Cannabinoid Receptor-Mediated Apoptosis

Win-55-212-2Cannabinoid Receptor-Mediated Apoptosis Induced by R(+)-Methanandamide and Win55,212-2 Is Associated with Ceramide Accumulation and p38 Activation in Mantle Cell Lymphoma

Kristin Gustafsson, Birger Christensson, Birgitta Sander and Jenny Flygare

Karolinska Institutet, Department of Laboratory Medicine, Division of Pathology, Karolinska University Hospital Huddinge, Stockholm, Sweden
Address correspondence to:
Birgitta Sander, Department of Laboratory Medicine, Division of Pathology, Karolinska University Hospital Huddinge, F-46, SE-14186 Stockholm, Sweden. E-mail: birgitta.sander@ki.se

Abstract:

We have recently shown that cannabinoids induce growth inhibition and apoptosis in mantle cell lymphoma (MCL), a malignant B-cell lymphoma that expresses high levels of cannabinoid receptor types 1 and 2 (CB1 and CB2). In the current study, the role of each receptor and the signal transduction triggered by receptor ligation were investigated. Induction of apoptosis after treatment with the synthetic agonists R(+)-methanandamide [R(+)-MA] and Win55,212-2 (Win55; (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo-[1,2,3-d,e]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone) was dependent on both cannabinoid receptors, because pretreatment with N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR141716A) and N-((1S)-endo-1,3,3-trimethyl bicyclo heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide) (SR144528), specific antagonists to CB1 and CB2, respectively, abrogated caspase-3 activity. Preincubation with the inhibitors 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580) and 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole (SB202190) showed that phosphorylation of MAPK p38 was implicated in the signal transduction leading to apoptosis. Treatment with R(+)-MA and Win55 was associated with accumulation of ceramide, and pharmacological inhibition of ceramide synthesis de novo prevented both p38 activation and mitochondria depolarization assessed by binding of 3,3′-dihexyloxacarbocyanine iodide (DiOC6). In contrast, the pancaspase inhibitor z-Val-Ala-Asp(Ome)-CH2F (z-VAD-FMK) did not protect the mitochondrial integrity. Taken together, these results suggest that concurrent ligation of CB1 and CB2 with either R(+)-MA or Win55 induces apoptosis via a sequence of events in MCL cells: accumulation of ceramide, phosphorylation of p38, depolarization of the mitochondrial membrane, and caspase activation. Although induction of apoptosis was observed in both MCL cell lines and primary MCL, normal B cells remained unaffected. The present data suggest that targeting CB1/CB2 may have therapeutic potential for the treatment of mantle cell lymphoma.

References:

http://molpharm.aspetjournals.org/content/70/5/1612.abstract

Arachidonyl ethanolamide apoptosis uterine cervix cancer

Arachidonyl ethanolamide induces apoptosis of uterine cervix cancer cells via aberrantly expressed vanilloid receptor-1.

Gynecol Oncol. 2004 Apr;93(1):182-8.

Contassot E, Tenan M, Schnüriger V, Pelte MF, Dietrich PY.
Oncology Division, Laboratory of Tumor Immunology, University Hospital, Geneva, Switzerland.

OBJECTIVES:
Delta(9)-Tetrahydrocannabinol, the active agent of Cannabis sativa, exhibits well-documented antitumor properties, but little is known about the possible effects mediated by endogenous cannabinoids on human tumors. In the present study, we analyzed the effect of arachidonyl ethanolamide (AEA) on cervical carcinoma (CxCa) cell lines.

METHODS:
To assess the sensitivity of CxCa cells to AEA, we selected three cell lines that were exposed to increasing doses of AEA with or without antagonists to receptors to AEA. DNA fragmentation and caspase-7 activity were used as apoptosis markers. The expression of receptors to AEA were analyzed in CxCa cell lines as well as CxCa biopsies.

RESULTS:
The major finding was that AEA induced apoptosis of CxCa cell lines via aberrantly expressed vanilloid receptor-1, whereas AEA binding to the classical CB1 and CB2 cannabinoid receptors mediated a protective effect. Furthermore, unexpectedly, a strong expression of the three forms of AEA receptors was observed in ex vivo CxCa biopsies.

CONCLUSIONS:
Overall, these data suggest that the specific targeting of VR1 by endogenous cannabinoids or synthetic molecules offers attractive opportunities for the development of novel potent anticancer drugs.

References:

http://www.ncbi.nlm.nih.gov/pubmed/15047233

Cannabinoid inhibits breast cancer

breast-cancer-ribbonThe endogenous cannabinoid anandamide inhibits human breast cancer cell proliferation

Luciano De Petrocellis,*† Dominique Melck,*‡ Antonella Palmisano,§ Tiziana Bisogno,‡ Chiara Laezza,¶ Maurizio Bifulco,¶ and Vincenzo Di Marzo‡||

†Istituto di Cibernetica and ‡Istituto per la Chimica di Molecole di Interesse Biologico (affiliated with the National Institute for the Chemistry of Biological Systems, Consiglio Nazionale delle Ricerche), Consiglio Nazionale delle Ricerche, Via Toiano 6, 80072 Arco Felice, Naples, Italy; § Istituto di Ricerche sull’Adattamento dei Bovini e dei Bufali all’Ambiente del Mezzogiorno, Consiglio Nazionale delle Ricerche, Ponticelli, 80147 Naples, Italy; and ¶Centro di Studio der l’Endocrinologia e l’Oncologia Sperimentale, Consiglio Nazionale Delle Richerche and Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli ‘Federico II,’ 80131 Naples, Italy
*L.D.P. & D.M. contributed equally to this work.

Anandamide was the first brain metabolite shown to act as a ligand of “central” CB1 cannabinoid receptors. Here we report that the endogenous cannabinoid potently and selectively inhibits the proliferation of human breast cancer cells in vitro. Anandamide dose-dependently inhibited the proliferation of MCF-7 and EFM-19 cells with IC50 values between 0.5 and 1.5 μM and 83–92% maximal inhibition at 5–10 μM. The proliferation of several other nonmammary tumoral cell lines was not affected by 10 μM anandamide. The anti-proliferative effect of anandamide was not due to toxicity or to apoptosis of cells but was accompanied by a reduction of cells in the S phase of the cell cycle. A stable analogue of anandamide (R)-methanandamide, another endogenous cannabinoid, 2-arachidonoylglycerol, and the synthetic cannabinoid HU-210 also inhibited EFM-19 cell proliferation, whereas arachidonic acid was much less effective. These cannabimimetic substances displaced the binding of the selective cannabinoid agonist [3H]CP 55,940 to EFM-19 membranes with an order of potency identical to that observed for the inhibition of EFM-19 cell proliferation. Moreover, anandamide cytostatic effect was inhibited by the selective CB1 receptor antagonist SR 141716A. Cell proliferation was arrested by a prolactin mAb and enhanced by exogenous human prolactin, whose mitogenic action was reverted by very low (0.1–0.5 μM) doses of anandamide. Anandamide suppressed the levels of the long form of the prolactin receptor in both EFM-19 and MCF-7 cells, as well as a typical prolactin-induced response, i.e., the expression of the breast cancer cell susceptibility gene brca1. These data suggest that anandamide blocks human breast cancer cell proliferation through CB1-like receptor-mediated inhibition of endogenous prolactin action at the level of prolactin receptor.

Anti-proliferative and apoptotic effects of anandamide in human prostatic cancer cell lines: Implication of epidermal growth factor receptor down-regulation and ceramide production

ANA induced a decrease of EGFR levels on LNCaP, DU145, and PC3 prostatic cancer cells by acting through cannabinoid CB1 receptor subtype and this leaded to an inhibition of the EGF-stimulated growth of these cells. Moreover, the G1 arrest of metastatic DU145 and PC3 growth was accompanied by a massive cell death by apoptosis and/or necrosis while LNCaP cells were less sensitive to cytotoxic effects of ANA. The apoptotic/necrotic responses induced by ANA on these prostatic cancer cells were also potentiated by the acidic ceramidase inhibitor, N-oleoylethanolamine and partially inhibited by the specific ceramide synthetase inhibitor, fumonisin B1 indicating that these cytotoxic actions of ANA might be induced via the cellular ceramide production. The potent anti-proliferative and cytotoxic effects of ANA on metastatic prostatic cancer cells might provide basis for the design of new therapeutic agents for effective treatment of recurrent and invasive prostatic cancers.

References:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC20983/

http://onlinelibrary.wiley.com/doi/10.1002/pros.10190/abstract

Journal of Neuroscience, Vol. 25

Journal of NeuroscienceFeb. 2005, Journal of Neuroscience, Vol. 25 Issue 8, Pages 1904-1913

Maria L. de Ceballos, PhD, Researcher and Group Leader of the Department of Neural Plasticity at the Cajal Institute, Spain, et al., wrote the following in a Feb. 2005 article titled “Prevention of Alzheimer’s Disease Pathology by Cannabinoids: Neuroprotection Mediated by Blockage of Microglial Activation,” published in the Journal of Neuroscience:

“Our results indicate that cannabinoid receptors are important in the pathology of AD [Alzheimer's Disease] and that cannabinoids succeed in preventing the neurodegenerative process occurring in the disease.”

Journal of Acquired Immune Deficiency Syndromes

Journal of Acquired Immune Deficiency SyndromesAug. 15, 2007, Journal of Acquired Immune Deficiency Syndromes, Vol. 45, Number 5, Pages 545-554

Margaret Haney, PhD, Associate Professor of Clinical Neuroscience at Columbia University, et al., wrote the following in their Aug. 15, 2007 study titled “Dronabinol and Marijuana in HIV-Positive Marijuana Smokers: Caloric Intake, Mood, and Sleep,” published in the Journal of Acquired Immune Deficiency Syndromes:

“Objectives: This placebo-controlled within-subjects study evaluated marijuana and dronabinol across a range of behaviors: eating topography, mood, cognitive performance, physiologic measures, and sleep.

METHODS: HIV-positive marijuana smokers (n = 10) completed 2 16-day inpatient phases. Each dronabinol (5 and 10 mg) and marijuana (2.0% and 3.9% [DELTA]9-tetrahydrocannabinol [THC]) dose was administered 4 times daily for 4 days, but only 1 drug was active per day, thereby maintaining double-blind dosing. Four days of placebo washout separated each active cannabinoid condition.

RESULTS: As compared with placebo, marijuana and dronabinol dose dependently increased daily caloric intake and body weight in HIV-positive marijuana smokers. All cannabinoid conditions produced significant intoxication, except for low-dose dronabinol (5 mg); the intoxication was rated positively (e.g., “good drug effect”) with little evidence of discomfort and no impairment of cognitive performance. Effects of marijuana and dronabinol were comparable, except that only marijuana (3.9% THC) improved ratings of sleep.

Conclusions: These data suggest that for HIV-positive marijuana smokers, both dronabinol (at doses 8 times current recommendations) and marijuana were well tolerated and produced substantial and comparable increases in food intake.”

Clinical Rehabilitation, Vol. 17

journal Clinical RehabilitationFeb. 2003, Clinical Rehabilitation, Vol. 17, Pages 18-26

Derick T. Wade, MD, Professor in the Department of Clinical Neurology at the University of Oxford, et al., wrote in a Feb. 2003 article titled “A Preliminary Controlled Study to Determine Whether Whole-Plant Cannabis Extracts Can Improve Intractable Neurogenic Symptoms” in the journal Clinical Rehabilitation:

“OBJECTIVES: To determine whether plant-derived cannabis medicinal extracts (CME) can alleviate neurogenic symptoms unresponsive to standard treatment, and to quantify adverse effects…

MEASURES USED: Patients recorded symptom, well-being and intoxication scores on a daily basis using visual analogue scales. At the end of each two-week period an observer rated severity and frequency of symptoms on numerical rating scales, administered standard measures of disability (Barthel Index), mood and cognition, and recorded adverse events.

RESULTS: Pain relief associated with both THC and CBD was significantly superior to placebo. Impaired bladder control, muscle spasms and spasticity were improved by CME in some patients with these symptoms. Three patients had transient hypotension and intoxication with rapid initial dosing of THC-containing CME.

CONCLUSIONS: Cannabis medicinal extracts can improve neurogenic symptoms unresponsive to standard treatments. Unwanted effects are predictable and generally well tolerated. Larger scale studies are warranted to confirm these findings.”

Neurology, Vol. 65

Sep. 2005, Neurology, Vol. 65, Issue 6, Pages 812-819

David J. Rog, PhD, from the Walton Centre for Neurology and Neurosurgery at the University of Liverpool, et al., wrote in a Sep. 2005 article titled “Randomized, Controlled Trial of Cannabis-Based Medicine in Central Pain in Multiple Sclerosis” in the journal Neurology:

“BACKGROUND: Central pain in multiple sclerosis (MS) is common and often refractory to treatment…

CONCLUSIONS: Cannabis-based medicine is effective in reducing pain and sleep disturbance in patients with multiple sclerosis related central neuropathic pain and is mostly well tolerated.”

Journal of Pain Vol. 9

the journal of painJune 2008, Journal of Pain Vol. 9, Issue 6, Pages 505-521

Barth Wilsey, MD, Director of the University of California at Davis Analgesic Research Center, et al., stated the following in their June 2008 study titled “A Randomized, Placebo Controlled Cross-Over Trial of Cannabis Cigarettes in Neuropathic Pain,” published in the Journal of Pain:

“This study’s objective was to examine the efficacy of two doses of smoked cannabis on pain in persons with neuropathic pain of different origins (e.g., physical trauma to nerve bundles, spinal cord injury, multiple sclerosis, diabetes). In a double-blind, randomized clinical trial participants received either lowdose, high-dose, or placebo cannabis cigarettes…

Thirty-eight patients underwent a standardized procedure for smoking either high-dose (7%), low-dose (3.5%), or placebo cannabis; of these, 32 completed all three smoking sessions. The study demonstrated an analgesic response to smoking cannabis with no significant difference between the low and the high dose cigarettes. The study concluded that both low and high cannabis doses were efficacious in reducing neuropathic pain of diverse causes.”

Neuropsychopharmacology Vol. 34

Aug. 2008, Neuropsychopharmacology Vol. 34, Pages 672-680

Ronald J. Ellis, MD, PhD, Professor In Residence in the Department of Neuroscience at the University of California at San Diego, et al., stated the following in their Aug. 2008 study titled “Smoked Medicinal Cannabis for Neuropathic Pain in HIV: A Randomized, Crossover Clinical Trial,” published in Neuropsychopharmacology:

“In a double-blind, randomized, clinical trial of the short-term adjunctive treatment of neuropathic pain in HIV-associated distal sensory polyneuropathy, participants received either smoked cannabis or placebo cannabis cigarettes…

Among completers, pain relief was significantly greater with cannabis than placebo. The proportion of subjects achieving at least 30% pain relief was again significantly greater with cannabis (46%) compared to placebo (18%). It was concluded that smoked cannabis was generally well-tolerated and effective when added to concomitant analgesic therapy in patients with medically refractory pain due to HIV-associated neuropathy.”

Independently Commissioned Reports

Marijuana-arrests-law“The [criminal] law’s implementation damages individuals in terms of criminal records and risks to jobs and relationships to a degree that far outweighs any harm that cannabis may be doing to a society. … [Therefore,] prison should no longer be a penalty for possession [of cannabis.]” – The Police Foundation (United Kingdom). 2000. Drugs and the Law: Report of the Independent Inquiry into the Misuse of Drugs Act of 1971. London.

“The current law prohibiting cannabis possession and trafficking appears to have a very limited deterrent effect, yet entails high social costs and diverts limited police resources from other pressing needs. … The severity of punishment for a cannabis possession charge should be reduced. Specifically, cannabis possession should be converted to a civil violation. … The available evidence indicates that removal of jail as a sentencing option would lead to considerable cost savings without leading to increases in the rates of cannabis use.” – Canadian Centre on Substance Abuse National Working Group on Addictions. 1998. Cannabis Control in Canada: Options Regarding Possession. CCSA, Ottawa.

“A number of studies have found experiencing contact with the police for a cannabis offense is likely to have a negative influence on young people’s confidence in the police … Reclassification [of marijuana so it is no longer a criminal offense] is likely to remove some of the friction between the police and communities that currently prevent more cooperative relationships.” – The Joseph Roundtree Foundation. 2002. The Policing of Cannabis as a Class B Drug. London.

“New Zealand politicians and the public should accept that cannabis has become part of our culture. Whatever harms are associated with cannabis are magnified by driving its use underground. A Tobacco, Alcohol, and Cannabis Authority should be created and charged with responsibility for developing and enforcing regulations concerning the production, distribution, sale, and use of these three substances.” – New Zealand Drug Policy Forum Trust. 1998. New Zealand Should Regulate and Tax Cannabis Commerce. Wellington.

“It appears clear that … a move toward more lenient laws for small scale cannabis offenses … will not lead to increased cannabis use. Thus, we can limit cannabis use without harsh penalties. … It may be that other governments, on reviewing the findings presented here and in other reports, will see fit to consider a similar approach for dealing with small scale cannabis offenses to the Cannabis Expiation System [decriminalization] of South Australia.” – Drug and Alcohol Services Council of South Australia, Monitoring, Evaluation and Research Unit. 1991. The Effects of Cannabis Legislation in South Australia on Levels of Cannabis Use. DASC Press: Parkside, South Australia.

Reference:

http://norml.org/index.cfm?Group_ID=3382