Induction of apoptosis is believed to be an important mechanism by which isothiocyanates (ITCs) inhibit carcinogenesis. A number of signaling pathways have been shown to link to this process;however, the upstream targets of ITCs have not been fully investigated. Our studies have shown that, as electrophiles, ITCs readily bind to glutathione and proteins once inside the cells and that intracellular proteins eventually serve as the predominant binding target of ITCs. We found that the total protein binding affinities of phenethyl ITC (PEITC) and sulforaphane (SFN) correlate well with their activities toward apoptosis induction. We identified tubulin as one of the protein targets of ITCs and its binding by ITCs can trigger conformational changes, disruption of microtubule network, selective degradation and, finally, cell cycle arrest and apoptosis. Recently, we made an intriguing observation that ITC treatment can deplete mutant p53 in human cancer cells, but not wild-type p53, and that depletion is accompanied by restoration of the wild-type-like activities. Due to gain of function, the mutant p53 cells are more proliferative and resistant to cell death. Therefore, the removal of mutant p53 and the restoration of its wild-type activity could be an important mechanism by which oncogenic cells can be eliminated by ITC-induced apoptosis. Because greater than 50% of human cancers contain p53 mutation, in this proposal we will focus our studies on the effects of ITCs on p53 protein. Our main hypothesis is that binding to the mutant p53 protein by ITCs can trigger conformational changes, leading to the mutant p53 depletion and restoration of the wild-type activities and consequently, causing cells to undergo apoptosis. We propose five aims to examine this hypothesis:
Aim 1 will investigate the mechanisms by which ITCs selectively deplete mutant p53 and restore the wild-type activity;
Aim 2 will determine the functional roles of mutant p53 depletion and its wild- type function restoration;
Aim 3 will examine Structure-Activity Relationships (SARs) of ITCs to discover more efficacious compounds for depleting mutant p53 and restoring the wild-type activities;
Aim 4 will study effects of ITCs on mutant p53 protein levels and lung tumor formation induced by benzo[a]pyrene in a transgenic mutant p53 A/J mouse model;
and Aim 5 will investigate the effects of drinking watercress juice (rich in PEITC) on mutant p53 and apoptosis in oral cells collected from heavy smokers.

Public Health Relevance

It is believed that apoptosis induction is an important mechanism by which isothiocyanates (ITCs) act as chemopreventive agents for various cancers. We observed that ITC treatment can deplete mutant p53, but not wild type p53, in cells and the depletion is accompanied by restoration of the wild-type-like activities. These findings suggest a mechanism by which ITCs can sensitize cells with mutant p53 to apoptosis induction. Because more than 50% human cancers have p53 mutation, in this program we will investigate the mechanisms by which ITCs deplete mutant p53 and restore its wild-type functions and understand the functional consequences. In addition, we will test the effects of ITCs in a mutant p53 transgenic animal model and by using oral cells collected from heavy smokers before and after drinking watercress juice rich in phenethyl ITCs. We also propose to carry out Structural-Activity Relationship (SAR) studies of ITCs for the discovery of new lead compounds. This research will allow design more effective small compounds related to ITCs that target to mutant p53 for cancer prevention and therapy.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Chemo/Dietary Prevention Study Section (CDP)
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Seifried, Harold E
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Georgetown University
Internal Medicine/Medicine
Schools of Medicine
United States
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Fu, Ying; Mi, Lixin; Sanda, Miloslav et al. (2014) A Click Chemistry Approach to Identify Protein Targets of Cancer Chemopreventive Phenethyl Isothiocyanate. RSC Adv 4:3920-3923
Xiao, Zhen; Mi, Lixin; Chung, Fung-Lung et al. (2012) Proteomic analysis of covalent modifications of tubulins by isothiocyanates. J Nutr 142:1377S-81S
Mi, Lixin; Gan, Nanqin; Chung, Fung-Lung (2011) Isothiocyanates inhibit proteasome activity and proliferation of multiple myeloma cells. Carcinogenesis 32:216-23
Wang, Xiantao; Govind, Sudha; Sajankila, Shyama P et al. (2011) Phenethyl isothiocyanate sensitizes human cervical cancer cells to apoptosis induced by cisplatin. Mol Nutr Food Res 55:1572-81
Wang, Xiantao; Di Pasqua, Anthony J; Govind, Sudha et al. (2011) Selective depletion of mutant p53 by cancer chemopreventive isothiocyanates and their structure-activity relationships. J Med Chem 54:809-16
Sun, Xiaoyun; Mi, Lixin; Liu, Jin et al. (2011) Sulforaphane prevents microcystin-LR-induced oxidative damage and apoptosis in BALB/c mice. Toxicol Appl Pharmacol 255:9-17
Mi, Lixin; Hood, Brian L; Stewart, Nicolas A et al. (2011) Identification of potential protein targets of isothiocyanates by proteomics. Chem Res Toxicol 24:1735-43
Mi, Lixin; Di Pasqua, Anthony J; Chung, Fung-Lung (2011) Proteins as binding targets of isothiocyanates in cancer prevention. Carcinogenesis 32:1405-13
Mi, Lixin; Xiao, Zhen; Veenstra, Timothy D et al. (2011) Proteomic identification of binding targets of isothiocyanates: A perspective on techniques. J Proteomics 74:1036-44
Mi, Lixin; Sirajuddin, Paul; Gan, Nanqin et al. (2010) A cautionary note on using N-acetylcysteine as an antagonist to assess isothiocyanate-induced reactive oxygen species-mediated apoptosis. Anal Biochem 405:269-71

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