Ying Fu, Lixin Mi, M. Aggarwal
Apr 15, 2013
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Journal
Cancer Research
Abstract
Isothiocyanates (ITCs) derived from cruciferous vegetables are inhibitors of carcinogenesis in animal models and can induce cell cycle arrest and apoptosis in a variety of cancer cell lines. It is known that ITCs can conjugate with glutathione (GSH) and induce cellular oxidative stress; ITCs can also bind to intracellular proteins (e.g. tubulin) which may constitute a mechanism underlying their cancer preventive effects. To fully understand the cellular responses to ITCs, it is crucial to develop an unbiased method that can rapidly identify their protein targets. In this study, the direct protein targets of phenethyl isothiocyanate (PEITC) were investigated by using click chemistry. An alkyne-tagged ITC was used as an analog of PEITC, a known cancer prevention agent. The tagged ITC (N-(2-(4-methoxyalkyne)phenethyl)isothiocyanate (NPPEITC) was designed and synthesized with a moiety for copper-catalyzed Huisgen 1,3 dipolar cycloaddition reaction (click chemistry), the tagged ITC, NPPEITC, was also found to show similar biological activities to PEITC; The click-chemistry reaction conditions of copper-catalyzed azide-alkyne cycloaddtion (CuAAC) for NPPEITC and N-(2-(4-methoxyalkyne)phenylethyl)acetamide (NPA, a negative control) with biotin azide were optimized and the reaction product was isolated and analyzed by mass spectrometry. A model click reaction between NPPEITC and tubulin was employed to validate this method. The western-blot showed that the tubulin protein is modified by biotin after NPPEITC treatment then followed by click reaction with biotin azide. In contrast, no biotin was found on NPA treated tubulin. Secondly, cancer cells exposed to the tagged ITC (NPPEITC or NPA) was lysed and exposed to click chemistry reagents to conjugate ITC-adducted proteins with biotin for subsequent affinity purification; the ITC conjugated proteins were identified and quantified using proteomic analysis by LC/MS/MS.(Supported by NCI grant CA100853) Citation Format: Ying Fu, Lixin Mi, Monika Aggarwal, Daniel M. Appella, Fung-Lung Chung. Methodology design for identifications of protein targets of chemopreventive isothiocyanates using click chemistry. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3656. doi:10.1158/1538-7445.AM2013-3656