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Apoptotic effect of ethyl-4-isothiocyanatobutanoate is associated with DNA damage, proteasomal activity and induction of p53 and p21cip1/waf1

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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.

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Abbreviations

E-4IB:

Ethyl 4-isothiocyanatobutanoate

ITC:

Isothiocyanate

MMR:

Mismatch repair

NSAID:

Nonsteroidal anti-inflammatory drugs

FITC:

Fluorescein isothiocyanate

FAP:

Familial adenomatous polyposis

HNPCC:

Hereditary non-polyposis colorectal cancer

DSB:

Double strand breaks

ATM:

Ataxia telangiectasia mutated

ATR:

Ataxia telangiectasia-mutated and Rad3-related

GADD45α :

Growth arrest and DNA-damage-inducible, α

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Authors and Affiliations

  1. Laboratory of Tumor Immunology, Cancer Research Institute, Slovak Academy of Sciences, Vlarska 7, 833 91, Bratislava, Slovak Republic

    Juraj Bodo, Jana Jakubikova & Jan Sedlak

  2. Carcinogenesis and Mutagenesis, Cancer Research Institute, Slovak Academy of Sciences, Vlarska 7, 833 91, Bratislava, Slovak Republic

    Ivan Chalupa

  3. Cancer Genetics, Cancer Research Institute, Slovak Academy of Sciences, Vlarska 7, 833 91, Bratislava, Slovak Republic

    Zdena Bartosova

  4. Department of Biochemistry and Microbiology, Slovak University of Technology, Radlinskeho 9, 812 37, Bratislava, Slovak Republic

    Katarina Horakova

  5. Department of Organic Chemistry, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinskeho 9, 812 37, Bratislava, Slovak Republic

    Lubomir Floch

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  1. Juraj Bodo
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  2. Jana Jakubikova
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  3. Ivan Chalupa
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  4. Zdena Bartosova
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  5. Katarina Horakova
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  6. Lubomir Floch
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  7. Jan Sedlak
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Correspondence to Jan Sedlak.

Additional information

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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Bodo, J., Jakubikova, J., Chalupa, I. et al. Apoptotic effect of ethyl-4-isothiocyanatobutanoate is associated with DNA damage, proteasomal activity and induction of p53 and p21cip1/waf1 . Apoptosis 11, 1299–1310 (2006). https://doi.org/10.1007/s10495-006-8760-5

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