Hyperthermia-induced apoptosis and its enhancement in the presence of a temperature-dependent
free radical initiator, 2,2'-azobis (2-aminopropane) dihydrochloride (
AAPH) were examined in human
uterine cervical cancer cell lines, CaSki and HeLa. When both cell lines were treated with
hyperthermia at 44 degrees C for 60 min, minimal apoptosis was observed. When combined with nontoxic
AAPH (50mM), significant enhancement of apoptosis was observed, where the initial rate of
free radical formation was about twice as high than that at 37 degrees C. Augmentation of the growth delay, lipid peroxidation (LPO), activation of
caspase-3 and increase in [Ca2+]i were also observed after the combined treatment. A water-soluble
vitamin E,
Trolox, blocked the increase in [Ca2+]i and an intracellular Ca2+
chelator,
BAPTA-AM, prevented the DNA fragmentation induced by the combination.
Cytochrome c release was also revealed by fluorescence microscopy. However, no significant change in mitochondrial membrane potential and expression of Bax and Bcl-2 was observed. A slight increase in Fas expression was observed only in CaSki cells after the combined treatment. These results indicate that
hyperthermia and
AAPH induce enhanced apoptosis and subsequent cell killing via two pathways; a pathway dependenton increase in LPO and [Ca2+]i, and a pathway associated with
cytochrome c release and subsequent
caspase activation without changes of mitochondrial membrane potential and Bax/Bcl-2 expression in these cell lines. Since it is known that
cancer cells are generally resistant to physical and chemical stress-induced apoptosis,
free radical generators like
AAPH appear to be a useful thermosensitizer for hyperthermic
cancer therapy.