Involvement of the CB₂ 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 (CB₁ and CB₂), comprising the endocannabinoid system (ECS). In this study, we investigated the ECS based on CB ₁ and CB ₂ 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 CB₁ and CB₂ 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 ₁ and CB ₂ gene expression levels were analyzed using real-time PCR. Flow cytometric, immunocytochemical and western blot analyses were performed to confirm CB₁ and CB₂ receptor protein expression. The anti-proliferative effects of synthetic cannabinoids were investigated on cell viability assay. The CB₁ and CB₂ 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 CB₂ receptor as compared to CB₁ in RCC cells. Immunocytochemical staining also confirmed the expression of the CB₁ and CB₂ 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 CB₂ 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 CB₂ in the in vitro inhibition of RCC cells, and future applications of CB₂ agonists in the prevention and management of RCC are discussed.