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.