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P66Shc is indispensable for phenethyl isothiocyanate-induced apoptosis in human prostate cancer cells.

ABSTRACT: Naturally occurring phenethyl isothiocyanate (PEITC) selectively inhibits growth of cancer cells by causing apoptosis, but the mechanism of cell death induction is not fully understood. We now show, for the first time, that growth factor adapter protein p66(Shc) is indispensable for PEITC-induced apoptosis. Mouse embryonic fibroblasts derived from p66(Shc) knockout mice were significantly more 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 Ser(36) phosphorylation of p66(Shc) in PC-3 and LNCaP human prostate cancer cells. Knockdown of p66(Shc) protein conferred significant protection against PEITC-mediated cytoplasmic histone-associated DNA fragmentation as well as production of reactive oxygen species in both PC-3 and LNCaP cells. The PEITC-treated PC-3 and LNCaP cells exhibited increased binding of p66(Shc) with prolyl isomerase Pin1, a protein implicated in translocation of p66(Shc) to mitochondria. Consistent with these results, treatment of PC-3 cells with PEITC resulted in translocation of p66(Shc) to the mitochondria as judged by immunoblotting using cytosolic and mitochondrial fractions and immunofluorescence microscopy. Growth suppression and apoptosis induction in tumor xenografts in vivo by oral administration of PEITC to the PC-3 tumor-bearing male athymic mice were accompanied by statistically significant increase in the level of Ser(36)-phosphorylated p66(Shc). Collectively, these results provide novel insight into the critical role of p66(Shc) in regulation of PEITC-induced apoptotic cell death in human prostate cancer cells.

SUBMITTER: Xiao D

PROVIDER: S-EPMC2855757 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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p66Shc is indispensable for phenethyl isothiocyanate-induced apoptosis in human prostate cancer cells.

Xiao Dong D Singh Shivendra V SV

Cancer research 20100330 8

Naturally occurring phenethyl isothiocyanate (PEITC) selectively inhibits growth of cancer cells by causing apoptosis, but the mechanism of cell death induction is not fully understood. We now show, for the first time, that growth factor adapter protein p66(Shc) is indispensable for PEITC-induced apoptosis. Mouse embryonic fibroblasts derived from p66(Shc) knockout mice were significantly more resistant to PEITC-mediated growth inhibition, cytoplasmic histone-associated apoptotic DNA fragmentati ...[more]

PMID: 20354186

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Project description:BACKGROUND:We reported previously that phenethyl isothiocyanate (PEITC), a dietary compound, can reactivate p53R175H mutant in vitro and in SK-BR-3 (p53R175H) breast xenograft model resulting in tumor inhibition. Because of the diversity of human cancers with p53 mutations, these findings raise important questions whether this mechanism operates in different cancer types with same or different p53 mutations. In this study, we investigated whether PEITC recuses mutant p53 in prostate cancer cells harboring different types of p53 mutants, structural and contact, in vitro and in vivo. METHODS:Cell proliferation, cell apoptosis and cell cycle arrest assays were performed to examine the effects of PEITC on prostate cancer cell lines with p53 mutation(s), wild-type p53, p53 null or normal prostate cells in vitro. Western blot analysis was used to monitor the expression levels of p53 protein, activation of ATM and upregulation of canonical p53 targets. Immunoprecipitation, subcellular protein fraction and qRT-PCR was performed to determine change in conformation and restoration of transactivation functions/ inhibition of gain-of-function (GOF) activities to p53 mutant(s). Mice xenograft models were established to evaluate the antitumor efficacy of PEITC and PEITC-induced reactivation of p53 mutant(s) in vivo. Immunohistochemistry of xenograft tumor tissues was performed to determine effects of PEITC on expression of Ki67 and mutant p53 in vivo. RESULTS:We demonstrated that PEITC inhibits the growth of prostate cancer cells with different "hotspot" p53 mutations (structural and contact), however, preferentially towards structural mutants. PEITC inhibits proliferation and induces apoptosis by rescuing mutant p53 in p53R248W contact (VCaP) and p53R175H structural (LAPC-4) mutant cells with differential potency. We further showed that PEITC inhibits the growth of DU145 cells that co-express p53P223L (structural) and p53V274F (contact) mutants by targeting p53P223L mutant selectively, but not p53V274F. The mutant p53 restored by PEITC induces apoptosis in DU145 cells by activating canonical p53 targets, delaying cells in G1 phase and phosphorylating ATM. Importantly, PEITC reactivated p53R175H and p53P223L/V274F mutants in LAPC-4 and DU145 prostate xenograft models, respectively, resulting in significant tumor inhibition. CONCLUSION:Our studies provide the first evidence that PEITC's anti-cancer activity is cancer cell type-independent, but p53 mutant-type dependent.

Project description:The aim of this study is to synthesize phenethyl-conjugated chitosan oligosaccharide (COS) (abbreviated as ChitoPEITC) conjugates and then fabricate chlorin E6 (Ce6)-incorporated nanophotosensitizers for photodynamic therapy (PDT) of HCT-116 colon carcinoma cells. PEITC was conjugated with the amine group of COS. Ce6-incorporated nanophotosensitizers using ChitoPEITC (ChitoPEITC nanophotosensitizers) were fabricated by dialysis method. 1H nuclear magnetic resonance (NMR) spectra showed that specific peaks of COS and PEITC were observed at ChitoPEITC conjugates. Transmission electron microscope (TEM) confirmed that ChitoPEITC nanophotosensitizers have spherical shapes with small hydrodynamic diameters less than 200 nm. The higher PEITC contents in the ChitoPEITC copolymer resulted in a slower release rate of Ce6 from nanophotosensitizers. Furthermore, the higher Ce6 contents resulted in a slower release rate of Ce6. In cell culture study, ChitoPEITC nanophotosensitizers showed low toxicity against normal CCD986Sk human skin fibroblast cells and HCT-116 human colon carcinoma cells in the absence of light irradiation. ChitoPEITC nanophotosensitizers showed a significantly higher Ce6 uptake ratio than that of free Ce6. Under light irradiation, cellular reactive oxygen species (ROS) production of nanophotosensitizers was significantly higher than that of free Ce6. Especially, PEITC and/or ChitoPEITC themselves contributed to the production of cellular ROS regardless of light irradiation. ChitoPEITC nanophotosensitizers showed significantly higher PDT efficacy against HCT-116 cells than that of free Ce6. These results indicate that ChitoPEITC nanophotosensitizers have superior potential in Ce6 uptake, ROS production and PDT efficacy. In the HCT-116 cell-bearing mice tumor-xenograft model, ChitoPEITC nanophotosensitizers efficiently inhibited growth of tumor volume rather than free Ce6. In the animal imaging study, ChitoPEITC nanophotosensitizers were concentrated in the tumor tissue, i.e., fluorescence intensity in the tumor tissue was stronger than that of other tissues. We suggest that ChitoPEITC nanophotosensitizers are a promising candidate for the treatment of human colon cancer cells.

Project description:Epigenetic silencing of tumor suppressor genes is a phenomenon frequently observed in multiple cancers. Ras-association domain family 1 isoform A (RASSF1A) is a well-characterized tumor suppressor that belongs to the Ras-association domain family. Several studies have demonstrated that hypermethylation of the RASSF1A promoter is frequently observed in lung, prostate, and breast cancers. Phenethyl isothiocyanate (PEITC), a phytochemical abundant in cruciferous vegetables, possesses chemopreventive activities; however, its potential involvement in epigenetic mechanisms remains elusive. The present study aimed to examine the role of PEITC in the epigenetic reactivation of RASSF1A and the induction of apoptosis in LNCaP cells. LNCaP cells were treated for 5days with 0.01% DMSO, 2.5 or 5μM PETIC or 2.5μM azadeoxycytidine (5-Aza) with 0.5μM trichostatin A (TSA). We evaluated the effects of these treatments on CpG demethylation using methylation-specific polymerase chain reaction (MSP) and bisulfite genomic sequencing (BGS). CpG demethylation was significantly enhanced in cells treated with 5μM PEITC and 5-Aza+TSA; therefore, the latter treatment was used as a positive control in subsequent experiments. The decrease in RASSF1A promoter methylation correlated with an increase in expression of the RASSF1A gene in a dose-dependent manner. To confirm that promoter demethylation was mediated by DNA methyltransferases (DNMTs), we analyzed the expression levels of DNMTs and histone deacetylases (HDACs) at the gene and protein levels. PEITC reduced DNMT1, 3A and 3B protein levels in a dose-dependent manner, and 5μM PEITC significantly reduced DNMT3A and 3B protein levels. HDAC1, 2, 4 and 6 protein expression was also inhibited by 5μM PEITC. The combination of 5-Aza and TSA, a DNMT inhibitor and a HDAC inhibitor, respectively, was used as a positive control as this treatment significantly inhibited both HDACs and DNMTs. The function of RASSF1A reactivation in promoting apoptosis and inducing G2/M cell cycle arrest was analyzed using flow-cytometry analysis with Annexin V and propidium iodide (PI). Growth inhibition effect on LNCaP cells were investigated by colony formation assay. In addition, we analyzed p21, caspase-3 and 7, Bax, and Cyclin B1 protein levels. Flow-cytometry analysis of cells stained with PI alone demonstrated that 5μM PEITC promotes early apoptosis and G2/M cell cycle arrest. Flow cytometry analysis of cells stained with Annexin V and PI also demonstrated an increased proportion of cells in early apoptosis in cells treated with 5μM PEITC or 5-Aza with TSA. PEITC and efficiently inhibit colony numbers and total area. In addition, 5μM PEITC significantly enhanced p21, caspase-3, 7 and Bax levels and reduced Cyclin B1 expression compared with the control group. Collectively, the results of our study suggest that PEITC induces apoptosis in LNCaP cells potentially by reactivating RASSF1A via epigenetic mechanisms.

Project description:BackgroundThe cytokine TRAIL (tumor necrotic factor-related apoptosis-inducing ligand) selectively induces apoptosis in cancer cells, but cancer stem cells (CSCs) that contribute to cancer-recurrence are frequently TRAIL-resistant. Here we examined hitherto unknown effects of the dietary anti-carcinogenic compound phenethyl isothiocyanate (PEITC) on attenuation of proliferation and tumorigenicity and on up regulation of death receptors and apoptosis in human cervical CSC.MethodsCancer stem-like cells were enriched from human cervical HeLa cell line by sphere-culture method and were characterized by CSC-specific markers' analyses (flow cytometry) and Hoechst staining. Cell proliferation assays, immunoblotting, and flow cytometry were used to assess anti-proliferative as well as pro-apoptotic effects of PEITC exposure in HeLa CSCs (hCSCs). Xenotransplantation study in a non-obese diabetic, severe combined immunodeficient (NOD/SCID) mouse model, histopathology, and ELISA techniques were further utilized to validate our results in vivo.ResultsPEITC attenuated proliferation of CD44(high/+)/CD24(low/-), stem-like, sphere-forming subpopulations of hCSCs in a concentration- and time-dependent manner that was comparable to the CSC antagonist salinomycin. PEITC exposure-associated up-regulation of cPARP (apoptosis-associated cleaved poly [ADP-ribose] polymerase) levels and induction of DR4 and DR5 (death receptor 4 and 5) of TRAIL signaling were observed. Xenotransplantation of hCSCs into mice resulted in greater tumorigenicity than HeLa cells, which was diminished along with serum hVEGF-A (human vascular endothelial growth factor A) levels in the PEITC-pretreated hCSC group. Lung metastasis was observed only in the hCSC-injected group that did not receive PEITC-pretreatment.ConclusionsThe anti-proliferative effects of PEITC in hCSCs may at least partially result from up regulation of DR4 and possibly DR5 of TRAIL-mediated apoptotic pathways. PEITC may offer a novel approach for improving therapeutic outcomes in cancer patients.

Project description:BackgroundThis study was undertaken to determine the chemopreventative efficacy of phenethyl isothiocyanate (PEITC), a bioactive constituent of many edible cruciferous vegetables, in a mouse model of prostate cancer, and to identify potential biomarker(s) associated with PEITC response.MethodsThe chemopreventative activity of dietary PEITC was investigated in Transgenic Adenocarcinoma of Mouse Prostate mice that were fed a control diet or one containing 3 μmol PEITC/g (n = 21 mice per group) for 19 weeks. Dorsolateral prostate tissue sections were stained with hematoxylin and eosin for histopathologic evaluations and subjected to immunohistochemistry for analysis of cell proliferation (Ki-67 expression), autophagy (p62 and LC3 protein expression), and E-cadherin expression. Autophagosomes were visualized by transmission electron microscopy. Apoptotic bodies were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Plasma proteomics was performed by two-dimensional gel electrophoresis followed by mass spectrometry to identify potential biomarkers of PEITC activity. All statistical tests were two-sided.ResultsAdministration of PEITC (3 μmol/g diet) decreased incidence (PEITC diet vs control diet, mean = 21.65 vs 57.58%, difference = -35.93%, 95% confidence interval = -45.48% to -13.10%, P = .04) as well as burden (affected area) (PEITC diet vs control diet, mean = 18.53% vs 45.01%, difference = -26.48%, 95% confidence interval = -49.78% to -3.19%, P = .02) of poorly differentiated tumors in the dorsolateral prostate of transgenic mice compared with control mice, with no toxic effects. PEITC-mediated inhibition of prostate carcinogenesis was associated with induction of autophagy and overexpression of E-cadherin in the dorsolateral prostate. However, PEITC treatment was not associated with a decrease in cellular proliferation, apoptosis induction, or inhibition of neoangiogenesis. Plasma proteomics revealed distinct changes in the expression of several proteins (eg, suppression of clusterin protein) in the PEITC-treated mice compared with control mice.ConclusionsIn this transgenic model, dietary PEITC suppressed prostate cancer progression by induction of autophagic cell death. Potential biomarkers to assess the response to PEITC treatment in plasma were identified.

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P66Shc is indispensable for phenethyl isothiocyanate-induced apoptosis in human prostate cancer cells.

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p66Shc is indispensable for phenethyl isothiocyanate-induced apoptosis in human prostate cancer cells.

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