Effects Of Phenethyl Isothiocyanate Research Articles

Phenethyl isothiocyanate Regulates the Cancer Stem Cell Phenotype of SNU449 Hepatocellular Carcinoma Cells via STAT3-CD44 Axis

Cancer stem cells play an important role in resistance to therapy, invasion, metastasis, and recurrence. CD44 is one of the well-known surface markers for hepatocellular carcinoma and its expression level is related to poor survival and a high recurrence rate. The effect of phenethyl isothiocyanate (PEITC) on SNU449 hepatocellular carcinoma cell line cancer stem cells is not known. The goal of the present study was to investigate whether PEITC regulates the cancer stem cell phenotype of SNU449 cells. Here, cell viability, colony formation, and wound healing assays were performed to determine proliferative and migratory characteristics. Additionally, Caspase 3, CD44, Akt/mTOR, and p38/STAT3 protein expression levels were measured by western blotting. We found that compared to control confluence, gap fill, and migration rate were increased while half gap time was decreased in PEITC-treated cells. Compared to control-treated cells CD44 (3.2 fold) and p-STAT3 (2.44 fold) protein expressions were upregulated in PEITC-treated cells. Results of this study suggest that STAT3-mediated upregulation of CD44 leads to the gain of cancer stem cell phenotype of PEITC-treated SNU449 cells.

Combinations of Phytochemicals More Efficiently than Single Components Activate Nrf2 and Induce the Expression of Antioxidant Enzymes in Pancreatic Cancer Cells

Cancer prevention particularly related to aging can be improved by the use of phytochemicals combinations. In this study, we evaluated the effect of phenethyl isothiocyanate (PEITC), xanthohumol (XAN), indole-3-carbinol (I3C), and resveratrol (RES) and their combinations on the Nrf2 signaling pathway. Human pancreatic cancer cells MIA-Pa-Ca-2 were treated with the phytochemicals alone or their equimolar mixture for 24 h and activation of Nrf2 and expression of its target genes were evaluated. Phytochemicals alone enhanced Nrf2 activation and expression, but their combinations were more efficient. The mixture of XAN and PEITC was found to be the most potent modulator of the Nrf2 pathway. Moreover, increased levels of P-Nrf2 and P-JNK and decreased level of P-GSK-3β suggested possible activation of Nrf2 through modulation of these kinases. The combinations of XAN with PEITC and RES with PEITC increased mostly the expression of SOD, GSTP, CAT, and GPx. XAN and PEITC mixture induced the cell cycle arrest in G0/G1 phase and increased apoptotic and autophagy markers. These results indicate that combinations of phytochemicals resembling that occurring in natural diets may efficiently modulate the signaling pathways, which proper function is important for pancreatic cancer prophylaxis or improving the results of conventional therapy.

Susceptibility to the acute toxicity of acrylonitrile in streptozotocin-induced diabetic rats: protective effect of phenethyl isothiocyanate, a phytochemical CYP2E1 inhibitor

Diabetes mellitus is a significant global public health issue. The diabetic state not only precipitates chronic disease but also has the potential to change the toxicity of drugs and chemicals. Acrylonitrile (AN) is a potent neurotoxin widely used in industrial products. This study used a streptozotocin (STZ)-induced diabetic rat model to examine the role of cytochrome P450 2E1 (CYP2E1) in acute AN toxicity. The protective effect of phenethyl isothiocyanate (PEITC), a phytochemical inhibitor of CYP2E1, was also investigated. A higher incidence of convulsions and loss of the righting reflex, and decreased rates of survival, as well as elevated CYP2E1 activity, were observed in diabetic rats treated with AN when compared to those in non-diabetic rats, suggesting that diabetes confers susceptibility to the acute toxicity of AN. Pretreatment with PEITC (20–80 mg/kg) followed by AN injection alleviated the acute toxicity of AN in diabetic rats as evidenced by the decreased incidence of convulsions and loss of righting reflex, and increased rates of survival. PEITC pretreatment at 40 and 80 mg/kg decreased hepatic CYP2E1 activity in AN-exposed diabetic rats. PEITC pretreatment (20 mg/kg) increased the glutathione (GSH) content and glutathione S-transferase (GST) activity and further decreased ROS levels in AN-exposed diabetic rats. Collectively, STZ-induced diabetic rats were more sensitive to AN-induced acute toxicity mainly due to CYP2E1 induction, and PEITC pretreatment significantly alleviated the acute toxicity of AN in STZ-induced diabetic rats. PEITC might be considered as a potential effective chemo-preventive agent against AN-induced acute toxicity in individuals with an underlying diabetic condition.

Transcriptomic Analysis of Histone Methyltransferase Setd7 Knockdown and Phenethyl Isothiocyanate in Human Prostate Cancer Cells.

Transcriptomic analysis was performed to evaluate the differential gene expression profiles of Setd7 knockdown (KD) and the effects of phenethyl isothiocyanate (PEITC) in human prostate cancer (PCa) LNCaP cells. RNA isolated from wild-type and Setd7-KD LNCaP cells in the presence or absence of PEITC was subjected to microarray analysis followed by Ingenuity® Pathway Analysis (IPA). Setd7 KD impacted a larger set of genes and caused a higher fold change compared to PEITC treatment. Several signaling pathways were altered particularly inflammation-related TNFR signaling and PTEN/PI3K/AKT signaling by Setd7 KD and PEITC. Interestingly, PEITC and Setd7 KD at a small subset of genes that could be potential molecular targets. This study offers new insights into the mechanisms of action of the epigenetic modifier Setd7 and the effects of PEITC treatment in PCa cells and enhances our understanding of the potential cancer preventive/treatment effects of isothiocyanate compounds such as PEITC in PCa.

Phenethyl Isothiocyanate Induces Apoptotic Cell Death Through the Mitochondria-dependent Pathway in Gefitinibresistant NCI-H460 Human Lung Cancer Cells In Vitro

Some lung cancer patients treated with gefitinib develop resistance to this drug resulting in unsatisfactory treatment outcomes. Phenethyl isothiocyanate (PEITC), present in our common cruciferous vegetables, exhibits anticancer activities in many human cancer cell lines. Currently, there is no available information on the possible modification of gefitinib resistance of lung cancer in vitro by PEITC. Thus, the effects of PEITC on gefitinib resistant lung cancer NCI-H460 cells were investigated in vitro. The total cell viability, apoptotic cell death, production of reactive oxygen species (ROS) and Ca2+, levels of mitochondria membrane potential (ΔΨm) and caspase-3, -8 and -9 activities were measured by flow cytometry assay. PEITC induced chromatin condensation was examined by DAPI staining. PEITC-induced cell morphological changes, decreased total viable cell number and induced apoptotic cell death in NCI-H460 and NCI-H460/G cells. PEITC decreased ROS production in NCI-H460 cells, but increased production in NCI-H460/G cells. PEITC increased Ca2+ production, decreased the levels of ΔΨm and increased caspase-3, -8 and -9 activities in both NCI-H460 and NCI-H460/G cells. Western blotting was used to examine the effect of apoptotic cell death associated protein expression in NCI-H460 NCI-H460/G cells after exposure to PEITC. Results showed that PEITC increased expression of cleaved caspase-3, PARP, GADD153, Endo G and pro-apoptotic protein Bax in NCI-H460/G cells. Based on these results, we suggest that PEITC induces apoptotic cell death via the caspase- and mitochondria-dependent pathway in NCI-H460/G cells.

Inhibition of autophagy potentiates the anti-metastasis effect of phenethyl isothiocyanate through JAK2/STAT3 pathway in lung cancer cells.

Phenethyl isothiocyanate (PEITC) is a natural compound abundant in cruciferous vegetables. PEITC possesses anti-tumor effect in various human malignances. Our previous study has shown that benzyl isothiocyanates (BITC) induce autophagy in lung cancer cells. However, whether autophagy play a role in the inhibitory effect of PEITC on lung cancer metastasis is unclear. In this study, we found that PEITC suppressed migration and invasion of lung cancer cells by regulating MMP2. It also induced autophagy, evidenced by the formation of acidic vesicular organelles (AVOs), the punctate pattern of LC3, the accumulation of LC3-II, and the expression of Beclin-1. Inhibition of autophagy by 3-MA and chloroquine (CQ) or knock down of Beclin-1 enhanced PEITC-caused metastasis inhibition. JAK2/STAT3 pathway was suppressed by PEITC, and further inhibited by 3-MA and CQ or Beclin-1 knock down, as a result of decreased expression of p-JAK2 and p-STAT3. Blocking JAK2/STAT3 pathway by inhibitor AG490 and Stattic suppressed cell migration and decreased the expression of MMP2, MMP9, Twist, and c-Myc. Further in vivo study showed that PEITC inhibited tumor growth, induced autophagy and suppressed JAK2/STAT3 pathway, and inhibitor CQ enhanced this effect. Taken together, our results demonstrate that PEITC inhibits metastasis potential of lung cancer cells, and induces autophagy. The autophagy induced by PEITC preserves metastasis potential of lung cancer cells, via activation of JAK2/STAT3 pathway. Inhibition of autophagy enhanced the inhibitory effect of PEITC on metastasis potential of lung cancer cells. Our finding suggests that targeting autophagy could be a promising strategy for anti-metastasis therapies.

Phenethyl isothiocyanate Triggers Apoptosis, Combats Oxidative Stress and Inhibits Growth of Ehrlich Ascites Carcinoma Mouse Model.

The aim of this study is to investigate the antitumor activity and possible molecular mechanism of Phenethyl isothiocyanate (PEITC) against Ehrlich ascites carcinoma in-vivo and in-vitro. In-vivo, ascetic fluid volume, body weight, serum malondialdehyde (MDA) level and total antioxidant capacity (TAC) were determined using Ehrlich ascites carcinoma (EAC) bearing mice. In-vitro, MTT assay was used. RT-PCR was used to investigate role of PEITC in apoptosis by analyzing the expression of Bax, caspase-9, and Bcl-2 genes. The effect of PEITC on caspase-9 enzyme activity was also tested. PEITC and/or Doxorubicin (Dox) treatment significantly suppressed EAC growth as compared to EAC/oil control mice. PEITC treatment showed a dose-dependent inhibition of EAC cells as indicated by MTT assay. We found that significant increase in MDA level and decrease in TAC caused by Dox treatment were significantly reduced by combination with PEITC treatment. Bax, caspase-9 genes' expression and caspase-9 enzymatic activity were significantly increased, while Bcl-2 gene expression was significantly decreased in PEITC treated mice. PEITC may act as a promising anticancer agent either alone or more effectively in combination with Dox through apoptotic cell death induction.

Phenethyl isothiocyanate suppresses cancer stem cell properties in vitro and in a xenograft model

BackgroundCancer stem cells (CSCs) are a subset of cells within the bulk of a tumor that have the ability to self-renew and differentiate, and are thus associated with cancer invasion, metastasis, and recurrence. Phenethyl isothiocyanate (PEITC) is a natural compound found in cruciferous vegetables such as broccoli and is used as a cancer chemopreventive agent; however, its effects on CSCs are little known. PurposeTo evaluate the effect of PEITC on CSCs in this study by examining CSC properties. MethodsNCCIT human embryonic carcinoma cells were treated with PEITC, and the expression of pluripotency factors Oct4, Sox-2, and Nanog were evaluated by luciferase assay and western blot. Effect of PEITC on self-renewal capacity and clonogenicity were assessed with the sphere formation, soft agar assay, and clonogenic assay in an epithelial cell adhesion molecule (EpCAM)-expressing CSC model derived from HCT116 colon cancer cells using a cell sorting system. The effect of PEITC was also investigated in a mouse xenograft model obtained by injecting nude mice with EpCAM-expressing cells. ResultsWe found that PEITC treatment suppressed expression of the all three pluripotency factors in the NCCIT cells, in which pluripotency factors are highly expressed. Moreover, PEITC suppressed the self-renewal capacity and clonogenicity in the EpCAM-expressing CSC model. EpCAM was used as a specific CSC marker in this study. Importantly, PEITC markedly suppressed both tumor growth and expression of three pluripotency factors in a mouse xenograft model. ConclusionThese results demonstrate that PEITC might be able to slow down or prevent cancer recurrence by suppressing CSC stemness.

PEITC-mediated inhibition of mRNA translation is associated with both inhibition of mTORC1 and increased eIF2α phosphorylation in established cell lines and primary human leukemia cells.

Increased mRNA translation drives carcinogenesis and is an attractive target for the development of new anti-cancer drugs. In this work, we investigated effects of phenethylisothiocyanate (PEITC), a phytochemical with chemopreventive and anti-cancer activity, on mRNA translation. PEITC rapidly inhibited global mRNA translation in human breast cancer-derived MCF7 cells and mouse embryonic fibroblasts (MEFs). In addition to the known inhibitory effects of PEITC on mTORC1 activity, we demonstrate that PEITC increased eIF2α phosphorylation. PEITC also increased formation of stress granules which are typically associated with eIF2α phosphorylation and accumulation of translationally stalled mRNAs. Analysis of genetically modified MEFs demonstrated that optimal inhibition of global mRNA translation by PEITC was dependent on eIF2α phosphorylation, but not mTORC1 inhibition. We extended this study into primary leukemic B cells derived from patients with chronic lymphocytic leukaemia (CLL). CLL cells were stimulated in vitro with anti-IgM to mimic binding of antigen, a major driver of this leukemia. In CLL cells, PEITC increased eIF2α phosphorylation, inhibited anti-IgM-induced mTORC1 activation and decreased both basal and anti-IgM-induced global mRNA translation. PEITC also inhibited transcription and translation of MYC mRNA and accumulation of the MYC oncoprotein, in anti-IgM-stimulated cells. Moreover, treatment of CLL cells with PEITC and the BTK kinase inhibitor ibrutinib decreased anti-IgM-induced translation and induced cell death to a greater extent than either agent alone. Therefore, PEITC can inhibit both global and mRNA specific translation (including MYC) via effects on multiple regulatory pathways. Inhibition of mRNA translation may contribute to the chemopreventive and anti-cancer effects of PEITC.

Abstract 1910: Reactivation of p53R175 mutant by dietary phenethyl isothiocyanate (PEITC) impairs tumor growth in vivo

Abstract 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

Effect of Phenethyl Isothiocyanate on p21Waf1/Cip1 Regulation of Cell Cycle Arrest in Pancreatic Cancer Cells

Pancreatic cancer continues to be a disease with a dismal prognosis primarily due to diagnosis occurring later in the progression of the disease. Once at an advanced stage, treatment of the tumor through conventional therapies often proves ineffective prompting the need for preventive strategies for this cancer. Phenethyl isothiocyanate (PEITC) is a natural compound found in cruciferous vegetables such as watercress. In this study we show that PEITC inhibited proliferation of MIAPaca2 and PL45 pancreatic cancer cells, and induced mitotic arrest within 8 and 16 h of treatment as evidenced by a dose‐dependent increase in expression of phospho‐histone H3 (Ser10), a marker of cells undergoing mitosis. Treatment with PEITC led to phosphorylation of p53 tumor suppressor protein, and modulated the p53‐inducible gene product p21Waf1/Cip1. PEITC induced a dose‐dependent upregulation in p21Waf1/Cip1 tumor suppressor protein in MIAPaca2 and PL45 pancreatic cancer cells within 4 and 8 h of treatment. In conclusion, the growth inhibitory effect of PEITC was associated with activation of p53, upregulation of cyclin‐dependent kinase inhibitor p21Waf1/Cip1, and induction of mitotic arrest in pancreatic cancer cells, and warrants further investigation of PEITC as a chemopreventive agent for pancreatic cancer. This study was supported in part by PUCCR ACS IRG#58‐006‐53 (awarded to SDS).

Site‐and promoter‐specific epigenetic alterations by a common dietary compound (271.5)

Effects of dietary compounds on site‐specific histone modifications, which are key epigenetic regulators of gene expression impacting cancer etiology, remain relatively unexplored. Phenethylisothiocyanate (PEITC) is a common dietary compound derived from cruciferous vegetables. Our previous research have shown promising anti‐inflammatory and chemo preventive properties of PEITC. The present study reports novel, dynamic, site‐specific chemical changes to histone H3 in a gene‐promoter‐specific manner, associated with PEITC exposure in human colon tumor‐derived epithelial cells. The time‐and concentration‐dependent effects of PEITC on histone modifications (di‐and tri‐methylations of lysine‐9 and lysine‐27 and acetylation) and gene expression changes were achieved at low, non‐cytotoxic concentrations, in contrast to the higher concentrations necessary to halt cancer cell proliferation likely mediated by caspase‐dependent apoptotic signaling. Increased understanding of specific epigenetic alterations by dietary compounds may provide molecular predictors for improved preventive strategies for reducing the healthcare burden of cancer and other human diseases.Grant Funding Source: The work is supported by the National Institutes of Health grant R00AT4245 and SDAES grant 328100/31

Differential hepatic GSTA2 expression of arylalkyl isothiocyanates in vivo and in vitro: The molecular mechanism of gene induction by phenethyl isothiocyanate

Naturally occurring isothiocyanates (ITCs) have been investigated for their chemopreventive actions. However, limited studies have determined the potential for regulation of hepatic glutathione S-transferase A2 (GSTA2) by arylalkyl or alkyl ITCs. The present study was designed to investigate the effects of ITCs on GSTA2 expression and upstream signaling analyzed by Northern blotting, RT-PCR, immunoblotting, and promoter-luciferase assay. A single dose of ethyl isothiocyanate and phenethyl isothiocyanate (PEITC) enhanced GSTA2 mRNA levels in rats. The three consecutive injections of arylalkyl ITCs resulted in the gene induction. PEITC was the most potent and sustained inducer of GSTA2 in vivo. PEITC induced GSTA2 mRNA and protein expression in H4IIE cells. PEITC increased the levels of nuclear factor erythroid 2 related factor 2 and CCAAT/enhancer binding protein β (C/EBPβ). The cells transfected with a pGL-ΔARE or pGL-ΔC/EBP-deleted promoter construct failed to increase GSTA2 promoter activity by PEITC. These effects of PEITC were downstream of multiple cellular signaling, including phosphoinositide 3-kinase (PI3K), c-jun N-terminal kinase (JNK), and/or protein kinase A (PKA). These results demonstrated that PEITC is the most efficacious of the arylalkyl ITCs for the induction of GSTA2 in vivo and in vitro, and that transactivation of C/EBP and nuclear factor erythroid 2 related factor 2 downstream of multiple signaling pathways is involved in this induction.

Phenethyl isothiocyanate suppresses EGF-stimulated SAS human oral squamous carcinoma cell invasion by targeting EGF receptor signaling

Phenethyl isothiocyanate (PEITC) is a natural compound that is involved in chemoprevention as well as inhibition of cell growth and induction of apoptosis in several types of cancer cells. Previous studies have revealed that PEITC suppresses the invasion of AGS gastric and HT-29 colorectal cancer cells. However, the effects of PEITC on the metastasis of SAS oral cancer cells remain to be determined. Our results showed that PEITC treatment inhibited the invasion of EGF-stimulated SAS cells in a concentration-dependent manner, but appeared not to affect the cell viability. The expression and enzymatic activities of matrix metalloprotease-2 (MMP-2) and matrix metalloprotease-9 (MMP-9) were suppressed by PEITC. Concomitantly, we observed an increase in the protein expression of both tissue inhibitor of metalloproteinase-1 (TIMP-1) and -2 (TIMP-2) in treated cells. Furthermore, PEITC treatments decreased the protein phosphorylation of epidermal growth factor receptor (EGFR) and downstream signaling proteins including PDK1, PI3K(p85), AKT, phosphorylated IKK and IκB to inactivate NF-κB for the suppression of MMP-2 and MMP-9 expression. In addition, PEITC can trigger the MAPK signaling pathway through the increase in phosphorylated p38, JNK and ERK in treated cells. Our data indicate that PEITC is able to inhibit the invasion of EGF-stimulated SAS oral cancer cells by targeting EGFR and its downstream signaling molecules and finally lead to the reduced expression and enzymatic activities of both MMP-2 and MMP-9. These results suggest that PEITC is promising for the therapy of oral cancer metastasis.

Phenethylisothiocyanate alters site- and promoter-specific histone tail modifications in cancer cells.

Site-specific histone modifications are important epigenetic regulators of gene expression. As deregulation of genes often results in complex disorders, corrective modulation of site-specific histone marks could be a powerful therapeutic or disease-preventive strategy. However, such modulation by dietary compounds and the resulting impact on disease risk remain relatively unexplored. Here we examined phenethylisothiocyanate (PEITC), a common dietary compound derived from cruciferous vegetables with known chemopreventive properties under experimental conditions, as a possible modulator of histone modifications in human colon cancer cells. The present study reports novel, dynamic, site-specific chemical changes to histone H3 in a gene-promoter-specific manner, associated with PEITC exposure in human colon tumor-derived SW480 epithelial cells. In addition, PEITC attenuated cell proliferation in a concentration- and time-dependent manner, likely mediated by caspase-dependent apoptotic signalling. The effects of PEITC on histone modifications and gene expression changes were achieved at low, non-cytotoxic concentrations, in contrast to the higher concentrations necessary to halt cancer cell proliferation. Increased understanding of specific epigenetic alterations by dietary compounds may provide improved chemopreventive strategies for reducing the healthcare burden of cancer and other human diseases.

Tumor regression by phenethyl isothiocyanate involves DDB2

Phenethyl isothiocyanate (PEITC) is a promising cancer chemopreventive agent commonly found in edible cruciferous vegetables. It has been implicated also for therapy, and is in clinical trial for lung cancer. Here, we provide evidence that the tumor suppressive effect of PEITC is related to its ability to induce expression of damaged DNA binding protein 2 (DDB2), a DNA repair protein involved also in apoptosis and premature senescence. DDB2 expression is attenuated in a wide variety of cancers including the aggressive colon cancers. We show that, in colon cancer cells, reactive oxygen species, which are induced by PEITC, augment expression of DDB2 through the p38MAPK/JNK pathway, independently of p53. PEITC-induced expression of DDB2 is critical for inhibition of tumor progression by PEITC. Tumors derived from DDB2-deficient colon cancer cells are refractory to PEITC-treatments, resulting from deficiencies in apoptosis and senescence. The DDB2-proficient tumors, on the other hand, respond effectively to PEITC. The results show that PEITC can be used to induce expression of DDB2, and that expression of DDB2 is critical for effective response of tumors to PEITC.

Chemopreventive and anti‐angiogenic effects of dietary phenethyl isothiocyanate in an N‐methyl nitrosourea‐induced breast cancer animal model

The effect of phenethyl isothiocyanate (PEITC), a component of cruciferous vegetables, on the initiation and progression of cancer was investigated in a chemically induced estrogen-dependent breast cancer model. Breast cancer was induced in female Sprague Dawley rats (8 weeks old) by the administration of N-methyl nitrosourea (NMU). Animals were administered 50 or 150 µmol/kg oral PEITC and monitored for tumor appearance for 18 weeks. The PEITC treatment prolonged the tumor-free survival time and decreased the tumor incidence and multiplicity. The time to the first palpable tumor was prolonged from 69 days in the control, to 84 and 88 days in the 50 and 150 µmol/kg PEITC-treated groups. The tumor incidence in the control, 50 µmol/kg, and 150 µmol/kg PEITC-treated groups was 56.6%, 25.0% and 17.2%, while the tumor multiplicity was 1.03, 0.25 and 0.21, respectively. Differences were statistically significant (p < 0.05) from the control, but there were no significant differences between the two dose levels. The intratumoral capillary density decreased from 4.21 ± 0.30 vessels per field in the controls to 2.46 ± 0.25 in the 50 µmol/kg and 2.36 ± 0.23 in the 150 µmol/kg PEITC-treated animals. These studies indicate that supplementation with PEITC prolongs the tumor-free survival, reduces tumor incidence and burden, and is chemoprotective in NMU-induced estrogen-dependent breast cancer in rats. For the first time, it is reported that PEITC has anti-angiogenic effects in a chemically induced breast cancer animal model, representing a potentially significant mechanism contributing to its chemopreventive activity.

Effect of phenethyl isothiocyanate on Ca2+ movement and viability in MDCK canine renal tubular cells

The effect of the natural compound phenethyl isothiocyanate (PEITC) on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and viability in MDCK renal cells is unknown. This study explored whether PEITC changed [Ca(2+)](i) in MDCK cells using the Ca(2+)-sensitive fluorescent dye fura-2. PEITC at 200-700 μM increased [Ca(2+)](i) in a concentration-dependent manner. The signal was reduced by removing extracellular Ca(2+). PEITC-induced Ca(2+) influx was inhibited by nifedipine, econazole, SK&F 96365 and protein kinase C modulators. In Ca(2+)-free medium, treatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) inhibited PEITC-induced rise in [Ca(2+)](i). Incubation with PEITC also inhibited TG or BHQ-induced rise in [Ca(2+)](i). Inhibition of phospholipase C with U73122 abolished PEITC-induced rise in [Ca(2+)](i). At 15-75 μM, PEITC decreased viability. The cytotoxic effect of PEITC was enhanced by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl ester. Annexin V-FITC data suggest that 20 and 50 μM PEITC induced apoptosis. At 10 and 15 μM, PEITC did not increase reactive oxygen species (ROS) production. Together, in renal tubular cells, PEITC-induced rise in [Ca(2+)](i) by inducing phospholipase C-dependent Ca(2+) release from endoplasmic reticulum and Ca(2+) entry via store-operated Ca(2+) channels. PEITC induced apoptosis in a concentration-dependent, ROS/Ca(2+)-independent manner.

Abstract 2584: Notch1 and Notch2 activation by phenethyl isothiocyanate hinders its inhibitory effect on prostate cancer cell migration

Abstract 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

Abstract 2410: Reduced expression of DDB2 promotes epithelial to mesenchymal transition in colon cancer

Abstract Invasion and metastasis are the hallmarks of malignant tumor progression and the principal cause of death related to solid tumors. Epithelial to mesenchymal transition (EMT) is intricately associated with cancer progression, as it confers cancer cells the ability to invade and metastasize to a distant organ. Here we identify a novel function of nucleotide excision repair protein DDB2 as an important antagonist of the EMT in colon carcinoma through its maintenance of epithelial phenotype. In human colon carcinoma samples, loss of DDB2 expression is highly correlated with the aggressiveness of the disease. Moreover, stimuli (TGF-beta and hypoxia) that induce EMT inhibit expression of DDB2; and loss of DDB2 is sufficient to promote EMT of colon cancer cells. The mechanism involves DDB2 mediated transcriptional repression of VEGF and MMP3. DDB2 represses these genes by histone deacetylation. We also observed that loss of DDB2 results in resistance to anoikis, an important barrier to metastasis. The lack of anoikis response is associated with increased Akt and ERK activity. Furthermore, treatment with Phenethyl Isothiocyanate (PEITC), a promising cancer chemopreventive component, augmented E-cadherin expression that is related to an increase in DDB2 expression. The present study suggests that anti-metastatic effect of PEITC involves important role of DDB2. Together, our results show that loss of DDB2 expression is both necessary and sufficient for EMT during colon carcinoma progression. 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 2410. doi:1538-7445.AM2012-2410