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Cannabinoid Receptor Activation Induces Apoptosis through Tumor Necrosis Factor ?–Mediated Ceramide De novo Synthesis in Colon Cancer Cells
Clinical Cancer Research Friday 17 Feb 2012 Purpose: Cannabinoids have been recently proposed as a new family of potential antitumor agents. The present study was undertaken to investigate the expression of the two cannabinoid receptors, CB1 and CB2, in colorectal cancer and to provide new insight into the molecular pathways underlying the apoptotic activity induced by their activation. Experimental Design: Cannabinoid receptor expression was investigated in both human cancer specimens and in the DLD-1 and HT29 colon cancer cell lines. The effects of the CB1 agonist arachinodyl-2'-chloroethylamide and the CB2 agonist N-cyclopentyl-7-methyl-1-(2-morpholin-4-ylethyl)-1,8-naphthyridin-4(1H)-on-3-carboxamide (CB13) on tumor cell apoptosis and ceramide and tumor necrosis factor (TNF)-? production were evaluated. The knockdown of TNF-? mRNA was obtained with the use of selective small interfering RNA. Results: We show that the CB1 receptor was mainly expressed in human normal colonic epithelium whereas tumor tissue was strongly positive for the CB2 receptor. The activation of the CB1 and, more efficiently, of the CB2 receptors induced apoptosis and increased ceramide levels in the DLD-1 and HT29 cells. Apoptosis was prevented by the pharmacologic inhibition of ceramide de novo synthesis. The CB2 agonist CB13 also reduced the growth of DLD-1 cells in a mouse model of colon cancer. The knockdown of TNF-? mRNA abrogated the ceramide increase and, therefore, the apoptotic effect induced by cannabinoid receptor activation. Conclusions: The present study shows that either CB1 or CB2 receptor activation induces apoptosis through ceramide de novo synthesis in colon cancer cells. Our data unveiled, for the first time, that TNF-? acts as a link between cannabinoid receptor activation and ceramide production. Cannabinoids, the active components of Cannabis sativa and their derivates, exert a wide spectrum of central and peripheral actions through the activation of specific receptors that are normally bound by a family of endogenous ligands, the endocannabinoid anandamide and 2-arachidonoylglycerol (1, 2). Two cannabinoid receptors have been characterized and cloned thus far: CB1 (3) and CB2 (4). CB1 receptors are particularly abundant in discrete areas of the brain, where they mediate cannabinoid psychoactivity, and are also expressed in peripheral nerve terminals and various extraneural sites, such as the testis, uterus, eye, and spleen (1–4). In contrast, CB2 receptors are believed to be almost exclusively expressed in the cells and organs of the immune system and are unrelated to cannabinoid psychoactivity (4). Nevertheless, they have also been recently shown in cells from other origins, especially tumor cells (5, 6). Experimental evidence has shown that cannabinoid administration can inhibit the growth of several models of tumor xenografts in rats and mice (7–11). This antitumor action of cannabinoids relies on the ability of these drugs to inhibit tumor angiogenesis (12) or directly induce apoptosis or cell cycle arrest in neoplastic cells (7–11). Cannabinoid receptors have been shown to modulate several signaling pathways involved in the control of cell survival, including extracellular signal-regulated kinase (ERK; ref. 13), c-Jun-NH2-kinase (14), p38 mitogen-activated protein kinase (MAPK; ref. 15), and the ceramide pathway (16, 17). These findings point to the potential application of cannabinoids as antitumor agents (5, 6). However, the majority of these studies have been done with the use of psychoactive cannabinoids, in particular ?9-tetrahydrocannabinol, the main active component of marijuana, and WIN, 55,212-2, a nonselective synthetic cannabinoid agonist. It would be preferable, instead, to have cannabinoid-based therapeutic strategies that are devoid of typical CB1 receptor–mediated psychotropic side effects. Hence, the recent synthesis of new, highly selective CB2 agonists (18) opens the very attractive clinical possibility of targeting this receptor selectively. Ceramide is a ubiquitous sphingolipid messenger that plays an important role in the control of tumor cell fate (19). CB1 receptor activation has been shown to induce sphingomyelin hydrolysis and acute ceramide production within minutes in both primary astrocytes (20) and C6 glioma cells (16). The functional coupling of CB1 receptors to sphingomyelinases might involve different adaptor proteins, one of which is the factor associated with neutral sphingomyelinase activation (FAN). FAN binds to a cytoplasmatic nine-amino-acid motif of the 55-kDa tumor necrosis factor (TNF) receptor, the neutral sphingomyelinase–activating domain, thereby coupling the receptor to sphingomyelin breakdown (21). On the contrary, sustained ceramide accumulation through enhanced de novo synthesis seems to play a major role in CB2 receptor activation–induced apoptosis in tumor cells, including glioma (8, 16, 22), leukemia (23), and pancreatic cancer (9) cells. However, the precise molecular mechanisms involved in the CB2 receptor–mediated generation of ceramide are still unknown. TNF-? is one of the most pleiotropic cytokines acting as a host defense factor in a number of immunologic responses and antitumor activity (24, 25). The deregulation of TNF-? signaling results in a wide spectrum of human diseases, including sepsis, multiple sclerosis, rheumatoid arthritis, and cancer (26). It has been shown that TNF-? exerts a cytostatic or cytotoxic effect in several types of cells, and this effect is mediated, at least in part, by the stimulated production of ceramide (27–29). A large body of evidence has shown that the cannabinoid system is involved in the regulation of the cytokine network (30). However, the effects of cannabinoids on cytokines and, in particular, TNF-? production are often conflicting and can be either stimulatory or inhibitory, depending on the experimental model system and the type of cell investigated. To our knowledge, the influence of cannabinoid receptor activation on TNF-? production in cancer cells has not yet been addressed. Although it is well known that the endogenous cannabinoid system and cannabinoid receptors regulate gastrointestinal functions, such as gastric emptying, secretion, and intestinal motility (31, 32), few studies have investigated the expression and role of CB1 and/or CB2 receptors in normal (33) or neoplastic (15, 34) epithelial cells of the colon in humans. The reported results have shown that both inhibition of proliferation (34) and induction of apoptosis (15) in colon cancer cells are mediated by CB1 receptor activation whereas little or no activity has been observed for the CB2 receptor. The aim of this study was to investigate whether even CB2 receptor activation is involved in the antitumor action of cannabinoids in colon cancer cells and to identify the possible molecular mechanisms underlying this effect. In particular, we explored the hypothesis that cannabinoid receptor activation might induce a proapoptotic effect through a TNF-?–mediated increase in ceramide production. Translational Relevance The present study shows that the antitumor actions of cannabinoid receptor agonists on colon cancer cells may be exerted either via the CB1 receptor or, more efficiently, via the CB2 receptor. The fact that selective targeting of CB2 receptor results in colorectal tumor growth inhibition is of potential clinical interest for future cannabinoid-based anticancer therapies because the use of CB2-selective ligands is not linked to the typical marijuana-like psychoactive effects of CB1 activation. Moreover, we showed that only the CB2 receptor is expressed by tumor cells in colorectal cancer human specimens and, thus, it is likely that only compounds with high selectivity for this receptor may be effective as anticancer agents in humans. The recent synthesis of new, highly selective CB2 agonists opens the very attractive clinical possibility of using these compounds as adjuvants to conventional chemotherapeutic regimens for the treatment of colorectal cancer. more at: http://clincancerres.aacrjournals.org/content/14/23/7691.long
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