Hyperactivated m-calpain affects acquisition of doxorubicin resistance in breast cancer cells

Background: Doxorubicin is commonly using chemotherapeutic agents for breast cancer. However, doxorubicin has limitations in clinical use because of dose-dependent cardiotoxicity and drug resistance. Despite of previously reported studies about mechanisms of doxorubicin resistance including overexpression of P-gp and abnormal expression and mutation of topoisomerase IIα, resistance to this agent still abundantly occur and is regarded as a major obstacle to successful treatment. Methods: We have established doxorubicin resistant T47D cells. Intracellular calcium and ROS levels and calpain activity were measured using fluorometric experiments. Cell viability assay, cell cycle analysis, immunofluorescence and western blot analysis were performed to evaluate m-calpain specific truncation of topoisomerase IIα and molecular mechanism in doxorubicin resistant cells. Results: We observed that doxorubicin treatment increased intracellular calcium and ROS (Reactive Oxygen Species) in parental and doxorubicin resistant T47D cells. The increases in intracellular calcium and ROS were much greater in doxorubicin resistant T47D cells, which led to higher activity of calpains. Hyperactivated m-calpain, but not μ-calpain, specifically induced cleavage of topoisomerase IIα and accumulation of truncated topoisomerase IIα in the cytoplasm. The increase in cytoplasmic truncated topoisomerase IIα reduced the efficacy of doxorubicin. Doxorubicin resistant T47D cells, with hyperactivated m-calpain and truncated cytosolic topoisomerase IIα, obtained cross-resistance to other topoisomerase II-targeting drugs. Conclusion: Hyperactivated m-calpain induced cytoplasmic accumulation of truncated topoisomerase IIα in doxorubicin resistant T47D cells. General significance: These data provide a new mechanism of doxorubicin resistance and suggest a novel strategy for overcoming drug resistance in topoisomerase IIα-targeting therapy.