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.