Enhancement of hyperthermia-induced apoptosis by sanazole in human lymphoma U937 cells

Sanazole has been tested clinically as a hypoxic cell radiosensitizer. The aim of the present study was to investigate whether sanazole enhances apoptosis induced by hyperthermia at 44°C for 20 min in human lymphoma U937 cells. Sanazole alone induced continuous increase in the intracellular superoxide generation in a time-dependent manner and transient increase in the peroxide formation, which further were enhanced at 1 hour after HT treatment. Moreover, when the cells were treated first with 10 mM sanazole for 40 min, exposed to HT at 44°C for 20 min and the cells were further treated with the drug at 37°C for 6 h, a significant enhancement of HT-induced apoptosis was evidenced by DNA fragmentation, morphological changes and phosphatidylserine externalization. Studying the apoptotic pathways involved in this enhancement, we found that loss of the mitochondrial membrane potential, release of cytochrome c from mitochondria to cytosol, and activation of caspase-3 and caspase-8 was enhanced significantly in the U937 cells after the combined treatment. Moreover, this combination enhanced activation of Bid, and down regulation of Hsp70. In addition, an increase in the intracellular Ca ² concentration ([Ca²]i), and externalization of Fas were observed immediately after sanazole and HT treatment. Our data indicate that sanazole can enhance the hyperthermia induced-apoptosis through the Fas-caspase-8- and [Ca²]i-dependent apoptotic pathways. In addition, the down regulation of Hsp70 contributed to this enhancement.