Effect of benzyl isothiocyanate (BITC) on STAT-3 DNA-binding and transcriptional activity in BxPC-3 cells. A) Effect of BITC on STAT-3 binding to its cognate DNA sequence. BxPC-3 cells were treated for 24 hours with or without 10 μM BITC, and cell extracts were tested for STAT-3 DNA-binding activity as measured by the Universal EZ-TFA transcription factor colorimetric assay, in which a biotinylated double-stranded oligonucleotide containing the consensus sequence for STAT-3 was used to link bound transcription factors to streptavidin-coated wells. The negative control contained only binding buffer and capture probe. Control wells contained non–BITC-treated cell lysates, with or without a competitor probe (CP) that was unbiotinylated. Means and 95% confidence intervals of two independent experiments performed in triplicate are shown. Differences between all the groups were compared by nonparametric analysis of variance with Bonferroni post hoc multiple comparison test. Statistical tests were two-sided. B) Effect of BITC on the expression of the STAT-3–regulated genes Mcl-1 and Bcl-2. Cells were treated with either dimethyl sulfoxide (DMSO) or 0–20 μM BITC for 24 hours. Whole-cell lysates were prepared, and 40 μg of protein was resolved by 10% SDS–PAGE, blotted, and analyzed for Mcl-1 and Bcl-2 proteins. β-Actin was used as a control for loading and transfer. The experiment was repeated twice and similar results were obtained. C) Effect of the proteosome inhibitor MG-132 on BITC-mediated reduction of STAT-3 protein levels. BxPC-3 cells were treated either with 10 μM BITC or DMSO alone (as a control) for 24 hours and with or without 10 μM MG-132 for 2 hours, and the expression of STAT-3 was determined by western blotting of whole-cell lysates. β-Actin was used as a control for loading and transfer. The experiment was repeated twice and similar results were obtained. D) Effect of the proteosome inhibitor MG-132 on BITC-mediated STAT-3 degradation. BxPC-3 cells were treated with or without BITC and MG-132 as described in (C). Cell lysates were immunoprecipitated overnight with anti–STAT-3 antibody and whole immunoprecipitates were resolved by 10% SDS–PAGE and immunoblotted with anti–STAT-3 and anti-ubiquitin antibodies. E) Effect of BITC on STAT-3–regulated luciferase reporter activity. STAT-3 luciferase transcriptional activity was determined in BxPC-3 cells cotransfected with 2 μg of a plasmid encoding firefly luciferase under the control of the STAT-3 promoter and with 0.2 μg of a plasmid expressing Renilla luciferase as a transfection efficiency control. At 24 hours after transfection, cells were treated with or without 10 μM BITC for 24 hours or pretreated with 10 μg/mL cycloheximide for 4 hours and then treated with or without 10 μM BITC for 24 hours. Whole-cell lysates were collected, and firefly luciferase activities were corrected for Renilla luciferase levels and then normalized relative to the DMSO control, which was considered as 1. A value less than 1.0 in this assay indicates attenuation of STAT-3–directed transcription by BITC. Means and 95% confidence intervals of two independent experiments performed in triplicate are shown. The differences between all the groups were compared by nonparametric analysis of variance with Bonferroni post hoc comparisons. Statistical tests were two-sided. F) Effect of cycloheximide on BITC-mediated reduction of STAT-3 mRNA content. Cells were pretreated with 10 μg/mL cycloheximide for 4 hours followed by treatment with 10 μM BITC for 24 hours. Total RNA was extracted by Trizol method and probed for STAT-3 expression. The same RNA samples were probed for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal loading control. The experiments were repeated three times and similar results were obtained.

Effect of benzyl isothiocyanate (BITC) on STAT-3 signaling in AsPC-1, Capan-2, and MiaPaCa-2 cells. AsPC-1, Capan-2, and MiaPaCa-2 cells were treated with either dimethyl sulfoxide or with varying concentrations of BITC. A) Cytotoxicity of BITC in these cell types, measured by the sulforhodamine B cell survival assay. The experiment was performed as described in Figure 1, A. It was repeated two times, each with eight replicates, and similar results were obtained. Means and 95% confidence intervals are shown. B) Apoptosis of these cell types in response to BITC measured by caspase-3 and PARP cleavage (caspase-3-CF and PARP-CF, respectively). The experiment was performed as described in Figure 1, B. It was repeated twice and similar results were obtained. C) Phosphorylation and expression of STAT-3 protein in these cell types in response to BITC. Western blots were performed as described in Figure 1, C. These experiments were repeated twice and similar results were obtained. D) Effect of BITC on STAT-3 DNA binding in these cell lines. DNA binding was measured by the Universal EZ-TFA transcription factor colorimetric assay as described in Figure 2, A. Means and 95% confidence intervals of two independent experiments performed in triplicate are shown. Differences between all the groups were compared by nonparametric analysis of variance with the Bonferroni post hoc multiple comparison test. Statistical tests were two-sided. E) Effect of BITC on STAT-3 transcriptional activity in these cell lines. Transcriptional activity was measured by luciferase assay as described in Figure 2, E. Means and 95% confidence intervals of two independent experiments performed in triplicate are shown. Differences between groups were compared using the Student t test (two-sided; ns denotes “not significant”). F) Effect of BITC on STAT-3 mRNA expression in these cell lines. Cells were treated for 24 hours with 0–20 μM BITC, and total RNA was analyzed for STAT-3 content by reverse transcription–polymerase chain reaction. The same RNA samples were analyzed for glyceraldehyde 3-phosphate (GAPDH) as an internal loading control. The experiment was repeated twice and similar results were obtained.

Effects of benzyl isothiocyanate (BITC) on growth, apoptosis, and STAT-3 expression in BxPC-3 human pancreatic tumor xenografts in nude mice. Tumor cells were implanted into athymic nude mice, and each mouse received 12 μmol BITC by oral gavage starting the day of tumor implantation and continuing 5 days a week for 6 weeks before the mice were killed. A) Effect of BITC on tumor volumes. Tumors were measured by vernier calipers and sizes were calculated as follows: volume (mm³) = length × (width²)/2. B) Effect on tumor weights. Tumors were excised on day 42 after implantation and weighed before use in immunohistochemical and western blotting experiments. C) Effect on average mouse weight. Tumor-bearing mice were weighed over the course of the experiment as a measure of possible BITC toxicity. Values in (A) are means of 10 (five sets of two) observations in each group with 95% confidence interval. Differences between control and BITC-treated groups in (A) were compared with exact two-sided Wilcoxon tests after summing the two log-transformed measurements on each mouse (P = .008). The weight of BITC-treated tumors was found to be statistically different from controls (P = .025). Differences between groups in (B) were compared by Student t test (two-sided) with 95% confidence intervals. D) Apoptosis and STAT-3 expression in tumor sections from BITC-treated and control mice. TdT-mediated dUTP-biotin nick end labeling (TUNEL), hematoxylin and eosin, and STAT-3 immunostaining were performed on representative tumor sections and photographs were taken at high (×100) magnification. E) STAT-3 expression and activation in lysates from the tumors of BITC-treated and control mice. Tumor lysates from tumor samples in (D) were further analyzed for phospho-Tyr705 STAT-3 and total STAT-3 protein by immunoblotting. Blots were stripped and reprobed with β-actin antibody to verify equal protein loading. Each lane represents a different tumor sample.

Effect of benzyl isothiocyanate (BITC) on the constitutive phosphorylation and expression of STAT-3 in BxPC-3 cells. A) Cytotoxicity of BITC in BxPC-3 cells as measured by the sulforhodamine B cell survival assay. Cells were treated with varying concentrations of BITC or with dimethyl sulfoxide (DMSO) as a control and surviving cells were quantified spectrophotometrically. The experiment was repeated four times, each time with eight replicates, and the data are expressed as the means of all experiments with 95% confidence intervals (error bars). B) Induction of apoptosis by BITC as assayed by caspase-3 and PARP cleavage. Whole-cell lysates were prepared, and samples containing 40 μg of protein were resolved by 10% SDS–PAGE and analyzed for the induction of apoptosis as assessed by caspase-3 and PARP cleavage (PARP-CF). β-Actin was used as a control for loading and transfer. The experiment was repeated three times with similar results. C) Effect of BITC on STAT-3 protein levels and phosphorylation. BxPC-3 cells were treated for 24 hours with 0–20 μM BITC, and cell lysates were examined on western blots for STAT-3 phosphorylation at Tyr705 and Ser727 and for STAT-3 protein expression. β-Actin was used as a control for loading and transfer. The experiment was repeated three times with similar results. D) Further examination of STAT-3 phosphorylation. Total STAT-3 was immunoprecipitated from control and BITC-treated BxPC-3 cells, and an equal amount of STAT-3 protein was resolved on 10% SDS–PAGE and analyzed for pSTAT-3 (Tyr705). The experiment was repeated three times and the mean of the three experiments was represented as the ratio of pSTAT-3 (Tyr705)/STAT-3 protein with 95% confidence intervals (error bars). E and F) Effect of BITC on STAT-3 mRNA levels as a function of concentration (E) and time (F). Cells were treated with 0–20 μM BITC for 24 hours (E) or with 10 μM BITC for 0–24 hours (F) before total RNA was extracted by the Trizol method and analyzed by reverse transcription– polymerase chain reaction. In each experiment the same RNA samples were analyzed for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control for loading and transfer. The experiment in (E) was repeated four times, the experiment in (F) twice, and similar results were obtained. G) Effect of BITC on JNK, ERK, and p38 phosphorylation. BxPC-3 cells were treated with varying concentrations of BITC for 24 hours and cell lysates were examined on western blots using antibodies specific for the activated pJNK (Thr183/Tyr185), pERK (Thr202/Tyr204), and pp38 (Thr180/Tyr182) proteins and for total protein of each type. β-Actin was used as a control for loading and transfer. The experiment was repeated twice and similar results were obtained.

Effects of benzyl isothiocyanate (BITC) in BxPC-3 cells in which STAT-3 has been activated by interleukin 6 (IL-6) or in which STAT-3 has been overexpressed. A) Effect of BITC on STAT-3 activation, STAT-3 protein expression, and apoptosis in IL-6–treated BxPC-3 cells. Cells were stimulated with 5 ng/mL of IL-6 for 15 minutes followed by treatment with 10 μM of BITC for 24 hours. Whole-cell lysates were resolved on 10% SDS–PAGE for the analysis of STAT-3 phosphorylation at Tyr705 and Ser727, STAT-3 expression, and cleavage of caspase-3 and PARP. β-Actin was used as a control for loading and transfer. The experiment was repeated three times and similar results were obtained. B) Effect of BITC on STAT-3 activation, STAT-3 expression, and apoptosis in BxPC-3 cells overexpressing STAT-3α. Cells were transfected with an empty vector or a plasmid expressing STAT-3α and 48 hours later were treated with 10 μM of BITC for 24 hours. Whole-cell lysates were analyzed as in (A). The experiment was repeated three times and similar results were obtained. C) Effect of STAT-3α overexpression on BITC-induced apoptosis in BxPC-3 cells. Apoptosis was further confirmed by the cell death detection assay, which uses monoclonal antibodies directed against DNA and histones to quantify apoptosis. Nontransfected BxPC-3 cells were treated with or without 10 μM BITC for 24 hours and analyzed for apoptosis. Separately, cells were transfected with empty vector or STAT-3α–expressing vector as described above and then treated with or without 10 μM BITC for 24 hours. Means and 95% confidence intervals of two independent experiments performed in triplicate are shown. Differences between all the groups were compared by nonparametric analysis of variance (ANOVA) with Bonferroni post hoc multiple comparison test. Statistical tests were two-sided. D) Effect of STAT-3α overexpression on BITC-associated reductions in cell survival. BxPC-3 cells were transfected with a STAT-3α−expressing vector as described above and treated with 10 μM BITC or 0.1% dimethyl sulfoxide (control) for 24 hours and the cell survival was evaluated by sulforhodamine B assay. Means and 95% confidence intervals of two independent experiments performed in triplicate are shown. Differences between all the groups were compared by nonparametric ANOVA with the Newman–Keuls post hoc multiple comparison test. Statistical tests were two-sided. E) Effect of IL-6 or STAT-3α overexpression on BITC-associated reductions in STAT-3 DNA binding. The Universal EZ-TFA transcription factor colorimetric assay was used to determine the DNA-binding activity of STAT-3 in BxPC-3 cells that were stimulated with 5 ng/mL IL-6 for 15 minutes or that overexpressed STAT-3α with and without treatment with 10 μM BITC for 24 hours. A negative control contained binding buffer and free probe without cell lysate, whereas free probe contained neither binding buffer nor cell lysate. Empty vector and control are defined above in (C). Means and 95% confidence intervals of two independent experiments performed in triplicate are shown. Differences between all the groups were compared by nonparametric ANOVA with the Newman–Keuls post hoc multiple comparison test. Statistical tests were two-sided. F) Effect of IL-6 or STAT-3α overexpression on BITC-associated reductions in STAT-3 transcriptional activity. Transcriptional activity of STAT-3 in BxPC-3 cells that were stimulated with 5 ng/ml IL-6 for 15 minutes or that overexpressed STAT-3α with and without 24-hour treatment with 10 μM BITC was determined by luciferase assays. Control cells were neither stimulated with IL-6 nor transfected with empty vector. Means and 95% confidence intervals of two independent experiments performed in triplicate are shown. Differences between all the groups were compared by nonparametric ANOVA with Newman–Keuls post hoc multiple comparison test. Statistical tests were two-sided. G) Effect of STAT-3α overexpression on BITC-associated reductions in Mcl-1 and Bcl-2 expression. At 48 hours after transfection with empty vector or a STAT-3α–expressing vector, BxPC-3 cells were treated with 10 μM BITC for 24 hours. Cell lysates were separated on 10% SDS–PAGE and blots were probed using Mcl-1– and Bcl-2–specific antibodies, then stripped and reprobed with β-actin antibody to ensure equal loading. The experiment was repeated twice and similar results were obtained.

Effect of benzyl isothiocyanate (BITC) on STAT-3 signaling in Panc-1 and immortalized normal pancreatic ductal epithelial cells. The effects of BITC on STAT-3 in Panc-1 and HPDE-6 cells were determined as described in Figure 4. A–F) Each experiment was repeated twice and similar results were obtained. The same blots were stripped and reprobed with β-actin antibody as a control for equal loading and transfer. The same RNA samples were analyzed for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression, as an internal control for loading and transfer. Means and 95% confidence intervals of two independent experiments performed in triplicate are shown. Differences between groups in (D) were compared by nonparametric analysis of variance with the Bonferroni post hoc multiple comparison test and in (E) were compared by the Student t test. Statistical tests were two-sided. Nonsignificance is denoted as “ns.”