176 Articles | JNCI Vol. 101, Issue 3 | February 4, 2009
Pancreatic cancer is one of the most common invasive malignan-
cies and is the fourth leading cause of cancer-related deaths in the
United States ( 1 ). The high mortality can be attributed to late
diagnosis, rapid disease progression, poor response to systemic
remedies, and resistance to chemotherapy and radiotherapy ( 2 , 3 ).
Therefore, the development of novel approaches to prevent and
treat pancreatic cancer is an important mission.
Evidence from epidemiological, pharmacological, and case –
control studies has indicated that isothiocyanates present in cruci-
ferous vegetables may have substantial chemopreventive activity
against human malignancies including pancreatic cancer ( 4 – 7 ).
The accumulated data from several in vitro models suggest that
isothiocyanates induce cell death by multiple signaling pathways
( 8 – 13 ). Benzyl isothiocyanate (BITC), an agent that is present in
cruciferous vegetables such as broccoli, watercress, cabbage, cauli-
fl ower, mustard, and horseradish, is widely consumed as part of a
routine diet and has been reported to inhibit the initiation, growth,
and metastasis of chemically induced human cancers in rodents
( 14 – 17 ). In previous studies, we demonstrated that BITC sup-
pressed the proliferation of human pancreatic cancer cells by
inducing DNA damage that caused G2/M cell cycle arrest
and apoptosis ( 18 ) and by inhibiting the activation of nuclear
factor kappa B ( 10 ). However, the mechanism by which BITC
might inhibit human pancreatic carcinogenesis was not fully
Members of the signal transducer and activator of transcription
(STAT) family of transcription factors have been identifi ed to play a
crucial role in the expression of genes that are involved in cell sur-
vival, proliferation, chemoresistance, and angiogenesis ( 19 – 26 ). The
Affiliation of authors: Department of Pharmaceutical Sciences and Cancer
Biology Center, School of Pharmacy, Texas Tech University Health Sciences
Center, Amarillo, TX (RPS, SKS) .
Correspondence to: Sanjay K. Srivastava, PhD, Department of Pharmaceutical
Sciences and Cancer Biology Center, School of Pharmacy, Texas Tech
University Health Sciences Center, Suite 404, 1300 S Coulter, Amarillo, TX
79106 (e-mail: firstname.lastname@example.org ).
See “Funding” and “Notes” following “References.”
© The Author 2009. Published by Oxford University Press. All rights reserved.
For Permissions, please e-mail: email@example.com.
The Role of STAT-3 in the Induction of Apoptosis in
Pancreatic Cancer Cells by Benzyl Isothiocyanate
Ravi P. Sahu , Sanjay K. Srivastava
Background Benzyl 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
Methods Human 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 µ M, cell survival was
evaluated by sulforhodamine B assay, and apoptosis by caspase-3 and poly-ADP ribose polymerase cleav-
age or by a commercial assay for cell death. Total and activated signal transducer and activator of tran-
scription-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.
Results BITC 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 µ mol/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 mm 3 , difference =162 mm 3 , 95% confidence interval = 118 to 204 mm 3 ; P = .008) and tumors
had increased apoptosis and reduced STAT-3 protein expression.
Conclusion BITC induces apoptosis in some types of pancreatic cancer cells by inhibiting the STAT-3 signaling
J Natl Cancer Inst 2009;101: 176 – 193
JNCI Journal of the National Cancer Institute Advance Access published January 27, 2009
JNCI | Articles 177
constitutive activation of STAT-3 has been reported in different
human tumors and cell lines and to some extent in pancreatic cancer
( 27 – 38 ), which makes STAT-3 an attractive molecular target for
cancer therapy ( 39 , 40 ). Interleukin 6 (IL-6), Janus-activated kinases,
epidermal growth factor receptor, and Src family kinases are among
the activators of STAT-3, all of which phosphorylate critical tyrosine
and serine residues, leading to STAT-3 dimerization, nuclear trans-
location of dimers, and initiation of transcription through their
specifi c binding to DNA response elements in the promoter region
of target genes ( 26 , 39 – 41 ). A few recent studies have shown that
several naturally occurring anticancer agents induce apoptosis in
human cancer cells via inhibition of STAT-3, although in these cases
the exact role of the STAT-3 molecule is not clear ( 27 , 36 , 38 ).
This study aimed to investigate the molecular mechanism by
which BITC inhibited the growth of pancreatic cancer cells. We
fi rst tested the BxPC-3 pancreatic cancer cell line for the effect of
BITC on apoptosis and on STAT-3 activation, protein and mRNA
levels, and DNA-binding and transcriptional activity. We exam-
ined whether overexpression of STAT-3 ? could prevent each of
these effects of BITC. We then extended these fi ndings to four
other pancreatic cancer cell lines, AsPC-1, Capan-2, MiaPaCa-2,
and Panc-1, and one relatively normal pancreatic cell line,
HPDE-6. Last, we looked at the effect of BITC on the growth of
BxPC-3 cells as xenografts in nude mice. Our results further eluci-
date the mechanism of BITC as an anticancer agent.
Materials and Methods
Cell Culture and Cell Survival Assays
The well-differentiated epithelial human pancreatic adenocarci-
noma 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 pan-
creatic 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 , 43 ).
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% CO 2 . 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/strep-
tomycin. 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 epider-
mal growth factor (GIBCO-BRL), 30 µ g/mL bovine pituitary
extract (GIBCO-BRL), and 1% (vol/vol) penicillin/streptomycin.
A 100 mM stock solution of BITC (Sigma-Aldrich) was pre-
pared in 100% dimethyl sulfoxide (DMSO) and subsequently
diluted in the cell culture medium so that the fi nal concentration
of DMSO was 0.1% in the medium. Cells were treated with BITC
at 5, 10, or 20 µ M concentration for 24 hours. The effect of BITC
on proliferation of BxPC-3 cells was determined by sulforhod-
amine 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 µ M) for 24 hours. For time-dependent experiments, cells were
treated with 10 µ M 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
CONTEXT AND CAVEATS
Benzyl isothiocyanate (BITC), a compound found in cruciferous
vegetables, has been reported to have anticancer properties. The
mechanism by which it inhibits proliferation of human pancreatic
cancer cells in culture was not well understood.
Human pancreatic cancer cell lines and a human nonmalignant,
but immortalized, pancreatic cell line were used to examine the
effects of BITC on proliferation and survival and on STAT-3 expres-
sion and activity in vitro. A mouse model of pancreatic cancer was
used to study the effects of BITC on tumor growth in vivo.
BITC treatment increased cell death in the pancreatic cancer cell
lines tested compared with the nonmalignant cell line. BITC-
sensitive cells also showed reduced levels of total and activated
STAT-3 protein. Overexpression of STAT-3 eliminated BITC-induced
apoptosis. Tumors grew more slowly in mice fed BITC than in
untreated control mice.
BITC induces apoptosis by a STAT-3 – dependent mechanism in
several human pancreatic cancer cell lines.
BITC promoted cell death and inhibited STAT-3 activation to vary-
ing degrees in several pancreatic cancer cell lines. Only one non-
malignant pancreatic cell line was studied. The in vivo experiments
were performed in a small number of mice that were continuously
fed BITC from the time of tumor cell implantation, and it is not clear
whether the protective dose would be practical in terms of human
consumption of vegetables.
From the Editors
178 Articles | JNCI Vol. 101, Issue 3 | February 4, 2009
BITC and with the proteasome inhibitor MG-132 (Calbiochem
EMD Chemicals, Gibbstown, NJ) at 10 µ M for 2 hours. In a sepa-
rate experiment, cells were pretreated with 10 µ g/mL cycloheximide,
a protein synthesis inhibitor (Sigma-Aldrich ), for 4 hours followed
by treatment with 10 µ M BITC. Cell lysates containing 20 – 40 µ g 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) accord-
ing 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 – ? -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 × 10 6 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 contain-
ing 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
? -glycerophosphate, 1 mM Na 3 VO 4 , and 1 µ g/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 14 000 g at 4°C, and the supernatant
was isolated. About 250 µ g 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
µ L of a 50% slurry) were added to each sample and incubated for 3
hours at 4°C. Lysates were centrifuged for 30 seconds at 1000 g , and
pellets were washed five times with 0.5 mL cell lysis buffer. STAT-3
protein from each sample was eluted with 40 µ L of 1% SDS and
quantified. About 3 µ g of pure STAT-3 protein was resolved on
10% SDS – PAGE and blotted with anti – pSTAT-3 (Tyr705) anti-
body. The same membrane was stripped and reprobed with anti –
STAT-3 antibody. In another experiment, cells were treated with
10 µ M BITC for 24 hours and with 10 µ M 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 µ M) 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 tran-
scription – 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 ′ -ATCCTGAAGCTGACCC
AGGTA-3 ′ ; STAT-3 reverse primer, 5 ′ - AGGTCGTTGGTGTC
ACACAGA-3 ′ ; GAPDH forward primer, 5 ′ - ACCACAGTCCATG
CCATCAC-3 ′ ; and GAPDH reverse primer, 5 ′ -TCCACCACCCTG
TTGCTGTA -3 ′ . Reactions were carried out in 50- µ L volumes
using 100 ng of total template RNA, 200 nM each of forward and
reverse primers, 25 µ L polymerase chain reaction (PCR) master mix,
and 1 µ L 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, exten-
sion 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 µ M BITC for 24 hours.
Then, nuclear extracts were prepared and incubated with 2 µ L 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 modi-
fications, 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 contain-
ing 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).
JNCI | Articles 179
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 µ g of pLuc-TK/STAT3 (a gen-
erous 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 µ g 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 µ M BITC or
with 0.1% DMSO for 24 hours or pretreated with 10 µ g/mL
cycloheximide for 4 hours and then treated with 10 µ M 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 activi-
ties 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 µ g pLuc-TK/STAT3 and with
0.2 µ g pRL-TK plasmid as a transfection effi ciency control.
Cells were again treated with 0.1% DMSO or with 10 µ M BITC
for 24 hours. In additional experiments, cells were 1) pretreated
with 5 ng/mL IL-6 for 15 minutes, 2) pretreated with 10 µ g/mL
cycloheximide for 4 hours, or 3) transfected for 48 hours with 2 µ g
STAT-3 ? , cloned in the pBabe Puro mammalian expression vec-
tor (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 µ M BITC for 24 hours, and lysates
were analyzed for luciferase activity as above.
Induction of STAT-3 by IL-6
Dishes containing 1 × 10 6 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 subse-
quent experiments to activate STAT-3.
Overexpression of STAT-3 ?
A plasmid that contained STAT-3 ? (Gene Accession number
U06922 ) cloned into the pBabe Puro mammalian expression vec-
tor (a generous gift from Dr J. F. Bromberg, Rockefeller University,
New York) was transfected to overexpress STAT-3 in BxPC-3
cells. Briefly 3 × 10 5 cells were transfected with 2 µ g of the STAT-
3 ? 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 µ M BITC for 24 hours. Cell lysates were
prepared, and 20 µ g of protein was analyzed by western blotting.
The same blots were stripped and reprobed with anti-actin anti-
body 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 apop-
totic cell death. Briefly, 1 × 10 4 BxPC-3 cells were seeded in
96-well plates and treated with either 10 µ M 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 trans-
fected with STAT-3 ? and then treated with BITC. Cell lysates
were mixed with biotinylated anti-histone antibodies and peroxide
POD conjugated anti-DNA antibodies in streptavidin-coated mul-
tiwell dishes, so that histone-bound DNA fragments that are char-
acteristic 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 experi-
ments 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 × 10 6 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 µ mol 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 CO 2 asphyxiation followed
by cervical dislocation in accordance with IACUC guidelines. The
tumors were removed aseptically from each mouse, and half of
180 Articles | JNCI Vol. 101, Issue 3 | February 4, 2009
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 Na 2 EDTA, 1 mM EGTA, 1% Triton X-100,
2.5 mM sodium pyrophosphate, 1 mM ? -glycerophosphate, 1 mM
Na 3 VO 4 , 1 µ g/mL leupeptin, and 1 mM phenylmethane sulfonyl
fluoride . The tumor lysate was cleared by centrifugation at 14 000 g
for 30 minutes. Lysate containing 60 µ g 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 dehy-
drated and embedded in paraffin, and sections that were 4 µ m
in thickness were prepared at every 100- µ m 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 µ g/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 µ L of biotinylated bromodeoxyu-
ridine 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 (dH 2 O). 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 dH 2 O three times for 5 minutes each,
then incubated in 3% hydrogen peroxide for 10 minutes followed
by two washes in dH 2 O 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 µ L 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 µ L 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 µ L
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 peroxi-
dase 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 Bonfer roni or Newman – Keuls post hoc multiple com-
parison 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 sig-
nificant 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 statisti-
cally 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
Effect of BITC on Constitutively Activated STAT-3 in Human
Pancreatic Cancer Cells
In our previous studies, we demonstrated that BITC strongly sup-
pressed the growth of human pancreatic cancer cells by causing
cell cycle arrest and apoptosis, but the exact mechanism by which
BITC induced apoptosis was not clear ( 18 ). Recent studies have
implicated STAT-3 in the promotion of cell survival and resistance
to apoptosis in a wide variety of human tumor cell lines
( 22 , 30 , 31 , 45 , 46 ). We hypothesized that BITC-induced apoptosis
in pancreatic cancer cells could be mediated by inhibiting STAT-3
activity; however, a role for STAT-3 in pancreatic cancer had not
yet been clearly determined. To test our hypothesis and to estab-
lish a role for STAT-3 in pancreatic tumorigenesis, the effects of
BITC treatment were evaluated in BxPC-3 human pancreatic can-
cer cells, in which STAT-3 is normally constitutively active.
Treatment of BxPC-3 cells with 0 – 40 µ M BITC for a 24-hour
period substantially reduced their survival in a concentration-
dependent manner, with an IC 50 of about 10 µ M ( Figure 1, A ).
JNCI | Articles 181
Figure 1 . Effect of benzyl isothiocyanate (BITC) on the constitutive phos-
phorylation 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 quanti-
fi ed 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% confi dence 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 µ g of pro-
tein were resolved by 10% SDS – PAGE and analyzed for the induction of
apoptosis as assessed by caspase-3 and PARP cleavage (PARP-CF).
? -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 µ M BITC, and cell lysates were examined on western
blots for STAT-3 phosphorylation at Tyr705 and Ser727 and for STAT-3
protein expression. ? -Actin was used as a control for loading and trans-
fer. 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% confi dence 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 µ M BITC for
24 hours (E) or with 10 µ M 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
specifi c for the activated pJNK (Thr183/Tyr185), pERK (Thr202/Tyr204),
and pp38 (Thr180/Tyr182) proteins and for total protein of each type.
? -Actin was used as a control for loading and transfer. The experiment
was repeated twice and similar results were obtained.
182 Articles | JNCI Vol. 101, Issue 3 | February 4, 2009
Figure 2 . 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 µ M 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% confi dence intervals of two independent experiments per-
formed 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
µ M BITC for 24 hours. Whole-cell lysates were prepared, and 40 µ g of
protein was resolved by 10% SDS – PAGE, blotted, and analyzed for
Mcl-1 and Bcl-2 proteins. ? -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 µ M BITC or DMSO alone (as a control) for 24 hours and
with or without 10 µ M MG-132 for 2 hours, and the expression of
STAT-3 was determined by western blotting of whole-cell lysates.
? -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
JNCI | Articles 183
In support of this fi nding, BxPC-3 cells that were treated with
0 – 20 µ M BITC showed cleavage of caspase-3 and PARP, both
indicative of apoptosis, when their lysates were separated on west-
ern blots ( Figure 1, B ). Treatment of BxPC-3 cells with 0 – 20 µ M
BITC for 24 hours not only substantially decreased STAT-3 acti-
vation, as refl ected by reduced phosphorylation of tyrosine 705
(Tyr705) and serine 727 (Ser727), but also reduced the total levels
of STAT-3 protein in a dose-dependent manner ( Figure 1, C ).
Therefore, the same concentrations of BITC that effectively
reduced STAT-3 expression and activation also substantially
induced apoptosis in this cell line ( Figure 1, C vs B).
To test whether BITC treatment was associated with reduced
STAT-3 phosphorylation in the absence of decreased STAT-3
protein levels, total STAT-3 protein was immunoprecipitated
from both 0.1% DMSO-treated and BITC-treated (10 µ M, 24
hours) BxPC-3 cells, using anti – STAT-3 antibody. Equal amounts
of STAT-3 protein were resolved by SDS – PAGE, blotted, and
probed with an anti – pSTAT-3 (Tyr705) antibody. Interestingly,
on a molar basis, no substantial change was observed in STAT-3
phosphorylation in response to BITC treatment ( Figure 1, D ).
These results suggested that the decreased STAT-3 phosphoryla-
tion that we observed in the presence of BITC is likely to be due
to reduced STAT-3 protein levels.
Because we observed substantial reductions in STAT-3 protein
expression in response to BITC treatment, we also tested whether
BITC might affect STAT-3 mRNA levels. When STAT-3 mRNA
transcripts were amplifi ed by RT-PCR from lysates of BxPC-3
cells that had been treated with BITC or 0.1% DMSO, BITC
substantially decreased STAT-3 mRNA levels in a dose- and time-
dependent manner as compared with controls ( Figure 1, E and F ).
To establish whether BITC specifi cally affected STAT-3 activa-
tion, the effect of BITC on MAPK signaling pathways was also
evaluated. We found that BITC treatment was not associated with
changes in the activation (phosphorylation) nor expression of JNK,
ERK, or p38 in BxPC-3 cells ( Figure 1, G ). Therefore, among the
pathways tested, BITC specifi cally affected synthesis and/or stabil-
ity of STAT-3.
Effect of BITC on DNA-Binding and Transcriptional
Activity of STAT-3
To perform its role in cell survival, activated STAT-3 translocates
to the nucleus and binds to specific response elements in the pro-
moters of its target genes. Because we observed substantially
reduced levels of STAT-3 in the nuclear fraction of BITC-treated
cells (data not shown), we expected that, in treated cells, decreased
amounts of STAT-3 would be able to bind to DNA response ele-
ments and promote transcription.
We determined the DNA-binding activity of STAT-3 using
the Universal EZ-TFA transcription factor colorimetric assay,
which combined the DNA-binding principle of the electropho-
retic mobility shift assay with the 96-well format of an enzyme-
linked immunosorbent assay. Compared with extracts from
DMSO-treated control cells, extracts from BxPC-3 cells treated
with 10 µ M BITC for 24 hours exhibited an approximately 48%
decrease in the STAT-3 DNA-binding activity (A 450 of STAT-3 –
DNA binding, control cells vs BITC-treated cells: 0.089 vs 0.049,
difference = 0.040, 95% CI = 0.033 to 0.046; P = .017; Figure 2, A ).
We also expected that if BITC inhibited the activation and
expression of STAT-3, it might inhibit transcription of STAT-3 –
responsive target genes. To examine this question, BxPC-3 cells
were treated with 0 – 20 µ M BITC for 24 hours and the whole-cell
lysate was resolved on 10% SDS – PAGE followed by western blot-
ting. As shown in Figure 2, B , BITC treatment was associated with
substantial and dose-dependent reductions in the expression of
Bcl-2 and Mcl-1 proteins, the products of two STAT-3 – responsive
genes that have been shown to play roles in apoptosis and cell
growth ( 27 , 36 ).
Because we had observed dramatic reductions of both STAT-3
mRNA and protein levels, we next asked whether STAT-3 protein
might have been inhibited through BITC-mediated activation of
the ubiquitin-proteasome pathway. To address this question, we
treated BxPC-3 cells with or without 10 µ M BITC for 24 hours and
with or without 10 µ M MG-132, a specifi c proteasome inhibitor, for
2 hours. In the blotted whole-cell lysates shown in Figure 2, C , the
BITC-mediated decline in the STAT-3 protein expression could be
almost completely prevented by MG-132 treatment. Furthermore,
when STAT-3 was fi rst immunoprecipitated from BITC- and/or
MG-132 – treated and untreated cells, then blotted, probed with
anti – STAT-3 antibody, and then reprobed with anti-ubiquitin anti-
bodies, high – molecular weight polyubiquitin conjugates were
observed in response to BITC treatment suggesting that STAT-3 is
degraded by the ubiquitin-proteasome pathway ( Figure 2, D ).
To delineate whether the mRNA levels for STAT-3 were
reduced due to decreased STAT-3 – mediated transcription after
BITC treatment, we transfected BxPC-3 cells with a plasmid that
encoded the STAT-3 promoter upstream of a luciferase reporter
gene, treated the cells with or without 10 µ M BITC, and per-
formed luciferase assays on extracts made 24 hours later. We
observed that BITC treatment statistically signifi cantly inhibited
STAT-3 – regulated luciferase reporter gene activity (luciferase
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-ubiq-
uitin antibodies. E ) Effect of BITC on STAT-3 – regulated luciferase
reporter activity. STAT-3 luciferase transcriptional activity was deter-
mined in BxPC-3 cells cotransfected with 2 µ g of a plasmid encoding
fi refl y luciferase under the control of the STAT-3 promoter and with 0.2
µ g of a plasmid expressing Renilla luciferase as a transfection effi ciency
control. At 24 hours after transfection, cells were treated with or without
10 µ M BITC for 24 hours or pretreated with 10 µ g/mL cycloheximide for
4 hours and then treated with or without 10 µ M BITC for 24 hours.
Whole-cell lysates were collected, and fi refl y 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% confi dence intervals of two independent experi-
ments 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 µ g/mL cycloheximide for 4 hours
followed by treatment with 10 µ M 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 dehy-
drogenase (GAPDH) as an internal loading control. The experiments
were repeated three times and similar results were obtained.
Figure 2 (continued).
184 Articles | JNCI Vol. 101, Issue 3 | February 4, 2009
Figure 3 . 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 treat-
ment with 10 µ M 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.
? -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-3 ? . Cells were transfected with an empty
vector or a plasmid expressing STAT-3 ? and 48 hours later were treated
with 10 µ M 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-3 ? overexpression on BITC-induced apoptosis
JNCI | Articles 185
activity in control vs BITC-treated cells: 1.08 vs 0.245, difference =
0.835, 95% CI = 0.415 to 1.264; P = .001) ( Figure 2, E ).
The transcription of the STAT-3 gene has been shown to be
autoregulated by STAT-3 protein through a composite IL-6
response element in its promoter that contains both a STAT-3 –
binding element and a cyclic AMP-responsive element ( 47 – 49 ).
To examine whether the results in Figure 2, E , which showed that
BITC treatment inhibited STAT-3 transcription, might be due to
transcriptional autoregulation by STAT-3, we pretreated the
BxPC-3 cells for 4 hours with 10 µ g/mL cycloheximide, a protein
synthesis inhibitor, before treating them with 10 µ M BITC for 24
hours and then evaluated STAT-3 – mediated luciferase reporter
activity in one experiment and STAT-3 mRNA levels in another.
Cycloheximide pretreatment blocked STAT-3 transcriptional
activity in the reporter assay and also blocked the BITC-mediated
decrease in STAT-3 mRNA levels, albeit incompletely ( Figure 2,
E and F ). However, STAT-3 mRNA levels were also consistently
reduced by cycloheximide treatment alone ( Figure 2, F ). The
explanation for this paradox is not clear at this time and warrants
further investigation. Taken together, our results consistently
show that BITC reduces STAT-3 mRNA and protein levels,
attenuates STAT-3 activation as demonstrated by reduced phos-
phorylation at Tyr705 and Ser727, and reduces DNA-binding and
STAT-3 – directed promoter activity.
Rescue of BITC-Induced Apoptosis by IL-6 – Induced STAT-3
Because IL-6 is a growth factor that has been shown to activate
STAT-3 ( 50 – 52 ), we next sought to determine whether IL-6 can
increase STAT-3 activity to higher levels than the constitutive
levels normally seen in BxPC-3 cells and whether the effect of
BITC on apoptosis is rescued under these conditions. BxPC-3 cells
were stimulated with 5 ng/mL of IL-6 for 15 minutes to 24 hours.
In lysates from cells treated with IL-6 for 15 minutes, we observed
20-fold increased phosphorylation of STAT-3 at Tyr705 com-
pared with constitutive levels, without any change in STAT-3
protein levels. In lysates from cells treated with IL-6 for 24 hours,
STAT-3 phosphorylation was increased 11-fold as compared with
DMSO-treated controls (data not shown).
Next, we determined whether BITC could inhibit IL-6 – induced
STAT-3 phosphorylation. BxPC-3 cells were fi rst stimulated with
5 ng/mL IL-6 for 15 minutes and then treated with either DMSO
or 10 µ M BITC for 24 hours. As shown in Figure 3, A , in the pres-
ence of IL-6 pretreatment, STAT-3 protein levels still appeared
to be reduced in association with BITC treatment. Moreover,
IL-6 – induced increases in STAT-3 phosphorylation at Tyr705 and
Ser727 (that were still visible 24 hours after IL-6 stimulation) were
markedly lessened after BITC treatment. Similar results were
obtained when BxPC-3 cells were fi rst treated for 24 hours with
BITC and then treated for 15 minutes with IL-6 (data not shown).
Furthermore, IL-6 – mediated enhancement of STAT-3 activation
was accompanied with a modest decrease in BITC-induced apopto-
sis, as indicated by reduced levels of caspase-3 and PARP cleavage
in lysates from BxPC-3 cells treated with both IL-6 and BITC com-
pared with those from cells treated with BITC alone ( Figure 3, A ).
Rescue of BITC-Induced Apoptosis by Overexpression of
Two isoforms of STAT-3, STAT-3 ? (p92) and STAT-3 ? (p83),
are derived from a single gene by alternative mRNA splicing and
demonstrate both distinct and overlapping functions in gene tran-
scription ( 53 , 54 ). STAT-3 ? is the predominant isoform expressed
in the cell lines used in this study (or, it is possible that the
STAT-3 antibody that we used was more specific to STAT-3 ? ,
because we detected a single band in our blots). To further con-
firm the role of STAT-3 ? in BITC-induced apoptosis, we overex-
pressed STAT-3 ? in BxPC-3 cells by transient transfection with a
STAT-3 ? – expressing plasmid. STAT-3 ? overexpression (2.6-fold
over control) completely protected BxPC-3 cells from BITC-
induced apoptosis as assessed by caspase-3 and PARP cleavage
( Figure 3, B ). Furthermore, STAT-3 ? overexpression protected
BxPC-3 cells from BITC-induced apoptosis as shown by quantita-
tive apoptosis ( Figure 3, C ), and cell survival assays ( Figure 3, D ).
STAT-3 ? overexpression (and, to a lesser extent, IL-6 – mediated
in BxPC-3 cells. Apoptosis was further confi rmed 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 µ M BITC for 24 hours and analyzed for
apoptosis. Separately, cells were transfected with empty vector or
STAT-3 ? – expressing vector as described above and then treated with or
without 10 µ M BITC for 24 hours. Means and 95% confi dence 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 com-
parison test. Statistical tests were two-sided. D ) Effect of STAT-3 ? over-
expression on BITC-associated reductions in cell survival. BxPC-3 cells
were transfected with a STAT-3 ? ? expressing vector as described above
and treated with 10 µ M BITC or 0.1% dimethyl sulfoxide (control) for 24
hours and the cell survival was evaluated by sulforhodamine B assay.
Means and 95% confi dence 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-3 ? overexpression on BITC-associated reductions in
STAT-3 DNA binding. The Universal EZ-TFA transcription factor colori-
metric 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-3 ? with and without treatment with 10 µ M
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 defi ned above in (C).
Means and 95% confi dence 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-3 ? 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-3 ? with and without 24-hour treatment with
10 µ M BITC was determined by luciferase assays. Control cells were
neither stimulated with IL-6 nor transfected with empty vector. Means
and 95% confi dence intervals of two independent experiments per-
formed 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-3 ? overexpression on BITC-associated reductions in Mcl-1 and
Bcl-2 expression. At 48 hours after transfection with empty vector or a
STAT-3 ? – expressing vector, BxPC-3 cells were treated with 10 µ M BITC
for 24 hours. Cell lysates were separated on 10% SDS – PAGE and blots
were probed using Mcl-1 – and Bcl-2 – specifi c antibodies, then stripped
and reprobed with ? -actin antibody to ensure equal loading. The experi-
ment was repeated twice and similar results were obtained.
Figure 3 (continued).
186 Articles | JNCI Vol. 101, Issue 3 | February 4, 2009
Figure 4 . 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 concen-
trations of BITC. A ) Cytotoxicity of BITC in these cell types, measured by
the sulforhodamine B cell survival assay. The experiment was per-
formed as described in Figure 1, A . It was repeated two times, each with
JNCI | Articles 187
activation of STAT-3) also abrogated BITC-associated reductions
in STAT-3 DNA-binding activity ( Figure 3, E ) and BITC-
associated reductions in STAT-3 transcriptional activity as mea-
sured by luciferase reporter assay ( Figure 3, F ) in BxPC-3 cells.
The ability of STAT-3 ? overexpression to reverse BITC-mediated
inhibition of STAT-3 transcriptional activity and cell survival was
also reflected by the restoration of Mcl-1 and Bcl-2 protein expres-
sion, levels of both of which were otherwise reduced in response to
BITC treatment ( Figure 3, G ).
Effect of BITC on Apoptosis, STAT-3 Activity, and STAT-3
mRNA and Protein Levels in Other Human Pancreatic
Cancer Cell Lines
To rule out the possibility that the observed effects of BITC were
specific to BxPC-3 cells, we also evaluated the effects of BITC on
the AsPC-1, Capan-2, MiaPaCa-2, and Panc-1 human pancreatic
cancer cell lines, which have varying degrees of baseline STAT-3
expression and activation. Treatment of AsPC-1, Capan-2, or
Mia-PaCa-2 cells for 24 hours with increasing concentrations of
BITC was associated with substantially reduced cell survival
( Figure 4, A ) and increased apoptosis as determined by caspase-3
and PARP cleavage ( Figure 4, B ) for each of these cell lines.
AsPC-1, Capan-2, and Mia-PaCa-2 cells treated with BITC also
showed substantially decreased levels of STAT-3 protein and its
phosphorylation at Tyr705 and Ser727 ( Figure 4, C ). Accordingly, the
DNA-binding activity of STAT-3 was substantially reduced in all
three cell lines after treatment with BITC for 24 hours ( Figure 4, D ).
The transcriptional activity of STAT-3 was decreased in AsPC-1
cells but not in Capan-2 or MiaPaCa-2 cells in response to BITC
treatment, as tested by the luciferase reporter assay ( Figure 4, E ).
Nevertheless, in contrast to what was seen in BxPC-3 cells,
STAT-3 mRNA levels were not altered in any of these cell lines
following treatment with up to 20 µ M BITC ( Figure 4, F ). We also
tested the effects of BITC treatment on Panc-1 pancreatic adeno-
carcinoma cells and HPDE-6 – immortalized “normal” pancreatic
cells. Exposure of Panc-1 cells to 0 – 40 µ M BITC for 24 hours
resulted in decreased survival of the cells, with an IC 50 of about 8
µ M ( Figure 5, A ). BITC also induced apoptosis in Panc-1 cells
( Figure 5, B ). However, to our surprise, BITC treatment had no
effect on the STAT-3 pathway in these cells. BITC failed to reduce
the phosphorylation or protein level of STAT-3 ( Figure 5, C ).
Similarly, BITC had no effect on the DNA-binding, transcrip-
tional activity or mRNA level of STAT-3 ( Figure 5, D – F), sug-
gesting that in Panc-1 cells, BITC reduces cell survival by affecting
pathways other than STAT-3.
The effects of BITC were then evaluated in HPDE-6 cells, a
line of immortalized human pancreatic ductal epithelial cells that
has been characterized extensively and is considered to be similar
to normal (nonmalignant) pancreatic cells in character ( 42 , 43 ).
The cells were exposed to 0 – 40 µ M BITC for 24 hours. As shown
in Figure 5, A , survival of HPDE-6 cells was not affected by BITC
treatment, even at concentrations that were very toxic to cancer
cells. Next, we determined the induction of apoptosis by cleavage
of caspase-3 and PARP. In contrast to what we found in pancreatic
cancer cells, BITC failed to induce apoptosis in HPDE-6 cells
( Figure 5, B ). We also observed no constitutive activation of
STAT-3, as indicated by the absence of phosphorylation at Tyr705
or Ser727, in HPDE-6 cells ( Figure 5, C ), consistent with the
general observation in the literature that STAT-3 is activated in
transformed cells and not in normal cells ( 37 ). Furthermore, BITC
treatment did not alter the protein level of STAT-3 in HPDE-6
cells ( Figure 5, C ). Similarly, BITC failed to inhibit DNA binding,
the transcriptional activity, or the mRNA level of STAT-3 ( Figure 5,
D – F), suggesting that HPDE-6 cells are altogether resistant to the
cytotoxic effects of BITC.
Effect of BITC Treatment on Apoptosis and STAT-3
Activity on BxPC-3 Pancreatic Cancer Cell Xenografts in
We next aimed to determine whether BITC could suppress the
growth of pancreatic tumors in vivo and if so, to examine the
mechanism. Based on our in vitro results, we hypothesized that
BITC treatment would inhibit in vivo tumor growth by reducing
STAT-3 expression in the tumor cells. To test our hypothesis,
human pancreatic tumor xenografts were implanted in athymic
nude mice by injecting 1 × 10 6 BxPC-3 cells subcutaneously in
both the left and right flanks of each mouse, so that each mouse
had two independent tumors. Mice were randomly divided into
two groups with five mice per group, and because each mouse had
two implanted tumors, each group had 10 tumors. Starting the
same day, after tumor cell implantation, one group of mice was fed
12 µ mol BITC dissolved 0.1 mL of PBS per day, 5 days a week,
and control mice received PBS alone. Tumors were measured
three times a week, and each mouse was weighed twice a week as
we described previously ( 8 ). By day 42, the growth of the tumors
in BITC-fed mice was substantially retarded compared with tumor
growth in control mice, and tumors also appeared to grow more
slowly in BITC-fed mice compared with control mice at all earlier
stages ( Figure 6, A ). For example, 6 weeks after treatment with
12 µ mol BITC (60 µ mol/wk), the average tumor volume in control
eight replicates, and similar results were obtained. Means and 95% con-
fi dence 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%
confi dence intervals of two independent experiments performed in trip-
licate are shown. Differences between all the groups were compared by
nonparametric analysis of variance with the Bonferroni post hoc multi-
ple 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% confi dence intervals of two independent experiments per-
formed in triplicate are shown. Differences between groups were com-
pared using the Student t test (two-sided; ns denotes “not signifi cant”).
F ) Effect of BITC on STAT-3 mRNA expression in these cell lines. Cells
were treated for 24 hours with 0 – 20 µ M BITC, and total RNA was ana-
lyzed 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.
Figure 4 (continued).
188 Articles | JNCI Vol. 101, Issue 3 | February 4, 2009
Figure 5 . Effect of benzyl isothiocya-
nate (BITC) on STAT-3 signaling in
Panc-1 and immortalized normal pan-
creatic 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 rep-
robed with ? -actin antibody as a con-
trol for equal loading and transfer.
The same RNA samples were ana-
lyzed for glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) expression,
as an internal control for loading and
transfer. Means and 95% confi dence
intervals of two independent experi-
ments performed in triplicate are
shown. Differences between groups
in (D) were compared by nonparamet-
ric analysis of variance with the
Bonferroni post hoc multiple com-
parison test and in (E) were compared
by the Student t test. Statistical tests
were two-sided. Nonsignifi cance is
denoted as “ns.”
JNCI | Articles 189
mice was about 1.92-fold higher than that in BITC-treated
mice (mean tumor volume, control vs BITC treated: 334 vs
172 mm 3 , difference = 162 mm 3 , 95% CI = 118 to 204 mm 3 ;
P = .008) ( Figure 6, A ).
At 42 days after the implantation of the tumors, all mice were
killed, and the tumors were excised, weighed, and processed for
immunohistochemistry and western blotting. In accordance with
the tumor volumes that were estimated from external measure-
ments, the weight of the tumors from BITC-treated mice was
approximately 53% less than the weight of tumors from control
mice (mean weight, control vs BITC treated: 0.425 vs 0.225 g, dif-
ference = 0.2 g, 95% CI = 0.181 to 0.219; P = .025) ( Figure 6, B ).
The average body weight of control and BITC-treated mice did
not change throughout the experiment ( Figure 6, C ), which sug-
gested that BITC was not associated with any discernible toxicity
to the mice.
To investigate the mechanism by which BITC reduced tumor
growth, tumors from control and BITC-treated mice were evalu-
ated by immunohistochemistry. As analyzed by TUNEL assay,
substantially increased numbers of apoptotic bodies were observed
in the tumor sections obtained from BITC-treated mice compared
with control mice ( Figure 6, D ), suggesting that the suppression of
tumor growth in the BITC-treated mice was due to increased apop-
tosis of the tumor cells. Furthermore, immunostaining of similar
tumor sections with antibodies to STAT-3 revealed substantially
reduced STAT-3 staining in the tumors of BITC-treated mice as
compared with controls ( Figure 6, D ) These observations were fur-
ther confi rmed by western blotting of protein lysates prepared from
tumors obtained from control and BITC-fed mice. BITC-treated
BxPC-3 tumors grown in mice contained lower levels of STAT-3
total protein and of STAT-3 phosphorylation than untreated
tumors as shown on blots using anti – STAT-3 anti – pSTAT-3
Figure 6 . 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 µ mol 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 (mm 3 ) = length × (width 2 )/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 toxic-
ity. Values in (A) are means of 10 (fi ve sets of two) observations in
each group with 95% confi dence 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% confi dence 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 sec-
tions and photographs were taken at high (×100) magnifi cation. 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
? -actin antibody to verify equal protein loading. Each lane represents a
different tumor sample.
190 Articles | JNCI Vol. 101, Issue 3 | February 4, 2009
(Tyr705) antibodies ( Figure 6, E ). Taken together, these in vivo
results are in agreement with our in vitro results and suggest that
STAT-3 is a critical signaling molecule responsible for BITC-
induced apoptosis both in vitro and in vivo.
STAT family proteins are cytoplasmic transcription factors that
are activated by receptor tyrosine kinases and intracellular kinases
through the phosphorylation of critical tyrosine and serine resi-
dues that regulate gene expression in response to cytokine and
growth factor receptor signaling ( 19 – 24 ). These proteins mediate
many diverse biological processes including cell survival, differen-
tiation, inflammation, immune response, and apoptosis. Among the
STAT proteins, STAT-3 has been extensively studied due to its
constitutive expression in a large proportion of human cancers
( 25 , 27 , 29 – 32 , 35 – 37 , 55 – 57 ) and its role in neoplastic development
and transformation ( 22 , 23 , 28 , 29 , 48 ) through the transcriptional
control of its regulated genes ( 19 , 20 , 24 , 58 ). Inhibition of STAT-3
activation has been shown to suppress the growth of human malig-
nant cells in experimental systems both in vitro and in vivo
( 25 , 27 , 30 , 32 , 35 , 36 , 55 – 57 ), and thus, targeted disruption of
STAT-3 could be one potential approach to treat human cancers
( 26 , 39 , 40 , 59 ).
It is well established that various cytokines and growth factors
can induce the activation of STAT-3 ( 44 , 60 , 61 ). Several lines of
evidence have suggested that IL-6 stimulates cancer cell growth by
the phosphorylation and activation of STAT-3 ( 50 – 53 ). Recent
studies have consistently demonstrated that phospho – STAT-3
levels are elevated in malignant prostate cells in vivo, further sup-
porting a role for activated STAT-3 in cancer ( 32 ). In agreement
with these fi ndings, in this study we fi nd differential levels of
constitutive STAT-3 phosphorylation in various pancreatic cancer
cell lines but no activating phosphorylation of STAT-3 in normal
Our fi ndings clearly demonstrate that apoptotic death of human
pancreatic cancer cells in the presence of BITC, a naturally occur-
ring anticancer agent, is associated with substantial reductions in
the levels of both activated STAT-3 (as represented by phospho-
rylation at Tyr705 and Ser727) and total STAT-3 protein. The
loss of STAT-3 expression in response to BITC treatment is both
dose and time dependent. In BxPC-3 pancreatic cancer cells,
STAT-3 mRNA levels are similarly reduced. The inhibition
of STAT-3 by BITC was associated with decreased amounts
of STAT-3 – mediated DNA-binding activity and decreased tran-
scription of the genes for Mcl-1 and Bcl-2, which are both known
to be downstream of STAT-3 activation ( 26 , 30 , 37 , 56 , 62 , 63 ). In
addition, our results show that overexpression of STAT-3 by gene
transfection completely protected BxPC-3 pancreatic cancer cells
from BITC-induced apoptosis, confi rming the role of STAT-3 in
BITC-induced apoptotic cell death. BITC treatment not only
blocked constitutive STAT-3 activation but also was able to block
tyrosine phosphorylation of STAT-3 induced by IL-6. Moreover,
BITC failed to cause any cytotoxic effects in HPDE-6 cells, which
are considered to be similar to normal human pancreatic cells.
Last, we provided evidence that orally feeding BITC to athymic
nude mice substantially suppressed the growth of BxPC-3 pancre-
atic tumor xenografts and that suppressed tumor growth was asso-
ciated with increased apoptosis and decreased STAT-3 expression.
Thus, we establish STAT-3 as a molecular target of BITC in pan-
creatic cancer cells. Our results are in agreement with recent stud-
ies that showed that the naturally occurring agents capsaicin and
silibinin suppress the growth of multiple myeloma and prostate
cancer cells, respectively, by blocking STAT-3 activation ( 27 , 36 ).
In another study, resveratrol was reported to cause cell cycle arrest
and apoptosis in breast and prostate cancer cells by inhibiting con-
stitutive STAT-3 activation and suppressing STAT-3 – regulated
cyclin D1, Bcl-xL, and Mcl-1 gene expression ( 38 ). It is notewor-
thy that our study demonstrates the degradation of STAT-3 pro-
tein by BITC, in contrast to some previously published reports in
which only phosphorylation was inhibited ( 27 , 36 ). In agreement
with other previous studies, degradation of STAT-3 protein in our
model involves the ubiquitin-proteasome pathway ( 64 , 65 ).
The identifi ed targets of STAT-3 transcriptional activation
include Bcl-2, Mcl-1, Bcl-xL, and cyclin D1 and refl ect the roles
of STAT-3 to promote cell survival and cell cycle progression
( 26 , 30 , 37 , 51 , 62 , 63 ). Moreover, constitutively active STAT-3 con-
tributes to resistance to apoptosis in colorectal tumors and multiple
myeloma cells, possibly through Bcl-2 and cyclin D1 expression
( 26 , 34 , 60 ). In our previous study, we showed that BITC treatment
decreased the expression of Bcl-2 and cyclin D1 in pancreatic can-
cer cells ( 10 ). In this study, inhibition of STAT-3 activation
together with reduced expression of Bcl-2 and Mcl-1 was associ-
ated with the ability of BITC to induce apoptosis in pancreatic
cancer cells. These results are consistent with previous observa-
tions which showed that transfection with dominant-negative
STAT-3 induced apoptosis in HeLa and SiHa cells that contained
constitutively active STAT-3 ( 59 ). Our results also showed that
overexpression of STAT-3 ? in BxPC-3 cells completely abrogated
the apoptosis-inducing effects of BITC. Expression of Bcl-2 and
Mcl-1 was not decreased after BITC treatment in cells that over-
expressed STAT-3 ? . These results support the notion that STAT-3 ?
is a critical target of BITC.
To rule out the possibility that BITC affects BxPC-3 cells spe-
cifi cally, the effects of BITC on apoptosis and STAT-3 activation
were also evaluated in AsPC-1, Capan-2, MiaPaCa-2, and Panc-1
pancreatic cancer cells and compared with normal HPDE-6 pan-
creatic cells. BITC inhibited the STAT-3 signaling pathway in
AsPC-1, Capan-2, and MiaPaCa-2 cells but not in Panc-1 cells.
Furthermore, normal HPDE-6 cells were totally resistant to the
deleterious effects of BITC. These results are in agreement with
our previous studies ( 18 ), in which we demonstrated that BITC
was least effective against acinar cells isolated from normal human
pancreas. Taken together, these results suggest that BITC may not
be as toxic to normal cells as to cancer cells. To extend the obser-
vations made in cultured cells and to evaluate the effect of BITC
in vivo, we determined the effect of BITC on the growth of
BxPC-3 pancreatic tumor xenografts in athymic nude mice. The
growth rate of BxPC-3 tumor xenografts was substantially retarded
in mice that were administered 12 µ mol BITC orally for each of
5 days a week for 6 weeks without causing detectable side effects.
The tumors obtained from BITC-treated mice exhibited substan-
tially enhanced apoptosis and decreased STAT-3 staining as
assessed by immunohistochemistry and immunoblotting. Our
JNCI | Articles 191
results in mice complement our in vitro results confi rming
STAT-3 as a potential therapeutic target of BITC.
BITC is a dietary agent that is abundant in many cruciferous
vegetables that are consumed by humans on a daily basis.
Epidemiological studies continue to support the notion that
dietary intake of cruciferous vegetables may reduce the risk of dif-
ferent types of malignancies, including pancreatic cancer ( 4 – 7 ).
Our data suggest that BITC is relatively safe to normal pancreatic
cells and also safe to mice as established previously ( 18 ) and in this
study. These data are consistent with previous in vitro studies of
other isothiocyanates such as allyl isothiocyanate, phenethyl iso-
thiocyanate (PEITC), and sulforaphane, in which cell growth
arrest and induction of apoptosis were observed at concentrations
lower than 40 µ M ( 8 , 9 , 66 , 67 ). Because the pharmacokinetics of
BITC in humans have not been determined, it is diffi cult to predict
how much cruciferous vegetable would need to be consumed to
clinically achieve a serum concentration of 10 µ M BITC, the con-
centration that was most effective in inhibiting STAT-3 activation
in our model. However, a very recent study suggested that orally
feeding male Sprague-Dawley rats with PEITC (an analog of
BITC) resulted in rapid absorption that reached a maximal plasma
concentration of 9.2 ± 0.6 µ M after 0.44 ± 0.1 hours of feeding
10 µ mol/kg PEITC and of 42.1 ± 11.4 µ M after 2.0 ± 1 hours of
feeding 100 µ mol/kg PEITC, suggesting that micromolar concen-
trations of isothiocyanates may be achieved in vivo ( 68 ). In another
pharmacokinetics study in which four human volunteers were fed
with a single dose of myrosinase-hydrolyzed extract from 3-day-
old broccoli sprouts (which contained about 200 µ mol of total
isothiocyanates), a peak concentration of 2.27 µ M isothiocyanates
was reached in the plasma at 1 hour after broccoli extract ingestion
( 69 ). Nevertheless, detailed pharmacokinetic studies of BITC are
required before conducting clinical testing of BITC as a cancer
There are some limitations to our study. Although we have
established the effi cacy of BITC in terms of limiting the growth of
human pancreatic cancer cells in vitro and in vivo and have dem-
onstrated that STAT-3 is a critical target, it remains to be deter-
mined whether BITC modulates signals upstream of STAT-3 to
inhibit STAT-3 transcription and protein levels. Furthermore, the
number of mice used in our animal studies was limited, and we did
not explore the growth of BxPC-3 cells with either stable overex-
pression of STAT-3 or with deletion of STAT-3 as tumor xeno-
grafts in mice. Such studies could better establish the role of
STAT-3 in pancreatic tumorigenesis. The effect of BITC treat-
ment under these conditions is not known and will be the focus of
our future studies.
In conclusion, our results demonstrate that BITC can suppress
both constitutive and inducible STAT-3 activation in human pan-
creatic cancer cells, block the DNA-binding and transcriptional
activity of STAT-3, reduce the expression of Bcl-2 and Mcl-1,
induce apoptosis, and inhibit cell proliferation. These effects of
BITC were not observed in normal pancreatic epithelial cells.
BITC-stimulated apoptosis was blocked when STAT-3 ? was over-
expressed in BxPC-3 cells. Taken together, these fi ndings may
provide the basis for further preclinical and clinical investigation of
BITC for the chemoprevention and/or chemotherapy of pancre-
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JNCI | Articles 193
This investigation was supported in part by United States Public Health Service
RO1 grant CA106953 (to SKS) awarded by the National Cancer Institute .
Funding from Texas Tech University Health Sciences Center , School of
Pharmacy (to SKS), and an instrument grant from Turner Biosystems Inc,
Sunnyvale, CA (to RPS), are also acknowledged.
The authors wish to thank Ruifen Zhang and Jeffery Richards for help in perform-
ing animal experiments; Dr J. F. Bromberg, Rockefeller University, New York, NY,
for providing the STAT-3 expression plasmid and pLuc-TK/STAT3 construct;
Dr Ming-Sound Tsao, University of Toronto, Toronto, Ontario, Canada, for pro-
viding HPDE-6 cells; and Dr Thomas L. Brown, Wright State University, Dayton,
OH, for providing Panc-1 cells. The authors also wish to thank Dr Doug Potter
(University of Pittsburgh) and Dr David Fike for statistical help; Dr Kalkunte
Srivenugopal and Dr Jayarama Gunaje, Texas Tech University Health Sciences
Center, for constructive suggestions during the revision of the manuscript; and
Christopher Adkins for technical help in performing fl uorescent microscopy.
The authors take sole responsibility for the design of the study; the collec-
tion, analysis, and interpretation of the data; the writing of the manuscript; and
the decision to submit the manuscript for publication.
Manuscript received January 28 , 2008 ; revised October 17 , 2008 ; accepted
November 19 , 2008 .