Natural and Synthetic Cannabinoids Reduce Cell Viability of Ewing Sarcoma TC‐71 Cells Potentially via Non‐canonical CB receptors

Abstract

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.