The combined use of platinum nanoparticles and hydrogen molecules induces caspase-dependent apoptosis

We previously reported electrochemically reduced water (ERW), produced near the cathode by electrolysis, exhibits reductive activity. We also revealed that ERW contains Pt nanoparticles (Pt nps) derived from Pt-coated titanium electrodes in addition to high concentration of dissolved molecular hydrogen (H2) by in vitro assay, and Pt nps exhibit powerful ROS scavenger activity and catalysis activity converting H2 to active hydrogen. Our study investigates apoptosis inducibility of H2 and synthesized Pt nps on human promyelocytic leukaemia HL60 cells. Human promyelocytic leukaemia cells (HL60) were cultured in RPMI 1640 medium supplemented with 10% FBS, 2.0 mM l-glutamine, 100 U/ml penicillin and 100 U/ml streptomycin. Cultures were incubated in an atmosphere of 75%(v/v) H2/20%(v/v) O2/5%(v/v) CO2, 75%(v/v) He/20%(v/v) O2/5%(v/v) CO2 atmosphere or 75%(v/v) N2/20%(v/v) O2/5%(v/v) CO2 atmosphere for 12-48 hr after incubated with Pt nps for 2 h. Untreated cultures were included as controls. Cytotoxicity was determined by cell-counter. Apoptosis pathway of HL60 cells was investigated by Sub G-1 assay. Growth suppression was not observed when cells were treated with Pt nps or H2 only. Analysis of cell cycle and activity of caspase-3 suggested that combination use of both Pt nps and H2 induced apoptosis in HL60 cells. Our caspase activity experimentation suggests that apoptosis was caused via caspase-8 activation. These results suggested that atomic hydrogen from H2 induces caspase-8 dependent apoptosis. The cytotoxicity was not detected in Pt nps or H2 separately treated cells. Apoptosis was determined only when cells were treated with both Pt nps and H2, suggesaspase-8 dependent apoptosis was caused by atomic hydrogen produced from H2 by catalyst activity of Pt nps.