title: Differential effects of vitamin E and three hydrophilic antioxidants on the actinomycin D-induced and colcemid-accelerated apoptosis in human leukemia CMK-7 cell line.
abstract: The actinomycin D (AD)-induced apoptosis in human leukemia CMK-7 cell line is greatly accelerated by microtubule disruption with colcemid (CL). We studied the effect of antioxidants on this apoptosis in order to learn how the universal signal mediators, reactive oxygen species (ROS), are involved. Caspase-3 activation and DNA fragmentation were both suppressed by vitamin E (VE), t-butylhydroxyanisole, and luteolin. The ROS formation in the AD treatment was evidenced by flow cytometry, and further supported by suppression of caspase-3 activation by superoxide radical-forming enzyme inhibitors (TTFA, rotenone, and DPI). The inhibition of apoptosis by VE was completed during the initial 1-h treatment with AD, but it did not appear when VE was added with CL to washed cells after AD treatment. Luteolin, an iron chelator PDTC, and a water-soluble VE analogue, trolox, inhibited the apoptosis when added with CL after the AD treatment. Western blot analysis showed that the proteolytic cleavage of procaspase-9 and procaspase-3 were both inhibited when VE was added with AD or when luteolin was added with CL, and that the cytochrome c liberation was suppressed by both antioxidants. This result implies that the ROS are initially formed in lipophilic environments (e.g. mitochondrial membrane) and then they diffuse into an aqueous environment (i.e. cytoplasm) where they promote the apoptotic process in combination with the cytoskeletal disruption. Thus, the different antioxidants are effective to scavenge ROS for preventing the apoptosis in its different phases.
authors.affiliation: Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.
authors.forename: Mie
authors.initials: M
authors.lastname: Tsuruga
authors.forename: Yong
authors.initials: Y
authors.lastname: Dang
authors.forename: Yoshihito
authors.initials: Y
authors.lastname: Shiono
authors.forename: Syuichi
authors.initials: S
authors.lastname: Oka
authors.forename: Yoshimitsu
authors.initials: Y
authors.lastname: Yamazaki
mesh_terms: D000975:Antioxidants / Q000494:pharmacology*; D017209:Apoptosis; D015153:Blotting, Western; D002083:Butylated Hydroxyanisole / Q000494:pharmacology; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002374:Catalase / Q000378:metabolism; D002395:Catecholamines / Q000494:pharmacology; D045744:Cell Line, Tumor; D002614:Chelating Agents / Q000494:pharmacology; D045304:Cytochromes c / Q000378:metabolism; D053938:DNA Fragmentation; D003609:Dactinomycin / Q000737:chemistry*; D003703:Demecolcine / Q000737:chemistry*; D004789:Enzyme Activation; D005419:Flavonoids / Q000378:metabolism / Q000494:pharmacology; D005434:Flow Cytometry; D005979:Glutathione Peroxidase / Q000378:metabolism; D006801:Humans; D048288:Imidazolines; D047311:Luteolin; D008954:Models, Biological; D008956:Models, Chemical; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D017382:Reactive Oxygen Species; D012402:Rotenone / Q000494:pharmacology; D013482:Superoxide Dismutase / Q000378:metabolism; D013997:Time Factors; D014810:Vitamin E / Q000378:metabolism*; D014867:Water / Q000737:chemistry
chemical_list: D000975:Antioxidants; D002395:Catecholamines; D002614:Chelating Agents; D005419:Flavonoids; D048288:Imidazolines; D019384:Nucleic Acid Synthesis Inhibitors; D017382:Reactive Oxygen Species; D012402:Rotenone; D014867:Water; D014810:Vitamin E; D003609:Dactinomycin; D002083:Butylated Hydroxyanisole; D045304:Cytochromes c; C011775:(3,4-dihydroxyphenylamino)-2-imidazoline; D002374:Catalase; D005979:Glutathione Peroxidase; D013482:Superoxide Dismutase; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases; D047311:Luteolin; D003703:Demecolcine