Cepharanthin (CEP) is a biscoclaurine alkaloid extracted from Stephania cepharantha Hayata. CEP is reported to inhibit drug resistance by inhibiting P-glycoprotein, a drug efflux pump, and recently to induce apoptosis. In the present study, we examined the effects of CEP as an inhibitor of adriamycin (ADR) resistance on ADR-induced apoptosis and necrosis. First, we established p53-deficient ADR-resistant osteosarcoma cell lines, SaOS2-AR and SaOS2 F-AR. Resistant cells showed a higher level of intracellular glutathione peroxidase activity than parent cells. P-glycoprotein was overexpressed in resistant cells. The intracellular ADR level of resistant cells was lower than that of parent cells. One µg/ml CEP eliminated the degradation of intracellular ADR of resistant cells; that is, to a level equivalent to that of the parent cells. CEP of 0.5 µg/ml, which was not cytotoxic when used alone, significantly increased the ADR sensitivity of resistant cells, to a level similar to the parent cell level. Isosorbide 5-mononitrate, a potential nitric oxide-generation agent, combined with CEP further increased the ADR sensitivity of resistant cells, indicating a synergistic effect of CEP and isosorbide 5-mononitrate on ADR cytotoxicity. Time-lapse microscopic observation revealed that ADR dominantly induced apoptosis much more than necrosis for both parent and resistant cells, and that the use of 0.5 µg/ml CEP with ADR synergistically accelerated apoptosis in resistant cells. Finally, we clarified the property by which CEP synergistically accelerates ADR-induced apoptosis. This property might be a new mechanism that explains how CEP overcomes ADR resistance.

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