MTBITC-induced apoptosis and cell cycle arrest of human hepatoma (HepG2) cells : a link between p53 and human telomerase?

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LamyEvelyn_2008_05_26.pdf (1.83 MB)

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2007

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DOI:
http://dx.doi.org/10.22029/jlupub-14136

Abstract

Experimental data provide strong evidence for the effective inhibition of tumorigenesis by isothiocyanates (ITCs), enzymatic cleavage products of glucosinolates in Brassica vegetables like e.g. cabbage, broccoli or brussel sprouts. A number of ITCs have been identified as strong inhibitors of cell proliferation and apoptosis inducing agents which presents the chance for therapeutic impact of ITC-treatment on malignant transformed cells. Although much is known about ITC-induced apoptosis, many questions remain to be answered. The present study aimed to investigate the chemopreventive properties of 4-methlythiobutylisothiocyanate (MTBITC), which are still largely unknown despite its substantial quantitative presence in food plants. Thereby, this study focused on the hypothesis that p53-dependent induction of apoptosis leads to suppression of telomerase. Human telomerase is stably expressed in the majority of cancer cells but absent in normal tissues which outlines the importance of this enzyme and its compounds in the process of carcinogenesis and provides a promising target for a selective therapeutic approach of malignancies. Proof of a connection between telomerase regulation and the induction of apoptosis by ITCs would present an important detail for a better understanding of the chemotherapeutic properties of these compounds. As a result, this study showed for the first time the suppression of the telomerase catalytic subunit hTERT and the holenzyme activity in malignant transformed cells mediated by an ITC. This was accompanied by a G2/M phase arrest of the cell cycle and apoptosis induction observed by internucleosomal DNA fragmentation, flow cytometry analysis and the detection of single stranded apoptotic DNA. A collapse of the mitochondrial membrane potential, as analysed by flow cytometry with the probe JC-1 and the suppression of the anti-apoptotic multi-domain Bcl-2 protein BclxL, were preceeded by glutathione depletion. Thereby, DNA strand breakage, induced by MTBITC-treatment probably presented the initial step in the growth suppression machinery. This DNA damage was followed by an increase in the protein level of the tumor suppressor p53 and subsequent by the initialization of p21WAF1 protein expression. But also, the activation of p63 expression by MTBITC-treatment could be shown for the first time. As a conclusion, the present findings support the idea of an existing extra-telomeric, cell cycle regulation function of telomerase in HepG2 cells. The results of this study also showed that irrespective of the intense degradation kinetics of MTBITC, the strong cytostatic effect of the ITC was not markedly affected by it and suggests that although ITCs are only present at maximum concentrations in a living system for a rather short time, this might be sufficient to exert their therapeutic effects.

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