Effects of antioxidants and caspase-3 inhibitor on the phenylethyl isothiocyanate-induced apoptotic signaling pathways in human PLC/PRF/5 cells.
Phenylethyl isothiocyanate (PEITC) is a well recognized potential chemopreventive compound against human cancers. In this study, the molecular mechanism of PEITC-induced apoptosis was examined with two antioxidants (N-acetyl-cysteine and vitamin E) and a caspase-3 inhibitor (z-DEVD-fmk). Results demonstrated that PEITC significantly induced human hepatoma PLC/PRF/5 (CD95-negative) cells undergoing apoptosis. Treatment with 0 approximately 10 microM PEITC-triggered cell apoptosis as revealed by the externalization of annexin V-targeted phosphatidylserine and the subsequent appearance of sub-G1 population. Results also displayed that PEITC-induced apoptosis involves the up-regulation of p53 and Bax protein, down-regulation of the XIAP, Bcl-2, Bcl-(XL) and Mcl-1 proteins, cleavage of Bid, and the release of cytochrome c and Smac/Diablo, which were accompanied by the activation of caspases -9, -3 and -8. PEITC-induced the generation of reactive oxygen species and the decrease of mitochondrial membrane potential (Deltapsim) in a time-dependent pattern. N-acetyl-cysteine and vitamin E at 100 microM, and z-DEVD-fmk at 50 microM markedly blocked PEITC-induced apoptosis, which was demonstrated by a decline in the reactive oxygen species generation and the release of the cytochrome c and Smac/Diablo from mitochondria to the cytosol. N-acetyl-cysteine, vitamin E and z-DEVD-fmk also prevented the PEITC in inducing the loss of Deltapsim. They also affected the activity of XIAP and Bax proteins. Taken together, these studies suggest that PEITC is an apoptotic inducer that acts on the mitochondria and the feedback amplification loop of caspase-8/Bid pathways in PLC/PRF/5 cells.
Wu
Shu-Jing
SJ
Graduate Institute of Natural products, College of Pharmacy, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan.
Ng
Lean Teik
LT
Lin
Chun-Ching
CC
D000111:Acetylcysteine / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins; D053477:Apoptotic Protease-Activating Factor 1; D051023:BH3 Interacting Domain Death Agonist Protein; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D002352:Carrier Proteins / Q000378:metabolism; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D045304:Cytochromes c / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D005434:Flow Cytometry; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects / Q000502:physiology; D047908:Intracellular Signaling Peptides and Proteins; D017879:Isothiocyanates / Q000494:pharmacology*; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D024101:Mitochondrial Proteins / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects; D014810:Vitamin E / Q000494:pharmacology; D051636:X-Linked Inhibitor of Apoptosis Protein; D051028:bcl-2-Associated X Protein; D019014:fas Receptor / Q000378:metabolism
C506592:APAF1 protein, human; D000975:Antioxidants; D051017:Apoptosis Regulatory Proteins; D053477:Apoptotic Protease-Activating Factor 1; C494826:BAX protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D002352:Carrier Proteins; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; C412403:DIABLO protein, human; D047908:Intracellular Signaling Peptides and Proteins; D017879:Isothiocyanates; D024101:Mitochondrial Proteins; D009842:Oligopeptides; D011506:Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D051028:bcl-2-Associated X Protein; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; D019014:fas Receptor; D014810:Vitamin E; C058305:phenethyl isothiocyanate; D045304:Cytochromes c; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D000111:Acetylcysteine