Cell Culture and Cell Survival Assays
The well-differentiated epithelial human pancreatic adenocarcinoma cell lines BxPC-3, Capan-2, AsPC-1, and MiaPaCa-2 were obtained from American Type Cell Culture (ATCC, Manassas, VA), and Panc-1 cells were a kind gift from Dr Thomas L. Brown (Wright State University, Dayton, OH). The normal human pancreatic duct epithelial cell line, HPDE-6, which was derived from normal (benign) adult human pancreata immortalized by infection with a retrovirus containing the E6 and E7 genes of the human papillomavirus 16, was a generous gift from Dr Ming-Sound Tsao (University of Toronto, Toronto, Ontario, Canada). Cultures of HPDE-6 cells were maintained as described previously (42
). Monolayer cultures of BxPC-3 and AsPC-1 cells were maintained in RPMI-1640 medium (Sigma-Aldrich, St Louis, MO), adjusted to contain 10% heat-inactivated fetal bovine serum (FBS) (Mediatech Inc, Herndon, VA), supplemented with 2 mM l
-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodium pyruvate, and 1% (vol/vol) penicillin and streptomycin (GIBCO-BRL, Carlsbad, CA) in a humidified incubator at 37°C and 5% CO2
. Capan-2 cells were maintained in McCoy’s 5A medium (Mediatech Inc) supplemented with 2 mM l
-glutamine and adjusted to contain 10% FBS and 1% (vol/vol) penicillin/streptomycin. MiaPaCa-2 cells were maintained in Dulbecco's Modified Eagle medium (DMEM; ATCC) that was supplemented with 4 mM l
-glutamine and adjusted to contain 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, 10% FBS, 2.5% horse serum (Mediatech Inc), and 1% (vol/vol) penicillin/streptomycin. Monolayer cultures of Panc-1 cells were maintained in DMEM supplemented with 4 mM l
-glutamine and adjusted to contain 10% FBS and 1% (vol/vol) penicillin/streptomycin. HPDE-6 cells were cultured in Keratinocyte serum-free medium (GIBCO-BRL) supplemented with 4 mM l
-glutamine and adjusted to contain 0.2 ng/mL epidermal growth factor (GIBCO-BRL), 30 μg/mL bovine pituitary extract (GIBCO-BRL), and 1% (vol/vol) penicillin/streptomycin.
A 100 mM stock solution of BITC (Sigma-Aldrich) was prepared in 100% dimethyl sulfoxide (DMSO) and subsequently diluted in the cell culture medium so that the final concentration of DMSO was 0.1% in the medium. Cells were treated with BITC at 5, 10, or 20 μM concentration for 24 hours. The effect of BITC on proliferation of BxPC-3 cells was determined by sulforhodamine B assay (Sigma-Aldrich), as described previously (44
). The plates were read at 590 nm using the Bio Kinetics plate reader EL-800 (BioTek Instrument Inc, Winooski, VA).
Western Blot Analysis
BxPC-3, AsPC-1, Capan-2, MiaPaCa-2, Panc-1, and HPDE-6 cells were treated with varying concentrations of BITC (0, 5, 10, and 20 μM) for 24 hours. For time-dependent experiments, cells were treated with 10 μM BITC for 0, 1, 8, and 24 hours. Whole-cell extracts were prepared as we described previously (18
). To examine whether degradation of STAT-3 protein was mediated by the ubiquitin-proteasome pathway, BxPC-3 cells were treated with BITC and with the proteasome inhibitor MG-132 (Calbiochem EMD Chemicals, Gibbstown, NJ) at 10 μM for 2 hours. In a separate experiment, cells were pretreated with 10 μg/mL cycloheximide, a protein synthesis inhibitor (Sigma-Aldrich), for 4 hours followed by treatment with 10 μM BITC. Cell lysates containing 20–40 μg of protein were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and proteins were transferred onto polyvinylidene fluoride membranes. After blocking with 5% nonfat dry milk, membranes were incubated overnight with the desired primary antibody, followed by an appropriate secondary antibody, and the immunoreactive bands were visualized using the enhanced chemiluminescence kit from Perkin-Elmer (Waltham, MA) according to the manufacturer's instructions. Two mouse monoclonal antibodies (anti-pp38 [Thr180/Tyr182] [#9261] and anti-ubiquitin Ub [#3936]) and all of the following rabbit antibodies were diluted 1:1000 in 50 mM Tris–HCl (pH 7.5) and 150 mM NaCl Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) before use on blots: anti–caspase-3 (#9664), anti-PARP (#9541), anti–pSTAT-3 (Tyr705) (#9131), anti–pSTAT-3 (Ser727) (#9134), anti–STAT-3 (total) (#9139), anti–Mcl-1 (#4572), anti–Bcl-2 (#2872), anti-pJNK (Thr183/Tyr185) (#9251), anti-JNK (#9252), anti-p38 (#9212), anti-pERK (Thr202/Tyr204) (#9101), and anti-ERK (#9102) (all from Cell Signaling Technologies, Danvers, MA). The same membranes were reprobed with a 1:50 000 dilution of mouse monoclonal anti–β-actin antibody (#A5441; Sigma-Aldrich) as a control for equal protein loading. The intensity of immunoreactive bands on western blots was determined using a densitometer (Molecular Dynamics, Sunnyvale, CA) equipped with Image QuaNT software. Unless otherwise stated, each experiment was repeated independently at least two to three times and expressed as mean values with 95% confidence intervals (CIs).
Briefly, 1 × 106 BxPC-3 cells were seeded and treated with different concentrations of BITC for 24 hours. The medium was removed and cells were rinsed with ice-cold 10 mM phosphate buffer (pH 7.4) containing 137 mM NaCl and 2.7 mM KCl phosphate-buffered saline (PBS) and then lysed with 0.5 mL of cell lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, and 1 μg/mL leupeptin for 5 minutes on ice. Cells were scraped, transferred to microcentrifuge tubes, and sonicated three times for 5 seconds. Cell extracts were centrifuged for 10 minutes at 14 000g at 4°C, and the supernatant was isolated. About 250 μg of total protein from control or BITC-treated cells were each incubated with anti–STAT-3 antibody overnight at 4°C with gentle rocking. Protein A agarose beads (20 μL of a 50% slurry) were added to each sample and incubated for 3 hours at 4°C. Lysates were centrifuged for 30 seconds at 1000g, and pellets were washed five times with 0.5 mL cell lysis buffer. STAT-3 protein from each sample was eluted with 40 μL of 1% SDS and quantified. About 3 μg of pure STAT-3 protein was resolved on 10% SDS–PAGE and blotted with anti–pSTAT-3 (Tyr705) antibody. The same membrane was stripped and reprobed with anti–STAT-3 antibody. In another experiment, cells were treated with 10 μM BITC for 24 hours and with 10 μM MG-132, a specific proteasome inhibitor, for 2 hours. The cells were lysed and STAT-3 protein was immunoprecipitated as described above. However, in this experiment, instead of quantifying and loading equal amounts of STAT-3 protein, total immunoprecipitated STAT-3 protein was resolved by SDS–PAGE and blotted, and the blots were probed with anti–STAT-3 and anti-Ub antibodies.
Determination of STAT-3 mRNA Transcripts
To determine the effects of BITC on STAT-3 gene transcription, BxPC-3, AsPC-1, Capan-2, MiaPaCa-2, Panc-1, or HPDE-6 cells were treated with varying concentrations of BITC (0, 5, 10, and 20 μM) for 24 hours. Total RNA was extracted from control and treated cells of each cell lines using Trizol RNA extraction reagent (Sigma-Aldrich), and RNA samples were prepared for reverse transcription–polymerase chain reaction (RT-PCR) by incubation with DNase I, then RT-PCR of STAT-3 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was performed using the Verso 1-Step RT-PCR kit from Thermo-Fisher Scientific (Surrey, UK), according to the manufacturer's protocol. The following primer sets were used: STAT-3 forward primer, 5′-ATCCTGAAGCTGACCCAGGTA-3′; STAT-3 reverse primer, 5′- AGGTCGTTGGTGTCACACAGA-3′; GAPDH forward primer, 5′- ACCACAGTCCATGCCATCAC-3′; and GAPDH reverse primer, 5′-TCCACCACCCTGTTGCTGTA -3′. Reactions were carried out in 50-μL volumes using 100 ng of total template RNA, 200 nM each of forward and reverse primers, 25 μL polymerase chain reaction (PCR) master mix, and 1 μL of Verso enzyme mixture. Reaction conditions for initial complementary DNA synthesis were 50°C for 15 minutes followed by 40 cycles of denaturation at 94°C for 30 seconds, annealing at 63–61°C for STAT-3 or at 55°C for GAPDH for 30 seconds, extension at 72°C for 45 seconds, and then final extension at 72°C for 5 minutes on a 7300 real time PCR system AB1 (Perkin-Elmer-Applied Biosystems, Foster City, CA). The products were separated on 2% agarose gels and visualized by staining with ethidium bromide.
STAT-3 DNA-Binding Assay
DNA binding of the STAT-3 transcription factor to the promoters of its target genes in pancreatic cancer cells was measured by Universal EZ-TFA transcription factor assay colorimetric kit (Upstate Biotechnology, Inc, Lake Placid, NY) according to the manufacturer's protocol. In these experiments, BxPC-3 cells were treated either with 0.1% DMSO or 10 μM BITC for 24 hours. Then, nuclear extracts were prepared and incubated with 2 μL of “capture probe,” a biotinylated double-stranded oligonucleotide that contained the consensus sequence for STAT-3. To determine the specificity of STAT-3 DNA binding, a mixture of capture probe and “competitor probe,” that contained the exact same sequence as the capture probe but did not include any biotin modifications, was also incubated in streptavidin-coated plates. The biotinylated oligonucleotide, along with any bound STAT-3, was then immobilized on the streptavidin-coated plate, and the inactive, unbound material was washed away. The bound transcription factor was then detected by incubation with a 1:500 dilution of rabbit anti–STAT-3 antibody and a 1:500 dilution of horseradish peroxidase (HRP)–conjugated secondary antibody. A negative control containing binding buffer and free probe without cell lysate was used in each assay. HRP activity was colorimetrically detected at 450 nm using an EL-800 ELISA plate reader (BioTek Instruments Inc).
STAT-3 Luciferase Reporter Assay
STAT-3 luciferase transcriptional activity was first determined in BxPC-3 cells cotransfected, using lipofectamine reagent (Invitrogen, Carlsbad, CA), with 2 μg of pLuc-TK/STAT3 (a generous gift from Dr J. F. Bromberg, Rockefeller University, New York), which encoded firefly luciferase under the control of the STAT-3 promoter, and with 0.2 μg of a pRL-TK (Promega Corp, Madison, WI), which constitutively expressed Renilla luciferase, the latter as a transfection efficiency control. Twenty-four hours after transfection, BxPC-3 cells were treated with 10 μM BITC or with 0.1% DMSO for 24 hours or pretreated with 10 μg/mL cycloheximide for 4 hours and then treated with 10 μM BITC for 24 hours. Whole-cell lysates were collected according to the dual luciferase reporter kit assay protocol, and the light output of lysates was measured with a luminometer. Firefly luciferase activities were corrected for Renilla values and then normalized relative to the DMSO control, which was considered as 1.0. A value less than 1 in this assay indicated attenuation of STAT-3–directed transcription by BITC.
In other experiments, human pancreatic cancer cells AsPC-1, MiaPaCa-2, Capan-2, Panc-1, and normal HPDE-6 cells were cotransfected for 24 hours with 2 μg pLuc-TK/STAT3 and with 0.2 μg pRL-TK plasmid as a transfection efficiency control. Cells were again treated with 0.1% DMSO or with 10 μM BITC for 24 hours. In additional experiments, cells were 1) pretreated with 5 ng/mL IL-6 for 15 minutes, 2) pretreated with 10 μg/mL cycloheximide for 4 hours, or 3) transfected for 48 hours with 2 μg STAT-3α, cloned in the pBabe Puro mammalian expression vector (a generous gift from Dr J. F. Bromberg, Rockefeller University, New York) followed by cotransfection for 24 hours with the pLuc-TK/STAT3 and pRL-TK plasmids. Transfected cells were then treated with or without 10 μM BITC for 24 hours, and lysates were analyzed for luciferase activity as above.
Induction of STAT-3 by IL-6
Dishes containing 1 × 106 subconfluent BxPC-3 cells were seeded 24 hours before stimulation with 5, 10, or 20 ng/mL human recombinant IL-6 (Sigma-Aldrich) for 0.25, 0.50, or 24 hours. Whole-cell extracts were prepared and analyzed for activated STAT-3 (pTyr705) and for total STAT-3 by western blotting with phospho-specific or anti–STAT-3 (total) antibodies. Substantial STAT-3 activation was observed after 15 minutes of exposure to 5 ng/mL IL-6. Therefore, these conditions were used in subsequent experiments to activate STAT-3.
Overexpression of STAT-3α
A plasmid that contained STAT-3α (Gene Accession number U06922) cloned into the pBabe Puro mammalian expression vector (a generous gift from Dr J. F. Bromberg, Rockefeller University, New York) was transfected to overexpress STAT-3 in BxPC-3 cells. Briefly 3 × 105 cells were transfected with 2 μg of the STAT-3α plasmid diluted in Opti-MEM serum-free medium to which lipofectamine reagent (Invitrogen) was added before the mixture was added to cells. Cells were incubated with plasmid–lipofectamine mixture for 5 hours and then replenished with normal growth medium for 48 hours. Transfected cells were treated with 0.1% DMSO or with 10 μM BITC for 24 hours. Cell lysates were prepared, and 20 μg of protein was analyzed by western blotting. The same blots were stripped and reprobed with anti-actin antibody for equal loading.
Apoptosis Detection by Cell Death Enzyme-Linked Immunosorbent Assay Method
In addition to caspase and PARP cleavage on western blots, the Cell Death Detection Enzyme-linked ImmunoSorbent Assay (ELISA) kit (Roche Applied Science, Mannheim, Germany) was used, following the manufacturer's instructions, to measure apoptotic cell death. Briefly, 1 × 104 BxPC-3 cells were seeded in 96-well plates and treated with either 10 μM BITC for 24 hours or with 0.1% DMSO as a control. In other experiments, similarly grown BxPC-3 cells were either stimulated with IL-6 or transfected with STAT-3α and then treated with BITC. Cell lysates were mixed with biotinylated anti-histone antibodies and peroxide POD conjugated anti-DNA antibodies in streptavidin-coated multiwell dishes, so that histone-bound DNA fragments that are characteristic of apoptosis could be detected and quantified. The plates were read at 405 nm and at 490 nm for background on EL-800 ELISA plate reader (BioTek Instruments Inc). Each sample was analyzed in triplicate, and the average values were subtracted from the background values.
In Vivo Xenograft Experiment
To assess the mechanism of BITC and its efficacy in vivo, BxPC-3 human pancreatic cancer cells were grown as xenografts in mice. The use of athymic nude mice and their treatment was approved by the Institutional Animal Care and Use Committee (IACUC), Texas Tech University Health Sciences Center, and all the experiments were carried out in strict compliance with their regulations. Six-week-old female athymic nude mice (NCR nu/nu, n = 10) were purchased from Tacomic (Germantown, NY). Mice were put on an antioxidant-free AIN-76A special diet (ICN Biomedicals, Aurora, OH) 1 week before starting the experiment. Tumor xenografts were implanted in athymic nude mice as described previously (8
). Briefly, 1 × 106
BxPC-3 tumor cells in 0.1 mL PBS were injected subcutaneously in both left and right flanks of the mice. Mice were divided randomly into two groups with five mice in each group. Because each mouse had two tumors, every group contained 10 tumors. In our past experience and in this experiment, the two tumors from the same mouse typically showed different growth patterns; however, a permutation-based Wilcoxon rank test was used to sum the two log-transformed measurements on each mouse. Treatment with BITC started the same day after tumor cell implantation. Group 1 served as controls and received 0.1 mL PBS as vehicle, whereas group 2 received 12 μmol BITC in 0.1 mL PBS five times a week (Monday to Friday) by oral gavage. Starting 8 days after tumor cell implantation and after each mouse started to develop palpable tumors, tumors were measured three times a week (Monday, Wednesday, and Friday) using vernier calipers. Each mouse was also weighed twice a week (Monday and Friday) from the day of tumor cell implantation until 42 days after that time. At day 42, at which time the tumors typically started to show signs of necrosis, mice were killed by CO2
asphyxiation followed by cervical dislocation in accordance with IACUC guidelines. The tumors were removed aseptically from each mouse, and half of each tumor was snap frozen in liquid nitrogen for western blotting while the remaining other half was fixed in 10% neutral buffered formalin overnight. For western blotting, tumors from control and BITC-treated mice were washed with ice-cold PBS, minced, and homogenized in lysis buffer containing 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM Na2
EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3
, 1 μg/mL leupeptin, and 1 mM phenylmethane sulfonyl fluoride. The tumor lysate was cleared by centrifugation at 14
for 30 minutes. Lysate containing 60 μg of protein was resolved by 10% SDS–PAGE, and immunoblots were probed with anti–pSTAT-3 (Tyr705) and anti–STAT-3 antibody.
Apoptosis Measurements from Human Tumor Xenografts
Tumor tissues fixed in 10% neutral buffered formalin were dehydrated and embedded in paraffin, and sections that were 4 μm in thickness were prepared at every 100-μm interval. Paraffin-embedded tumor tissues were stained using hematoxylin and eosin (Anatech Ltd, Battle Creek, MI). Apoptosis was measured using a TdT-mediated dUTP-biotin nick end labeling (TUNEL)-based In Situ Apoptosis detection kit (#4828-30-DK; Trevigen, Inc, Gaithersburg, MD) according to the manufacturer's instructions. Briefly, tissue sections were deparaffinized and hydrated by sequential incubation in xylene, absolute alcohol, 70% alcohol, deionized water, and PBS at room temperature. Sections on slides were then incubated in proteinase K (20 μg/mL in 10 mM Tris–HCl, pH 7.4) for 15 minutes at 37°C, quenched in 3% hydrogen peroxide, and washed with PBS. Slides were incubated with TdT labeling buffer in a humidifying chamber at 37°C for 30 minutes followed by immersion in TdT stop buffer for 5 minutes at room temperature, incubation with 50 μL of biotinylated bromodeoxyuridine antibody at 37°C for 1 hour, and washed with PBS. Finally, apoptotic staining was developed with (diluted 1:50 in PBS) streptavidin-conjugated HRP and (diluted 1:200 in PBS) 3,3′-diaminobenzidene, an HRP substrate. After washing, sections were counterstained with methyl green and analyzed under a phase-contrast Olympus microscope (Olympus America Inc, Central Valley, PA).
Immunohistochemistry for STAT-3 Localization
Paraffin-embedded tissue sections for immunohistochemistry were deparaffinized and rehydrated by incubating sections in three washes of xylene for 5 minutes each, two washes of 100% ethanol for 10 minutes each followed by two washes of 95% ethanol for 10 minutes, and the sections were given two 5-minute washes in double-distilled water (dH2O). Antigens were unmasked by boiling the sections in 10 mM sodium citrate buffer (pH 6.0) and then reducing the temperature to below the boiling point at around 95°C for 10 minutes. The slides were cooled on the bench top for 30 minutes and washed in dH2O three times for 5 minutes each, then incubated in 3% hydrogen peroxide for 10 minutes followed by two washes in dH2O for 5 minutes each. Then, tumor sections were washed twice in wash buffer (PBS with 0.1% Tween-20) for 5 minutes each, blocked in 200 μL of blocking solution (5% horse serum diluted in TBST) for 1 hour at room temperature, and incubated with anti–STAT-3 antibody (1:300 in TBST) overnight at 4°C. After removal of the primary antibody, sections were washed three times in wash buffer for 5 minutes each followed by incubation with 200 μL of HRP-conjugated secondary antibody diluted 1:5000 in blocking solution for 30 minutes. Subsequently, sections were washed with wash buffer and incubated with 200 μL of avidin-biotin conjugate (ABC) reagent containing avidin and biotinylated HRP for 30 minutes at room temperature using ABC staining kit according to the manufacturer's instructions (Santa Cruz Biotechnology Inc, Santa Cruz, CA). Three drops of peroxidase substrate was added to each section and incubated until the desired color developed. The sections were counterstained with hematoxylin and mounted and analyzed under a phase-contrast Olympus microscope (Olympus America Inc).
All statistical calculations were performed using InStat software and GraphPad Prizm 4.0. Nonparametric analysis of variance followed by Bonferroni or Newman–Keuls post hoc multiple comparison tests were used to test the statistical significance of STAT-3 DNA-binding assay, luciferase reporter assay, apoptosis and cell survival assays between multiple control and treated groups. The Student t test was used to compare the control and treated groups in the STAT-3 luciferase reporter assay. Experiments were usually repeated three times unless otherwise indicated with three replicates each. The data represent mean values with 95% confidence intervals. Differences were considered statistically significant when the P value was less than .05. To analyze the mouse tumor size, we computed the geometric mean of the right and left flank tumor size measurements for each mouse. The control and treatment groups were compared at each time point using a two-sided exact Wilcoxon rank sum test (no corrections for multiple comparisons were made because measurements at successive time points are highly correlated). Differences were considered statistically significant when the P value was less than .05. Mean tumor sizes were based on the within-group means of the geometric means for each of the five mice, and 95% confidence intervals were computed.