Experimental validation of deuterium oxide—mediated antitumoral activity as it relates to apoptosis in murine malignant astrocytoma cells

Abstract

Object. Deuterium oxide (D2O), or heavy water, affects a variety of biological activities different from those of water. The authors examined the antitumoral effect of D2O on brain neoplasms and demonstrated D2O-mediated cytotoxicity by using a Rous sarcoma virus—induced murine malignant astrocytoma cell line, RSVM. The mechanism of the observed cytotoxicity may involve D2O-induced apoptosis and cell-cycle modulation. Methods. The authors performed an assay with methylthiazol tetrazolium bromide and a trypan blue dye exclusion test to confirm in vitro D2O-mediated cytotoxicity for RSVM cells. At D2O concentrations of 10 to 50%, the cytotoxic effect was dose and time dependent. Flow cytometry analysis revealed programmed cell death (apoptosis) and the accumulation of RSVM cells during the G2/M phase. By applying the terminal deoxynucleotidyl transferase—mediated deoxyuridine triphosphate nick-end labeling method, fluorescein isothiocyanate—annexin V and propidium iodide double staining, and caspase-family protease activity analysis, the authors demonstrated both DNA fragmentation and enhancement of caspase activity after a 48-hour treatment with D2O, thus indicating that D2O induces apoptosis in RSVM cells. Apoptotic DNA fragmentation was completely abolished by the caspase inhibitor Z-VAD-FMK (benzyloxycarbonil-Val-Ala-Aps-fluoromethylketone). The findings indicate that the caspase activation pathway may be involved in D2O-induced apoptosis. Conclusions. The authors found that D2O is cytotoxic to malignant astrocytoma cells. The mechanism of D2O-mediated cytotoxicity involved the induction of apoptosis and cell accumulation during the G2/M phase. This D2O-induced apoptosis is modulated through the caspase activation pathway.