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Opposing Actions of Endocannabinoids On Cholangiocarcinoma Growth

Julie Gardener

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Opposing Actions of Endocannabinoids on Cholangiocarcinoma Growth​
Sharon DeMorrow, Shannon Glaser, Heather Francis, Julie Venter, Bradley Vaculin, Shelley Vaculin and Gianfranco Alpini
The Journal of Biological Chemistry-April 27, 2007


Cholangiocarcinomas are devastating cancers of biliary origin with limited treatment options. Modulation of the endocannabinoid system is being targeted to develop possible therapeutic strategies for a number of cancers; therefore, we evaluated the effects of the two major endocannabinoids, anandamide and 2-arachidonylglycerol, on numerous cholangiocarcinoma cell lines. Although anandamide was antiproliferative and proapoptotic, 2-arachidonylglycerol stimulated cholangiocarcinoma cell growth. Specific inhibitors for each of the cannabinoid receptors did not prevent either of these effects nor did pretreatment with pertussis toxin, a Gi/o protein inhibitor, suggesting that anandamide and 2-arachidonylglycerol did not exert their diametric effects through any known cannabinoid receptor or through any other Gi/o protein-coupled receptor. Using the lipid raft disruptors methyl-β-cyclodextrin and filipin, we demonstrated that anandamide, but not 2-arachidonylglycerol, requires lipid raft-mediated events to inhibit cellular proliferation. Closer inspection of the lipid raft structures within the cell membrane revealed that although anandamide treatment had no observable effect 2-arachidonylglycerol treatment effectively dissipated the lipid raft structures and caused the lipid raft-associated proteins lyn and flotillin-1 to disperse into the surrounding membrane. In addition, anandamide, but not 2-arachidonylglycerol, induced an accumulation of ceramide, which was required for anandamide-induced suppression of cell growth. Finally we demonstrated that anandamide and ceramide treatment of cholangiocarcinoma cells recruited Fas and Fas ligand into the lipid rafts, subsequently activating death receptor pathways. These findings suggest that modulation of the endocannabinoid system may be a target for the development of possible therapeutic strategies for the treatment of this devastating cancer.

Cholangiocarcinomas are devastating cancers of intrahepatic and extrahepatic origin that are increasing in both their worldwide incidence and mortality rates. The challenges posed by these often lethal biliary tract cancers are daunting; conventional treatment options are limited, and the only hope for long term survival is that of complete surgical resection of the tumor. Conventional chemotherapy and radiation therapy are not effective in prolonging long term survival; therefore it is important to understand the cellular mechanisms of cholangiocarcinoma cell growth with a view to develop novel chemopreventive strategies.

Marijuana and its derivatives have been used in medicine for many centuries, and presently there is an emerging renaissance in the study of the therapeutic effects of cannabinoids. Ongoing research is determining that regulation of the endocannabinoid system may be effective in the treatment of pain, glaucoma, and neurodegenerative disorders such as Parkinson disease and multiple sclerosis. In addition, cannabinoids might be effective antitumoral agents because of their ability to inhibit the growth of various types of cancer cell lines in culture and in laboratory animals.

The finding in the early 1990s of specific G-protein-coupled receptors for the psychoactive component of Cannabis sativa (-)-Δ9-tetrahydrocannabinol led to the discovery of a whole endogenous signaling system now known as the endocannabinoid system. This system consists of the cannabinoid receptors (to date there are two, Cb12 and Cb2, as well as a putative involvement of the vanilloid receptor VR1), their endogenous ligands (endocannabinoids), and the proteins for their synthesis and inactivation. The cannabinoid receptors are seven-transmembrane domain proteins coupled to the Gi/o type of G-proteins. Cb1 receptors are found predominantly in the central nervous system but also in most peripheral tissues including immune cells, the reproductive system, the gastrointestinal tract, and the lung. On the other hand, Cb2 receptors are found predominantly in the immune system, i.e. in tonsils, spleen, macrophages, and lymphocytes. To date, many endocannabinoids have been identified with varying affinities for the receptors. Anandamide (AEA) was the first endogenous ligand to be identified; it acts as a partial Cb1 agonist and weak Cb2 agonist. Another endocannabinoid is 2-arachidonylglycerol (2-AG), which activates both Cb1 and Cb2 receptors. The chemical structures of these two endocannabinoids are shown in Fig. 1. Although the physiological role of the many other ligands has not yet been fully clarified, AEA and 2-AG have been implicated in a wide variety of physiological and pathological processes.

In addition to their cannabinoid receptor-mediated effects, endocannabinoids, and in particular AEA, are capable of mediating a plethora of receptor-independent cell signaling effects, most of which are via an interaction with cholesterolrich microdomains of the cell membrane, also known as lipid rafts, and the synthesis of the raft-associated, sphingolipid moiety ceramide. These lipid rafts are thought to provide a platform for signaling molecules to concentrate and efficiently interact thereby facilitating the subsequent signal transduction process. Indeed the subcellular localization of death receptors such as tumor necrosis factor (TNF) receptor 1, Fas, Fas-associated death domain protein (FADD), and other receptors of the TNF-related apoptosis-inducing ligand pathway are redistributed to lipid rafts in response to many apoptosis-inducing factors including a number of chemotherapeutic agents.

In the present study, we show the opposing effects of AEA and 2-AG on cholangiocarcinoma cell growth that were cannabinoid receptor-independent. In addition, we show that AEA mediated its inhibitory effects on cell growth by stimulating the accumulation of ceramide, stabilization of lipid rafts, and the recruitment of Fas and Fas ligand (FasL) to the lipid rafts. Conversely 2-AG increased cholangiocarcinoma cell growth and disrupted the organization of the lipid raft structures in the cell membrane.

Source with Charts, Graphs and Links: Opposing Actions of Endocannabinoids on Cholangiocarcinoma Growth
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