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Benzyl Isothiocyanate (BITC) Induces Reactive Oxygen Species-dependent Repression of STAT3 Protein by Down-regulation of Specificity Proteins in Pancreatic Cancer.

ABSTRACT: The antineoplastic agent benzyl isothiocyanate (BITC) acts by targeting multiple pro-oncogenic pathways/genes, including signal transducer and activator of transcription 3 (STAT3); however, the mechanism of action is not well known. As reported previously, BITC induced reactive oxygen species (ROS) in Panc1, MiaPaCa2, and L3.6pL pancreatic cancer cells. This was accompanied by induction of apoptosis and inhibition of cell growth and migration, and these responses were attenuated in cells cotreated with BITC plus glutathione (GSH). BITC also decreased expression of specificity proteins (Sp) Sp1, Sp3, and Sp4 transcription factors (TFs) and several pro-oncogenic Sp-regulated genes, including STAT3 and phospho-STAT3 (pSTAT3), and GSH attenuated these responses. Knockdown of Sp TFs by RNA interference also decreased STAT3/pSTAT3 expression. BITC-induced ROS activated a cascade of events that included down-regulation of c-Myc, and it was also demonstrated that c-Myc knockdown decreased expression of Sp TFs and STAT3 These results demonstrate that in pancreatic cancer cells, STAT3 is an Sp-regulated gene that can be targeted by BITC and other ROS inducers, thereby identifying a novel therapeutic approach for targeting STAT3.

SUBMITTER: Kasiappan R

PROVIDER: S-EPMC5207142 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Benzyl Isothiocyanate (BITC) Induces Reactive Oxygen Species-dependent Repression of STAT3 Protein by Down-regulation of Specificity Proteins in Pancreatic Cancer.

Kasiappan Ravi R Jutooru Indira I Karki Keshav K Hedrick Erik E Safe Stephen S

The Journal of biological chemistry 20161115 53

The antineoplastic agent benzyl isothiocyanate (BITC) acts by targeting multiple pro-oncogenic pathways/genes, including signal transducer and activator of transcription 3 (STAT3); however, the mechanism of action is not well known. As reported previously, BITC induced reactive oxygen species (ROS) in Panc1, MiaPaCa2, and L3.6pL pancreatic cancer cells. This was accompanied by induction of apoptosis and inhibition of cell growth and migration, and these responses were attenuated in cells cotreat ...[more]

PMID: 27875298

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Project description:In our previous studies, we have shown that benzyl isothiocyanate (BITC) inhibits the growth of human pancreatic cancer cells by inducing apoptosis. In the present study, we demonstrate the activation of all the three (MAPK) family members [extracellular signal-regulated protein kinase (ERK), c-jun N-terminal kinase (JNK) and P38] in response to BITC treatment. Exposure of Capan-2 cells with varying concentrations of BITC for 24 h resulted in the phosphorylation (activation) of ERK at Thr202/Tyr204, JNK at Thr183/Tyr185 and P38 at Thr180/Tyr182, leading to the induction of apoptosis. Similar MAPK activation was also observed in MiaPaCa-2 cells in response to BITC treatment. However, normal human pancreatic ductal epithelial cells did not show the activation of MAPK's and remained unaffected by BITC treatment. To confirm the role of ERK, JNK and P38 in BITC-induced G(2)/M arrest and apoptosis, Capan-2 cells were pre-treated with MAPK-specific inhibitors or MAPK8-short hairpin RNA (shRNA) prior to BITC treatment. Significant protection from BITC-induced G(2)/M arrest was observed in the cells pre-treated with MAPK kinase (MEK-1) but not JNK or P38 inhibitors. On the other hand, BITC-induced apoptosis was almost completely abrogated in the cells pre-treated with MEK-1, JNK or P38 inhibitors. Similarly, MAPK8-shRNA also offered almost complete protection against BITC-induced G(2)/M arrest and apoptosis. Furthermore, we observed that BITC treatment leads to the generation of reactive oxygen species (ROS) in Capan-2 and MiaPaCa-2 cells, which in part was orchestrated by depletion of reduced glutathione (GSH) level. Blocking ROS generation with N-acetyl-L-cysteine (NAC) significantly prevented GSH depletion and activation of ERK and JNK but not P38. Further, NAC or tiron prevented G(2)/M arrest by blocking G(2)/M regulatory proteins and completely protected the cells from BITC-induced apoptosis. Taken together, our results suggest that BITC-mediated G(2)/M arrest is mediated through ERK activation, whereas apoptosis is via ERK, JNK and P38.

Project description:BackgroundBenzyl isothiocyanate (BITC), a compound found in cruciferous vegetables, has been reported to have anticancer properties, but the mechanism whereby it inhibits growth of human pancreatic cancer cells is incompletely understood.MethodsHuman pancreatic cancer cells (BxPC-3, AsPC-1, Capan-2, MiaPaCa-2, and Panc-1) and immortalized human pancreatic cells (HPDE-6) were treated with vehicle or with BITC at 5-40 microM, cell survival was evaluated by sulforhodamine B assay, and apoptosis by caspase-3 and poly-ADP ribose polymerase cleavage or by a commercial assay for cell death. Total and activated signal transducer and activator of transcription-3 (STAT-3) protein expression in the cells were examined by western blotting, STAT-3 mRNA levels by reverse transcription-polymerase chain reaction, and STAT-3 DNA-binding and transcriptional activity by commercially available binding and reporter assays. The effects of BITC treatment on tumor growth, apoptosis, and STAT-3 protein expression in vivo were studied in xenografts of BxPC-3 pancreatic tumor cells in athymic nude mice. All statistical tests were two-sided.ResultsBITC treatment reduced cell survival and induced apoptosis in BxPC-3, AsPC-1, Capan-2, and MiaPaCa-2 cells, and to a much lesser extent in Panc-1 cells, but not in HPDE-6 cells. It also reduced levels of activated and total STAT-3 protein, and as a result, STAT-3 DNA-binding and transcriptional activities. Overexpression of STAT-3 in BxPC-3 cells inhibited BITC-induced apoptosis and restored STAT-3 activity. In mice that were fed BITC (60 micromol/wk, five mice, 10 tumors per group), growth of BxPC-3 pancreatic tumor xenografts was suppressed compared with control mice (at 6 weeks, mean tumor volume of control vs BITC-treated mice = 334 vs 172 mm3, difference =162 mm3, 95% confidence interval = 118 to 204 mm3; P = .008) and tumors had increased apoptosis and reduced STAT-3 protein expression.ConclusionBITC induces apoptosis in some types of pancreatic cancer cells by inhibiting the STAT-3 signaling pathway.

Dataset Information

Benzyl Isothiocyanate (BITC) Induces Reactive Oxygen Species-dependent Repression of STAT3 Protein by Down-regulation of Specificity Proteins in Pancreatic Cancer.

Publications

Benzyl Isothiocyanate (BITC) Induces Reactive Oxygen Species-dependent Repression of STAT3 Protein by Down-regulation of Specificity Proteins in Pancreatic Cancer.

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