Two-Step Cell Death Induction by the New 2-Arachidonoyl Glycerol Analog and Its Modulation by...

2-arachnadoyl glycerol (2-AG) is one of the most common endocannabinoid molecules with anti-proliferative, cytotoxic, and pro-proliferative effects on different types of tumors. Typically, it induces cell death via cannabinoid receptor 1/2 (CB1/CB2)-linked ceramide production. In breast cancer, ceramide is counterbalanced by the sphingosine-1-phosphate, and thus the mechanisms of 2-AG influence on proliferation are poorly understood. We evaluated the mechanism of the anti-proliferative action by 2-AG and the influence of lysophaosphatidylinositol (LPI) on it in six human breast cancer cell lines of different tumor degree (MCF-10A, MCF-7, BT-474, BT-20, SK-BR-3, and MDA-MB-231) using resazurin test, inhibitor, blocker, and anti-oxidant analysis, and siRNA interference. To avoid acyl migration in 2-AG, we replaced it with the analog 2-arachidonoyl-1,3-difluoropropanol (2-ADFP) newly synthesized by us. Using a molecular docking approach, we showed that at the CB2 receptor, 2-ADFP and 2-AG were very close to each other. However, 2-ADFP demonstrated a stronger affinity towards CB1 in the antagonist-bound conformation. 2-ADFP was anti-proliferative in all the cell lines tested. The toxicity of 2-ADFP was enhanced by LPI. 2-ADFP activity was reduced or prevented by the CB2 and vanilloid receptor 1 (TRPV1) blockers, inositol triphosphate receptor, CREB, and cyclooxygenase 2 inhibitor, and by anti-oxidant addition. Together with the literature data, these results indicate CB2- and TRPV1-dependent COX-2 induction with concomitant cell death induction by the oxidized molecule's metabolites.