Inhibition of Rat C6 Glioma Cell Proliferation by Endogenous and Synthetic Cannabinoids.Stig O. P. Jacobsson1,2, Thomas Wallin1 and Christopher J. Fowler
Relative Involvement of Cannabinoid and Vanilloid Receptors
Relative Involvement of Cannabinoid and Vanilloid Receptors
JPET December 1, 2001
The effects of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) upon rat C6 glioma cell proliferation were examined and compared with a series of synthetic cannabinoids and related compounds. Cells were treated with the compounds each day and cell proliferation was monitored for up to 5 days of exposure. AEA time- and concentration-dependently inhibited C6 cell proliferation. After 4 days of treatment, AEA and 2-AG inhibited C6 cell proliferation with similar potencies (IC50 values of 1.6 and 1.8 μM, respectively), whereas palmitoylethanolamide showed no significant antiproliferative effects at concentrations up to 10 μM. The antiproliferative effects of both AEA and 2-AG were blocked completely by a combination of antagonists at cannabinoid receptors (SR141716A and SR144528 or AM251 and AM630) and vanilloid receptors (capsazepine) as well as by α-tocopherol (0.1 and 10 μM), and reduced by calpeptin (10 μM) and fumonisin B1 (10 μM), but not byL-cycloserine (1 and 100 μM). CP 55,940, JW015, olvanil, and arachidonoyl-serotonin were all found to affect C6 glioma cell proliferation (IC50 values of 5.6, 3.2, 5.5, and 1.6 μM, respectively), but the inhibition could not be blocked by cannabinoid + vanilloid receptor antagonists. It is concluded that the antiproliferative effects of the endocannabinoids upon C6 cells are brought about by a mechanism involving combined activation of both vanilloid receptors and to a lesser extent cannabinoid receptors, and leading to oxidative stress and calpain activation. However, there is at present no obvious universal mechanism whereby plant-derived, synthetic, and endogenous cannabinoids affect cell viability and proliferation.
The antimitotic effects of the cannabinoid Δ9-tetrahydrocannabinol (Δ9-THC), the principal psychoactive component of hashish and marijuana, have been known since the 1970s (Munson et al., 1975). More recent studies have shown that Δ9-THC and/or the synthetic cannabinoid (CB) receptor agonist WIN 55,212-2 induces apoptosis in various mouse, rat, and human cells (SÃ¡nchez et al., 1998; Zhu et al., 1998; Ruiz et al., 1999; Jacobsson et al., 2000; Sarker et al., 2000) and have been shown to induce regression of malignant gliomas in Wistar rats and mice (Galve-Roperh et al., 2000), a result also seen with the CB2 receptor-selective agonist JWH-133 (SÃ¡nchez et al., 2001). In rat C6 glioma cells, the effect of Δ9-THC upon cell viability, which takes ∼5 days to become apparent (SÃ¡nchez et al., 1998), is brought about by a pathway involving CB receptors followed by sustained ceramide accumulation (Galve-Roperh et al., 2000). Blockade of both CB1 and CB2 receptors by the selective antagonists SR141716A and SR144528 is required to protect the cells against the deleterious effects of Δ9-THC upon cell viability (Galve-Roperh et al., 2000). In contrast, in human PC-3 prostate cells, the apoptotic effects of Δ9-THC are not mimicked by WIN 55,212-2 and are not prevented by pertussis toxin treatment of the cells, suggestive of a CB receptor-independent mechanism (Ruiz et al., 1999).
The endogenous cannabinoid (“endocannabinoid”) arachidonoylethanolamide (anandamide, AEA) is a partial agonist with similar affinity at CB1 and CB2 receptors but lower efficacy at the CB2 receptor. In addition, AEA can activate vanilloid receptors (Zygmunt et al., 1999; Smart et al., 2000). AEA has been shown to induce apoptosis in several cell types, but the molecular mechanism behind this action appears to be rather different from that of Δ9-THC. Thus, the proapoptotic effects of AEA on human CHP-100 neuroblastoma and U-937 lymphoma cells (Maccarrone et al., 2000) were suggested to be mediated via vanilloid receptors. These authors proposed that AEA induces a rise in intracellular calcium, mitochondrial uncoupling, and cytochrome c release, and activation of the caspase cascade. Furthermore, Sarker et al. (2000) found that AEA induces apoptosis in rat PC-12 pheochromocytoma cells by increasing the superoxide anion formation and caspase-3 activation. AEA has also been shown to inhibit the proliferation of human breast and prostate cancer cell lines in vitro (De Petrocellis et al., 1998) secondary to a CB1 receptor-mediated inhibition of prolactin action, at the level of the prolactin receptor.
The different mechanisms proposed for the effects of, on the one hand plant-derived and synthetic cannabinoids, and on the other hand AEA, upon cell survival present a somewhat confusing picture. In the present study, we have investigated, in the same cells and under the same conditions, the receptors involved in the antiproliferative effects of synthetic and endogenous cannabinoids, including compounds that are selective for the CB1 or CB2 receptors. In addition, we have investigated whether the antiproliferative effects of AEA are mimicked by the endocannabinoid 2-arachidonoylglycerol (2-AG) and the metabolically stable AEA analog R-(+)-methanandamide.
Rat glioma C6 cells are ideal for this study because they express both functional cannabinoid and vanilloid receptors (SÃ¡nchez et al., 1997; Bı́rÃ³ et al., 1998) and respond to AEA (Maccarrone et al., 2000). These cells, however, do not in our hands show a mitogenic response to prolactin (T. Wallin and S.O.P Jacobsson, unpublished data), thereby precluding investigation into effects of AEA upon proliferation stimulated by this hormone. We reported previously that single administrations of AEA (≤10 μM) failed to affect C6 glioma cell viability (Jacobsson et al., 2000), although this result presumably reflected the rapid removal of AEA from the culture medium. To reduce the cellular metabolism of AEA, we have used a sensitive assay to minimize the number of cells required per well, and in addition treated the cells daily with AEA (or the other test compounds). Because the effects of Δ9-THC upon cell viability are more apparent at low culturing serum contents (Jacobsson et al., 2000), we have elected to investigate the effects of the compounds upon cell proliferation at 1% fetal bovine serum.
Source with Charts, Graphs and Links:Inhibition of Rat C6 Glioma Cell Proliferation by Endogenous and Synthetic Cannabinoids. Relative Involvement of Cannabinoid and Vanilloid Receptors