Cholesteryl-hemisuccinate-induced apoptosis of promyelocytic leukemia HL-60 cells through a cyclosporin A-insensitive mechanism |
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Authors: | Yamada Katsuhiko Arita Kayo Kobuchi Hirotsugu Yamamoto Shinji Yoshioka Tamotsu Tamai Hiroshi Utsumi Kozo |
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Affiliation: | Department of Pediatrics, Osaka Medical College, 2-7 Daigakucho, Takatsuki 569-8686, Osaka, Japan. |
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Abstract: | We reported previously that alpha-tocopheryl-succinate (VES) induced apoptosis of cultured human promyelocytic leukemia cells (HL-60) (Free Radic Res 2000;33:407-18). We have now studied the effect of cholesteryl-hemisuccinate (CS) on the fate of HL-60 cells to clarify whether CS has an effect similar to that of VES. CS inhibited the growth of HL-60 cells without differentiation to granulocytes and induced DNA fragmentation and ladder formation. CS inhibited the phosphorylation of pleckstrin homology domain-containing protein kinase B (Akt) and initiated the activation of a caspase cascade. CS triggered the reaction leading to the cleavage of Bid and also released cytochrome c (Cyt. c) from mitochondria. In addition, CS induced mitochondrial membrane depolarization and translocation of Bax to mitochondria in HL-60 cells. However, CS did not induce an increase in the concentration of intracellular calcium ions in HL-60 cells. The membrane depolarization, Cyt. c release, and DNA fragmentation were inhibited by z-VAD-fluoromethylketone (z-VAD-fmk), a pan-caspase inhibitor, but not by cyclosporin A, an inhibitor of membrane permeability transition. These results suggested that CS-induced apoptosis of HL-60 cells might be caused by inhibiting Akt phosphorylation following cleavage of Bid through caspase-8 activation and subsequently via an Apaf complex-caspase cascade pathway. |
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Keywords: | Akt, pleckstrin homology domain-containing protein kinase B [Ca2+]i, intracellular concentration of Ca2+ CS, cholesteryl-hemisuccinate CsA, cyclosporin A Cyt. c, ferricytochrome c JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzinidazol carbocyanine iodide KRP, Krebs-Ringer-phosphate buffer MPT, membrane permeability transition pCPT-cAMP, 8-(4-chlorophenylthio)adenosine 3′:5′-cyclic monophosphate PKC, protein kinase C ROS, reactive oxygen species VES, α-tocopheryl-succinate z-VAD-fmk, z-Val-Ala-Asp(OMe) fluoromethylketone. |
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