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1.  Phenethyl isothiocyanate sensitizes human cervical cancer cells to apoptosis induced by cisplatin 
Molecular nutrition & food research  2011;55(10):1572-1581.
Naturally-occurring chemopreventive agent phenethyl isothiocyanate (PEITC), derived primarily from watercress, has been shown to inhibit cell growth and induce apoptosis in cancer cells. In this study, we examined the potential of PEITC in enhancing cisplatin-induced apoptosis in cervical cancer cells. HeLa cells were exposed to PEITC, cisplatin or both. Pretreatment of cells with PEITC strongly enhanced cisplatin-induced cytotoxicity. PEITC activated the mitogen-activated protein kinases, including JNK, ERK, and p38. The synergistic induction of apoptosis was significantly attenuated by MEK1/2 inhibitor U0126, but not by JNK or p38 inhibitor, suggesting that ERK activation is responsible for the synergistic effect. We found that NF-κB signaling pathway is not involved in the synergistic effect. Sulforaphane and benzyl isothiocyanate, two other members of the isothiocyanate family, also sensitize HeLa cells to apoptosis induced by cisplatin. Furthermore, we found that the synergistic effect was not seen in normal cells. Finally, we demonstrated that Noxa induction was associated with apoptosis induced by PEITC plus cisplatin. Taken together, this study shows that PEITC can sensitize cancer cells to apoptosis induced by cisplatin and this effect is mediated through ERK activation, suggesting the potential of PEITC to be used as an adjuvant with cisplatin in combination therapeutic treatments.
PMCID: PMC3561716  PMID: 21595016
Phenethyl isothiocyanate; cisplatin; apoptosis; ERK activation; sensitization
2.  Identification of Potential Protein Targets of Isothiocyanates by Proteomics 
Chemical research in toxicology  2011;24(10):1735-1743.
Isothiocyanates (ITCs), such as phenethyl isothiocyanate (PEITC) and sulforaphane (SFN), are effective cancer chemopreventive compounds. It is believed that a major mechanism for the cancer preventive activity of ITCs is through induction of cell cycle arrest and apoptosis. However, the upstream molecular targets of ITCs have been underexplored until recently. To identify proteins that are covalently modified by ITCs, human non-small cell lung cancer A549 cells were treated with 14C-PEITC and 14C-SFN and the cell lysates were extracted for analysis by 2-D gel electrophoresis and mass spectrometry. After superimposing the colloidal Coomassie blue protein staining pattern with the pattern of radioactivity obtained from X-ray films, it was clear that only a small fraction of cellular proteins contained radioactivity, presumably resulting from selective binding with PEITC or SFN via thiocarbamation. More than 30 proteins with a variety of biological functions were identified with high confidence. Here we report the identities of these potential ITC target proteins and discuss their biological relevance. The discovery of the protein targets may facilitate studies of the mechanisms by which ITCs exert their cancer preventive activity and provide molecular basis for designing more efficacious ITC compounds.
PMCID: PMC3493163  PMID: 21838287
3.  Increased Phagocyte-Like NADPH Oxidase and ROS Generation in Type 2 Diabetic ZDF Rat and Human Islets 
Diabetes  2011;60(11):2843-2852.
To determine the subunit expression and functional activation of phagocyte-like NADPH oxidase (Nox), reactive oxygen species (ROS) generation and caspase-3 activation in the Zucker diabetic fatty (ZDF) rat and diabetic human islets.
Expression of core components of Nox was quantitated by Western blotting and densitometry. ROS levels were quantitated by the 2′,7′-dichlorofluorescein diacetate method. Rac1 activation was quantitated using the gold-labeled immunosorbent assay kit.
Levels of phosphorylated p47phox, active Rac1, Nox activity, ROS generation, Jun NH2-terminal kinase (JNK) 1/2 phosphorylation, and caspase-3 activity were significantly higher in the ZDF islets than the lean control rat islets. Chronic exposure of INS 832/13 cells to glucolipotoxic conditions resulted in increased JNK1/2 phosphorylation and caspase-3 activity; such effects were largely reversed by SP600125, a selective inhibitor of JNK. Incubation of normal human islets with high glucose also increased the activation of Rac1 and Nox. Lastly, in a manner akin to the ZDF diabetic rat islets, Rac1 expression, JNK1/2, and caspase-3 activation were also significantly increased in diabetic human islets.
We provide the first in vitro and in vivo evidence in support of an accelerated Rac1–Nox–ROS–JNK1/2 signaling pathway in the islet β-cell leading to the onset of mitochondrial dysregulation in diabetes.
PMCID: PMC3198065  PMID: 21911753
4.  Selective depletion of mutant p53 by cancer chemopreventive isothiocyanates and its structure-activity relationships 
Journal of medicinal chemistry  2011;54(3):809-816.
Isothiocyanates (ITCs) derived from cruciferous vegetables induce apoptosis in cancer cells. We demonstrate that certain naturally-occurring ITCs selectively deplete mutant p53, but not the wild type, and do so via a transcription-independent mechanism. Direct p53 binding followed by conformational changes appears to be a mechanism by which mutant p53 is depleted. Structure-activity relationship studies (SARs) using naturally-occurring and synthetic ITCs show that depletion is influenced by the ITC side chain moiety. Furthermore, we show that cells with p53 mutations are more sensitive to cytotoxicity induced by phenethyl isothiocyanate (PEITC) than those with the wild type. 2,2-diphenylethyl ITC, a synthetic ITC and one of the most potent depletors of mutant p53 studied, induces apoptosis to the greatest extent in mutant p53 breast cancer cells. Collectively, this study shows that mutant p53 depletion may be an important novel target for cancer chemoprevention and therapy by natural and synthetic ITCs.
PMCID: PMC3139710  PMID: 21241062
Isothiocyanate(s); p53; protein depletion; structure-activity relationships
5.  Simultaneous Recruitment of Coactivators by Gcn4p Stimulates Multiple Steps of Transcription In Vivo 
Molecular and Cellular Biology  2005;25(13):5626-5638.
Transcriptional activation by Gcn4p is dependent on the coactivators SWI/SNF, SAGA, and Srb Mediator, which are recruited by Gcn4p and stimulate assembly of the preinitiation complex (PIC) at the ARG1 promoter in vivo. We show that recruitment of all three coactivators is nearly simultaneous with binding of Gcn4p at ARG1 and is followed quickly by PIC formation and elongation by RNA polymerase II (Pol II) through the open reading frame. Despite the simultaneous recruitment of coactivators, rapid recruitment of SWI/SNF depends on the histone acetyltransferase (HAT) subunit of SAGA (Gcn5p), a non-HAT function of SAGA, and on Mediator. SAGA recruitment in turn is strongly stimulated by Mediator and the RSC complex. Recruitment of Mediator, by contrast, occurs independently of the other coactivators at ARG1. We confirm the roles of Mediator and SAGA in TATA binding protein (TBP) recruitment and demonstrate that all four coactivators under study enhance Pol II recruitment or promoter clearance following TBP binding. We also present evidence that SWI/SNF and SAGA stimulate transcription elongation downstream from the promoter. These functions can be limited to discrete time intervals, providing evidence for multiple stages in the induction process. Our findings reveal a program of coactivator recruitment and PIC assembly that distinguishes Gcn4p from other yeast activators studied thus far.
PMCID: PMC1156971  PMID: 15964818

Results 1-5 (5)