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

BMC Cancer. 2018 May 23;18(1):583. doi: 10.1186/s12885-018-4496-1.

Abstract

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.

Keywords: CB1 and CB2 receptors; Endocannabinoid system (ECS); JWH-133; Renal cell carcinoma; WIN 55,212–2 mesylate.

MeSH terms

  • Apoptosis / drug effects
  • Benzoxazines / pharmacology*
  • Benzoxazines / therapeutic use
  • Cannabinoid Receptor Agonists / pharmacology*
  • Cannabinoid Receptor Agonists / therapeutic use
  • Cannabinoid Receptor Antagonists / pharmacology
  • Carcinoma, Renal Cell / drug therapy*
  • Carcinoma, Renal Cell / pathology
  • Cell Line, Tumor
  • Cell Proliferation / drug effects
  • Drug Screening Assays, Antitumor
  • G1 Phase Cell Cycle Checkpoints / drug effects
  • Humans
  • Kidney Neoplasms / drug therapy*
  • Kidney Neoplasms / pathology
  • Morpholines / pharmacology*
  • Morpholines / therapeutic use
  • Naphthalenes / pharmacology*
  • Naphthalenes / therapeutic use
  • Receptor, Cannabinoid, CB1 / agonists
  • Receptor, Cannabinoid, CB1 / antagonists & inhibitors
  • Receptor, Cannabinoid, CB1 / metabolism
  • Receptor, Cannabinoid, CB2 / agonists
  • Receptor, Cannabinoid, CB2 / antagonists & inhibitors
  • Receptor, Cannabinoid, CB2 / metabolism*
  • Signal Transduction / drug effects

Substances

  • Benzoxazines
  • Cannabinoid Receptor Agonists
  • Cannabinoid Receptor Antagonists
  • Morpholines
  • Naphthalenes
  • Receptor, Cannabinoid, CB1
  • Receptor, Cannabinoid, CB2
  • Win 55212-2