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Small Molecule Therapeutics
Bazedoxifene as a Novel GP130 Inhibitor forPancreatic Cancer
TherapyXiaojuan Wu1,2, Yang Cao2,3, Hui Xiao2, Chenglong Li4, and
Jiayuh Lin5
Abstract
The IL6/GP130/STAT3 pathway is crucial for tumorigenesis
inmultiple cancer types, including pancreatic cancer, and presents
asa viable target for cancer therapy. We reported
Bazedoxifene,which is approved as a selective estrogen modulator by
FDA, asa novel inhibitor of IL6/GP130 protein–protein
interactionsusingmultiple ligand simultaneous docking and drug
reposition-ing approaches. STAT3 is one of the major downstream
effectorsof IL6/GP130. Here, we observed Bazedoxifene inhibited
STAT3phosphorylation and STAT3 DNA binding, induced apoptosis,and
suppressed tumor growth in pancreatic cancer cells withpersistent
IL6/GP130/STAT3 signaling in vitro and in vivo. Inaddition, IL6,
but not INFg , rescued Bazedoxifene-mediatedreduction of cell
viability. Bazedoxifene also inhibited STAT3
phosphorylation induced by IL6 and IL11, but not by OSM orSTAT1
phosphorylation induced by INFg in pancreatic cancercells,
suggesting that Bazedoxifene inhibits the GP130/STAT3pathway
mediated by IL6 and IL11. Furthermore, Bazedoxifenecombinedwith
paclitaxel or gemcitabine synergistically inhibitedcell viability
and cell migration in pancreatic cancer cells. Theseresults
indicate that Bazedoxifene is a potential agent and cangenerate
synergism when combined with conventional chemo-therapy in human
pancreatic cancer cells and tumor xenograftin mice. Therefore, our
results support that Bazedoxifene as anovel inhibitor of GP130
signaling and may be a potentialand safe therapeutic agent for
human pancreatic cancer therapy.Mol Cancer Ther; 15(11); 2609–19.
�2016 AACR.
IntroductionHuman pancreatic cancer is one of the deadly
malignant
diseases with very poor clinical outcome. The median
overallsurvival is approximately 6 months after surgical and
chemor-adiotherapies for locally advanced and metastatic stages
ofpancreatic cancer and the 5-years overall survival rate is
lessthan 5%. Because of the absence of specific symptoms, the
lackof early detection techniques, pancreatic cancer is
usuallydiagnosed at advanced and metastatic stages and is not
resectedby surgery (1, 2). To date, chemo- and radiotherapy have
onlylimited success because of high resistance (3).
Unfortunately,only less than 15% of all pancreatic cancer patients
have achance for surgical resection, after which 5-year survival
rarelysucceed to 20% to 25% (4, 5).
The IL6/GP130/STAT3 signaling pathway is frequently acti-vated
in many human cancer and contributes to oncogenesisand cancer
progression (6, 7). The present review is to highlightthe role of
IL6 in pancreatic cancer development and progres-sion (8). It is
well-established that IL6 is elevated in the serum
of pancreatic cancer patients compared with healthy controlsand
those with chronic pancreatitis (9–13). Several studiesraised
strong evidence that elevated levels of IL6 protein andmRNA in
serum and tumor samples of patients with pancreaticcancer is
associated with increased tumor size and poor prog-nosis (14). IL6
binds a nonsignaling a-receptor IL6R to form abinary complex
(IL6/IL6Ra), which, after dimerization withGP130, leads to
activation of receptor-associated JAKs. In turn,these several
phosphorylate downstream targets, includingcytoplasmic STAT3, which
after dimerization rapidly translo-cate to the nucleus and promotes
pancreatic cancer progressionthrough transcriptional regulation of
antiapoptotic and pro-proliferative genes (14). STAT3 has been
identified as a keyoncogenic factor in a number of human cancers
and is requiredfor oncogenesis in mouse model of cancers (15, 16).
In pan-creatic cancers, constitutive activation of STAT3 by
phosphor-ylation of Tyr705 has been reported in 30% to 100% of
humantumor specimens, as well as in many pancreatic cancer cell
lines(17, 18). In contrast, this pathway is inactive in normal
pan-creas, and correspondingly STAT3 is not required for
pancreaticdevelopment or homeostasis (19). These studies suggest
thatSTAT3 activation by IL6/GP130 signaling pathway plays
animportant role in human pancreatic cancer development
andprogression.
Inhibiting IL6/GP130 signaling might be a new therapeuticoption
for pancreatic cancer. One possibility would be the treat-ment with
the humanized monoclonal anti–IL6R antibodies,which is already
approved for the treatment of some inflamma-tion disease (20).
However, its potential therapeutic effect onpancreatic cancer has
not yet examined. Selective inhibitors ofIL6/GP130/STAT3 are more
effective options for treatment ofpancreatic cancer. Our previous
study explored that a small-molecular inhibitor of STAT3, LLL12,
was proposed selectivelyblocking exogenous IL6-induced STAT3
phosphorylation andnuclear translocation in two human pancreatic
cancer cell lines
1Department of Pediatric Surgery, Tongji Hospital, Huazhong
Univer-sity of Science and Technology,Wuhan, China. 2Department of
Pedi-atrics, Center for Childhood Cancer and Blood Diseases, the
ResearchInstitute at Nationwide Children's Hospital, College of
Medicine, TheOhio State University, Columbus, Ohio. 3Department of
Hematology,Tongji Hospital, Huazhong University of Science and
Technology,Wuhan, China. 4Division of Medicinal Chemistry and
Pharmacognosy,College of Pharmacy, The Ohio State University,
Columbus, Ohio.5Department of Biochemistry and Molecular Biology,
University ofMaryland School of Medicine, Baltimore, Maryland.
Corresponding Author: Jiayuh Lin, University of Maryland School
of Medicine,655 W Baltimore S, Baltimore, MD 21201. Phone:
410-706-7469; Fax: 410-706-8297; E-mail: [email protected]
doi: 10.1158/1535-7163.MCT-15-0921
�2016 American Association for Cancer Research.
MolecularCancerTherapeutics
www.aacrjournals.org 2609
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Research. mct.aacrjournals.org Downloaded from
Published OnlineFirst August 17, 2016; DOI:
10.1158/1535-7163.MCT-15-0921
http://mct.aacrjournals.org/
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(21). Because of the importance of small molecules in
drugdiscovery, and as there are no any examples of small
moleculesinhibiting the IL6/GP130 pathway associated in pancreatic
cancertreatment, it would be a good strategy to use small
molecularinhibitors for the same.
Bazedoxifene is known as a selective estrogen modulator
andcommonly used for the prevention for osteoporosis. Recently,we
have discovered Bazedoxifene as a novel small molecularGP130
inhibitor, which binds to GP130 D1 domain (22). Itmay be expected
to speed up the development of clinicaltherapies for the
IL6/GP130/STAT3–dependent cancers. In thisstudy, we report the new
role of Bazedoxifene as a GP130inhibitor to inhibit the GP130/STAT3
signaling pathway medi-ated by IL6 and IL11, induce apoptosis in
pancreatic cancercells, and suppress the tumor growth in human
pancreaticcancer xenograft, suggesting that Bazedoxifene may serve
as anovel therapeutic drug for pancreatic cancer by targeting
theGP130/STAT3 signaling pathway.
Materials and MethodsCell lines and reagents
Human pancreatic cancer cell lines (AsPC-1, PANC-1,
HPAF-II,BxPC-3, HPAC, Capan-1) were purchased from the ATCC.HPAF-II
cells were cultured in Eagle's Minimum EssentialMedium (DMEM),
Capan-1 cells were maintained in Iscove'sModified Dubecco's Medium
(IMDM) supplemented with 20%FBS and the others in DMEM supplemented
10% FBS and 1%penicillin/streptomycin. HPAF-II was purchased within
2months before the experiments about HPAF-II was performed.AsPC-1,
PANC-1, BxPC-3, Capan-1, and HPAC cell lines werefrozen within 2
months of receipt and were resuscitated fromearly passage liquid
nitrogen stocks as needed. Cells werecultured for less than 3
months before reinitiating cultures andwere routinely inspected
microscopically for stable phenotype.All cell lines were cultured
in a humidified 37�C incubator with5% CO2.
IL6, IL11, OSM, and IFNg were purchased from Cell
SignalingTechnology. The powder was dissolved in sterile PBS to
make a100 ng/mL stock solution. Bazedoxifene was purchased
fromAcesys Pharmatech, and paclitaxel and gemcitabine were
boughtfrom LC Laboratories. These three drugs were dissolved in
sterileDMSO tomake a 20mmol/L stock solution. Aliquots of the
stocksolution were stored at �20�C.
Western blotting assayHuman pancreatic cancer cell lines
(HPAF-II, BxPC-3, Capan-1,
and HPAC) were harvested after treatment with Bazedoxifeneor
DMSO at 50% to 60% confluence overnight, then lysed incold RIPA
lysis buffer containing protease inhibitors cocktailand phosphatase
inhibitor cocktail. The lysates were subjectedto 10% or 12%
SDS-PAGE gel and transferred to a PVDFmembrane. Membranes were
probed with a 1:1,000 dilutionof specific primary antibody and
1:10,000 horseradish perox-idase–conjugated secondary antibody.
Primary antibodiesagainst phosphorylated STAT3 (Tyr705), STAT3,
phosphorylat-ed STAT1 (Tyr701), STAT1, cleaved caspase-3,
phospho-specificextracellular signal-regulated kinase (ERK) 1/2
(Threonine 202/Tyrosine 204), P-AKT (Ser473), GAPDH and secondary
anti-body are all from Cell Signaling Technology. Membranes
wereanalyzed using enhanced chemiluminescence plus reagents and
scanned with the Storm Scanner (Amersham Pharmacia Bio-tech
Inc.).
STATs phosphorylation induced by cytokines or growth
factorsPANC-1, AsPC-1, and HPAF-II pancreatic cancer cells were
seeded in 10-cm plates and allowed to adhere overnight.
Thefollowing night, the cells were serum starved. The cells were
thenleft untreated orwere treatedwith Bazedoxifene (5–20mmol/L)
orDMSO. After 2 hours, the untreated and Bazedoxifene-treatedcells
were stimulated by IL6 (50 ng/mL), IL11 (50 ng/mL), OSM(50 ng/mL),
or INFg (50 ng/mL) for 30 minutes. The cells wereharvested and
analyzed by Western blot analysis for p-STAT3Y705
or p-STAT1Y701.
Reverse transcriptase-PCRCells were treated with Bazedoxifene
(5–20 mmol/L) or DMSO
at 50% to 60% confluence in the presence of 10% FBS for 24hours.
RNA from the cells was then extracted using RNeasy Kits(Qiagen)
according to the manufacturer's instruction. Reversetranscription
was done using an Omniscript reverse transcriptionkit (Qiagen). PCR
amplification was performed under the fol-lowing conditions: 5
minutes at 94�C followed by 30 cycles of 30seconds at 94�C, 30
seconds at 48–55�C, and 60 seconds at 72�Cwith a final extension of
10 minutes at 72�C. The followingprimers were used: Cyclin D1,
annealing at 52�C (For): 50-GCTGGAGCCCGTGAAAAAGA-30 (Rev):
50-CTCCGCCTCTGG-CATTTTG-30; Bcl-Xl, annealing at 48�C (For):
50-TTGGACAATG-GACTGGTTGA-30 (Rev): 50-GTAGAGTGGATGGTCAGTG-30;
Sur-vivin, annealing at 52�C (For): 50-ACCAGGTGAGAAGTGAGG-GA-30
(Rev): 50-AACAGTAGAGGAGCCAGGGA-30; GAPDH,annealing at 52�C (For):
50-TGATGACATCAAGAAGGTGGT-GAAG-30 (Rev):
50-TCCTTGGAGGCCATGTGGGCAT-30 (integrat-ed DNA Technologies).
MTT cell viability assayHuman pancreatic cancer cell lines
(HPAC, PANC-1, HPAF-II,
BxPC-3, and Capan-1), were seeded in 96-well plates at a
densityof 3,000 cells per well. The next day, IL6 (50 ng/mL), INFg
(50 ng/mL), or Bazedoxifene (10 mmol/L) alone, or combination of
IL6or INFg with Bazedoxifene were added in triplicate to the plates
inthe presence of 0% FBS in HPAF-II cells for 24 hours.
Differentconcentrations of Bazedoxifene (5–10 mmol/L), paclitaxel
(1–2.5mmol/L), or gemcitabine (5 mmol/L) alone, or Bazedoxifene
pluspaclitaxel or gemcitabine were add in triplicate to the plates
in thepresence of 10% FBS in BxPC-3 or Capan-1 cells or
paclitaxelplus gemcitabine were add in the plates in PANC-1,
HPAC,BxPC-3 and Capan-1 cells. The cells were incubated at 37�Cfor
a period of 24 to 48 hours. BxPC-3 and Capan-1 cells wereseeded in
96-well plates at a density of 3,000 cells per welland cultured at
37�C. The next day, GP130 siRNA (100 nmol/L) or negative control
siRNA was transfected into cells intriplicate using lipofectamine
2000 for 72 hours. Twenty-fivemL of
3-(4,5-Dimethylthiazolyl)-2,5-diphenyltetrazolium bro-mide (MTT,
Sigma) was added to each sample in a volumeof 100 mL and incubated
for 4 hours. Then 150 mL of N, N-dimethylformamide (Sigma)
solubilization solution wasadded to each well. The absorbance was
read at 595 nm.Combination index (CI) was performed using data
obtainedfrom MTT assay with CompuSyn software. The CI
valuesindicate a synergistic effect when 1, and an additive effect
when equal to 1 (23).
Wu et al.
Mol Cancer Ther; 15(11) November 2016 Molecular Cancer
Therapeutics2610
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Research. mct.aacrjournals.org Downloaded from
Published OnlineFirst August 17, 2016; DOI:
10.1158/1535-7163.MCT-15-0921
http://mct.aacrjournals.org/
-
STAT3 DNA binding assayBxPC-3 cells were seeded in a 10-cm plate
and treated with
Bazedoxifene (5–10 mmol/L) or DMSO for 24 hours. The
NuclearExtract Kit (Clontech Inc.) was used to prepare cell nuclear
extractsfollowing the manufacturer's protocol. Nuclear extracts
wereanalyzed for STAT3 DNA–binding activity using a STAT3
DNAbinding ELISA kit (Active Motif) with an ELISA-based
method.Absorbance was read at 450 nm.
Wound-healing/cell migration assayWhen HPAC cells were 100%
confluent, the monolayer was
scratched in same width using a pipette tip. After washing,
HPACcells were then treated with different concentrations of
Bazedox-ifene or DMSO. In addition, we treated HPAC cells with
Bazedox-ifene, paclitaxel alone, or combination of them. After 24
hoursculture, when thewound in theDMSO control was closed,
imageswere captured by Leica Microsystems.
ImmunofluorescenceHPAF-II cells were seeded on glass coverslips
in 6-well plate.
The next day, the cells were cultured in serum free medium for24
hours and pretreated with Bazedoxifene (20 mmol/L) for2 hours,
followed by induction with 50 ng/mL IL6 for 30 min-utes. Cells were
fixed with cold methanol for 15 minutes andblocked with 5% normal
goat serum and 0.3% Triton X-100 inPBS for 1 hour. The cells were
incubated with primary anti-bodies of p-STAT3Y705 (Cell Signaling
Technology, 1:100)overnight at 4�C. After incubation with
anti-rabbit FIFC-con-jugated secondary antibody (Invitrogen, 1:
200), the cells weremounted with Vectashield Hardset mounting
medium withDAPI (Vector Laboratories). Photomicrographs were
capturedby Leica Microsystems.
Mouse xenograft tumor modelAll animal studies were conducted in
accordance with the
principles and standard procedures approved by IACUC of
theResearch Institute at Nationwide Children's Hospital.
Capan-1(3�106) andHPAF-II (3�106) cells inMatrigel
(BDBiosciences)were injected subcutaneously into the both side of
flank area of6-week-old female athymic nude mice which were
purchasedfrom Harlan. After Capan-1 tumor development, which was
1week after initial implantation, mice were divided into
twotreatment groups consisting of four mice (tumors: n¼ 8):
DMSOvehicle control and gavage injection of Bazedoxifene
(5mg/kg/d).Mice bearing HPAF-II tumor were irrigated with
Bazedoxifene(5 mg/kg/d) and/or injected via abdomen with paclitaxel
(15mg/kg, 2/w). Tumor growth was determined by measured thelength
(L) and width (W) of the tumor every other day with acaliper, and
tumor volume was calculated on the basis of thefollowing formula:
volume ¼ 0.52 � LW2. After 21 days oftreatment,
tumorswereharvested, snap-frozen indry ice, andstoredat�80�C.
Tumors tissue homogenates were lysed and separated bySDS-PAGE to
examine the expression of STAT3 phosphorylation,P-ERK1/2, P-AKT
(Ser473), and cleaved caspase-3.
Statistical analysisSignificance of correlations was done using
GraphPad Prism
software. Unpaired t tests were used for analyses assuming
Gauss-ian populations with a 95% confidence interval. Data are
pre-sented asmean� SE.Differences were analyzedwith the Student
t
test, and significance was set at P < 0.05; �, P < 0.05;
��, P < 0.01;and ���, P < 0.001, respectively.
ResultsBazedoxifene, a novel small molecule inhibitor that
targetsGP130
IL6/IL6Ra or IL11/IL11R binds to the GP130 D1 domainthrough a
few hot residues to form the IL6/IL6Ra/GP130 orIL11/IL11R/GP130
heterotrimers and dimerization of the trimersactivates
IL6/GP130/STAT3 or IL11/GP130/STAT3 signalingpathway, which is
crucial for the progression in multiple humancancers. We discovered
Bazedoxifene as a novel molecule inhib-itor of IL6/GP130 or
IL11/GP130 protein–protein interactions(PPI) using multiple ligand
simultaneous docking (MLSD).We identified that Bazedoxifene
disables the dimerization of theIL6/IL6Ra/GP130 or IL11/IL11R/GP130
heterotrimers usingMLSD method (Fig. 1; ref. 22). Bazedoxifene
targets the humanestrogen receptor (ER) and is approved by FDA as a
drug for theprevention of osteoporosis. In our study, we confirmed
thatBazedoxifene inhibits STAT3 phosphorylation induced by IL6and
IL11 in GP130/STAT3 pathway signaling.
Bazedoxifene inhibits STAT3 phosphorylation induced bycytokines
in human pancreatic cancer cells
IL6 family cytokines such as IL6, IL11, and OSM can induceSTAT3
phosphorylation. AsPC-1, HPAF-II, and PANC-1 pancre-atic cancer
cells, which do not express phosphorylated STAT3 inserum-free
medium for 24 hours, were used to examine if Baze-doxifene can
inhibit IL6, IL11, or OSM induced STAT3 phosphor-ylation. In this
study, we found IL6 as well as IL11 could stimulatephosphorylation
of STAT3 and Bazedoxifene could decrease thephosphorylation in a
dose-dependent manner. However, Baze-doxifene could not suppress
p-STAT3 induced by OSM (Fig. 2A–C) or phosphorylation of STAT1
stimulated by INFg in AsPC-1andHPAF-II cells (Fig. 2D). These
results indicate Bazedoxifene isan inhibitor of GP130/STAT3
signaling pathway mediated by IL6and IL11 in pancreatic cancer
cells.
Figure 1.
Bazedoxifene (ball-and-stick) binds to GP130 D1 domain (ribbon
model). Ile83and Phe36 offer important hydrophobic interaction with
bazedoxifene'sazepanylring; and Tyr94 and Asn92 form aromatic and
hydrogen-bondinginteractions with bazedoxifene, respectively.
Bazedoxifene as a Novel GP130 Signaling Inhibitor
www.aacrjournals.org Mol Cancer Ther; 15(11) November 2016
2611
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Published OnlineFirst August 17, 2016; DOI:
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Bazedoxifene inhibits phosphorylation of STAT3,
inducesapoptosis, and suppresses transcription of STAT3
downstreamtargets in human pancreatic cancer cells
Bazedoxifene was assessed for its inhibitory effect of
GP130/STAT3 signaling on Capan-1, BxPC-3, HPAF-II, and HPAChuman
pancreatic cancer cells which expressed persistentSTAT3
phosphorylation. The results showed that Bazedoxifenedecreased
expression of p-STAT3Y705 in dose-dependent man-ner in all four
cell lines (Fig. 2F). The inhibition of phosphor-ylated STAT3 by
Bazedoxifene was consistent with the induc-tion apoptosis evidenced
by the increasing caspase-3 in all celllines (Fig. 2F). However,
Bazedoxifene could impact differentdownstream targets of GP130 in
different pancreatic cell lines.
As shown in Fig. 2C, p-AKT (S473) was downregulated
byBazedoxifene in BxPC-3, HPAF-II, and HPAC except Capan-1cells,
but only in BxPC-3, Capan-1, and HPAC cells suppressionof p-ERK1/2
(T202/Y204) was seen. In addition, to examine theinfluence of
Bazedoxifene on the inhibition of STAT3 pathway,we detected the RNA
expression of its downstream targets byreverse transcription
(RT)-PCR, including CyclineD1, Bxl-cL,and Survivin. We found these
STAT3 targets were decreased inCapan-1, BxPC-3, and HPAF-II cells
when they were treatedwith different concentration of Bazedoxifene
(5–20 mmol/L;Fig. 2E). This indicates that Bazedoxifene is an
effective GP130inhibitor through donwregulation of its downstream
targets inpancreatic cancer cells.
A
IL6 0
Bazedoxifene (μmol/L) +IL6
10 20 IL11 0 10 20
Bazedoxifene (μmol/L) +IL11
Bazedoxifene (μmol/L) +OSM
OSM 0 10 20P-STAT3 (Y705)STAT3
GAPDH
P-ERK1/2 (T202/Y204)P-AKT (S473)
AsPC-1 B
0
Bazedoxifene (μmol/L) +IL6
10 20
PANC-1
IL6 IL11 0 10 20
Bazedoxifene (μmol/L) +IL11
Bazedoxifene (μmol/L) +OSM
OSM 0 10 20P-STAT3 (Y705)STAT3
GAPDH
P-ERK1/2 (T202/Y204)P-AKT (S473)
C HPAF-II
IL6 10 200
Bazedoxifene (μmol/L) +IL6
IL11 10 200
Bazedoxifene (μmol/L) +IL11
Bazedoxifene (μmol/L) +OSM
OSM 10 200 P-STAT3 (Y705)STAT3
GAPDH
P-ERK1/2 (T202/Y204)
D
P-STAT1 (Y701)STAT1
GAPDH
0 INFγ
Bazedoxifene (μmol/L) +INFγ10 20
AsPC-1
0 INFγ
Bazedoxifene (μmol/L) +INFγ10 20
HPAF-II
Capan-10 10 20
BxPC-30 5 10
HPAF-II0 10 20
P-STAT3 (Y705)STAT3P-AKT (S473)P-ERK1/2 (T202/Y204)
GAPDH
Cleaved caspase-3
Bazedoxifene (μmol/L) HPAC
0 10 15
E
G
F
Capan-1
**
10500
50
100
150
Rela
tive
STAT
3 DN
A bi
ndin
g ac
tivity
(%)
Bazedoxifene (μmol/L)
BxPC-3
0
50
100
150
Rela
tive
STAT
3 DN
A bi
ndin
g ac
tivity
(%)
**
Bazedoxifene (μmol/L) 20100
H
0
50
100
150
Cell
viab
ility
(%)
DMSO IL6 INFγ Ba IL6+Ba INFγ+Ba
*
IL6 & INFγ: 50 ng/mLBa: Bazedoxifene (10 μmol/L)
HPAF-II
Capan-10 10 20
BxPC-30 5 10
HPAF-II0 10 20
CyclinD1
Bcl-xL
Survivin
GAPDH
Bazedoxifene(μmol/L)
Figure 2.
Bazedoxifene inhibits STAT3 phosphorylation induced by IL6 and
IL11, downregulates expression of STAT3 downstream target genes,
and decreasesthe STAT3 DNA–binding activity in pancreatic cancer
cells. P-STAT3Y705, P-AKT, and P-ERK were analyzed by Western blot
analysis in AsPC-1 (A), PANC-1 (B),and HPAF-II (C) cells after
induction by IL6, IL11, and OSM (50 ng/mL). D, AsPC-1 and HPAF-II
cells were stimulated by INFg (50 ng/mL) and thecells were
harvested and analyzed for p- STAT1Y701. E, the mRNA expression of
CyclinD1, Bcl-xL, and Survivin was detected at by RT-PCR in
Capan-1,BxPC-3, and HPAF-II after treatment with Bazedoxifene. F,
Western blot analysis of p-STAT3Y705, p-AKT (S473), p-ERK
(T202/Y204), and cleaved caspase-3were performed after treatments
with Bazedoxifene at the indicated concentrations in Capan-1,
BxPC-3, HPAF-II, and HPAC cells. G, STAT3 DNA–bindingactivity in
BxPC-3 or Capan-1 cells treated with Bazedoxifene for 24 hours was
detected by DNA-binding assay (� , P < 0.05; �� , P < 0.01).
H, HPAF-IIcells were treated with cytokines, including IL6 and INFg
(50 ng/mL), with or without Bazedoxifene (10 mmol/L) in serum-free
condition for 24 hours(� , P < 0.05). Cell viability was
detected by MTT assay.
Wu et al.
Mol Cancer Ther; 15(11) November 2016 Molecular Cancer
Therapeutics2612
on June 5, 2021. © 2016 American Association for Cancer
Research. mct.aacrjournals.org Downloaded from
Published OnlineFirst August 17, 2016; DOI:
10.1158/1535-7163.MCT-15-0921
http://mct.aacrjournals.org/
-
Bazedoxifene inhibits STAT3 DNA binding and STAT3
nucleartranslocation induced by IL6; IL6 partially
rescuesbazedoxifene-mediated decrease of cell viability in
humanpancreatic cancer cells
IL6 activates GP130/STAT3 signaling pathway throughSTAT3 nuclear
translocation to bind DNA. To evaluate theinhibition of STAT3 DNA
binding activity, BxPC-3 and Capan-1 cells were treated with
Bazedoxifene (5–20 mmol/L) over-night and were harvested and
performed STAT3 DNA-bindingactivity assay as described in Materials
and Methods. We foundthat Bazedoxifene caused a significant
different inhibitionof STAT3 DNA-binding activity in BxPC-3 and
Capan-1 cells(Fig. 2G). Furthermore, HPAF-II cells were starved in
serum-free medium for 24 hours and pretreated with Bazedoxifene(20
mmol/L) for 2 hours followed by IL6 stimulation.
Immu-nofluorescence results showed STAT3 nuclear
translocationinduced by IL6 was blocked by Bazedoxifene (Fig. 3A).
How-ever, Bazedoxifene could not inhibit STAT1 nuclear
translo-cation induced by INFg (Fig. 3B). On the other hand,
toexplore whether exogenous IL6 could rescue the decreased
cellviability by Bazedoxifene, we treated HPAF-II cells with
10mmol/L Bazedoxifene with or without IL6 or INFg (50 ng/mL)in
serum-free medium for 24 hours. The data in Fig. 2Hshowed IL6 could
partially rescue Bazedoxifene-mediatedinhibition in HPAF-II
cells.
Inhibition of GP130/STAT3 using Bazedoxifene andSiRNA-sensitized
pancreatic cancer cells to anticancer drugs,gemcitabine, or
paclitaxel
To evaluate the synergistic effect of suppression of GP130/STAT3
with other anticancer drugs, we first treated pancreaticcancer
cells in combination of Bazedoxifene with gemcitabineor paclitaxel,
which are the standards of care for humanpancreatic cancer at
present. As shown in Fig. 4A, the combi-nation effect of
Bazedoxifene with gemcitabin or paclitaxel for48 hours in BxPC-3
and Capan-1 cells showed cell viability wasmore significantly
decreased in the combination treatmentgroup than single drug group.
The CI values of all the combi-nation treatments were less than 1,
suggesting there was syn-ergism in the combination treatments of
Bazedoxifene withgemcitabin. To further determine the role of
inhibition of theIL6/GP130/STAT3 pathway in combination treatment
withother anticancer drugs, BxPC-3 and Capan-1 cells were
trans-fected with GP130 or IL6 SiRNA for 48 hours and treated
withgemcitabine or paclitaxel for more 24 hours. Knockdown ofGP130
or IL6 was confirmed by western blot, as shownin Fig. 4B,
p-SATA3Y705, GP130, or IL6 was decreased. Cellviability was further
reduced in GP130-knockdown-cells andIL6-knockdown-cells treated
with gemcitabine or paclitaxelthan control groups (Fig. 4C).
Furthermore, our results showedthe greater inhibition was seen in
the combination treatment of
HPAF-IIDMSO
IL6 (50 ng/mL)
IL6 (50 ng/mL) +Bazedoxifene (20 µmol/L)
DMSO
INFγ (50 ng/mL)
INFγ (50 ng/mL) +Bazedoxifene (20 µmol/L)
P-STAT1 DAPI Merge
P-STAT3 DAPI MergeA
B
Figure 3.
Bazedoxifene inhibits STAT3 nucleartranslocation induced by IL6,
but notp-STAT1 by INFg . HPAF-II cells withlower expression of
p-STAT3Y705 werepretreated with bazedoxifene(20 mmol/L) for 2
hours. After that, thecells were stimulated by IL6 or INFg
(50ng/mL) for half an hour, and STAT3 (A)or STAT1 (B) nuclear
translocation wasdetected by immunofluorescence.
Bazedoxifene as a Novel GP130 Signaling Inhibitor
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-
Bazedoxifene with paclitaxel or gemcitabine than paclitaxelplus
gemcitabine in HPAC, Capan-1, HPAF-II and BxPC-3 cells(Fig. 4D).
These results revealed that suppression of GP130/STAT3 signaling
pathway could sensitize pancreatic cancer cellsto the first-line
chemotherapeutic agents, gemcitabine, andpaclitaxel.
Bazedoxifene inhibits cell migrationGP130/STAT3 activation is
involved in cell migration, so sup-
pression ofGP130might block cellmigration in pancreatic
cancercells. Therefore, we next evaluated whether Bazedoxifene
couldblock cell migration which is an important process in
tumorinvasion and metastasis. As shown in Fig. 5A,
Bazedoxifenetreatment reduced migration ability in a dose-dependent
mannerinHPAC cells.Moreover, combination treatment blocked the
cellsmigration more significantly than single group (Fig. 5B).
Theseresults suggest that inhibition of GP130 by Bazedoxifene
blocksthe cellsmigration in pancreatic cancer cells and the
cellmigration
was more significantly inhibited in the combination
treatmentgroup than single-agent groups.
Bazedoxifene inhibits capan-1 tumor growth in mouse modelin
vivo
We further verified whether Bazedoxifene suppressed thetumor
growth in vivo as in vitro. Capan-1 cells (3 � 106)injection was
performed as previously described in Materialsand Methods. One week
after initial implantation, when thetumors reached a size of 0.05
to 0.1cm3, the mice were given5 mg/kg Bazedoxifene in the treated
group or DMSO in vehiclegroup daily for 18 days. As shown in Fig.
6A, Bazedoxifenesignificantly suppressed tumor growth compared with
thevehicle group. P-STAT3Y705 of tumor tissue sample in
Bazedox-ifene-treated group was reduced, and caspase-3 was
induced(Fig. 6A), suggesting that Bazedoxifene could suppress
pancre-atic cancer xenograft tumor growth and induce apoptosis
intumor cells.
B
GP130
Capan-1BxPC-3Con GP130
P-STAT3 (Y705)
GAPDH
P-ERK1/2 (T202/Y204)
Con GP130
STAT3
P-AKT (S473)
siRNA
C
Con IL6
Capan-1 Con IL6
BxPC-3 siRNA
P-STAT3 (Y705)
GAPDH
P-ERK1/2 (T202/Y204)
IL6
STAT3
P-AKT (S473)
Con: negative control siRNA (100 mmol/L)GP130: siRNA (100
mmol/L)IL6: siRNA (100 mmol/L)
BxPC-3
GP130 Con+P1 GP130+P1Con0
50
100
150*
*
Con: Negative control siRNA (100 mmol/L) GP130: siRNA (100
mmol/L) IL6: siRNA (100 mmol/L) P: Paclitaxel (μmol/L)
Capan-1
Con+P1 IL6+P1IL6Con0
50
100
150 ****
Capan-1BxPC-3
Cell
viab
ility
(%)
GP130+P10Con+P10GP130Con
50
100
150 ***
Cell
viab
ility
(%)
0 Cell
viab
ility
(%)
IL6+P10Con+P10IL6Con0
50
100
150 ***
Cell
viab
ility
(%)
DHPAC
P1+G5P1B10DMSOG5
B10+G5
B10+P1
0
50
100
150
*****
Capan-1
P1+G5P1B10DMSO G5
B10+G5
B10+P1
0
50
100
150 *****
P1+G5P1B10DMSOG5
B10+G5
B10+P1
0
50
100
150 HPAF-II
******
P1+G5P1B10DMSOG5
B10+G5
B10+P1
0
50
100
150 BxPC-3
*****
B: Bazedoxifene (μmol/L) G: Gemcitabine (μmol/L) P: Paclitaxel
(μmol/L)
Cell
viab
ility
(%)
Cell
viab
ility
(%)
Cell
viab
ility
(%)
Cell
viab
ility
(%)
A BxPC-3
B10+G5G5B10DMSO 0
50
100
150***
B: Bazedoxifene (μmol/L)G: Gemcitabine (μmol/L)
B10+P2.5B5+P2.5 B10P2.5P2.5B5DMSO 0
50
100
150 ***
****
B: Bazedoxifene (μmol/L)P: Paclitaxel (μmol/L)
BxPC-3
B10+P1P1B10B5+P1P1B5DMSO 0
50
100
150***
*******
B: Bazedoxifene (μmol/L)P: Paclitaxel (μmol/L)
B: Bazedoxifene (μmol/L)G: Gemcitabine (μmol/L)
***
*
B5+G5G5B5DMSO 0
50
100
150Capan-1 Capan-1
Cell
viab
ility
(%)
Cell
viab
ility
(%)
Cell
viab
ility
(%)
Cell
viab
ility
(%)
Figure 4.
Blocking GP130 signaling using Bazedoxifene, GP130 SiRNA, or IL6
SiRNA enhances the effect of treatment with paclitaxel and
gemcitabine in pancreaticcancer cells. A, cell viability was
performed by MTT after treatment with bazedoxifene, paclitaxel,
gemcitabine alone or combination in BxPC-3 and Capan-1 cells(� , P
< 0.05; �� , P < 0.01; ��� , P < 0.001). B, BxPC-3 and
Capan-1 cells were transfected with GP130 SiRNA or IL6 SiRNA for 72
hours. IL6, GP130, p-STAT3Y705,p-AKT (S473), and p-ERK (T202/Y204)
were assessed by Western blot analysis. C, BxPC-3 and Capan-1 cells
were transfected with GP130 SiRNA or IL6SiRNA for 48hours and
treatedwith paclitaxel for another 24hours. Cell
viabilitywasdetected (�,P
-
Finally, we tested whether the combined Bazedoxifene
andpaclitaxel had stronger inhibitory effects than single drug
treat-ment in HPAF-II xenograft tumor growth. HPAF-II
tumor-bearingmice were treated with Bazedoxifene and paclitaxel
either indi-vidually or in combination as described in Materials
and Meth-ods. Compared with vehicle-treated mice, tumor volumewas
significantly decreased in mice treated with Bazedoxifene(P <
0.05) or paclitaxel alone (P < 0.05). Furthermore, combi-nation
of Bazedoxifene and paclitaxel dramatically decreasedtumor growth
compared with both vehicle and monotherapy(P < 0.05, Fig. 6B).
Correspondingly, the combination therapywas well tolerated and did
not result in any significant in vivotoxicity (Fig. 6B). As Fig. 6B
shown, P-STAT3Y705 of tumor tissuesample in Bazedoxifene- and
combination-treated groups wasreduced, and caspase-3 was more
induced in the combination-treated group than the
Bazedoxifene-treated group.
DiscussionPancreatic cancer is an extremely aggressive malignant
tumor
characterized by extensive invasion and early metastasis
(24).Pancreatitis is known as the most common precursor lesions
ofpancreatic cancer. Recent evidences indicate several
inflamma-tory cytokines, including IL6, express abnormally highly
inchronic pancreatitis, at all stages of human pancreatic
carcino-genesis in mouse models of this disease (7, 25–28). Given
bythe fact that IL6 plays important role during the
initiation,maintenance, and progression of pancreatic cancer (14,
26).
Several studies have revealed that the inhibition of IL6
provedanti–IL6–blocking antibodies or selective molecule sgp130Fcto
inhibit IL6 signaling and induce cell apoptosis in pancreaticcancer
cells and animal models (20, 29). However, antibodytreatment led to
massive systemic elevations in IL6 (30). Toovercome such
difficulties, inhibitors of GP130, as an impor-tant part of
receptor signaling complexes of IL6/IL6R/GP130,are required. SC144,
a GP130 inhibitor, was reported that itinhibits the GP130/STAT3
pathway through decrease constitu-tive STAT3 phosphorylation and
its downstream genes expres-sion in ovarian cancer (31, 32). Though
their study showed thatGP130 is directly inhibited by SC144, the
domain that binds toGP130 was not examined and still unclear.
Existing drug,Bazedoxifene, which is approved by FDA as an estrogen
recep-tor modulator and commonly used as treatment for
osteopo-rosis (33, 34). As shown in Fig. 1, Bazedoxifene binds to
GP130D1 domain through spots Ile83, Phe36, Tyr94 and Asn92,which
suggesting that Bazedoxifene could be a novel inhibitorof IL6/GP130
signaling (22). Because the IL6/GP130/STAT3signaling pathway is
involved in cancer growth, progression,and drug resistance in a
variety of human cancers, includingpancreatic cancer (26, 35, 36),
targeting this signaling pathwaywould be a promising therapy for
the treatment of pancreaticcancer (37). The in vitro and in vivo
results obtained in thisstudy, confirmed that the inhibition of
persistent STAT3 acti-vation by Bazedoxifene, including suppressing
the STAT3 phos-phorylation induced by IL6, reducing the downstream
genesexpression, inhibiting cell migration in pancreatic cancer
cells,
(μmol/L)B: Bazedoxifene
Bazedoxifene 20 μmol/L0 Bazedoxifene 10 μmol/L
0 h
24 h
AHPAC
B20B10DMSO0
50
100
150 **
**
HPAC
Mig
ratin
g ce
lls (%
)
B
0 h
24 h
Bazedoxifene: 5 μmol/LPaclitaxel: 0.5 μmol/L
+ –– ++ +––
HPAC
(μmol/L)B: BazedoxifeneP: Paclitaxel (μmol/L)
B5+P0.5P0.5B5DMSO0
50
100
150 **
*
HPAC
Mig
ratin
g ce
lls (%
)
Figure 5.
Wound-healing assay was done in HPAC cells. A, HPAC cells were
treated with different concentrations of bazedoxifene or DMSO (�� ,
P < 0.01). B, HPAC cellswere treated by bazedoxifene and
paclitaxel alone or combination of both drugs (� , P < 0.05; ��
, P < 0.01).
Bazedoxifene as a Novel GP130 Signaling Inhibitor
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-
as well as suppress the pancreatic tumor growth in mousemodel in
vivo.
Pancreatic cancer cell lines, BxPC-3,Capan-1,HPACandHPAF-IIcells
were reported to secrete IL6 (38, 39). Therefore, BxPC-3,Capan-1,
HPAC and HPAF-II cells expressing persistent IL6/GP130/STAT3
signaling were used to explore the inhibitory effecton the
IL6/GP130/STAT3 signaling pathway by Bazedoxifene. Asthe major
downstream effector of IL6/GP130 pathway, phosphor-ylation of STAT3
was downregulated by Bazedoxifene in all fourpancreatic cancer cell
lines and the downstream target genes ofSTAT3, including cyclinD1,
Bcl-xL, and survivin, were decreasedusing RT-PCR in Capan-1, BxPC-3
and HPAF-II cells, which con-firmed that Bazedoxifene is an
effective inhibitor of IL6/GP130signaling. Our results also showed
that Bazedoxifene could inhibitP-AKTandP-ERK1/2 in threeout of
fourpancreatic cancer cell lines.We found P-AKT (S473) was much
lower in Capan-1 cells than theother three cancer cell lines, and
Bazedoxifene, IL6, and GP130siRNA all induced P-AKT insteadof
inhibited P-AKT in this cell line.The observation that Bazedoxifene
could inhibit P-AKT in BXPC-3cells and P-ERK1/2 in BXPC-3 and
Capan-1 cells but IL6 or GP130siRNAcouldnot,
suggestingapossiblemechanism:P-ERK inBXPC-3 and Capan-1 cells and
P-AKT in BXPC-3 cells are not mainlydependent on IL6 or GP130
signaling. In addition, to investigate
whether suppression of IL6/GP130 signaling could induce
cellapoptosis as reported in vitro and in vivo (40–43),
apoptoticmarker cleaved caspase-3 was examined in
Bazedoxifene-treatedpancreatic cancer cells. The results showed
that Bazedoxifenetreatment induced cell apoptosis in pancreatic
cancer cells. Onthe other hand, IL6 partially rescued
Bazedoxifene-mediatedinhibition of cell viability inHPAF-II cells.
Our results, therefore,support the idea that Bazedoxifene is a
potent inhibitor ofGP130, which is consistent with suppression of
GP130 inhibitsSTAT3 activity and induces cell apoptosis (42, 44,
45). Bazedox-ifene also inhibits pancreatic cancer cell migration.
Pancreaticcancer cell lines we tested here secrete IL6. IL6 can
induceP-STAT3 and other downstream target (such as AKT or
ERK)through the autocrine pathway. Therefore, the ability of
Baze-doxifene to inhibit cell viability and migration is likely due
to itsability to inhibit one of the pathways ormore than one
pathwayscombined: (i) autocrine IL6 induction of P-STAT3; (ii)
autocrineIL6 and non-GP130 pathway(s) induction of P-ERK and
P-AKT;(iii) other pathway(s) in addition to STAT3, ERK, and
AKT.Furthermore, Bazedoxifene suppresses human pancreatic
tumorgrowth in a mouse xenograft model, which is showing
Bazedox-ifene as a potent inhibitor of pancreatic cancer cells
expressingpersistent GP130/STAT3 signaling.
A
0 2 4 6 8 10 12 14 16 180
500
1,000
Days after treatment
VehicleBazedoxifene (5 mg/Kg)
** *****
***** ***
Capan-1 tumor model
Vehicle Bazedoxifene(5 mg/Kg)P-STAT3(Y705)
STAT3
Cleaved caspase-3
GAPDH
B: Bazedoxifene (5 mg/Kg) ; P: Paclitaxel (15 mg/Kg)
Vehicle B P B+PP-STAT3(Y705)
STAT3
Cleaved caspase-3
GAPDH0
100
200
300
400
500
Days after treatment
Tum
or v
olum
e (m
m3 )
Tum
or v
olum
e (m
m3 )
Vehicle
B
P
B+P
**
**
***
0 2 4 6 8 10 12 14 16
HPAF-II tumor modelB
0 2 4 6 8 10 12 14 1616
18
20
22
24
Vehicle
B
P
B+P
Days after treatment
Bod
y w
eigh
t (g)
P-ERK1/2(T202/Y204)P-AKT (S473)
P-ERK1/2(T202/Y204)P-AKT (S473)
Figure 6.
Inhibitory efficacy of Bazedoxifene on tumor growth in the
Capan-1 tumor xenograft mouse model. Combined Bazedoxifene and
paclitaxel treatment results inrobust efficacy in HPAF-II tumor
xenograft model. A, growth rate of the Capan-1 tumor xenograft in
mouse model treated with vehicle and Bazedoxifene (�� , P <
0.01;��� , P < 0.001). B, growth rate of HPAF-II tumor xenograft
in mouse model treated with vehicle, Bazedoxifene, paclitaxel, or
Bazedoxifene/paclitaxel treatment(� , P < 0.05; �� , P <
0.01; ���, P < 0.001). P-STAT3Y705 and caspase-3 of Capan-1
tumor tissue samples from these mice were detected by Western blot
analysis.
Wu et al.
Mol Cancer Ther; 15(11) November 2016 Molecular Cancer
Therapeutics2616
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http://mct.aacrjournals.org/
-
IL6 family cytokines, IL6 and IL11, act on the cells
usingreceptor GP130 by similar molecular interactions, which
leadsto the intracellular signal (46, 47). In this study,
Bazedoxifeneinhibited STAT3 phosphorylation induced by IL6 and
IL11, butnot STAT1 phosphorylation induced by INFg was not
inhibitedfurther indicating its selectivity on STAT3 over STAT1.
IL6 binds toIL6Ra to form a binary complex and then recruits GP130
to formthe IL6/IL6Ra/GP130 heterotrimer. In addition,
homodimeriza-tion of the IL6/IL6Ra/GP130 heteotrimers occurs by
interactionsbetween IL6 of one trimer and the D1 domain of GP130 of
theother trimer, forming a hexamer (48, 49). The reciprocal
homo-dimerization of the IL6/IL6Ra/GP130 trimers triggers a
signalingcascade downstream such as JAK/STAT3. Interestingly, from
ourcomputational modeling, we also found that IL11 exhibits
verysimilar hexamer formation as IL6. IL11 also binds to IL11Ra
toform a binary complex and then recruits GP130 to form
theIL11/IL11Ra/GP130 heterotrimer. Homodimerization of
theIL11/IL11Ra/GP130 heteotrimers also occurs by
interactionsbetween IL11 of one trimer and the D1 domain of GP130
of theother trimer to form a hexamer. Bazedoxifene specifically
bindsto D1 domain of GP130 (but not D2 and D3 domains) andblocking
IL6 or IL11 of one trimer to bind to the D1 domain ofGP130 on the
other trimer, and thus further blocking hexamerformation and the
signaling cascade downstream to STAT3.Our computational modeling is
further supported by Bazedox-ifene can inhibit P-STAT3 induced by
IL6 and IL11. IL11 isreported to be involved in tumorigenesis in
gastric and breastcancers and is also a potential cancer
therapeutic target (50–52). The expression of IL11 is also elevated
in pancreatic cancer,suggesting that it may play a role in the
oncogenesis of pan-creatic cancer (53). Furthermore, from our
computationalmodeling, LIF and OSM, however, only form trimer to
signal-ing downstream and do not form hexamerization through
D1domain of GP130 protein. Therefore, Bazedoxifene did notinhibit
P-STAT3 induced by OSM (this study) or LIF (22). Theseresults
support the selectivity of Bazedoxifene for inhibitingGP130/STAT3
signaling mediated by IL6 and IL11 but not OSMand LIF. These
results also suggest that Bazedoxifene could be adual inhibitor of
IL6 and IL11 for cancer therapy.
Majority of the pancreatic cancer patients are relying on
theneoadjuvant chemotherapy or chemoradiotherapy may beused in
cases that are considered to be borderline resectableto reduce the
cancer to such a level where surgery is could bedelayed and this in
turn, given the fact that operation is notpreferable in many cases.
However, this strong limitation ofconventional treatment is mainly
due to the drug resistanceon the current standard-of-care treatment
(54). The IL6/GP130/STAT3 pathway is involved in drug resistance in
avariety of human cancers, including pancreatic cancer (55,56). In
this study, we show that Bazedoxifene or knockdownof GP130 works
synergistically with gemcitabine or paclitaxel
in BxPC-3 and Capan-1 pancreatic cancer cells,
respectively.Furthermore, the experiment of cell migration with the
com-bination treatment of Bazedoxifene and paclitaxel in HPACcells
provides more evidences that Bazedoxifene sensitizespancreatic
cancer to other anticancer drugs. In vivo, combina-tion of
Bazedoxifene and paclitaxel was superior to bothvehicle and
monotherapy in HPAF-II tumor xenograft mice.The ability of
Bazedoxifene to generate stronger inhibition ofcell viability when
combined with gemcitabine or paclitaxel,and is likely due to its
ability to inhibit one of the pathway ormore than one pathways
combined: (i) autocrine IL6 induc-tion of P-STAT3; (ii) autocrine
IL6 and non-GP130 pathway(s) induction of P-ERK in BXPC-3 and
Capan-1 cells andinduction of P-AKT in BXPC-3 cells; (iii) other
pathway(s) inaddition to STAT3, ERK, and AKT. These results
indicate thatIL6/GP130 signaling contributes to the drug resistance
andBazedoxifene has stronger inhibition effect in combinationwith
paclitaxel or gemcitabine than as a single agent.
On thebasis of ourfindings,we suggested that Bazedoxifene is
apotent inhibitor of GP130/STAT3 signaling mediated by IL6 andIL11.
Not only effectively it blocks activation of STAT3, but
alsosuppresses pancreatic cancer growth in vitro and in vivo
andsensitizes pancreatic cancer cells to paclitaxel and
gemcitabine.Thus, Bazedoxifene is a potential therapeutic small
molecularagent that could be useful for the treatment of human
pancreaticcancer when combined with paclitaxel and gemcitabine.
Disclosure of Potential Conflicts of InterestNo potential
conflicts of interest were disclosed.
Authors' ContributionsConception and design: X. Wu, C. Li, J.
LinDevelopment of methodology: X. Wu, C. Li, J. LinAcquisition of
data (provided animals, acquired and managed patients,provided
facilities, etc.): X. Wu, Y. Cao, C. Li, J. LinAnalysis and
interpretation of data (e.g., statistical analysis,
biostatistics,computational analysis): X. Wu, J. LinWriting,
review, and/or revision of the manuscript: X. Wu, H. Xiao, J.
LinAdministrative, technical, or material support (i.e., reporting
or organizingdata, constructing databases): X. Wu, J. LinStudy
supervision: X. Wu, J. Lin
Grant SupportThis work was supported by the grants NIH/NCI/R21,
CA173473-01 (PI: J.
Lin). NIH/R01, NS087213-01A1 (PI: J. Lin). AACR-Pancreatic
Cancer Networkresearch grants (PI: J. Lin). National Natural
Science Funding of China,81500395 (PI: X. Wu).
The costs of publication of this articlewere defrayed inpart by
the payment ofpage charges. This article must therefore be hereby
marked advertisement inaccordance with 18 U.S.C. Section 1734
solely to indicate this fact.
Received November 17, 2015; revised August 2, 2016; accepted
August 4,2016; published OnlineFirst August 17, 2016.
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2619
Bazedoxifene as a Novel GP130 Signaling Inhibitor
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2016;15:2609-2619. Published OnlineFirst August 17, 2016.Mol
Cancer Ther Xiaojuan Wu, Yang Cao, Hui Xiao, et al.
TherapyBazedoxifene as a Novel GP130 Inhibitor for Pancreatic
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