Hydrogen peroxide in the reactions of cancer cells to cisplatin.

Background: Hydrogen peroxide (HO) is thought to be one of the key components involved in the responses of tumor cells to chemotherapy. The aim of this study was to reveal the pathways and the phases of cisplatin-induced cell death that are characterized by changes of HO level.

Methods: The genetically encoded cytosolic fluorescent sensor HyPer2 was used for flow cytometric analysis of the cisplatin-induced changes in HO level in HeLa Kyoto cells. Using a vital dye and the apoptotic markers PE Annexin V or TMRE the pathways and stages of cell death were investigated simultaneously with HyPer2 response. The HO level was studied separately in viable and early apoptotic cells after 12, 18, 24 h's incubation with cisplatin at several concentrations with or without the scavenger of reactive oxygen species NAC.

Results: Cisplatin causes dose- and time-dependent increase of HO level in TMRE-positive and PE Annexin V-negative cancer cells. The scavenging of ROS by NAC decreased the HO level and restored cell viability.

Conclusion: НО generation begins in cells that have already lost mitochondrial membrane potential but have not yet externalized phosphatidylserine. Prevention of apoptosis by NAC confirmed the role of HO in apoptosis induction.

General Significance: This is the first time that the sensor HyPer2 has been used in parallel with apoptotic markers and vital dye to demonstrate the role of HO in different stages and types of tumor cell death under chemotherapeutic action.