A free radical initiator, 2,2'-azobis (2-aminopropane) dihydrochloride enhances hyperthermia-induced apoptosis in human uterine cervical cancer cell lines.
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.
Yuki
Hiroyoshi
H
Department of Obstetrics and Gynecology, Yukiguniyamato General Hospital, 4415 Urasa, Yamato-machi, Niigata 949-7302, Japan.
Kondo
Takashi
T
Zhao
Qing-Li
QL
Fujiwara
Yoshisada
Y
Tanabe
Kiyoshi
K
Ogawa
Ryohei
R
Nakashima
Akitoshi
A
Fushiki
Hiroshi
H
Fujimura
Masaki
M
Saito
Shigeru
S
D000578:Amidines / Q000494:pharmacology*; D017209:Apoptosis; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D053938:DNA Fragmentation; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D005260:Female; D005334:Fever; D005434:Flow Cytometry; D005609:Free Radicals; D006367:HeLa Cells; D006801:Humans; D007477:Ions; D015227:Lipid Peroxidation; D008856:Microscopy, Fluorescence; D016877:Oxidants / Q000494:pharmacology*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D002583:Uterine Cervical Neoplasms / Q000378:metabolism*; D051028:bcl-2-Associated X Protein; D019014:fas Receptor / Q000096:biosynthesis
D000578:Amidines; C494826:BAX protein, human; D005609:Free Radicals; D007477:Ions; D016877:Oxidants; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D019014:fas Receptor; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; C046728:2,2'-azobis(2-amidinopropane); D045304:Cytochromes c; D002118:Calcium