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

ABSTRACT: 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.

SUBMITTER: Eisa NH

PROVIDER: S-EPMC6269550 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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

Eisa Nada H NH Said Heba S HS ElSherbiny Nehal M NM Eissa Laila A LA El-Shishtawy Mamdouh M MM

Iranian journal of pharmaceutical research : IJPR 20180101 4

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 ap ...[more]

PMID: 30568691

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Project description:Mutations in the p53 tumor-suppressor gene are prevalent in human cancers. The majority of p53 mutations are missense, which can be classified into contact mutations (that directly disrupts the DNA-binding activity of p53) and structural mutations (that disrupts the conformation of p53). Both of the mutations can disable the normal wild-type (WT) p53 activities. Nevertheless, it has been amply documented that small molecules can rescue activity from mutant p53 by restoring WT tumor-suppressive functions. These compounds hold promise for cancer therapy and have now entered clinical trials. In this study, we show that cruciferous-vegetable-derived phenethyl isothiocyanate (PEITC) can reactivate p53 mutant under in vitro and in vivo conditions, revealing a new mechanism of action for a dietary-related compound. PEITC exhibits growth-inhibitory activity in cells expressing p53 mutants with preferential activity toward p53(R175), one of the most frequent 'hotspot' mutations within the p53 sequence. Mechanistic studies revealed that PEITC induces apoptosis in a p53(R175) mutant-dependent manner by restoring p53 WT conformation and transactivation functions. Accordingly, in PEITC-treated cells the reactivated p53(R175) mutant induces apoptosis by activating canonical WT p53 targets, inducing a delay in S and G2/M phase, and by phosphorylating ATM/CHK2. Interestingly, the growth-inhibitory effects of PEITC depend on the redox state of the cell. Further, PEITC treatments render the p53(R175) mutant sensitive to degradation by the proteasome and autophagy in a concentration-dependent manner. PEITC-induced reactivation of p53(R175) and its subsequent sensitivity to the degradation pathways likely contribute to its anticancer activities. We further show that dietary supplementation of PEITC is able to reactivate WT activity in vivo as well, inhibiting tumor growth in xenograft mouse model. These findings provide the first example of mutant p53 reactivation by a dietary compound and have important implications for cancer prevention and therapy.

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.

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

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

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