Apoptotic effect of ethyl-4-isothiocyanatobutanoate is associated with DNA damage, proteasomal activity and induction of p53 and p21cip1/waf1 |
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| Authors: | Juraj Bodo Jana Jakubikova Ivan Chalupa Zdena Bartosova Katarina Horakova Lubomir Floch Jan Sedlak |
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| Institution: | (1) Laboratory of Tumor Immunology, Cancer Research Institute, Slovak Academy of Sciences, Vlarska 7, 833 91 Bratislava, Slovak Republic;(2) Carcinogenesis and Mutagenesis, Cancer Research Institute, Slovak Academy of Sciences, Vlarska 7, 833 91 Bratislava, Slovak Republic;(3) Cancer Genetics, Cancer Research Institute, Slovak Academy of Sciences, Vlarska 7, 833 91 Bratislava, Slovak Republic;(4) Department of Biochemistry and Microbiology, Slovak University of Technology, Radlinskeho 9, 812 37 Bratislava, Slovak Republic;(5) Department of Organic Chemistry, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinskeho 9, 812 37 Bratislava, Slovak Republic |
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| 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 (γ-H2AX), mitotic fraction (phospho-histone H3), cell cycle modulation, apoptosis induction (sub-G0 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, p21cip1/waf1 and GADD45α 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 (IC50 8 μM vs. 4 μM). In both cell lines E-4IB treatment induced phosphorylation of H2AX, increase of p53 protein level, phospho-histone
H3 staining, and G2/M arrest. The sub-G0 fragmentation was accompanied by PARP degradation, decreased mitochondrial transmembrane potential, and diminished p21cip1/waf1 protein expression in TK6 cells. Caspase inhibitor Z-VAD-fmk decreased E-4IB induced sub-G0 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.
This work was supported in part by Slovak Governmental Research and Development sub-program Food-quality and safety No. 2003SP270280E010280E01,
National Program “Use of Cancer Genomics to Improve the Human Population Health”, project 2003 SP 510280800/0280801, European
Commission project (QLG1-CT-2000-01230), and VEGA projects 2/4069 and 2/3161/23. |
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| Keywords: | Apoptosis Double strand breaks Isothiocyanate E-4IB Mismatch repair Mitotic block |
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