抄録
Sanazole (AK-2123, 3-nitrotriazole derivative, N1-(3-methoxypropyl)-2-(3-nitro-1 H-1, 2, 4-triazol-1-yl)acetamide) has been tested clinically as a hypoxic cell radiosensitizer. The aim of this study was to examine whether sanazole enhances apoptosis induced by hyperthermia (HT) or ionizing radiation (IR) in air condition. The combined effects of HT (44°C, 20 min) or IR (X-rays, 10 Gy) and sanazole on apoptosis in human lymphoma U937 cells were investigated. When the cells were treated first 10 mM sanazole for 40 min, and exposed to HT or IR afterwards, a significant enhancement of HT- or IR-induced apoptosis was evidenced by DNA fragmentation and phosphatidylserine externalization at 6 h after treatment. Flow cytometry revealed rapid and sustained increase of intracellular superoxide due to sanazole, and showed subsequent and transient increase in intracellular peroxide formation. Mitochondrial membrane potential was decreased and the activation of caspase-3 and caspase-8 was enhanced in the cells treated with the combined treatment. The activation of Bid, but no change of Bax and Bcl-2 were observed after the combined treatment. The release of cytochrome c from mitochondria to cytosol, which was induced by HT or IR, was enhanced by sanazole. An increase in the intracellular Ca2+ concentration [Ca2+]i, externalization of Fas, and decrease in Hsp70 were observed following the combined treatment. These results indicate that the intracellular superoxide and peroxides generated by sanazole are involved in the enhancement of apoptosis through Fas-mitochondria caspase and [Ca2+]i-dependent pathways, and a decrease in Hsp70 also contributed to the enhancement of apoptosis.
Keywords: sanazole; apoptosis; hyperthermia; X-irradiation; reactive oxygen species; calcium.
