Experimental validation of deuterium oxide-mediated antitumoral activity as it relates to apoptosis in murine malignant astrocytoma cells.
OBJECTIVE
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 D2Oinduced 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.
Uemura
Takeshi
T
Department of Neurosurgery, Shimane Medical University, Izumo, Japan. uemura@shimane-med.ac.jp
Moritake
Kouzo
K
Akiyama
Yasuhiko
Y
Kimura
Yoriyoshi
Y
Shingu
Takashi
T
Yamasaki
Toshiki
T
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001254:Astrocytoma; D001932:Brain Neoplasms; D020169:Caspases / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002462:Cell Membrane / Q000737:chemistry; D003603:Cytotoxins / Q000494:pharmacology; D017666:Deuterium Oxide / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D005434:Flow Cytometry; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008809:Mice, Inbred C3H; D010718:Phosphatidylserines / Q000032:analysis; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects
D000970:Antineoplastic Agents; D003603:Cytotoxins; D010718:Phosphatidylserines; D020169:Caspases; D017666:Deuterium Oxide