Proliferation of RCC spheres was halted and size of spheres was reduced. and WIN 55,212C2 (WIN-55)] in RCC cell lines. Methods Human RCC cell lines were used for this study. The and 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 and 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. have been used for medicinal and recreational purposes. Cannabinoids, the active components of and receptor genes were compared with that of the (123?bp) gene as an endogenous control. As a negative control, no cDNA was added to the PCR tubes containing the FastStart Essential DNA Green Master Mix to determine whether all of the reagents were free of the target sequence. The total RNA from ASE-5063 MW-150 dihydrochloride dihydrate cells MW-150 dihydrochloride dihydrate was used as a positive control for the and genes. The data were obtained using LightCycler? Nano software 1.0 (Roche, Basel, Switzerland). The relative mRNA expression levels were then normalized using the mRNA level of the reference gene (value 0.05 was considered to indicate statistical significance. Results mRNA expression of and in RCC cells The primary goal of this experiment was to investigate the mRNA expression of the cannabinoid receptors and in RCC cells. Our real-time PCR results revealed the expression of and genes. The amplified cDNA products of the (66?bp) and (141?bp) genes were detected by agarose gel electrophoresis (Table?1). Figure?2a and b shows the MW-150 dihydrochloride dihydrate mRNA expression levels for and in RCC and ASE-5063 cells. Table 1 Primer sequences used for and genes and in different RCC cell lines. a The quantitative data indicate the expression of the and receptor genes in RCC cells. ASE-5063 (ASE) cells were used as a control for the and receptor genes. b Two agarose gels showing the presence of mRNA expression MW-150 dihydrochloride dihydrate of (66?bp), (141?bp) and (123?bp) (endogenous control gene) in the RCC cell lines ACHN, Caki-1, Erg 786-O, Caki-2, SMKT-R2, SMKT-R3, 769-P, and RCC-6, as well as in the healthy kidney cell line ASE-5063. M indicates the molecular marker Expression of the cannabinoid receptor CB2 in RCC MW-150 dihydrochloride dihydrate cells We used flow cytometry to analyze the expression of the membrane receptor proteins CB1 and CB2 in 8 different RCC cell lines. The objective of this experiment was to determine which of these proteins was highly expressed in RCC cells. Our flow cytometry analysis confirmed the expression of the CB1 and CB2 proteins in all the cell lines analyzed; however, more cells expressed the CB2 protein than the CB1 protein (Fig.?3a and b). Figure?3a and b displays representative histograms for the CB1 and CB2 protein expression, and the quantitative analysis of the CB1 and CB2 receptors in RCC cells is shown in Fig. ?Fig.3c.3c. The western blot analysis also revealed the protein expression of the CB1 and CB2 receptors in RCC cells. The receptors expressed in RCC cells had estimated molecular masses of approximately 55?kDa for CB1 and 62?kDa for CB2 (Fig. ?(Fig.3d3d and e). As a control for the CB1 and CB2 proteins,.