Hydrogen peroxide detection in viable and apoptotic tumor cells under action of cisplatin and bleomycin

Abstract Objective: A flow cytometric approach is proposed to assess the hydrogen peroxide (H2O2) level under chemotherapy action separately in viable and apoptotic tumor cells. Materials and methods: For studying the involvement of H2O2 in the process of cell death, the genetically encoded fluorescent sensor HyPer2, apoptosis marker PE Annexin V and vital dye 7-AAD were employed. The approach was used for testing the capacity of two cytotoxic drugs, cisplatin and bleomycin, to change the intracellular H2O2 concentration, depending on the stage of cell death. Results: An increase in HyPer2 fluorescence has been revealed in cells undergoing apoptosis under cisplatin action. This finding indicates that accumulation of H2O2 accompanies the cisplatin-induced apoptotic reaction. HyPer2 response was also revealed in negative to PE Annexin V viable cells which can be explained either by participation of H2O2 in the earliest stages of apoptosis or in a cell response to a non-fatal injury. Under bleomycin action, neither an apoptotic reaction nor changes of fluorescence intensity HyPer2 were detected, allowing one to assume that H2O2 is not involved in the reaction of tumor cells to bleomycin. Conclusion: The proposed approach can be used for studying the mechanisms of cell death under action of any types of antitumor drugs.