Pharmaceutical Sciences Faculty Publications

Title

Covalent Binding to Tubulin by Isothiocyanates: A Mechanism of Cell Growth Arrest and Apoptosis

Document Type

Article

Publication Date

8-8-2008

Journal Title

The Journal of Biological Chemistry

ISSN

0021-9258

Volume

283

Issue

32

First Page

22136

Last Page

22146

DOI

10.1074/jbc.M802330200

PubMed ID

18524779

PubMed Central® ID

PMC2494917

Abstract

Isothiocyanates (ITCs) found in cruciferous vegetables, including benzyl-ITC (BITC), phenethyl-ITC (PEITC), and sulforaphane (SFN), inhibit carcinogenesis in animal models and induce apoptosis and cell cycle arrest in various cell types. The biochemical mechanisms of cell growth inhibition by ITCs are not fully understood. Our recent study showed that ITC binding to intracellular proteins may be an important initiating event for the induction of apoptosis. However, the specific protein target(s) and molecular mechanisms were not identified. In this study, two-dimensional gel electrophoresis of human lung cancer A549 cells treated with radiolabeled PEITC and SFN revealed that tubulin may be a major in vivo binding target for ITC. We examined whether binding to tubulin by ITCs could lead to cell growth arrest. The proliferation of A549 cells was significantly reduced by ITCs, with relative activities of BITC > PEITC > SFN. All three ITCs also induced mitotic arrest and apoptosis with the same order of activity. We found that ITCs disrupted microtubule polymerization in vitro and in vivo with the same order of potency. Mass spectrometry demonstrated that cysteines in tubulin were covalently modified by ITCs. Ellman assay results indicated that the modification levels follow the same order, BITC > PEITC > SFN. Together, these results support the notion that tubulin is a target of ITCs and that ITC-tubulin interaction can lead to downstream growth inhibition. This is the first study directly linking tubulin-ITC adduct formation to cell growth inhibition.

Keywords

Apoptosis, cell cycle, tumor, cell proliferation, chemical precipitation, cysteine, isothiocyanates, microtubules, molecular sequence data, protein binding, protein structure, tertiary, sulfhydryl compounds, tubulin

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