Involvement of the CB cannabinoid receptor in cell growth inhibition and G0/G1 cell cycle arrest via the cannabinoid agonist WIN 55,212-2 in renal cell carcinoma.

BACKGROUND

The anti-tumor properties of cannabinoids have been investigated in many in vitro and in vivo studies. Many of these anti-tumor effects are mediated via cannabinoid receptor types 1 and 2 (CB and CB), comprising the endocannabinoid system (ECS). In this study, we investigated the ECS based on CB and CB receptor gene and protein expression in renal cell carcinoma (RCC) cell lines. In view of their further use for potential treatments, we thus investigated the roles of CB and CB receptors in the anti-proliferative action and signal transduction triggered by synthetic cannabinoid agonists [such as JWH-133 and WIN 55,212-2 (WIN-55)] in RCC cell lines.

METHODS

Human RCC cell lines were used for this study. The CB and CB gene expression levels were analyzed using real-time PCR. Flow cytometric, immunocytochemical and western blot analyses were performed to confirm CB and CB receptor protein expression. The anti-proliferative effects of synthetic cannabinoids were investigated on cell viability assay. The CB and CB receptors were blocked pharmacologically with the antagonists SR141716A and AM-630, respectively, to investigate the effects of the agonists JWH-133 and WIN-55. Cell cycle, apoptosis and LDH-based cytotoxicity were analyzed on cannabinoid-treated RCC cells.

RESULTS

The CB1 and CB2 genes expression was shown by real-time PCR and flow cytometric and western blot analysis indicating a higher level of CB receptor as compared to CB in RCC cells. Immunocytochemical staining also confirmed the expression of the CB and CB proteins. We also found that the synthetic cannabinoid agonist WIN-55 exerted anti-proliferative and cytotoxic effects by inhibiting the growth of RCC cell lines, while the CB agonist JWH-133 did not. Pharmacologically blocking the CB1 and CB2 receptors with their respective antagonists SR141716A and AM-630, followed by the WIN-55 treatment of RCC cells allowed uncovering the involvement of CB2, which led to an arrest in the G0/G1 phase of the cell cycle and apoptosis.

CONCLUSIONS

This study elucidated the involvement of CB in the in vitro inhibition of RCC cells, and future applications of CB agonists in the prevention and management of RCC are discussed.