indicate that these compounds similarly effect the uptake of 14C-uridine into the acid-precipitable fraction. Ara-C markedly inhibited 3H-TDR uptake more dramatically than did the cannabinoids (table 7). Note that delta-9-THC exhibited inhibitory properties in the isolated Lewis lung tumor and L1210 cells at concentrations that did not interfere with thymidine uptake into bone marrow cells.

At certain concentrations of CBD (2,5 X 10 -6 and 2.5 X 10 -7M), radiolabel uptake was consistently stimulated in bone marrow cells and in several experiments with the isolated Lewis lung cells.

Discussion

We investigated four cannabinoids for antineoplastic activity against three animal tumor models in vivo and for cytotoxic or cystostatic activity in two tumor cell lines and bone marrow cells in vitro.

The cannabinoids (delta-9-THC, delta-8-THC, and CBN) active in vivo against the Lewis lung tumor cells are also active in the in vitro systems. The differential sensitivity of delta-9-THC against Lewis lung cells versus bone marrow cells is unique in that delta-8-THC and CBN are equally active in these systems. Johnson and Wiersma (5) reported that delta-9-THC administered iv caused a reduction in bone marrow metamyelocytes and an increase in lymphocytes.

It is unclear from the data whether this is a depression of myelopoiesis or if it represents a lymphocyte infiltration into the bone marrow. The use of isolated bone marrow cells, which represent a nonneoplastic rapidly proliferating tissue, enables the rapid evaluation and assessment of drug sensititity and specificity, and thereby may predict toxicity related to bone marrow suppression.

CBD showed noninhibitory activity either against the Lewis lung cells in vivo or Lewis lung and bone marrow cells in vitro at 10 -5M an 10 -6M, respectively. Indeed, the tumor growth rate in mice treated with CBD was significantly increased over controls.

This may, in part, be the consequence of the observation made in vitro (i.e., 10 -7M CBD stimulated thymidine uptake), which may be reflected by an increased rate of tumor growth. One problem related to the use of cannabinoids is the development of tolerance to many of its behavioral effects.

It also appears that tolerance functions in the chemotherapy of neoplsms in that the growth of the Lewis lung tumor is initially markedly inhibited but, by 3 weeks, approaches that of vehicle-treated mice (tables 1, 3).

This, in part, may reflect drug regimens, doses used, increased drug metabolism, or conversion to metabolites with antagonistic actions to delta-9-THC. It may also represent some tumor cell modifications rendering the cell insensitive to these drugs.

Of further interest was the lack of activity of delta-9-THC against the L1210 in vivo, whereas the invitro L1210 studies indicated that delta-9-THC could effectively inhibit thymidine uptake.

The apparent reason for this discrepancy may be related to the high growth fraction and the short doubling time of this tumor. The in vitro data does not indicate that the cannabinids possess that degree of activity; e.g., ara-C, which "cures" L1210 mice, is several orders of magnitude more potent on a molar basis than delta-9-THC in vitro.

Inhibition of tumor growth and increased animal survival after treatment with delta-9-THC may, in part, be due to the ability of the drug to inhibit nucleic acid synthesis. Preliminary data with Lewis lung cells grown in tissue culture indicate that 10 -5M delta-9-THC inhibits by 50% the uptake of 3H-TDR into acid-precipitable counts over a 4-hour incubation period. Simultaneous determination of acid-soluble fractions did not show any inhibitory effects on radiolabeled uptake.

Therefore, delta-9-THC may be acting at site(s) distal to the uptake of precursor. We are currently evaluating the acid-soluble pool to see if phosphorylation of precursor is involved in the action of delta-9-THC.

These results lend further support to increasing evidence that, in addition to the well-known behavioral effects of delta-9-THC, this agent modifies other cell responses that may have greater biologic significance in that they have antineoplastic activity.

The high doses of delta-9-THC (i.e., 200 mg/kg) are not tolerable in humans. On a body-surface basis, this would be about 17 mg/m(2) for mice.

Extrapolation to a 60-kg man would require 1,020 mg for comparable dosage. The highest doses administered to man have been 250-300 mg. Whether only cannabinoids active in the central nervous system (CNS) exhibit this antineoplastic property is not the question, since CBN, which lacks marihuana-like psychoactivity, is quite active in our systems.

With structure-activity investigations, more active agents may be designed and synthesized which are devoid of or have reduced CNS activity. That these compounds readily cross the blood-brain barrier and do not possess many of the toxic manifestations of presently used cytotoxic agents, makes them an appealing group of drugs to study.

REFERENCES

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