Enhancement of hyperthermia-induced apoptosis by a new synthesized class of furan-fused tetracyclic compounds

The combined effects of hyperthermia (44 degrees C, 20 min) or X-rays (10 Gy) and a new class of furan-fused tetracyclic synthesized compounds (DFs), on apoptosis in human lymphoma U937 cells were investigated. Among the tested compounds (DF1 approximately 6), the combined treatment of 10 microM DF with TIPS (triisopropylsilyloxy) (Designated #3 DF3) and hyperthermia showed the largest potency to induce DNA fragmentation at 6 h after hyperthermia but no enhancement was observed if it was combined with X-rays. Enhancement of hyperthermia-induced apoptosis by DF3 in a dose-dependent manner was observed. When the cells were treated first with DF3 at a nontoxic concentration of 20 microM, and exposed to hyperthermia afterwards, a significant enhancement of heat-induced apoptosis was evidenced by DNA fragmentation, morphological changes and phosphatidylserine externalization. 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 hyperthermia, was enhanced by DF3. Mitochondrial transmembrane potential was decreased and the activation of caspase-3 and caspase-8 was enhanced in the cells treated with the combination. Externalization of Fas was observed following the combined treatment. Flow cytometry revealed rapid and sustained increase of intracellular superoxide due to DF3, and showed subsequent and transient increase in the formation of intracellular hydrogen peroxide (H(2)O(2)), which was further increased when hyperthermia was combined. These results indicate that the intracellular superoxide and H(2)O(2) generated by DF3 enhance the hyperthermia-induced apoptosis via the Fas-mediated mitochondrial caspase-dependent pathway.