Natural And Synthetic Cannabinoids Reduce Cell Viability Of Ewing Sarcoma Tc-71 Cells Potentially Via Non-Canonical Cb Receptors

Ewing Sarcoma (EWS) is the second most common bone tumor in children, characterized by rapid tumor growth and extensive bone destruction. Treatment for EWS is multimodal: surgery, radiation, and chemotherapy. However, these approaches are effective in only 70% of the patients with localized disease and thus new drugs are needed. WIN-55,212-2 (WIN-2), a synthetic cannabinoid in the aminoalkyindole (AI) class, has been shown to slow tumor growth in several types of cancer and human Kaposi sarcoma via cannabinoid (CB) and non-CB mechanisms. Therefore, we hypothesize that WIN-2 and other synthetic CBs will exhibit anti-proliferative effects in EWS TC-71 cells via CB receptors. Competitive binding screens using TC-71 membranes showed that binding of the nonselective CB1/CB2 radiolabeled agonist [3H]CP-55,940 could not be displaced by selective CB1 or CB2 ligands, or even non-radioactive CP-55,940 itself. However, binding of a second nonselective CB1/CB2 radiolabeled agonist [3H]WIN-2 could be displaced with a CB1 but not CB2 antagonist, and non-radioactive WIN-2 itself. This suggests that TC-71 cells express a novel non-canonical CB binding site (WIN-2 site). Subsequent homologous receptor binding studies with [3H]WIN-2 showed that TC-71 cells do indeed express a WIN-2 site at a density (Bmax) of 3.7 +/- 1.6 pmole/mg, to which WIN-2 binds with an affinity (Ki) of 4.5 +/- 0.6 nM. Next, thirty-eight different synthetic CBs were screened for potential binding to WIN-2 receptors in TC-71 cells. Only four compounds exhibited a high affinity for the WIN-2 receptor in the low to the mid-nanomolar range. Interestingly, four other compounds increased [3H]WIN-2 binding, indicating that these ligands might act as positive allosteric modulators of the WIN-2 site. When compounds with the highest affinity were examined in G-protein activation assays for functional activity, two of them reduced [35S]GTPgS binding below basal levels indicating that they may act as inverse agonists at the novel WIN-2 site. Most importantly, WIN-2 and all four compounds with high affinity for the WIN-2 site produced a significant time-dependent reduction in viability of EWS TC-71 cells as measured by trypan blue exclusion and lactate dehydrogenase (LDH) release. In conclusion, WIN-2 and other synthetic CBs reduce EWS cell viability potentially via a non-canonical CB binding site. These results suggest that the WIN-2 site might be a novel therapeutic target for drug development to treat EWS and perhaps other types of cancer as well.