title: Apoptotic effect of ethyl-4-isothiocyanatobutanoate is associated with DNA damage, proteasomal activity and induction of p53 and p21cip1/waf1.
abstract: The effect of synthetic isothiocyanate ethyl-4-isothiocyanatobutanoate (E-4IB) on survival of mismatch repair-proficient TK6 and -deficient MT1 cell lines as well as the influence of proteasomal inhibitor MG132, caspase inhibitor Z-VAD-fmk, and ATM inhibitor caffeine on E-4IB modulation of cell cycle and apoptosis was evaluated. Flow cytometric analyses of DNA double strand breaks (gamma-H2AX), mitotic fraction (phospho-histone H3), cell cycle modulation, apoptosis induction (sub-G(0) fraction and fluorescein diacetate staining), and dissipation of transmembrane mitochondrial potential (JC-1 staining) were performed. Western blotting was used for the evaluation of ERK activation, expression of p53, p21(cip1/waf1) and GADD45alpha proteins, as well as PARP fragmentation. Analysis of mitotic nuclei was performed for chromosomal aberrations assessment. MT1 cells were more resistant to E-4IB treatment then TK6 cells (IC(50) 8 muM vs. 4 muM). In both cell lines E-4IB treatment induced phosphorylation of H2AX, increase of p53 protein level, phospho-histone H3 staining, and G(2)/M arrest. The sub-G(0) fragmentation was accompanied by PARP degradation, decreased mitochondrial transmembrane potential, and diminished p21(cip1/waf1) protein expression in TK6 cells. Caspase inhibitor Z-VAD-fmk decreased E-4IB induced sub-G(0) fragmentation and extent of apoptosis in TK6 cells, while proteasome inhibitor MG132 increased number of apoptotic cells in both cell lines tested. A number of aberrant metaphases and clastogenic effect of high E-4IB concentration was observed. The synthetic isothiocyanate E-4IB induced DNA strand breaks, increased mitotic fraction and apoptosis potentiated by MG132 inhibitor in both mismatch repair-proficient and -deficient cell lines.
authors.affiliation: Laboratory of Tumor Immunology, Cancer Research Institute, Slovak Academy of Sciences, Vlarska 7, 833 91 Bratislava, Slovak Republic.
authors.forename: Juraj
authors.initials: J
authors.lastname: Bodo
authors.forename: Jana
authors.initials: J
authors.lastname: Jakubikova
authors.forename: Ivan
authors.initials: I
authors.lastname: Chalupa
authors.forename: Zdena
authors.initials: Z
authors.lastname: Bartosova
authors.forename: Katarina
authors.initials: K
authors.lastname: Horakova
authors.forename: Lubomir
authors.initials: L
authors.lastname: Floch
authors.forename: Jan
authors.initials: J
authors.lastname: Sedlak
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D064007:Ataxia Telangiectasia Mutated Proteins; D002087:Butyrates / Q000008:administration & dosage / Q000494:pharmacology*; D002110:Caffeine / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors; D002460:Cell Line; D002869:Chromosome Aberrations / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000096:biosynthesis*; D004249:DNA Damage; D005434:Flow Cytometry; D006801:Humans; D017879:Isothiocyanates / Q000008:administration & dosage / Q000494:pharmacology*; D007976:Leupeptins / Q000494:pharmacology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis*
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D002087:Butyrates; C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D017879:Isothiocyanates; D007976:Leupeptins; D016159:Tumor Suppressor Protein p53; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C082832:ethyl 4-isothiocyanatobutanoate; D002110:Caffeine; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde