Background: Prostate cancer is a common malignancy and a leading cause of death. Despite advances in prostate cancer treatment, there is a need for novel therapeutic approaches. Cannabinoid compounds have attracted attention as potential anticancer drugs. Preclinical studies showed that cannabinoids have effects on cancer progression, inhibition of proliferation, invasion, chemoresistance, apoptosis, autophagy and the immune system. We investigated synthetic cannabinoids AM-251 and AM-1241 in cannabinoid receptor-expressing DU145 and PC3 prostate cancer cells. AM-251 is an inverse agonist of the CB1 receptor while AM-1241 is a selective agonist for CB2receptor. We examined the pro-apoptotic and anti-proliferative effect of AM-251 and AM-1241 in cancer cells.

Materials and methods: MTT proliferation assay was used to assess the cytotoxicity in DU145 and PC3 cells. To assess apoptosis and effects on cell cycle, we applied Annexin V/Propidium iodide staining and FACS analysis. The ability to cause DNA fragmentation was measured with an ELISA assay. Mechanism of action was evaluated with Western Blot.

Results: These show that AM-251 and AM-1241 inhibit the proliferation of DU-145 and PC3 prostate cancer cells with AM-251 been more potent than AM-1241 in reducing viable cell number in DU145 and PC3 cells.. Both AM-1241 and ΑΜ-251 increased DNA fragmentation in DU145 cells 18-fold compared to untreated control. In PC3 cells DNA fragmentation was not observed. DU145 treated cells stained positive for Annexin V indicating early apoptosis. A late apoptotic effect was observed at 72 h, staining double positive for AnV/PI following treatment with both agents.In PC3 cells, a late apoptotic effect was observed at 48h, staining double positive for AnV/PI following treatment with both agents. AM-251 induced cleavage of caspase 3, caspase 8 and PARP, supporting the induction of caspase-dependent apoptosis in DU145 cells. In PC3 cells, the levels of LC3B-II were increased suggesting autophagy induced after treatment with both drugs. Autophagy may be induced via the PI3K pathway / p-AKT / mTOR, as suggested by decrease in p-AKT levels in PC3 cells. Apoptosis i was also evident by an increase of the sub-G1 fraction in cells using flow cytometry. The pan-caspase inhibitor z.vad.fmk partially restored the viability of cells treated with ΑΜ251, while the viability was completely restored in cells treated with AM1241. This suggests that the mechanism of action involves the induction of caspase-dependent apoptosis. Consistently, DNA fragmentation was completely abolished in the presence of the pan-caspase inhibitor. The pan-caspase inhibitor z.vad.fmk did not restore the viability in PC3 cells, confirming that the cells are driven to cell death via a caspase-independent mechanism.

Conclusions: Synthetic cannabinoids AM-251 and AM-1241 induce caspase-dependent apoptosis in DU145 cells while autophagy is likely involved in the mode of action of PC3 cells. Further studies will focus on further elucidating the effects and mechanism of action of these compounds in prostate cancer and normal cells.

Citation Format: Sotiroula Louka, Christiana Neophytou, Andreas Constantinou. Synthetic cannabinoids AM-251 and AM-1241 induce cell death in prostate cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4030.