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J. Biol. Chem., Vol. 283, Issue 32, 22136-22146, August 8, 2008

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Covalent Binding to Tubulin by Isothiocyanates

A MECHANISM OF CELL GROWTH ARREST AND APOPTOSIS^*

Lixin Mi¹, Zhen Xiao, Brian L. Hood^¶, Sivanesan Dakshanamurthy, Xiantao Wang, Sudha Govind, Thomas P. Conrads^¶, Timothy D. Veenstra, and Fung-Lung Chung²

From the Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, D. C. 20057, Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick Inc., NCI-Frederick, Frederick, Maryland 21702, and the ^¶Department of Pharmacology and Chemical Biology and the Clinical Proteomics Facility, University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213

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.

Received for publication, March 25, 2008 , and in revised form, June 3, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant CA100853 from NCI. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. A–C.

¹ To whom correspondence may be addressed: Dept. of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, 3800 Reservoir Road, LL 129, Box 571465, Washington, D. C. 20057. Tel.: 202-687-3648; Fax: 202-687-1068; E-mail: lm293{at}georgetown.edu. ² To whom correspondence may be addressed: Dept. of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, 3800 Reservoir Road, LL 128A, Box 571465, Washington, D. C. 20057. Tel.: 202-687-3021; Fax: 202-687-1068; E-mail: flc6{at}georgetown.edu.

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