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Cancer is a complex disease and cancer development takes 10–50 years involving epigenetics. Evidence suggests that approximately 80% of human cancers are linked to environmental factors impinging upon genetics/epigenetics. Because advanced metastasized cancers are resistant to radiotherapy/chemotherapeutic drugs, cancer prevention by relatively nontoxic chemopreventive “epigenetic modifiers” involving epigenetics/epigenomics is logical. Isothiocyanates are relatively nontoxic at low nutritional and even higher pharmacologic doses, with good oral bioavailability, potent antioxidative stress/antiinflammatory activities, possess epigenetic-modifying properties, great anticancer efficacy in many in vitro cell culture and in vivo animal models. This review summarizes the latest advances on the role of epigenetics/epigenomics by isothiocyanates in prevention of skin, colon, lung, breast, and prostate cancers. The exact molecular mechanism how isothiocyanates modify the epigenetic/epigenomic machinery is unclear. We postulate “redox” processes would play important roles. In addition, isothiocyanates sulforaphane and phenethyl isothiocyanate, possess multifaceted molecular mechanisms would be considered as “general” cancer preventive agents not unlike chemotherapeutic agents like platinum-based or taxane-based drugs. Analogous to chemotherapeutic agents, the isothiocyanates would need to be used in combination with other nontoxic chemopreventive phytochemicals or drugs such as NSAIDs, 5-α-reductase/aromatase inhibitors targeting different signaling pathways would be logical for the prevention of progression of tumors to late advanced metastatic states.

2-Phenethyl isothiocyanate (PEITC) is a natural product found as a conjugate in cruciferous vegetables. It has been reported to have preventative properties against lung cancer and to inhibit metabolic activation of tobacco carcinogens. In this study, we evaluated the ability of PEITC to influence the metabolism of the human carcinogen 1,3-butadiene in current smokers in a phase II clinical trial with a crossover design. Urinary mercapturic acids of 1,3-butadiene were quantified at baseline and during PEITC treatment. Seventy-nine smokers were randomly assigned to one of two arms: PEITC followed by placebo or placebo followed by PEITC. During the 1-week treatment period, each subject took PEITC (10 mg in 1 mL of olive oil, 4 times per day). There was a 1-week washout period between the PEITC and placebo periods. Oral ingestion of PEITC increased urinary levels of BD-mercapturic acids (MHBMA and DHBMA) by 11.1% and 3.7%, respectively, but these increases were not statistically significant (P = 0.17 and 0.64, respectively). A much stronger effect was observed among subjects with the null genotype of both GSTM1 and GSTT1: in these individuals, PEITC increased urinary levels of MHBMA by 58.7% (P = 0.004) and 90.0% (P = 0.001), respectively, but did not have a significant effect on urinary DHBMA. These results reveal a potentially protective effect of PEITC treatment with respect to the detoxification of 1,3-butadiene in cigarette smokers, specifically in those null for GSTT1, and provide further evidence in support of stronger chemopreventive effects from consumption of dietary isothiocyanates in these individuals.

We have shown previously that dietary administration of phenethyl isothiocyanate (PEITC), a small molecule from edible cruciferous vegetables, significantly decreases the incidence of poorly differentiated prostate cancer in Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice without any side effects. In this study, we investigated the role of c-Myc–regulated glycolysis in prostate cancer chemoprevention by PEITC. Exposure of LNCaP (androgen-responsive) and 22Rv1 (castration-resistant) human prostate cancer cells to PEITC resulted in suppression of expression as well as transcriptional activity of c-Myc. Prostate cancer cell growth inhibition by PEITC was significantly attenuated by stable overexpression of c-Myc. Analysis of the RNA-Seq data from The Cancer Genome Atlas indicated a significant positive association between Myc expression and gene expression of many glycolysis-related genes, including hexokinase II and lactate dehydrogenase A. Expression of these enzyme proteins and lactate levels were decreased upon PEITC treatment in prostate cancer cells, and these effects were significantly attenuated by ectopic expression of c-Myc. A normal prostate stromal cell line (PrSC) was resistant to lactic acid suppression by PEITC treatment. Prostate cancer chemoprevention by PEITC in TRAMP mice was associated with a significant decrease in plasma lactate and pyruvate levels. However, a 1-week intervention with 10 mg PEITC (orally, 4 times/day) was not sufficient to decrease lactate levels in the serum of human subjects. These results indicated that although prostate cancer prevention by PEITC in TRAMP mice was associated with suppression of glycolysis, longer than 1-week intervention might be necessary to observe such an effect in human subjects. Cancer Prev Res; 11(6); 337–46. ©2018 AACR.

Cigarette smoke contains relatively large quantities of volatile organic toxicants or carcinogens such as benzene, acrolein, and crotonaldehyde. Among their detoxification products are mercapturic acids formed from glutathione conjugation, catalyzed in part by glutathione S-transferases (GST). A randomized phase II clinical trial with a crossover design was conducted to evaluate the effect of 2-phenethyl isothiocyanate (PEITC), a natural product formed from gluconasturtiin in certain cruciferous vegetables, on the detoxification of benzene, acrolein, and crotonaldehyde in 82 cigarette smokers. Urinary mercapturic acids of benzene, acrolein, and crotonaldehyde at baseline and during treatment were quantified. Overall, oral PEITC supplementation increased the mercapturic acid formed from benzene by 24.6% (P = 0.002) and acrolein by 15.1% (P = 0.005), but had no effect on crotonaldehyde. A remarkably stronger effect was observed among subjects with the null genotype of both GSTM1 and GSTT1: in these individuals, PEITC increased the detoxification metabolite of benzene by 95.4% (P < 0.001), of acrolein by 32.7% (P = 0.034), and of crotonaldehyde by 29.8% (P = 0.006). In contrast, PEITC had no effect on these mercapturic acids in smokers possessing both genes. PEITC had no effect on the urinary oxidative stress biomarker 8-iso-prostaglandin F2α or the inflammation biomarker prostaglandin E2 metabolite. This trial demonstrates an important role of PEITC in detoxification of environmental carcinogens and toxicants which also occur in cigarette smoke. The selective effect of PEITC on detoxification in subjects lacking both GSTM1 and GSTT1 genes supports the epidemiologic findings of stronger protection by dietary isothiocyanates against the development of lung cancer in such individuals. Cancer Prev Res; 9(7); 598–606. ©2016 AACR.

2-Phenethyl isothiocyanate (PEITC), a natural product found as a conjugate in watercress and other cruciferous vegetables, is an inhibitor of the metabolic activation and lung carcinogenicity of the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in F344 rats and A/J mice. We carried out a clinical trial to determine whether PEITC also inhibits the metabolic activation of NNK in smokers. Cigarette smokers were recruited and asked to smoke cigarettes containing deuterium-labeled [pyridine-D4]NNK for an acclimation period of at least 1 week. Then subjects were randomly assigned to one of two arms: PEITC followed by placebo, or placebo followed by PEITC. During the 1-week treatment period, each subject took PEITC (10 mg in 1 mL of olive oil, 4 times per day). There was a 1-week washout period between the PEITC and placebo periods. The NNK metabolic activation ratio [pyridine-D4]hydroxy acid/total [pyridine-D4]NNAL was measured in urine samples to test the hypothesis that PEITC treatment modified NNK metabolism. Eighty-two smokers completed the study and were included in the analysis. Overall, the NNK metabolic activation ratio was reduced by 7.7% with PEITC treatment (P = 0.023). The results of this trial, while modest in effect size, provide a basis for further investigation of PEITC as an inhibitor of lung carcinogenesis by NNK in smokers. Cancer Prev Res; 9(5); 396–405. ©2016 AACR.

Androgen receptor (AR) over-expression, mutation and the emergence of AR splice variants (AR-Vs) promote disease-driving AR signaling in castration-resistant prostate cancer (CRPC). In addition to the sustained AR activation, apoptosis evasion adds extra hurdle for treating hormone refractory prostate cancer (PCa). To overcome these challenges, we firstly designed isothiocyanate (ITC)-containing hybrid AR antagonist (ITC-ARi) and then rationally combined ITC-ARi with glutathione (GSH) biosynthesis inhibitor buthionine sulphoximine (BSO) to efficiently downregulate AR/AR-V and induce ferroptosis in enzalutamide (Enz)-resistant PCa cells. Naturally occurring ITCs (e.g., sulforaphane and phenylethyl isothiocyanate) display pleotropic anti-PCa activities. A representative ITC-ARi 2-63 is designed by incorporating ITC into an AR ligand scaffold. The sulfhydryl reactivity of ITC is transiently masked as N-acetyl cysteine (NAC) conjugate that gradually releases parental free ITC in aqueous solution. 2-63 antagonizes AR transactivation, downregulates AR/AR-V7, and upregulates cellular stress markers Hsp70 and heme oxygenase-1 (HO-1), indicating the disruption of heat shock proteins (Hsps) and the activation of Nrf2 pathway. Because the anticancer effects of ITC/ITC-NAC conjugate are attenuated by GSH-participated conjugation or thiol exchange, we rationally combined 2-63 with BSO for enhanced anti-PCa activities. 2-63 and BSO synergistically reduce viability of multiple PCa cell lines (VCaP, C4-2 and 22Rv-1), and the noncancerous prostatic RWPE-1 cell is much less affected. Because 2-63 plus BSO does not upregulate typical apoptosis markers, we investigated alternative cell death mechanism in combinatorial treatment. Drug combination-caused cell viability loss is effectively rescued by iron chelator deferoxamine (DFO), antioxidants α-tocopherol (α-Toc) and ferrostatin-1 (Fer-1), or the inhibitor of HO-1 Zinc (II) protoporphyrin IX (ZnPP). Both Fer-1 and α-Toc are potent radical scavengers blocking lipid hydroperoxide generation, and ZnPP suppresses HO-1-catalyzed upregulation of intracellular Fe2+. The reverse of growth suppression by DFO, antioxidants and ZnPP supports the induction of ferroptosis, a regulated non-apoptotic cell death caused by reactive oxygen species (ROS)- and iron-dependent lipid peroxidation. 2-63 and BSO also more efficiently downregulate AR/AR-V than 2-63 alone in Enz-resistant PCa cells. Interestingly, ferroptosis-preventing DFO and Fer-1 do not rescue AR/AR-V7 and are unable to suppress the upregulation of Hsp70 and HO-1. Our results suggest that the synergism of 2-63 and BSO occurs through increasing drug accessibility to cellular targets, expanding availability of iron and potentially affecting glutathione peroxidase 4 (GPX4) activity, the most important cellular defense to eliminate lipid hydroperoxide. Combining ITC-ARi and GSH-depleting agents could be a new concept leading to effective CRPC treatment. Citation Format: Zhihui Qin, Liping Xu, Siyu Ou. Isothiocyanate-containing hybrid androgen receptor (AR) antagonist downregulates AR and induces ferroptosis in GSH–deficient prostate cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A110. doi:10.1158/1535-7163.TARG-19-A110

Isothiocyanates (ITCs) derived from cruciferous vegetables, including phenethyl isothiocyanate (PEITC) and sulforaphane (SFN), exhibit in vivo activity against prostate cancer in a xenograft and transgenic mouse model, and thus are appealing for chemoprevention of this disease. Watercress constituent PEITC and SFN-rich broccoli sprout extract are under clinical investigations but the molecular mechanisms underlying their cancer chemopreventive effects are not fully understood. The present study demonstrates that chemokine receptor CXCR4 is a novel target of ITCs in prostate cancer cells. Exposure of prostate cancer cells (LNCaP, 22Rv1, C4-2, and PC-3) to pharmacologically applicable concentrations of PEITC, benzyl isothiocyanate (BITC), and SFN (2.5 and 5 μmol/L) resulted in downregulation of CXCR4 expression. None of the ITCs affected secretion of CXCR4 ligand (stromal-derived factor-1). In vivo inhibition of PC-3 xenograft growth upon PEITC treatment was associated with a significant decrease in CXCR4 protein level. A similar trend was discernible in the tumors from SFN-treated TRAMP mice compared with those of control mice, but the difference was not significant. Stable overexpression of CXCR4 in PC-3 cells conferred significant protection against wound healing, cell migration, and cell viability inhibition by ITCs. Inhibition of cell migration resulting from PEITC and BITC exposure was significantly augmented by RNAi of CXCR4. This study demonstrates, for the first time, that cancer chemopreventive ITCs suppress CXCR4 expression in prostate cancer cells in vitro as well as in vivo. These results suggest that CXCR4 downregulation may be an important pharmacodynamic biomarker of cancer chemopreventative ITCs in prostate adenocarcinoma. Cancer Prev Res; 8(5); 365–74. ©2015 AACR.

Mutations in the p53 gene occur in a wide variety of human cancers at remarkably high frequencies. Prostate cancer (PCa), the most commonly diagnosed cancer and the second leading cause of cancer-related death in men in the United States, show a significantly high frequency of p53 mutations (~30%-70%). However, the role of mutant p53 as a target for the treatment or prevention of PCa remained to be investigated. In this study, we showed that cruciferous vegetable-derived phenethyl isothiocyanate (PEITC) can reactivate p53 mutants, irrespective of its site of mutation, in PCa cells in vitro. Importantly, this study showed that dietary supplementation of PEITC can significantly inhibit DU145 p53 mutant xenograft tumor in a nu/nu Balb/c mice. Interestingly, the animals in the PEITC-treated group showed a significant decrease in the mutant p53 expression levels as compared to the animals in the control group. DU145 cells harboring p53V274F/P223L mutant display maximum sensitivity to PEITC-induced growth inhibition as compared to the wild-type p53 LNCaP and p53 null PC-3 cells. PEITC treatment also caused a significant decrease in the p53 expression level in DU145 cells. No significant inhibition of cell proliferation was detected for the normal prostate epithelial RWPE1 cells treated with PEITC. Mechanistic studies revealed that PEITC induces a conformation change and restores the transactivation functions to the p53 mutant. Accordingly, in PEITC-treated cells the reactivated p53 mutant(s) induces apoptosis by activating canonical wild-type p53 targets, inducing a G1 and S phase delay and by phosphorylating ATM. PEITC treatment of isogenic PC-3 p53 null cells revealed that PC-3 cells expressing p53P223L mutant exhibit maximum inhibition of cell proliferation, suggesting that PEITC acts in a p53P223L mutant-dependent manner. Furthermore, PEITC treatment restores the transactivation functions to p53P223L mutant as shown by the activation of p53 regulated downstream target genes. Interestingly p73, a member of the p53 gene family that shares high functional homology and regulate cell-growth/ cell-death pathways in a manner similar to p53, does not play a role in p53-mediated pathway activation in PEITC treated cells. We further showed that PEITC inhibits cell proliferation and induces apoptosis in prostate cancer cells with hotspot mutants R248W (VCaP) and R175H (LAPC-4). Importantly, PEITC can induce a conformation change, reactivates p53 mutants and induces G1 and S-phase delays in these cells. These results demonstrate that PEITC can reactivate mutant p53, irrespective of the site of p53 mutation, in human prostate cancer cells. These studies provide the first example of targeting mutant p53 in prostate cancer cells by a dietary-related compound that may have impact on the prevention and treatment strategies of this disease. Citation Format: Monika Aggarwal, Rahul Saxena, Nasir Asif, Elizabeth Sinclair, Judy Tan, Idalia Cruz, Deborah Berry, Bhaskar Kallakury, Quynhchi Pham, Thomas Ty Wang, Fung-Lung Chung. Reactivation of mutant p53 in human prostate cancer cells as a critical mechanism for inhibition of tumor growth by phenethyl isothiocyanate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 269.

Prostate cancer (PCa) is the most common male malignancy in U.S.. In contrast to the localized early-stage disease, prognosis of castration-resistant prostate cancer (CRPC), i.e., the relapsed/metastatic advanced disease phenotype, is extremely poor. Because the sustained androgen receptor (AR) signaling remains one of the central driving forces of CRPC, new-generation nonsteroid AR antagonist has been developed in recent years, exemplified by the approval of enzalutamide (Enz) in 2012 for post-chemo and in 2014 for chemotherapy-naïve CRPC. However, drug resistance inevitably develops and survival benefits of Enz in CRPC patients are short-lived. Isothiocyanates (ITCs), e.g., sulforaphane (SFN) and phenethyl isothiocyanate (PEITC), are active metabolites of dietary glucosinolate from cruciferous vegetables with broad anti-PCa activities, such as suppressing AR expression, downregulating antiapoptotic proteins, inducing cell cycle arrest and apoptosis, and inhibiting PCa stem-like cells, etc. SFN-rich extract was recently tested in recurrent PCa patients and notably prolonged PSA doubling time without severe adverse events. By modifying Enz structure, we designed and synthesized AR ligand-ITC hybrid drug, aimed at antagonizing androgen-stimulated AR transcriptional activity and meanwhile downregulating AR expression and inducing other anticancer effects in PCa cells. To increase water solubility, ITCs were further reacted with N-acetyl cysteine (NAC) to obtain NAC conjugates. A representative hybrid drug NAC conjugate 2-63 was selected for in vitro study. In PBS buffer (pH 7.4), 2-63 gradually decomposes and releases free ITC with a half-life of about 3.4h. Measured using MTT assay, 2-63 was more tolerable in noncancerous RWPE-1 cell, significant differences were observed between RWPE-1 and PCa LNCaP cells, suggesting NAC conjugate could be a water-soluble carrier of AR ligand-ITC hybrid drug. The slower release of ITC constrains the concentration of electrophilic ITC within limit that is tolerated in healthy tissue. The newly designed hybrid drug chemical scaffold retains AR antagonist activity, evidenced by the inhibition of ARE-regulated luciferase activity in kb2 cell and the suppression of DHT-stimulated proliferation of LNCaP cell. 2-63 also dose- and time-dependently inhibited growth of CRPC VCaP cell and disrupted mitochondrial membrane potential, more effectively than SFN and Enz. Analyzed using Western blot, 2-63 downregulated the expression of full-length AR (fAR) and AR splice variants 7 (AR-V7). The AR downregulating properties were also observed in LNCaP and in 22Rv1 cells carrying different AR mutants. Since AR-V expression is one of the mechanisms causing clinical resistance to Enz treatment, targeting both fAR and AR-Vs using the newly discovered AR ligand-ITC hybrids may provide a new modality to improve the prognosis in CRPC patients. Citation Format: Siyu Ou, Liping Xu, Zhihui Qin. Synthesis and in vitro anti-prostate cancer activities of androgen receptor ligand-isothiocyanate hybrid drug [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A197.

Phenethyl isothiocyanate (PEITC) is a promising cancer chemopreventive agent but the mechanism of its anticancer effect is not fully understood. We now show, for the first time, that PEITC treatment triggers Atg5-dependent autophagic and apoptotic cell death in human prostate cancer cells. Exposure of PC-3 (androgen independent, p53 null) and LNCaP (androgen responsive, wild-type p53) human prostate cancer cells to PEITC resulted in several specific features characteristic of autophagy, including appearance of membranous vacuoles, formation of acidic vesicular organelles, and cleavage and recruitment of microtubule-associated protein 1 light chain 3 (LC3) to autophagosomes. A normal human prostate epithelial cell line (PrEC) was markedly more resistant toward PEITC-mediated cleavage and recruitment of LC3 compared with prostate cancer cells. Although PEITC treatment suppressed activating phosphorylations of Akt and mammalian target of rapamycin (mTOR), which are implicated in regulation of autophagy by different stimuli, processing and recruitment of LC3 was only partially/marginally reversed by ectopic expression of constitutively active Akt or overexpression of mTOR-positive regulator Rheb. The PEITC-mediated apoptotic DNA fragmentation was significantly attenuated in the presence of a pharmacologic inhibitor of autophagy (3-methyl adenine). Transient transfection of LNCaP and PC-3 cells with Atg5-specific small interfering RNA conferred significant protection against PEITC-mediated autophagy as well as apoptotic DNA fragmentation. A xenograft model using PC-3 cells and Caenorhabditis elegans expressing a lgg-1:GFP fusion protein provided evidence for occurrence of PEITC-induced autophagy in vivo. In conclusion, the present study indicates that Atg5 plays an important role in regulation of PEITC-induced autophagic and apoptotic cell death. [Cancer Res 2009;69(8):3704–12]

There is little information on early molecular events in the development of N-nitrosomethylbenzylamine (NMBA)–induced rat esophageal tumorigenesis and of the effects of chemopreventive agents on these events. In this study, we identified genes in rat esophagus that were differentially expressed in response to short-term NMBA treatment and modulated by cotreatment with phenylethyl isothiocyanate (PEITC). Rats were fed AIN-76A diet or AIN-76A diet containing PEITC for 3 weeks. During the 3rd week of dietary treatment, they were administered three s.c. doses of NMBA (0.5 mg/kg body weight). Rats were sacrificed 24 h after the last treatment; esophagi were excised and processed for histologic grading, microarray and real-time PCR analysis. Histopathologic analysis showed that treatment of rats with PEITC had a protective effect on NMBA-induced preneoplastic lesions in the rat esophagus. We identified 2,261 genes that were differentially expressed in the NMBA-treated versus control esophagi and 1,936 genes in the PEITC + NMBA versus NMBA-treated esophagi. The intersection of these two sets resulted in the identification of 1,323 genes in NMBA-treated esophagus, the vast majority of which were modulated by PEITC to near-normal levels of expression. Measured changes in the expression levels of eight selected genes were validated using real-time PCR. Results from 12 microarrays indicated that PEITC treatment had a genome-wide modulating effect on NMBA-induced gene expression. Samples obtained from animals treated with PEITC alone or cotreated with PEITC + NMBA were more similar to controls than to samples treated with NMBA alone. [Cancer Res 2007;67(13):1–9]

Previous studies, including those from our laboratory, have revealed that phenethyl isothiocyanate (PEITC), a constituent of many edible cruciferous vegetables, not only affords significant protection against chemically induced cancer in animal models but also inhibits growth of cancer cells in culture and in vivo by causing cell cycle arrest and apoptosis induction. We now report a novel response to PEITC involving inhibition of angiogenesis in vitro and ex vivo at pharmacologically achievable concentrations. The PEITC treatment caused a decrease in survival of human umbilical vein endothelial cells (HUVEC) in a concentration- and time-dependent manner. The capillary-like tube structure formation (in vitro neovascularization) and migration (invasion potential) by HUVEC was also inhibited significantly in the presence of PEITC at pharmacologically relevant concentrations (

The up-regulation of phase II detoxifying and stress-responsive genes is believed to play an important role in cancer prevention, and many natural compounds have been shown to be potent inducers of these genes. Previous studies showed that the antioxidant responsive element (ARE), found in these genes, can be bound by the transcription factor Nrf2, and is responsive to the activation by chemopreventive compounds and by oxidative stress. In the present study, we investigated the roles of extracellular signal-regulated kinase (ERK) and c-Jun-NH2-kinase (JNK) in the regulation of phenethyl isothiocyanate (PEITC)–induced and Nrf2-dependent ARE activity and ARE-driven heme oxygenase-1 (HO-1) gene expression in PC-3 cells. ARE activity and HO-1 expression were strongly increased after treatment with PEITC. PEITC also increased the phosphorylation of ERK1/2 and JNK1/2 and caused release of Nrf2 from sequestration by Keap1, and its subsequent translocation into the nucleus. Importantly, Nrf2 was also translocated into the nucleus after transfection with ERK or JNK and that these activated ERK and JNK colocalized with Nrf2 in the nucleus. Activation of ERK and JNK signaling also resulted in the elevation of ARE activity and HO-1 expression. Importantly, PEITC-induced ARE activity was attenuated by inhibition of ERK and JNK signaling. In vitro kinase assays showed that both ERK2 and JNK1 could directly phosphorylate glutathione S-transferase–Nrf2 protein. Taken together, these results strongly suggest a model in which PEITC treatment of PC-3 cells activates ERK and JNK, which, in turn, phosphorylate Nrf2 and induce its translocation to the nucleus. Nuclear Nrf2 activates ARE elements and induces expression of stress-responsive genes, including HO-1. [Mol Cancer Ther 2006;5(8):1918–26]

We previously showed that dietary treatment with the N-acetylcysteine conjugate of phenethyl isothiocyanate (PEITC-NAC) inhibited benzo(a)pyrene-induced lung tumorigenesis in A/J mice, and that tumor inhibition was associated with induction of activator protein-1 (AP-1) activity and stimulation of apoptosis in the lungs of mice. In the present study, we show that PEITC-NAC also induces apoptosis and AP-1 activity in human lung adenocarcinoma A549 cells, and that activation of AP-1 is important in PEITC-NAC induced apoptosis in these cells. PEITC-NAC induced AP-1 binding activity in A549 cells in a dose- and time-dependent manner; peak activity appeared at 10 μmol/L after 24 hours. At that time, flow cytometric analysis showed a sub-G1 peak, indicating that ∼4.5% of the cells had undergone apoptosis. When wild-type c-jun cDNA was transfected into A549 cells, PEITC-NAC–mediated apoptosis was greatly increased in the c-jun–transfected cells compared with the control vector–transfected cells, based on cell morphology and analysis of DNA fragmentation. Furthermore, cells that were pretreated with 100 nmol/L 12-O-tetradecanoyl phorbol-13-acetate, and then treated with 25 μmol/L PEITC-NAC, underwent enhanced apoptosis compared with cells that were treated with PEITC-NAC alone; cells treated with 12-O-tetradecanoyl phorbol-13-acetate alone showed active cell growth without apoptosis. Bivariate flow cytometric analysis of DNA strand breaks versus DNA content showed that apoptosis induced by PEITC-NAC occurred predominantly in the G2-M phase. These findings suggest that growth-stimulated cells with an elevated basal AP-1 activity, i.e., A549 cells transfected with wild-type c-jun or treated with a tumor promoter, were more sensitive to PEITC-NAC–mediated apoptosis. The observation that PEITC-NAC induces apoptosis predominantly in growth-promoted cells, such as neoplastic cells, suggests a selective mechanism by which PEITC-NAC inhibits lung carcinogenesis.

We have shown previously that naturally occurring isothiocyanates derived from cruciferous vegetables and their N-acetylcysteine conjugates inhibit lung adenoma formation induced by tobacco carcinogens in A/J mice at the post-initiation stage. The tumor-inhibitory activity by these compounds is linked with activation of activator protein and induction of apoptosis in lung tissues, suggesting that these compounds may also inhibit the development of adenomas to adenocarcinomas in lung. In this study, the chemopreventive activity of phenethyl isothiocyanate and sulforaphane and their N-acetylcysteine conjugates during progression of lung adenomas to malignant tumors was investigated in A/J mice. Mice were divided into 14 groups and treated with a mixture of 3 μmol benzo(a)pyrene [B(a)P] and 3 μmol 4-(methylnitrosamino)-1-(3-pyridyl)–1-butanone (NNK) given by gavage once weekly for 8 weeks. Twenty weeks after the beginning of carcinogen administration, a total of 20 mice in the treatment groups were sacrificed with an average yield of 7.3 ± 4.5 lung adenomas per mouse. The remaining mice in each group were fed diets containing phenethyl isothiocyanate (3 and 1.5 mmol/kg diet), sulforaphane (3 and 1.5 mmol/kg diet), phenethyl isothiocyanate-N-acetylcysteine (8 and 4 mmol/kg diet), sulforaphane-N-acetylcysteine (8 and 4 mmol/kg diet) during weeks 21 to 42. Four mice in each of the high-dose treatment groups were sacrificed during weeks 28 and 36 and the bioassay was terminated during week 42; lung tissues were harvested for histopathologic examination of tumors and for cell proliferation (proliferating cell nuclear antigen) and apoptosis (caspase-3) assays using immunohistochemical staining. At termination, the incidence of adenocarcinoma in the 3 mmol/kg diet phenethyl isothiocyanate group and 8 mmol/kg diet phenethyl isothiocyanate-N-acetylcysteine group was reduced to 19% and 13%, respectively, compared with 42% in the carcinogen-treated control group. At the lower doses, phenethyl isothiocyanate and its N-acetylcysteine conjugate also inhibited the incidences of lung adenocarcinoma, however, the decreases were not statistically significant. The lung tumor incidences in groups treated with sulforaphane-N-acetylcysteine in the diet were also significantly reduced to 11% or 16%. Furthermore, the malignant lung tumor multiplicity was significantly reduced from 1.0 tumor/mouse in the carcinogen-treated control group to 0.3 in the sulforaphane low-dose group, 0.3 and 0.4 in the two sulforaphane-N-acetylcysteine groups, and 0.4 in the phenethyl isothiocyanate high-dose group. The malignant tumor multiplicities in other treatment groups were also reduced (0.5-0.8 tumors/mouse), but not significantly. Unlike lung adenocarcinomas, both incidences and multiplicities of lung adenomas were not much affected by treatment with isothiocyanates or their conjugates. Immunohistochemical examination of the lung tumors from all time points indicated that significant reduction in proliferating cell nuclear antigen and induction of apoptosis (terminal nucleotidyl transferase–mediated nick end labeling and caspase-3) were observed in the isothiocyanate and isothiocyanate-N-acetylcysteine–treated groups that showed inhibition of the development of lung adenocarcinomas. The results of the study provide a basis for future evaluation of the potential of phenethyl isothiocyanate and sulforaphane and their conjugates as chemopreventive agents in smokers and ex-smokers with early lung lesions.

Purpose: The present study was undertaken to gain insights into the molecular mechanism of apoptosis induction by phenethyl isothiocyanate (PEITC) using prostate cancer cell lines derived from transgenic adenocarcinoma mouse prostate (TRAMP) mice (TRAMP-C1 and TRAMP-C2). Experimental Design and Results: The viability of TRAMP-C1 and TRAMP-C2 cells was reduced significantly in the presence of PEITC in a concentration-dependent manner as determined by sulforhodamine B and trypan blue dye exclusion assays. Treatment of TRAMP-derived cells with PEITC revealed features characteristic of apoptosis induction, including appearance of subdiploid cells (determined by flow cytometry), cytoplasmic histone-associated DNA fragmentation (determined by an ELISA assay), and cleavage of caspase-3 (determined by immunoblotting). The PEITC-induced apoptosis in TRAMP-derived cells was associated with a marked increase in the level of proapoptotic protein Bak and/or a decrease in the levels of antiapoptotic protein Mcl-1 or Bcl-xL and disruption of mitochondrial membrane potential. The SV40 immortalized mouse embryonic fibroblasts derived from Bak and Bax double knockout mice were significantly more resistant to PEITC-induced DNA fragmentation compared with wild-type or Bak−/− mouse embryonic fibroblasts. The PEITC-induced apoptosis in both cell lines was significantly attenuated in the presence of caspase inhibitors zVAD-fmk, zLEHD-fmk, and zIETD-fmk. Oral administration of PEITC (9 or 12 μmol PEITC/d, Monday-Friday) significantly retarded growth of TRAMP-C1 xenografts in nude mice without causing weight loss or any other side effects. Conclusion: The results of the present study indicate that caspase-dependent apoptosis by PEITC is mediated by Bak and Bax proteins.

Cabbage contains the glucosinolate sinigrin, which is hydrolyzed by myrosinase to allyl isothiocyanate. Isothiocyanates are thought to inhibit the development of cancer cells by a number of mechanisms. The effect of cooking cabbage on isothiocyanate production from glucosinolates during and after their ingestion was examined in human subjects. Each of 12 healthy human volunteers consumed three meals, at 48-h intervals, containing either raw cabbage, cooked cabbage, or mustard according to a cross-over design. At each meal, watercress juice, which is rich in phenethyl isothiocyanate, was also consumed to allow individual and temporal variation in postabsorptive isothiocyanate recovery to be measured. Volunteers recorded the time and volume of each urination for 24 h after each meal. Samples of each urination were analyzed for N-acetyl cysteine conjugates of isothiocyanates as a measure of entry of isothiocyanates into the peripheral circulation. Excretion of isothiocyanates was rapid and substantial after ingestion of mustard, a source of preformed allyl isothiocyanate. After raw cabbage consumption, allyl isothiocyanate was again rapidly excreted, although to a lesser extent than when mustard was consumed. On the cooked cabbage treatment, excretion of allyl isothiocyanate was considerably less than for raw cabbage, and the excretion was delayed. The results indicate that isothiocyanate production is more extensive after consumption of raw vegetables but that isothiocyanates still arise, albeit to a lesser degree, when cooked vegetables are consumed. The lag in excretion on the cooked cabbage treatment suggests that the colon microflora catalyze glucosinolate hydrolysis in this case.

We have already found that a nuclear receptor PXR can modulate the cytotoxicity of CDDP for cancer cells, and ketoconazole, a PXT antagonist, can be an agent enhancing the antitumor activity of CDDP due to increase of the intracellular platinum content as the mechanism. However, nuclear receptor ligands have a variety of functions, and there is a possibility that PXR antagonist may enhance the cytotoxicity of CDDP through the mechanism other than the inhibition of transporter-mediated efflux of CDDP. The goal of this study was to compare the enhancing ability of three PXR antagonists, ketoconazole (KTZ), phenethyl isothiocyanate (PEITC) and metformin (MFM), for the antitumor effect of CDDP and clarify the contribution of inhibition of the efflux transporter as the mechanism of PXR antagonist. Cell lines used in this study were human hepatoma cell line HepG2. Changes in mRNA expression were assessed by real-time RT-PCR, and the ability of apoptosis induction was assessed as caspase-3 activity. Total intracellular platinum contents were determined by ICP-AES. When HepG2 cells were treated for 24 hr with 10 μM of CDDP, no change of caspase-3 activity was observed when compared to control cells. Also, 30 μM of KTZ, 30 μM of PEITC or 5 mM of MFM had no affect on the caspase-3 activity in treated cells compared with control cells. When KTZ, PEITC or MFM was exposed to HepG2 cells from 24 hr before treatment and during 24-hr treatment with 10 μM of CDDP, the caspase-3 activity was significantly increased to 461%, 355% and 326% of the control, respectively, in comparison with that in CDDP alone. When HepG2 cells were treated for 8 hr with 25 μM of CDDP, the intracellular platinum content was 110 ng/mg protein. On the other hand when KTZ, PEITC or MFM was exposed to HepG2 cells from 24 hr before treatment and during 8-hr treatment with 25 μM of CDDP, the intracellular platinum content was apparently increased to 227%, 491% and 161% of CDDP alone, respectively. Three PXR antagonists resulted in an increase in the intracellular platinum content that seems to be due to inhibition of the platinum efflux transporter, but there is no correlation between increase in intracellular platinum content and enhancement of antitumor activity by PXR antagonists. These results concluded that PXR antagonist can be an agent enhancing the antitumor activity of CDDP, but the ability of PXR antagonist for enhancing antitumor activity of CDDP can not be determined from only the increase in the intracellular platinum content. Citation Format: Shuichi Kishimoto, Megumi Yasuda, Megumi Tomita, Yuki Yamane, Ryosuke Suzuki, Shoji Fukushima. Comparison of enhancement of the antitumor effect of CDDP by PXR antagonists. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4742.

Background: Almost 90% of all breast cancers (BCs) express a splice variant of HER2 lacking exon 16 (d16HER2), which promotes the generation of stable and active d16HER2 homodimers on the tumor cell surface. Comparison of the tumorigenic potential of human d16HER2 and the full-length HER2 (WTHER2) in transgenic mice revealed a significantly shorter latency period and a higher incidence of tumors in the d16HER2 line, suggesting enrichment of this variant in HER2+ BC stem cells. Based on reports that cruciferous vegetable-derived compounds such as isothiocyanates (ITCs) mediate strong anti-tumorigenic effects on numerous oncotypes and target BC stem cells, we tested the effect of the phenethyl ITC (PEITC), alone or together with Trastuzumab (T), which reportedly targets HER2+ BC stem cells, on d16HER2-positive tumor growth and progression in transgenic mice. Methods: The percentage of tumor cells expressing high levels of CD29 and CD24 and low levels of SCA-1 (CD29high/CD24+/SCA-1low) murine stem cell markers was evaluated by multiparametric flow cytometry in cell models derived from spontaneous d16HER2 and WTHER2 transgenic lesions. Mammosphere forming efficiency (MFE%) was calculated in d16HER2- and WTHER2-positive tumor cells. The therapeutic activity of PEITC, T and their combination was assessed in vitro based on their ability to decrease the MFE% and the expression of stem cell markers in d16HER2- vs. WTHER2-positive tumor cells, and in vivo in d16HER2 transgenic mice. Analyses by western blot, histology/immunohistochemistry and magnetic resonance spectroscopy (MRS) are ongoing to assess apoptosis, necrosis and metabolomic profiles of treated tumors vs. controls. Results: d16HER2 tumor cells showed a significantly higher MFE% and higher expression of stem cell markers than did WTHER2 tumor cells. PEITC and T significantly reduced MFE% as compared to controls (p = 0.0342 and p = 0.0083, respectively), and their combination exerted significantly stronger inhibition than T (p = 0.001) or PEITC (p = 0.0003) alone only in d16HER2 cells. The percentage of tumor cells expressing CD29high/CD24+/SCA-1low in d16HER2 tumor cells after treatment with T, PEITC or both was also reduced. Whereas PEITC monotherapy did not significantly reduce mammary tumor incidence compared to controls, while T effectively suppressed d16HER2-driven tumor growth (p = 0.0002), the combination of PEITC and T significantly heightened the impairment of mammary lesion development compared to T alone (p = 0.0046). Ex vivo metabolomic analyses by MRS of d16HER2-positive murine and human BC cell lines showed an increase in glycolytic pathway as compared to the respective WTHER2 cells. Conclusions: Our results provide in vivo evidence that the combination of T and PEITC mediates a significantly greater anti-tumor effect than either alone, most likely targeting BC stem cells. Supported by the Italian Ministry of Health and AIRC. Citation Format: Ada Koschorke, Lorenzo Castagnoli, Gaia C. Ghedini, Tiziana Triulzi, Claudia Chiodoni, Rossella Canese, Egidio Iorio, Manuela Iezzi, Patrizia Nanni, Elda Tagliabue, Serenella M. Pupa. Phenethyl isothiocyanate hampers growth and progression of HER2-positive breast cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3826.

Mutations in the p53 gene occur in a variety of human cancers with remarkably high frequencies (as high as 80%), depending on the type and stage of the tumors (www-p53.iarc.fr). The majority of mutations are missense that are localized to six “hotspot” residues, which play a role either in the structural integrity (structural mutants, e.g., R175H) or in the DNA binding domain (contact mutants, e.g., R273H). Previously, we showed that phenethyl isothiocyanate (PEITC), a naturally occurring dietary constituent present abundantly in watercress, selectively depletes p53 mutant protein, but not the WT p53, and induces apoptosis in tumor cells expressing p53 mutants. In this study we demonstrated that PEITC can reactivate p53R175 mutant protein and inhibit tumor growth in a mutant p53R175H -dependent manner. PEITC displayed a 175 allele preference of inhibition of cell proliferation as compared to other hotspot mutants as detected by WST-1 assay and induced apoptosis in a p53R175 mutant -dependent manner. Immunofluorescence and co-immunoprecipitation assay on PEITC treated p53R175 cells using conformation-specific antibodies (PAB1620 and PAB240) showed restoration of the “WT-like” conformation to p53 mutant. Reactivation of the WT like functions to the p53R175 mutant was established by its ability to bind DNA, transactivate p53 regulated downstream target genes (p21, PUMA, MDM2, NOXA, BAX and BCL2), and overexpression of p21 protein. PEITC treatment induced DNA damage response as detected by pATM-S1981 and pCHK2-Thr68, G2/M and S-phase cell-cycle arrest and oxidative stress as measured by decrease in the GSH levels in the cells. In contrast, co-treatment of cells with PEITC and reducing agents alleviated the inhibitory effects of PEITC on cell proliferation. Elevated oxidative stress due to the combined effect of PEITC and elevated ROS levels in p53 mutant cells might be responsible for the activation of restored “WT-like” p53R175 mutant protein and induction of apoptosis. PEITC induced proteasomal degradation and autophagy of the p53R175 mutant. Animals fed with PEITC diet showed a statistically significant reduction in xenograft tumor volumes, proliferation marker Ki67, and mutant p53 stained cells. Elevated mRNA levels of p53 regulated genes from animals fed PEITC diet provide in vivo evidence for the p53R175H mutant reactivation and inhibition of xenograft growth in a mutant p53-dependent manner. These are important findings because they demonstrated that a dietary compound can inhibit the growth of tumor cells in vivo by restoring the tumor suppressor functions to mutant p53, and thus provide a target for the development of novel chemopreventive strategies through dietary interventions. Citation Format: Monika Aggarwal, Elizabeth Sinclair, Anna Jacobs, Ying Fu, Marcin Dyba, Xiantao Wang, Idalia M. Cruz, Deborah Berry, Kallakury Bhaskar, Susette C. Mueller, Maria Laura Avantaggiati, Fung-Lung Chung. Reactivation of p53R175 mutant by dietary phenethyl isothiocyanate (PEITC) impairs tumor growth in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1910. doi:10.1158/1538-7445.AM2015-1910

Isothiocyanates (ITCs) derived from cruciferous vegetables, including phenethyl isothiocyanate (PEITC) and sulforaphane (SFN), exhibit in vivo activity against prostate cancer in xenograft and transgenic mouse models, and thus are appealing for chemoprevention of this disease. Watercress constituent PEITC and SFN-rich broccoli sprout extract are under clinical investigation but the molecular mechanisms underlying their cancer chemopreventive effects are not fully understood. The present study demonstrates that chemokine receptor CXCR4 is a novel target of ITCs in prostate cancer cells. Exposure of prostate cancer cells (LNCaP, 22Rv1, C4-2, and PC-3) to pharmacologically applicable concentrations of PEITC, benzyl isothiocyanate (BITC), and SFN (2.5 and 5 μmol/L) resulted in downregulation of CXCR4 expression. None of the isothiocyanates affected secretion of CXCR4 ligand (stromal-derived factor-1). In vivo inhibition of PC-3 xenograft growth upon PEITC treatment was associated with a significant decrease in CXCR4 protein level. A similar trend was discernible in the tumors from SFN-treated TRAMP mice compared with those of control mice, but the difference was not significant. Stable overexpression of CXCR4 in PC-3 cells conferred significant protection against wound healing, cell migration, and cell viability inhibition by ITCs. Inhibition of cell migration resulting from PEITC and BITC exposure was significantly augmented by RNA interference of CXCR4. This study demonstrates, for the first time, that cancer chemopreventive ITCs suppress CXCR4 expression in prostate cancer cells in vitro as well as in vivo. These results suggest that CXCR4 downregulation may be an important pharmacodynamic biomarker of cancer chemopreventative ITCs in prostate adenocarcinoma. Citation Format: Ali I. Amjad, Kozue Sakao, Avani R. Vyas, Sreenivasa R. Chinni, Rahul Parikh, Shivendra V. Singh. CXCR4 is a novel target of cancer chemopreventative isothiocyanates in prostate cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1906. doi:10.1158/1538-7445.AM2015-1906

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the United States and Europe. Despite recent advance in new therapeutic agents that have improved treatment outcomes, CLL remains incurable. CLL patients with loss of p53 due to unfavorable cytogenetic alterations such as chromosome 17p deletion often exhibit more malignant disease phenotype, are resistant to many standard therapeutic agents, and have poor clinical outcomes. Thus, finding effective treatment for CLL with p53 deficiency represents a significant challenge, and development new agents and therapeutic strategies to effectively kill CLL cells with loss of p53 function would have profound clinical implications in CLL treatment. We have recently created a mouse model with Tcl1-Tg:p53-/- genotype that resembles the aggressive human CLL disease with p53 deficiency (17p-deletion). We showed that leukemia cells isolated from this mouse model as well as primary human leukemia cells with 17p deletion isolated from CLL patients are resistant to standard anti-CLL agents. Because p53 plays important roles in regulation of apoptosis and in maintaining mitochondrial function/normal metabolism/redox homeostasis, we hypothesized that a loss of p53 function in CLL with 17p- or p53 mutations would on one hand lead to drug resistance and poor therapeutic response to conventional treatment, and would on the other hand also cause mitochondrial dysfunction and high ROS stress. Our study tested the possibility to use the high oxidative stress as a biochemical basis to effectively target the drug-resistant CLL cells with p53 deletion using redox-modulating strategy in vitro and in vivo. Our results showed that ROS-mediating strategies could overcome drug resistance of CLL cells with p53 deficiency, and was able to effectively eliminate fludarabine-resistant CLL cells using phenethyl isothiocyanate (PEITC), which induced rapid glutathione depletion and caused severe ROS accumulation leading to massive death of CLL cells in culture. Importantly, PEITC also exhibited promising in vivo therapeutic activity in the CLL mouse model as a single agent, leading to a significant prolongation of the mouse survival time. Since PEITC is a natural compound found in vegetables with very low toxicity to normal cells, the results of this study may have important therapeutic implications. Our study suggests that PEITC may potentially be used for clinical treatment of CLL with 17p deletion and improve the outcome of this subpopulation of CLL patients, who would otherwise refractory to standard therapeutics. Citation Format: Jinyun Liu, Gang Chen, Helen Pelicano. Targeting chronic lymphocytic leukemia with p53 deficiency with phenethyl isothiocyanate. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4560. doi:10.1158/1538-7445.AM2015-4560

The goal of this study was to evaluate the enhancing ability of PXR antagonists for the antitumor effect of platinum complex. Cell lines used in this study were human hepatoma cell line HepG2, human ovarian cancer cell line 2008 and human colon cancer cell line HCT116. Changes in mRNA expression were assessed by real-time RT-PCR, and the ability of apoptosis induction was assessed as caspase-3 activity. Cytotoxicity of platinum complexes was assessed by SRB assay, and IC75 and IC90 values of them were determined. They did not induce apoptosis at IC75 but did it markedly at IC90. Previously, we reported that though apoptosis in HepG2 was induced by IC90 dose of CDDP, addition of a PXR agonist, rifampicin, from 24-hr before treatment and during treatment with CDDP was almost suppressed. Moreover, we found that apoptosis in 2008 and HCT116 induced by IC90 dose of CDDP or oxaliplatin was suppressed by rifampicin. That suggests a nuclear receptor PXR affects on the cytotoxicity of platinum complexes for cancer cells other than HepG2. Ketoconazole (KTZ) and phenethyl isothiocyanate (PEITC) were selected as PXR antagonists that were added with platinum complex. Both drugs at 30 μM induced cytostatic cell growth inhibition in HepG2, 2008 and HCT116 but did not affect on caspase-3 activity compared with control in those cells. When these PXR antagonists at 30 μM were added to cancer cell lines from 24-hr before treatment and during treatment with CDDP at IC75, caspase-3 activity was significantly increased compared with that in CDDP alone. Also, when cells were exposed to CDDP and a PXR antagonist without PXR antagonist pretreatment, caspase-3 activity was increased but the level was reduced compared with that in the combination with pretreatment. Similarly, oxaliplatin in combination with KTZ or PEITC markedly induced apoptosis in HCT116. In HepG2, KTZ reduced the level of MRP2 mRNA. That suggests that pretreatment with KTZ might increase the intracellular platinum content and enhance the cytotoxicity of platinum complex. We concluded that PXR antagonist can be an agent enhancing the antitumor activity of platinum complex, and one of the mechanism is to suppress the expression of the platinum efflux transporter. Citation Format: Shuichi Kishimoto, Erika Bou, Kaho Higashi, Ryosuke Suzuki, Shoji Fukushima. Enhancement of antitumor effect of platinum complexes by PXR antagonist. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 840. doi:10.1158/1538-7445.AM2014-840

Phenethyl isothiocyanate (PEITC), a constituent of many edible cruciferous vegetables (eg, watercress), exhibits in vivo preventive efficacy against cancer in rodents induced by structurally diverse chemical carcinogens. We have also shown recently that dietary administration of PEITC (3 μmol PEITC/g diet) confers significant protection against breast and prostate cancer development in MMTV-neu and TRAMP model, respectively. Prevention of prostate cancer by PEITC administration in TRAMP mice was associated with induction of E-cadherin protein expression. Because suppression of E-cadherin protein level concomitant with induction of mesenchymal markers (eg, vimentin) is a biochemical hallmark of epithelial-mesenchymal transition, a process implicated in cancer metastasis, we hypothesized that PEITC treatment was likely to suppress vimentin protein expression. Contrary to this prediction, exposure of cultured human breast (MDA-MB-231) and prostate cancer cells (PC-3 and DU145) to PEITC resulted in a dose-dependent increase in vimentin protein level, which was observed as early as 6 hours post-treatment and persisted for the duration of the experiment (24 hours). RNA interference of vimentin resulted in a modest augmentation of PEITC-mediated inhibition of MDA-MB-231 and PC-3 cell migration as well as cell viability. Furthermore, the PEITC-induced apoptosis was moderately increased upon siRNA knockdown of vimentin protein level in MDA-MB-231 and PC-3 cells. To our surprise, PEITC administration caused a marked decrease in vimentin protein expression in vivo in tumors of MMTV-neu (breast) and TRAMP mice (prostate). The results of the present study indicate that cancer chemoprevention by PEITC in vivo is associated with suppression of vimentin protein expression. The present study was supported by the grant CA101753-09 awarded by the National Cancer Institute. Citation Format: Kozue Sakao, Eun-Ryeong Hahm, Shivendra V. Singh. Chemoprevention of mammary and prostate cancer by phenethyl isothiocyanate in vivo is associated with suppression of vimentin protein expression. [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 3669. doi:10.1158/1538-7445.AM2013-3669

Phenethyl isothiocyanate (PEITC), a promising chemopreventive compound originated from cruciferous vegetables, is known to inhibit proliferation of colon cancer cells through inducing apoptosis and cell cycle arrest. Although exact mechanisms are unclear, it seems that PEITC may have multiple molecular targets and play multiple roles in a variety of events. Some of our recent studies indicate that a key mechanism of apoptosis induction by PEITC may be through covalent modification of its target proteins, such as tubulin. Numerous studies also reported that PEITC may induce apoptotic signals through gene transcriptional regulation. However, systematic studies on PEITC-induced gene expression profile, particularly in colon cancer cells, are rare. In this study, we examined overall gene expression profile of SW480 cells at different time points after PEITC treatment using cDNA microarray. Results showed that PEITC induced significant changes in expression of several apoptosis-related genes, such as BIM, DEDD2, GADD45B, HSPB8, SYK, PIK3C2A, CTNNB1, LGALS8, and NOXA. Furthermore, these changes were confirmed by real-time polymerase chain reaction at transcript level and immunoblot at protein level. Considering significant roles of these genes in signaling transduction leading to downstream events, such as apoptosis, the findings of this study may shed lights on the molecular mechanisms by which PEITC exert its anti-carcinogenic effects. (Supported by NCI grant CA100853) Citation Format: Lixin Mi, Xiantao Wang, Xin Li, Fung-Lung Chung. Global gene expression regulated by phenethyl isothiocyanate in human colon cancer SW480 cells. [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 2591. doi:10.1158/1538-7445.AM2013-2591

Phenethyl isothiocyanate (PEITC) derived from cruciferous vegetables has been shown to inhibit tumor growth by induction of apoptosis. Previously, we demonstrated that PEITC can selectively deplete mutant p53, but not the wild type. However, whether p53, wild type or mutant, plays functional roles in PEITC-induced growth inhibition, remains unsettled. We hypothesize that the direct binding to mutant p53 by PEITC followed by conformational changes, subsequently leading to its depletion or/and restoration to the wild type may constitute a mechanism by which PEITC induces apoptosis. To determine whether PEITC can deplete mutant p53 in vivo and whether its depletion is correlated with tumor inhibition and apoptosis, in this xenograft model study BALB-C Nu/Nu mice carrying prostate tumor cells with either mutant (DU145) or wild type p53 (LNCap) were fed diets with and without PEITC (5 micromol per gram diet) for 8 weeks. Tumors were harvested at the end of bioassay for immunohistochemistry and immunoblot studies. Results showed that the tumor volumes of both LNCap and DU145 were significantly decreased in the PEITC-treated animals, compared with that on a diet without PEITC. The p53 level detected by immunoblot showed a moderate, yet significant, decrease in DU145 tumors, but not in LNCap tumors. Immunohistochemistry assays were carried out to examine the apoptotic markers, such as TUNEL, and anti-proliferation marker Ki67 in tumors from the PEITC-treated vs. untreated mice. This study provides the first in vivo evidence that mutant p53 can be depleted in tumors of mice fed PEITC-containing diet and its depletion seems to correlate with growth inhibition in the mutant p53 tumors. These findings suggest that mutant p53 maybe further investigated as a possible novel target for cancer chemoprevention and therapy by PEITC. (Supported by NCI grant CA100853) Citation Format: Lixin Mi, Thomas T.Y. Wang, Haiqiu Huang, Arnetta Fletcher, Lu Yu, Xiantao Wang, Fung-Lung Chung. Mutant p53 depletion: a possible mechanism of inhibition of human prostate cancer by dietary phenethyl isothiocyanate. [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 4858. doi:10.1158/1538-7445.AM2013-4858

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

Biomarker(s) predictive of efficacy is necessary for clinical development of promising cancer chemopreventive agents because cancer incidence is too rigorous of an end point for malignancies with long latency such as breast cancer. The present study was undertaken to identify biomarker(s) associated with phenethyl isothiocyanate (PEITC)-mediated mammary cancer prevention in MMTV-neu transgenic mouse model. The PEITC is a promising cancer chemopreventive constituent of cruciferous vegetables (e.g., watercress). Two-dimensional gel electrophoresis-mass spectrometry of tumor specimens from control and PEITC-fed mice (n= 3) revealed alterations in expression of several proteins, including hexokinase-1, isoform CRA_f (1.36-fold increase by PEITC administration), L-lactate dehydrogenase A chain isoform 1 (1.57-fold increase by PEITC administration), pyruvate kinase isozymes M1/M2 (1.33-fold decrease by PEITC administration), and mitochondrial ATP synthase, H+ transporting F1 complex beta subunit (1.38-fold decrease by PEITC administration). Interestingly, the PEITC-induced apoptotic cell death in cultured cancer cells is associated with inhibition of oxidative phosphorylation leading to production of reactive oxygen species. Down-regulation of mitochondrial ATP synthase protein in tumors of PEITC-fed mice was confirmed by immunohistochemistry. Expression of mitochondrial ATP synthase was reduced by about 66% in the tumors from PEITC-fed mice in comparison with control (P= 0.05 by two-sided Student's t-test, n= 6-7). In addition, plasma level of transthyretin was significantly higher in the PEITC treatment group compared with control (P= 0.04 by two-sided Student's t-test, n= 5). In conclusion, the present study not only underscores the power of proteomics in cancer chemoprevention research but also suggests that mitochondrial ATP synthase, and possibly hexokinase-1, pyruvate kinase isozymes M1/M2, and/or L-lactate dehydrogenase A chain isoform 1, may be useful biomarkers to assess the biological activity of PEITC in future clinical trials. This study was supported in part by the US PHS grants CA101753 and CA129347, awarded by the National Cancer Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 568. doi:1538-7445.AM2012-568

Bioactive food components continue to gain traction for possible chemoprevention of prostate cancer, which is a leading cause of cancer-related death among American men. Phenethyl isothiocyanate (PEITC) is one such compound that is under active investigation for chemoprevention of prostate cancer as well as other types of malignancies, including colon, lung, and breast cancers. PEITC occurs naturally as a thioglucoside conjugate (gluconasturtiin) in a variety of edible cruciferous vegetables (e.g., watercress), and is generated upon cutting or chewing of cruciferous vegetables due to myrosinase-catalyzed breakdown of the gluconasturtiin. Initial evidence supporting prostate cancer protective effect of cruciferous vegetables emerged from population-based case control studies. In vivo chemopreventive efficacy of PEITC against prostate cancer has now been demonstrated in a transgenic mouse model (Transgenic Adenocarcinoma of Mouse Prostate mice; hereafter abbreviated as TRAMP mice). Post-initiation cancer chemopreventive response to PEITC is associated with apoptosis induction but the mechanism of cell death is not fully understood. The present study was undertaken to determine the role of inhibitor of apoptosis (IAP) family proteins in regulation of PEITC-induced apoptosis. The IAP family proteins play critical roles in apoptosis regulation by inhibiting caspases. Elevated expression of IAP proteins, including X-linked inhibitor of apoptosis (XIAP) and Survivin has been reported in human prostate cancers. Exposure of PC-3 and LNCaP human prostate cancer cells to PEITC resulted in downregulation of XIAP and Survivin proteins and suppression of Survivin mRNA expression. PEITC administration to Transgenic Adenocarcinoma of Mouse Prostate mice caused modest but significant downregulation of XIAP and Survivin proteins in the dorsolateral prostate. Proapoptotic response to PEITC was significantly attenuated by ectopic expression of XIAP and Survivin proteins. In addition, Survivin overexpression conferred modest but significant protection against PEITC-mediated inhibition of PC-3 cell migration. In conclusion, these results indicate that cellular responses to PEITC, including apoptosis induction and inhibition of cell migration, in prostate cancer cells are mediated by downregulation of XIAP and/or Survivin, which may serve as valid biomarkers to assess PEITC response in future clinical investigations. This investigation was supported by the USPHS grant RO1 CA101753-08. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 587. doi:1538-7445.AM2012-587

Drug resistance is a major problem in clinical treatment of malignant melanoma. A characteristic feature of melanoma is the abnormally high levels of basal free radicals produced in transformed melanosomes when compared to normal melanocytes. Reactive oxygen species (ROS) activate pro-oncogenic signaling pathways that promote cancer progression, metastasis and contribute to chemotherapeutic drug resistance. However, an excessive overload of ROS in cells beyond a certain tolerance limit may trigger oxidative damage and cell death. Thus cellular ROS modulation presents a valuable therapeutic strategy for melanoma. Based on the above observations, we hypothesize that causing ROS agglomeration in cells, either through depletion of antioxidants or by initiating uncontrolled ROS production, is a plausible strategy to selectively kill melanoma cells, and that α-phenylethyl isothiocyanate (PEITC), a natural compound known to disable cellular GSH antioxidant system, will be effective for this purpose. We showed that that PEITC exhibited potent inhibitory effect on malignant melanoma cells with an average IC50 value of approximately 3 µM. Interestingly, MEL624 and HS294T cells, which are resistant to existing antitumor agents used in melanoma treatment such as AZD6244 and Vemurafenib, were highly sensitive to PEITC. The cell death was primarily apoptotic; the effect of PEITC could be partially reversed using the pan-caspase inhibitor Z-VAD. Furthermore, treatment of cells with PEITC lead to an aberrant increase in reactive oxygen species levels, as evidenced by FACS analysis using the fluorescent ROS dyes DCF-DA, DAF-FM and dihyroethidium. ROS increase upon PEITC treatment occurred as early as one hour, indicating that seemed to a primary event leading to subsequent cell damage. This increase in ROS was associated with a decrease in the glutathione levels in cells, suggesting that depletion of the glutathione antioxidant could be the reason for the abnormal ROS elevation induced by PEITC. This was further verified by using PEITC in combination with the antioxidant N-acetyl cysteine, which completely rescued the cells from the toxic effect of PEITC. Importantly, normal melanocytes were minimally sensitive to PEITC. In addition, combination of PEITC with other standard melanoma drugs like carmustine and temozolomide exhibited synergistic effect, suggesting the possibility of using this compound to overcome drug resistance. This was further validated through in vivo studies in mice, where a combination of PEITC with BCNU seemed to reduce the tumor burden. The cytotoxic effect of PEITC was also validated in primary cancer cells isolated from melanoma specimens from patients. Thus PEITC proves to be an effective antimelanoma agent that warrants further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 920. doi:1538-7445.AM2012-920

Practical and safe modalities for chemoprevention of prostate cancer are clinically attractive because of high mortality associated with this malignancy in American men. Plant products, including constituents of fruits and vegetables, continue to attract attention for the discovery of novel cancer chemopreventive agents. Phenethyl isothiocyanate (PEITC) is one such promising cancer chemopreventive agent abundant in edible cruciferous vegetables such as watercress. Evidence for protective effect of cruciferous vegetables and their components, including PEITC, against prostate cancer derives from population-based observational studies as well as laboratory investigations. For example, we have shown previously that PEITC administration in the diet (3 µmol/g diet) significantly inhibits incidence as well as burden of poorly-differentiated prostate cancer in the dorsolateral prostate of Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) transgenic mice. Cancer chemopreventive response to PEITC is attributed to its ability to inhibit multiple oncogenic signaling pathways, including nuclear factor-βB, Akt, and androgen receptor. The present study demonstrates, for the first time, that PEITC treatment activates Notch1 and Notch2 in malignant as well as normal human prostate cells. The Notch1 and Notch2 belong to a family of transmembrane receptors implicated in prostate cancer development and metastasis. Exposure of human prostate cancer cells (LNCaP, PC-3, and DU145) and a normal human prostate epithelial cell line (PrEC) to PEITC resulted in cleavage (active form) of Notch1 and Notch2, which was associated with an increase in transcriptional activity of Notch. PEITC treatment caused induction of Notch ligands Jagged1 and Jagged2, overexpression of γ-secretase complex components Presenilin1 and Nicastrin, nuclear enrichment of cleaved Notch2, and/or up-regulation of Notch1, Notch2, Jagged1, and/or Jagged2 mRNA in cancer cells. PEITC-induced apoptosis in LNCaP and PC-3 cells was significantly attenuated by RNA interference of Notch2, but not by pharmacological inhibition of Notch1. Inhibition of PC-3 and LNCaP cell migration resulting from PEITC exposure was significantly augmented by RNA interference of Notch2 as well as pharmacological inhibition of Notch1 activation. Nuclear expression of cleaved Notch2 protein was significantly higher in PC-3 xenografts from PEITC-treated athymic mice and dorsolateral prostates from PEITC-fed TRAMP mice compared with the respective control. Because Notch signaling is implicated in epithelial-mesenchymal transition and metastasis, the present study suggests that anti-metastatic effect of PEITC may be augmented by a combination regimen involving PEITC and a Notch inhibitor. This investigation was supported by the US PHS grant RO1 CA101753-08. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2584. doi:1538-7445.AM2012-2584

We have shown previously that phenethyl isothiocyanate (PEITC), which is a highly promising cancer chemopreventive constituent of edible cruciferous vegetables (e.g., watercress), inhibits growth of cultured breast cancer cells in association with apoptosis induction. The present study was undertaken to determine the efficacy of PEITC for prevention of breast cancer using MMTV-neu mouse model. PEITC administration in the diet (3 mmol PEITC/kg AIN76A diet) for 29 weeks caused an approximate 27% decrease in incidence of mammary hyperplasia compared with control, but the difference between control and PEITC treatment groups did not reach statistical significance. On the other hand, incidence of palpable tumors was reduced by about 53% by PEITC administration (P= 0.07 by two-sided Fisher's exact test). Moreover, size of microscopic carcinoma lesions was significantly lower in mice fed PEITC-supplemented diet compared with those fed basal diet (P= 0.05 by two-sided Fisher's exact test). Average body weight of the PEITC-fed mice was modestly but significantly higher than that of control mice at earlier time points (weeks 1-7) but this differential was not observed at later time points (weeks 8-29). Diet consumption was comparable for mice placed on basal diet and PEITC-supplemented diet. Mean PEITC concentration in the plasma and tumor tissue was 901 nM (95% confidence interval- 741-1060 nM) and 87 nmol/kg (95% confidence interval- 67-107 nmol/kg), respectively. Expression of HER-2 oncoprotein as well as number of CD31-positive blood vessels in the tumor did not differ between control and PEITC treatment groups. On the other hand, tumors from PEITC treatment group exhibited about 46% decrease in Ki-67 expression in comparison with control (P= 0.01). Consistent with cellular observations, number of TUNEL-positive apoptotic bodies was about 4.4-fold higher in the tumors of PEITC-fed mice as compared with controls (P= 0.01). Pulmonary metastasis was evident in 3/35 control mice but only 1/32 mice placed on PEITC-supplemented diet exhibited microscopic evidence of metastasis. Size of pulmonary metastasis was also smaller in the PEITC treatment group compared with control. The results indicate that PEITC administration in the diet inhibits carcinoma development in MMTV-neu mice without any signs of overt toxicity. This investigation was supported in part by the US PHS grants CA101753 and CA129347 awarded by the National Cancer Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 563. doi:1538-7445.AM2012-563

Pancreatic cancer is the fourth leading cause of cancer-related deaths in the U.S. with a 5-year survival rate of less than 5%. Low survival rate for patients with pancreatic cancer points towards an increased need for novel chemoprevention, early detection, and therapeutic strategies for this disease. This study was undertaken to determine the efficacy of phenethyl isothiocyanate (PEITC), a naturally occurring isothiocyanate found in cruciferous vegetables, against pancreatic cancer cells in vitro and in vivo. Exposure of human pancreatic cancer cells (MIAPaca2, PL-45, and BxPC3) to PEITC resulted in growth inhibition, which was accompanied by apoptotic cell death regardless of the K-ras status. Apoptotic cell death by PEITC treatment was characterized by an increase in cytoplasmic histone-associated DNA fragmentation and enrichment of sub-diploid fraction. PEITC-induced apoptosis correlated with induction of pro-apoptotic protein Bak, down-regulation of anti-apoptotic proteins Bcl-2 and Bcl-xL, and cleavage of caspase-3 and PARP. Oral administration of 12 µmol PEITC/mouse (five times per week) significantly retarded the growth of MIAPaca2 xenografts in male nude mice without causing weight loss or any other signs of toxicity. PEITC administration significantly reduced proliferation as determined by PCNA and Ki-67 stainings and increased apoptosis in tumors as shown by TUNEL assay. In conclusion, the present study demonstrates, for the first time, that PEITC administration retards the growth of human pancreatic cancer xenografts by inducing apoptotic cell death. The present study provides pre-clinical evidence for development of PEITC as a chemopreventive agent for pancreatic cancer. This investigation was supported in part by the Wayne Fusaro Pancreatic Cancer Research Fund (DCW), the Shirley Hobbs Martin Memorial Fund (REB), and the NCI grants CA101753 and CA113363 (SVS). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4596. doi:10.1158/1538-7445.AM2011-4596

Phenethyl isothiocyanate (PEITC) is a promising cancer chemopreventive constituent of cruciferous vegetables (e.g., watercress) as evidenced by efficacy against chemically-induced cancer in experimental rodents. The PEITC is also empowered to inhibit growth of cultured and xenografted human prostate cancer cells, which correlates with apoptotic and autophagic cell death. We have shown previously that reactive oxygen species (ROS) play a critical role in proapoptotic signal transduction by PEITC in human prostate cancer cells. Interestingly, a representative normal human prostate epithelial cell line (PrEC) is significantly more resistant to PEITC-induced apoptosis compared with cancerous prostate cells including PC-3 and LNCaP. However, the mechanism behind this differential response remains elusive. The present study was undertaken to test a hypothesis that differential sensitivity of normal versus cancerous human prostate cells to PEITC-mediated proapoptotic response is determined by differences in ROS production and/or basal or inducible expression of antioxidant defense genes. The PEITC treatment resulted in ROS production in PC-3 human prostate cancer cells as judged by flow cytometry and fluorescence microscopy using a chemical probe (MItoSOX Red) and EPR using a cell permeable sin probe (1-hydroxy-3-methoxy-carbonyl-2,2,5,5-tetramethylpyrrolidine). These effects were not observed in PrEC cells similarly treated with the PEITC. Basal antioxidant defense gene expression signature was strikingly different between PC-3 and PrEC cells. Moreover, PEITC treatment (2.5 μM, 6 h) caused up-regulation of 29 genes and down-regulation of 2 genes in PC-3 cells. Conversely only 4 genes were up-regulated and 10 genes were down-regulated by a similar PEITC treatment in the PrEC cell line. The results of the present study clearly indicate that differential sensitivity of PC-3 versus PrEC cells to proapoptotic effect of PEITC is likely attributable to both differences in ROS production and basal as well as altered expression of antioxidant defense genes. This investigation was supported by the USPHS grant CA101753-07, awarded by the National Cancer Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5583. doi:10.1158/1538-7445.AM2011-5583

Background: Renal Cell Carcinoma (RCC) has low 5 year survival rate and is resistant to radiation and chemotherapy. Phenethyl Isothiocyanate (PEITC) is a naturally occurring phytochemical that has a variety of anti-cancer properties. Here we explore two anti-cancer properties of PEITC: induction of apoptosis and induction of cell cycle arrest in RCC cell lines and the underlying mechanisms. Methods: We used two human RCC cell lines Caki-1 and Caki-2. Survival and cell proliferation was assayed using Calcein AM. Annexin V staining was used to measure apoptosis. Caspase-3/7 induction was measured using a fluorescent substrate. Cell Cycle was studied using Propidium Iodide staining. DNA damage was determined using H2AX-γ antibody. Protein expression and phosphorylation was determined using immunoblotting. Results: PEITC significantly reduced survival of Caki-1 and Caki-2 cells and inhibited their proliferation as determined by Calcein AM. 15 and 20 μM PEITC induced apoptosis (revealed by Annexin V staining) in both cell lines and induced caspase-3/7 activity. Western blot analysis revealed caspase-8, caspase-9 and Bid cleavage suggesting that the apoptosis is mediated through the extrinsic pathway. Lower doses (up to 10 μM) arrested Caki-1 cells in G2/M phase, and this was associated with an increase p38 and MK2 (Thr334) phosphorylation (as revealed by Western Blot). The p38 inhibitor SB203850 inhibited this G2 arrest induced by PEITC. In order to determine if DNA damage was responsible for the apoptosis and G2 arrest, we used an antibody to H2AX-γ phosphorylation. 15 and 20 µM treatment resulted in increased H2AX-γ phosphorylation, but this was completely blocked by caspase-3 inhibitor. Conclusion: PEITC induces apoptosis in Caki-1 and Caki-2 cells by a mechanism that likely involves the extrinsic pathway. We will use immunoblotting and siRNA to determine which of the extrinsic receptors (DR4, DR5, TRAIL or FAS) is responsible for this apoptosis induction. PEITC does not cause direct DNA damage to the cells; the observed DNA damage is a result of the apoptotic process and is blocked by caspase-3 inhibitors. PEITC induces G2/M arrest in Caki-1 cells and the mechanism is dependent on p38 phosphorylation which activates MK2. Inducing cell cycle arrest and apoptosis may play an important role in the anti-cancer properties of PEITC. Fully understanding the mechanism by which PEITC induces apoptosis and cell cycle arrest in RCC cells may lead to development of novel chemotherapeutic drugs against RCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4222. doi:10.1158/1538-7445.AM2011-4222

Her2 is a well established marker of breast cancer and found to be overexpressed in around 20-30% of cases. It plays role in cell proliferation, angiogenesis, metastasis and drug resistance leading to poor prognosis and survival. Therefore development of novel agents targeting Her2 is highly imminent. Based on several epidemiological and experimental evidences, isothiocyanates have been shown to have anticancer effects. We evaluated the effect of phenethyl isothiocyanate (PEITC) in breast cancer cells. Treatment with PEITC for 24h significantly suppressed the survival of both MCF-7 (estrogen dependent) and MDA-MB-231 (estrogen independent) cells in a concentration dependent manner with an IC50 of about 12µM and 8µM respectively. Substantial apoptosis by PEITC treatment was observed in both the cell lines. Interestingly, PEITC treatment after 24h drastically reduced Her2 expression. The decrease in Her2 expression by PEITC treatment correlated with the cleavage of Her2. The low molecular weight fragments of Her2 increased with increasing PEITC concentration. Caspase-3 specific inhibitor blocked PEITC-mediated cleavage of Her2 and apoptosis indicating the involvement of caspase-3 in the cleavage of Her2. Her2 fragments have been shown to induce mitochondria-mediated apoptosis. In agreement, our results show the cleavage of Bid and increase in Bax level, whereas the expression of Bcl-XL was down regulated. Further, PEITC treatment resulted in the release of cytochrome c from the mitochondria into the cytosol, activating caspase 9/3 and PARP cleavage. Taken togther, our results suggests that cleavage of Her2 by PEITC activates mitochondrial death pathway in breast cancer cells. Further mechanistic studies are in progress. [Supported in part by R01 grants CA106953 and CA129038 (to S.K.S) awarded by the National Cancer Institute]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4624. doi:10.1158/1538-7445.AM2011-4624

We have shown previously that phenethyl isothiocyanate (PEITC), a constituent of edible cruciferous vegetables (e.g., watercress), suppresses growth of cultured and xenografted human prostate cancer cells in association with apoptotic and autophagic cell death. The goals of the present study were to: (a) determine effect of dietary administration of PEITC on prostate cancer development using a transgenic mouse model (Transgenic Adenocarcinoma of Mouse Prostate; TRAMP); (b) elucidate whether PEITC utilizes the same mechanisms in vivo as it does in cultured prostate cancer cells; and (c) identify biomarker(s) predictive of PEITC response for future clinical applications. Administration of PEITC in the diet (3 μmol PEITC/g diet for 19 weeks) significantly inhibited burden (affected area) of poorly-differentiated cancer in the dorsolateral prostate (DLP) of TRAMP mice without causing weight loss or any other side effects. PEITC was detectable in plasma (∼1451 nM) as well as in the prostate (∼1138 nmol/kg) of non-transgenic littermates placed on PEITC-supplemented diet. The PEITC-mediated inhibition of prostate cancer progression correlated with a decrease in the expression of cell proliferation marker Ki-67. To our surprise, number of apoptotic bodies was comparable in the DLP of Control and PEITC-fed mice. On the other hand, the DLP from mice placed on PEITC-supplemented diet exhibited 2-3 fold increase in number of autophagosomes (judged by transmission electron microscopy) and elevated expression of autophagy regulator microtubule-associated protein 1 light chain 3 (judged by immunohistochemistry), as compared with that of Control group. Consistent with these observations, level of selective autophagy substrate p62 (also known as sequestosome 1) was reduced in the DLP of PEITC-fed mice in comparison with Control. The PEITC-mediated inhibition of prostate cancer development was accompanied by a marked increase in expression of E-cadherin. Additionally, 2-dimensional gel electrophoresis coupled with mass spectrometry revealed a marked decrease in plasma clusterin in PEITC-treated mice compared with Controls. Together these results indicate that PEITC administration inhibits prostate cancer progression in TRAMP mice in association with reduced cell proliferation and induction of autophagy. Furthermore, the present study discovers plasma clusterin as a potential biomarker of PEITC response. This investigation was supported by the USPHS grant 2 RO1 CA101753-07, awarded by the National Cancer Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5563. doi:10.1158/1538-7445.AM2011-5563

Ovarian cancer has the highest mortality rate of all the gynecological cancers in the United States, and is highly resistant to current forms of treatment. Metformin, a type II diabetes drug, and phenethyl isothiocyanate (PEITC), a component of cruciferous vegetables, were examined for their effects on ovarian cancer cell growth and survival. Although the exact mechanism of both drugs is not known, both have been found to inhibit complexes within the electron transport chain. Metformin is also known to activate the AMPK-LKB1 pathway, possibly downstream of complex I inhibition. We examined the effects of these compounds in vitro to evaluate the potential of electron transport chain inhibitors as novel therapies for ovarian cancer. To assess cell growth and death, hemacytometry with trypan blue exclusion was used in the ovarian cancer cell lines: SKOV3, OVCAR3, CAOV3, and PA-1. Induction of apoptosis was measured by Western blotting for intact and cleaved poly-ADP ribose polymerase (PARP). Western blotting was also used to determine endogenous levels of enzymes responsible for clearing reactive oxygen species (ROS). Levels of mitochondrial ROS production were measured by flow cytometry and confocal microscopy using the fluorescent probe MitoSOX. Metformin treatment was found to induce cell death in PA-1, arrest growth in OVCAR3 and CAOV3, and slow growth in SKOV3. PEITC treatment induced cell death through apoptosis in all tested cell lines at low micromolar concentrations. Metformin's inhibitory effects were partially reversed when growth medium was supplemented with succinate to bypass complex I. Low expression levels of anti-oxidant enzymes were also found to correspond with higher sensitivity to metformin and PEITC. Additionally, levels of mitochondrial ROS increased with treatment. Metformin and PEITC both show potential as ovarian cancer therapies. Complex I inhibition likely contributes to metformin's anti-cancer effects, as succinate supplementation was able to reverse growth inhibition. Additional work is necessary to determine if the increase in mitochondrial ROS is caused by electron transport chain complex inhibition and whether it is essential to the anti-cancer effects observed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4073. doi:10.1158/1538-7445.AM2011-4073

Cruciferous vegetables, such as broccoli and watercress, have been studied extensively for their chemopreventive activities in animal models. It has been demonstrated that the thiol-reactive isothiocyanates (ITCs) are primarily responsible for their chemopreventive activity. While ITCs are known to induce Phase II enzymes and hence detoxification of certain carcinogens to prevent initiation of carcinogenesis, they also exhibit antitumor activity at a post-initiation phase, suggesting their additional role(s) in cancer prevention. In vitro studies have demonstrated that ITCs induce apoptosis which could be linked to their chemopreventive activity in the post-initiation phase. A number of studies have suggested that IKK inhibition, NF-κB suppression and/or STAT3 down-regulation may contribute to ITC-induced apoptosis. Here, we provide evidence that topoisomerase II (Top2) is involved in ITC-induced apoptosis in cultured tumor cells: (1) BITC (benzyl isothiocyanate) and PEITC (phenethyl isothiocyanate) induced much reduced apoptosis in HL-60 cells and T-antigen transformed MEFs transfected with Top2α siRNA. (2) Similarly, the induction of the DSB signal, γ-H2AX, was also reduced in HL-60 cells transfected with Top2α siRNA. (3) BITC and PEITC induced Top2-mediated DNA damage in vitro through the formation of topoisomerase II-DNA covalent complexes. Together, these results suggest a possible role of Top2 in ITC-induced cell death, presumably through the formation of topoisomerase II-DNA cleavage complexes. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A36.

Background. Isothiocyanates (ITCs) found in cruciferous vegetables have demonstrated cancer preventive activity in animals. Increased dietary intake of ITCs has been shown to be associated with a reduced cancer risk in humans. ITCs exert their cancer chemopreventive action by multiple mechanisms, for example, by modulating the activities of phase I and phase II drug metabolism enzymes, by inhibiting the cell cycle and histone deacetylase, and by causing apoptotic cell death. In cells, protein adducts account for most of total cellular ITC-uptake at 4h after treatment. The time course of this protein binding correlates well with the inhibition of proliferation and the induction of apoptosis. In animal studies, glutathione conjugates are the major products of ITCs. The major urinary excretion products of ITCs in human are N-acetyl cysteine conjugates. Urinary metabolites might provide the exposure history of the last 24h, if the urine of the full next day is collected. Therefore, stable biomarkers such as reaction products with blood proteins are needed which reflect a larger time span of the ITC-exposure history. Results. We developed a method to determine serum albumin adducts of allyl isothiocyanate (AITC), benzylisothiocyanate (BITC), phenylethyl isothiocyanate (PEITC), and sulforaphane (SFN) in humans. The feasibility of the method was tested on biological samples collected previously for a diet-study. The volunteers were randomly assigned to three groups: Group A (n=29): the diet was rich in flavonoids, but not supplemented. Group B (n=29), the diet was a normal iso-caloric diet with an adequate administration of fruit and vegetables. Group C (n=27): the diet was based on supplementation of the normal diet with additional flavonoids in the form of green tea and soy products. All subjects were male smokers. Blood samples were collected at the beginning and after 4 weeks. After enzymatic digestion of albumin we determined the adducts of the ITCs with lysine (Lys) using LC-MS/MS and isotope dilution method. At the beginning of the study (and after 4 weeks) 5.9% (2.4%), 83.5% (96.5%), 11.8% (15.3%), and 31.8% (32.9%) samples were found positive for AITC-Lys, BITC-Lys, PEITC-Lys, and SFN-Lys respectively. Significant differences among the groups were found at the beginning of the study. SFN-Lys levels in group C were significantly higher than in group A and B, and BITC-levels in group C were significantly lower than in group A and B. These differences disappear after 4 weeks. Conclusions. This new method enables to quantify ITC adducts in blood proteins from large prospective studies about diet and cancer. Protein adducts are involved in the chemopreventive effects of ITCs. Therefore, blood protein adducts are a potential surrogate marker for the effects of ITCs at the cellular level. This new technique will improve the assessment of ITC exposure and the power of studies on the relationship between ITC intake and cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5701.

p53 mutations occur in more than 50% of all human tumor. Mutant p53 is incapable of activating downstream target genes necessary for cell cycle arrest and apoptosis. Phenethyl isothiocyanate (PEITC), a cruciferous vegetable-derived compound, has been shown to possess anti-cancer property by causing cell growth arrest or apoptosis in tumor cell lines and animal models. Recently, we have found that PEITC can selectively reduce mutant p53, but not wild type protein levels in a variety of tumor cell lines. Here, we demonstrate that PEITC can increase sequence specific DNA binding of p53 in NSCLC H596 cells expressing mutant p53 and induce the expression of p53-dependent target proteins, such as p21 and Noxa. Similar effects were observed in human glioblastoma T98G cells which also express mutant p53. H596 cells incubated with sulforaphane (SFN), another widely studied ITC compound with much weaker cytotoxicity, showed no increased specific DNA binding of p53. In addition, PEITC induced p53-responsive p21 and Noxa promoter luciferase activity. Blocking mutant p53 protein expression with shRNA in T98G cells entirely prevented p21 protein induction triggered by PEITC. Immunoprecipitation studies with conformation-specific p53 antibodies (Pab1620 and Pab240) show that treatment with PEITC alters mutant p53 to wild type conformation in H596 cells. Currently, we are investigating the significance of function restoration to mutant p53 by phenethyl isothiocyanate in the regulation of apoptosis by PEITC or other conventional chemotherapeutic drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1629.

Phenethyl isothiocyanate (PEITC), a constituent of cruciferous vegetables (e.g., watercress), not only affords significant protection against chemically-induced cancer in experimental animals but also inhibits growth of human cancer cells in association with induction of apoptosis and autophagy. However, the mechanism by which PEITC causes cell death is not fully understood. Using LNCaP and PC-3 human prostate cancer cells as a model, we now demonstrate that the PEITC-induced cell death is initiated by reactive oxygen species (ROS) production due to inhibition of oxidative phosphorylation. Exposure of prostate cancer cells to pharmacologic concentrations of PEITC resulted in ROS production, which was accompanied by inhibition of complex-III activity, suppression of oxidative phosphorylation, and ATP depletion. These effects were not observed in a normal human prostate epithelial cell line (PrEC). The Rho-0 variants of LNCaP and PC-3 cells lacking normal oxidative phosphorylation were significantly more resistant to PEITC-mediated ROS generation, apoptotic DNA fragmentation, caspase-3 activation, and collapse of mitochondrial membrane potential compared with respective wild-type cells. The PEITC treatment resulted in activation of Bax in wild-type LNCaP and PC-3 cells, but not in their respective Rho-0 variants. Furthermore, Bax and Bak knockdown conferred significant protection against PEITC-induced apoptosis. The Rho-0 variants of LNCaP and PC-3 cells also resisted autophagy induction by PEITC treatment. In conclusion, the present study provides concrete experimental evidence to implicate ROS in initiation of PEITC-induced apoptosis and autophagy in human prostate cancer cells. This investigation was supported by the National Cancer Institute grant 2 RO1 CA101753-06. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3792.

Phenethyl isothiocyanate (PEITC), a cancer chemopreventive constituent of edible cruciferous vegetables such as watercress, suppresses growth of human prostate cancer cells by causing apoptosis but the mechanism of cell death induction is not fully understood. Here, we provide experimental evidence to implicate adapter protein p66shc in regulation of PEITC-induced apoptotic cell death. Mouse embryonic fibroblasts lacking p66shc isoform were resistant to PEITC-mediated growth inhibition, cytoplasmic histone-associated apoptotic DNA fragmentation, and caspase-3 activation compared with wild-type fibroblasts. The PEITC treatment resulted in induction as well as increased Ser36 phosphorylation of p66shc in PC-3 (an androgen-independent cell line lacking functional p53) and LNCaP (an androgen-responsive cell line with intact p53) human prostate cancer cells. Moreover, siRNA knockdown of p66shc protein in PC-3 and LNCaP cells conferred significant protection against PEITC-induced cytoplasmic histone-associated DNA fragmentation, caspase-3 activation, and production of reactive oxygen species. Immunoprecipitation-immunoblotting experiments revealed increased binding of p66shc with prolyl isomerase 1 (Pin1), which is implicated in translocation of p66shc to mitochondria, upon treatment of PC-3 and LNCaP cells with PEITC compared with vehicle-treated control cells. Consistent with these results, treatment of PC-3 cells with PEITC resulted in translocation of p66shc to the mitochondria as judged by immunoblotting using cytosolic and mitochondrial fractions and immunofluorescence microscopy. Growth of PC-3 cells subcutaneously implanted in male athymic mice was significantly retarded by gavage with 9 μmol PEITC five times per week. The PEITC-mediated inhibition of PC-3 xenograft growth correlated with reduced cell proliferation, increased apoptosis, hyperphosphorylation of p66shc, and down-regulation of Pin1 protein level. Collectively, these results provide novel insight into the mechanism of PEITC-induced apoptosis to indicate critical role of p66shc in regulation of cell death. This investigation was supported by the National Cancer Institute grant 2 RO1 CA101753-06. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3788.

To establish animal models for identifying mechanisms of urinary bladder cancer (UBC) invasion, male human c-Ha-ras proto-oncogene transgenic rats (Hras 128) were divided into 5 groups and treated with 0.05% N-butyl-N-(hydroxybutyl)nitrosameine (BBN) in drinking water and/or 0.1% phenylethyl isothiocyanate (PEITC) in diet, respectively, as follows: BBN (8 wks)→PEITC (8 wks); PEITC (8 wks)→BBN (8 wks); PEITC alone (16 wks); BBN alone (16 wks); non-treatment. At the end of week 16, incidences of invasive UBC in BBN→PEITC and BBN alone groups were 77% (10/13 rats) and 56% (5/9 rats), respectively. The average number of invasive UBC in BBN→PEITC and BBN alone groups were 1.3 ± 0.9 and 0.7 ± 0.7/rat, respectively. Both incidence and numbers of invasive UBC tended to be higher in BBN→PEITC group compared to the BBN alone group. Furthermore, non-invasive UBC were also found in all rats of above 2 groups. No tumors were found in other groups. p53 positive staining were observed in 58% of invasive UBC and 9% of non-invasive UBC in BBN→PEITC group that is similar to positive incidences in human UBCs. These findings indicated that treatment of Hras 128 rats with BBN followed by PEITC induced high incidences of both non-invasive and invasive UBC, and therefore provide a useful comparison model for explore the mechanisms of UBC invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3237.

Epidemiological studies have demonstrated a protective association between dietary intake of cruciferous vegetables and the incidence of renal cell carcinoma. Isothiocyanates (ITCs) are a class of sulfur-containing phytochemicals found uniquely and abundantly in cruciferous vegetables (broccoli, cauliflower, brussel sprouts, etc). We hypothesize that the inhibition of renal carcinoma cell growth and viability by ITCs underlies the protective effect of cruciferous vegetables on renal cell carcinoma. To test this hypothesis, we examined the effects of two ITCs: the naturally-occurring phenethyl ITC (PEITC) and the synthetic 4-phenylbutyl ITC (PBITC) on the Caki-1 human renal carcinoma cell line. Both PEITC and PBITC inhibited the proliferation of Caki-1 cells in a concentration-dependent manner. Cell cycle analysis revealed that PEITC and PBITC caused an arrest in the G2/M phase. Additionally, higher concentrations of PEITC and PBITC were cytotoxic to Caki-1 cells (IC50 of 7.0 and 4.8 µM, respectively). PEITC and PBITC induced caspase-3/7 activity in a concentration- and time-dependent manner, indicating that the ITCs induce apoptosis in the cells. These results indicate that PEITC and PBITC may be efficacious against renal cell carcinoma and suggest that the preventive effect of diets rich in cruciferous vegetables is due to their ITC content. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B74.

A37 Modulation of GSTP1 and NQO1 by plant-derived chemopreventive agents in human lung normal, immortalized, and malignant bronchial cells Xiang-Lin Tan 1, 2 Shengli Xiong 1, 2 Miao Shi 1 , Weiguo Han 1, 2 Simon D. Spivack 1, 2, 3, 4, 5 1 Division of Pulmonary Medicine, Albert Einstein College of Medicine, Bronx, NY, 2 Laboratory of Human Toxicology and Molecular Epidemiology, Wadsworth Center, New York State Department of Health, Albany, NY 3 Environmental Health Sciences, School of Public Health, University at Albany, Albany, NY 4 Department of Epidemiology, Albert Einstein College of Medicine, Bronx, NY, 5 Department of Genetics, Albert Einstein College of Medicine, Bronx, NY, Many phytochemicals possess putative cancer-preventive properties. However, none have yet demonstrated clear benefit for lung cancer prevention in clinical trials. In this study, we applied an in vitro system of assay to systematically investigate the influence of putative anti-mutagen, phase II metabolism inducing naturally-occurring mixtures and single compounds, including green tea extracts (GTE), broccoli sprout ITC extracts (ITCs), epigallocatechin gallate (EGCG), sulforaphane (SFN), phenethyl isothiocyanate (PEITC) and benzyl isothiocyanate (BITC), for phase II enzymes GSTP1 and NQO1 in the mRNA and protein level. Primary normal human bronchial epithelial cells (NHBE), immortalized human bronchial epithelial cells (HBEC), and overtly malignant lung adenocarcinoma cells (A549) were incubated with 0.5, 1.0, 5.0 µg/ml mixture or 0.5, 1.0, 2.0 µM single index compounds for 24h, 48h and 7 days, respectively. The proliferation of normal and malignant cells determined by MTT assay was significantly reduced by all studied agents, with relative activities of BITC > PEITC > ITCs > SFN > EGCG > GTE. We then determined the mRNA expression by RNA-specific quantitative RT-PCR previously developed in the laboratory, and the protein expression by conventional Western blot. Overall, the mixtures GTE and ITC increased GSTP1 and NQO1 mRNA levels up to two-fold (1ug/ml) to four-fold (5 ug/ml) increased. However, these mRNA levels were decreased by single compounds PEITC and BITC, in a time and dose dependent manner. EGCG and SFN did not show a consistent effect on the mRNA expression of both GSTP1 and NQO1 genes, although SFN showed two- to four-fold induction activity for NQO1 mRNA expression at 0.5-1.0 uM, uniquely for NHBE. The results were largely confirmed at the protein level, although there was some mRNA-protein discordance. Our results suggest that further in vitro and in vivo screening studies are needed to discover new, more potent phase II-inducing chemopreventive agents for lung cancer, along with mechanistic studies of gene regulation for these key upstream anti-carcinogenesis genes. Such studies are in process in the laboratory. 1 Citation Information: Cancer Prev Res 2008;1(7 Suppl):A37.