HHS Public Access Author manuscript Author Manuscript
Mol Cancer Ther. Author manuscript; available in PMC 2017 November 01. Published in final edited form as: Mol Cancer Ther. 2016 November ; 15(11): 2609–2619. doi:10.1158/1535-7163.MCT-15-0921.
Bazedoxifene as a Novel GP130 Inhibitor for Pancreatic Cancer Therapy Xiaojuan Wu1,2, Yang Cao2,3, Hui Xiao2, Chenglong Li4, and Jiayuh Lin5,6 1Department
of Pediatric Surgery, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China
Author Manuscript
2Center
for Childhood Cancer and Blood Diseases, the Research Institute at Nationwide Children's Hospital, Department of Pediatrics, College of Medicine, the Ohio State University, Columbus OH, USA
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 OH, USA
5Department
of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, USA.
Abstract Author Manuscript Author Manuscript
The IL-6/GP130/STAT3 pathway is crucial for tumorigenesis in multiple cancer types, including pancreatic cancer and presents as a viable target for cancer therapy. We reported Bazedoxifene, which is approved as a selective estrogen modulator by FDA, as a novel inhibitor of IL-6/GP130 protein−protein interactions using multiple ligand simultaneous docking and drug repositioning approaches. STAT3 is one of the major downstream effectors of IL-6/GP130. Here, we observed Bazedoxifene inhibited STAT3 phosphorylation and STAT3 DNA binding, induced apoptosis, and suppressed tumor growth in pancreatic cancer cells with persistent IL-6/GP130/STAT3 signaling in vitro and in vivo. In addition, IL-6 but not INF-γ rescued Bazedoxifene-mediated reduction of cell viability. Bazedoxifene also inhibited STAT3 phosphorylation induced by IL-6 and IL-11, but not by OSM or STAT1 phosphorylation induced by INF-γ in pancreatic cancer cells, suggesting that Bazedoxifene inhibits GP130/STAT3 pathway mediated by IL-6 and IL-11. Furthermore, Bazedoxifene combined with paclitaxel or gemcitabine synergistically inhibited cell viability and cell migration in pancreatic cancer cells. These results indicate that Bazedoxifene is a potential agent and can generate synergism when combined with conventional chemotherapy in human pancreatic cancer cells and tumor Xenograft in mice. Therefore, our results support that Bazedoxifene as a novel inhibitor of GP130 signaling and may be a potential and safe therapeutic agent for human pancreatic cancer therapy.
6
Correspondence should be sent to Jiayuh Lin, Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Green Street, Baltimore, MD 21201, Tel:410-706-7469, [email protected]. Conflict of interest: The authors declare no conflict of interest.
Wu et al.
Page 2
Author Manuscript
Keywords Pancreatic Cancer; IL-6/GP130/STAT3; Bazedoxifene; STAT3 phosphylation; Drug resistance
Introduction
Author Manuscript
Human pancreatic cancer is one of the deadly malignant diseases with very poor clinical outcome. The median overall survival is approximately six months after surgical and chemoradiation therapies for locally advanced and metastatic stages of pancreatic cancer and the five-years overall survive rate is less than five percent. D ue to the absence of specific symptoms, the lack of early detection techniques, pancreatic cancer is usually diagnosed at advanced and metastatic stages and is not resected by surgery (1, 2). To date, chemo- and radiation-therapy has only limited success because of high resistance (3). Unfortunately, only less than 15% of all pancreatic cancer patients have a chance for surgical resection, after which 5-year survival rarely succeed to 20-25% (4, 5).
Author Manuscript
IL-6/GP130/STAT3 signaling pathway is frequently activated in many human cancer and contributes to oncogenesis and cancer progression (6,7). The present review is to highlight the role of IL-6 in pancreatic cancer development and progression (8). It is well-established that IL-6 is elevated in the serum of pancreatic cancer patients compared with healthy controls and those with chronic pancreatitis (9-13). Several studies raised strong evidence that elevated levels of IL-6 protein and mRNA in serum and tumor samples of patients with pancreatic cancer is associated with increased tumor size and poor prognosis (14). IL-6 binds a nonsignaling α-receptor IL-6R to form a binary complex (IL-6/IL-6Rα), which, after dimerization with GP130, leads to activation of receptor-associated JAKs. In turn, these several phosphorylate downstream targets including cytoplasmic STAT3, which after dimerization rapidly translocate to the nucleus and promotes pancreatic cancer-progression through transcriptional regulation of anti-apoptotic and pro-proliferative genes (14). STAT3 has been identified as a key oncogenic factor in a number of human cancers and is required for oncogenesis in mouse model of cancers (15, 16). In pancreatic cancers, constitutive activation of STAT3 by phosphorylation of Tyr705 has been reported in 30% to 100% of human tumor specimens, as well as in many pancreatic cancer cell lines (17, 18). In contrast, this pathway is inactive in normal pancreas, and correspondingly STAT3 is not required for pancreatic development or homeostasis (19). These studies suggest STAT3 activation by IL-6/GP130 signaling pathway play an important role in human pancreatic cancer development and progression.
Author Manuscript
Inhibiting IL-6/GP130 signaling might be a new therapeutic option for pancreatic cancer. One possibility would be the treatment with the humanized monoclonal anti-IL-6R antibodies, which is already approved for the treatment of some inflammation disease (20). However, its potential therapeutic effect on pancreatic cancer has not yet examined. Selective inhibitors of IL-6/GP130/STAT3 are more effective options for treatment of pancreatic cancer. We previous study explored a small molecular inhibitor of STAT3, LLL12, was proposed selectively blocking exogenous IL-6-induced STAT3 phosphorylation and nuclear translocation in two human pancreatic cancer cell lines (21). Due to the
Mol Cancer Ther. Author manuscript; available in PMC 2017 November 01.
Wu et al.
Page 3
Author Manuscript
importance of small molecules in drug discovery, and as there are no any examples of small molecules inhibiting the IL-6/GP130 pathway associated in pancreatic cancer treatment, it would be a good strategy to use small molecular inhibitor for the same. Bazedoxifene is known as a selective estrogen modulator and commonly used for the prevention for osteoporosis. Recently, we have discovered Bazedoxifene as a novel small molecular GP130 inhibitor, which binds to GP130 D1 domain (22). It may be expected to speed up the development of clinical therapies for the IL-6/GP130/STAT3 dependent cancers. In this study, we report the new role of Bazedoxifene as a GP130 inhibitor to inhibit GP130/STAT3 signaling pathway mediated by IL-6 and IL-11, induce apoptosis in pancreatic cancer cells, and suppress the tumor growth in human pancreatic cancer xenograft, suggesting that Bazedoxifene may serve as a novel therapeutic drug for pancreatic cancer by targeting GP130/STAT3 signaling pathway.
Author Manuscript
Materials and Methods Cell Lines and Reagents
Author Manuscript
Human pancreatic cancer cell lines (AsPC-1, PANC-1, HPAF-II, BxPC-3, HPAC, Capan-1) were purchased from ATCC (the American Type Culture Collection, Manassas, VA, USA). HPAF-II cells were cultured in Eagle's Minimum Essential Medium (DMEM), Capan-1 cells were maintained in Iscove's Modified Dubecco's Medium (IMDM) supplemented with 20% FBS and the others in Dulbecco's Modified Eagle Medium (DMEM) supplemented 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin. HPAF-II was purchased within two months before the experiments about HPAF-II was performed. AsPC-1, PANC-1, BxPC-3, Capan-1, and HPAC cell lines were frozen within two months of receipt and were resuscitated from early passage liquid nitrogen stocks as needed. Cells were cultured for less than 3 months before reinitiating cultures and were routinely inspected microscopically for stable phenotype. All cell lines were cultured in a humidified 37 °C incubator with 5% CO2. IL-6, IL-11, OSM and IFN-γ were purchased from Cell Signaling. The powder was dissolved in sterile PBS to make a 100ng/μl stock solution. Bazedoxifene was purchased from Acesys Pharmatech (USA) and paclitaxel and gemcitabine were bought from LC Laboratories (Woburn, MA, USA). These three drugs were dissolved in sterile dimethyl sulfoxide (DMSO) to make a 20mM stock solution. Aliquots of the stock solution were stored at −20°C. Western Blotting Assay
Author Manuscript
Human pancreatic cancer cell lines (HPAF-II, BxPC-3, Capan-1, HPAC) were harvested after treatment with Bazedoxifene or DMSO at 50-60% confluence overnight, then lysed in cold RIPA lysis buffer containing protease inhibitors cocktail and phosphatase inhibitor cocktail. The lysates were subjected to 10% or 12% SDS-PAGE gel and transferred to a PVDF membrane. Membranes were probed with a 1:1000 dilution of specific primary antibody and 1:10000 HRP conjugated secondary antibody. Primary antibodies against phosphorylated STAT3 (Tyr705, p-STAT3Y705), STAT3, phosphorylated STAT1 (Tyr701), STAT1, cleaved caspase-3, phospho-specific extracellular signal-regulated kinase (ERK) 1/2
Mol Cancer Ther. Author manuscript; available in PMC 2017 November 01.
Wu et al.
Page 4
Author Manuscript
(Threonine 202/Tyrosine 204), P-AKT (Ser473), GAPDH and secondary antibody are all from Cell Signaling Technology (Beverly, MA, USA). Membranes were analyzed using enhanced chemiluminescence plus reagents and scanned with the Storm Scanner (Amersham Pharmacia Biotech Inc, Piscataway, NJ). STATs Phosphorylation Induced by Cytokines or Growth Factors PANC-1, AsPC-1, and HPAF-II pancreatic cancer cells were seeded in 10cm plates and allowed to adhere overnight. The following night, the cells were serum-starved. The cells were then left untreated or were treated with Bazedoxifene (5-20μM) or DMSO. After 2 hours, the untreated and Bazedoxifene-treated cells were stimulated by IL-6 (50ng/ml), IL-11 (50ng/ml), OSM (50ng/ml), or INF-γ (50ng/ml) for 30 minutes. The cells were harvested and analyzed by western blot for p-STAT3Y705 or p-STAT1Y701.
Author Manuscript
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Author Manuscript
Cells were treated with Bazedoxifene (5-20μM) or DMSO at 50-60% confluence in the presence of 10% FBS for 24 hours. RNA from the cells was then extracted using RNeasy Kits (Qiagen) according to the manufacturer's instruction. Reverse transcription was done using an Omniscript reverse transcription kit (Qiagen). Polymerase chain reaction (PCR) amplification was performed under the following conditions : 5 min at 94 °C followed by 30 cycles of 30 seconds at 94 °C, 30 sec at 48-55 °C, and 60 seconds at 72 °C with a final extension of 10 min at 72 °C. The following primers were used: Cyclin D1, annealing at 52 °C, (For): 5’-GCTGGAGCCCGTGAAAAAGA-3’, (Rev): 5’CTCCGCCTCTGGCATTTTG-3’; Bcl-Xl , annealing at 48 °C, (For): 5’TTGGACAATGGACTGGTTGA-3’, (Rev): 5’-GTAGAGTGGATGGTCAGTG-3’; Survivin, annealing at 52 °C, (For): 5’-ACCAGGTGAGAAGTGAGGGA-3’, (Rev): 5’AACAGTAGAGGAGCCAGGGA-3’; GAPDH, annealing at 52 °C, (For): 5’TGATGACATCAAGAAGGTGGTGAAG-3’, (Rev): 5’TCCTTGGAGGCCATGTGGGCAT-3’ (integrated DNA Technologies, USA). MTT Cell Viability Assay
Author Manuscript
Human pancreatic cancer cell lines (HPAC, PANC-1, HPAF-II, BxPC-3, and Capan-1), were seeded in 96-well plates at a density of 3000 cells per well. The next day, IL-6 (50ng/ml), INF-γ (50ng/ml), or Bazedoxifene (10μM) alone, or combination of IL-6 or INF-γ with Bazedoxifene were added in triplicate to the plates in the presence of 0% FBS in HPAF-II cells for 24 hours. Different concentrations of Bazedoxifene (5-10μM), paclitaxel (1-2.5 μM), or gemcitabine (5 μM) alone, or Bazedoxifene plus paclitaxel or gemcitabine were add in triplicate to the plates in the presence of 10% FBS in BxPC-3 or Capan-1 cells or paclitaxel plus gemcitabine were add in the plates in PANC-1, HPAC, BxPC-3 and Capan-1 cells. The cells were incubated at 37°C for a period of 24-48 hours. BxPC-3 and Capan-1 cells were seeded in 96-well plates at a density of 3000 cells per well and cultured at 37 °C. The next day, GP130 siRNA (100 nM) or negative control siRNA was transfected into cells in triplicate using lipofectamine 2000 for 72 hours. 25μl of 3-(4,5-Dimethylthiazolyl)-2,5diphenyltetrazolium bromide (MTT, Sigma) was added to each sample in a volume of 100 μl and incubated for 4 hours. Then 150 μl of N, N-dimethylformamide (Sigma) solubilization solution was added to each well. The absorbance was read at 595 nm. Combination index Mol Cancer Ther. Author manuscript; available in PMC 2017 November 01.
Wu et al.
Page 5
Author Manuscript
(CI) was performed using data obtained from MTT assay with CompuSyn software. The CI values indicate a synergistic effect when 1, and an additive effect when equal to 1 (23). STAT3 DNA Binding Assay BxPC-3 cells were seeded in a 10-cm plate and treated with Bazedoxifene (5, 10μM) or DMSO for 24 hours. The nuclear extract kit (Clontech Inc., Mountain View, CA) was used to prepare cell nuclear extracts following the manufacturer's protocol. Nuclear extracts were analyzed for STAT3 DNA binding activity using a STAT3 DNA binding ELISA kit (Active Motif, Carlsbad, CA, USA) with an ELISA-based method. Absorbance was read at 450 nm. Wound Healing/Cell Migration Assay
Author Manuscript
When HPAC cells were 100% confluent, the monolayer was scratched in same width using a pipette tip. After washing, HPAC cells were then treated with different concentrations of Bazedoxifene or DMSO. In addition, we treated HPAC cells with Bazedoxifene, paclitaxel alone, or combination of them. After 24 hours culture, when the wound in the DMSO control was closed, images were captured by Leica Microsystems. Immunofluorescence
Author Manuscript
HPAF-II cells were seeded on glass coverslips in 6-well plate. The next day, the cells were cultured in serum free medium for 24 hours and pretreated with Bazedoxifene (20 μM) for 2 hours, followed by induction with 50ng/mL IL-6 for 30 minutes. Cells were fixed with cold methanol for 15 minutes and blocked with 5% normal goat serum and 0.3% Triton X-100 in PBS for 1 hour. The cells were incubated with primary antibodies of p-STAT3 Y705 (Cell Signaling, 1:100) overnight at 4 °C. After incubation with anti-rabbit FIFC-conjugated secondary antibody (Invitrogen, 1: 200), the cells were mounted with Vectashield Hardset mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Photomicrographs were captured by Leica Microsystems (Bannockburn, IL, USA). Mouse Xenograft Tumor Model
Author Manuscript
All animal studies were conducted in accordance with the principles and standard procedures approved by IACUC of the Research Institute at Nationwide Children's Hospital. Capan-1 (3 × 106) and HPAF-II (3 × 106) cells in Matrigel (BD Science Franklin Lakes, NJ) were injected subcutaneously into the both side of flank area of 6- week-old female athymic nude mice which were purchased from Harlan (Indianapo lis, IN, USA). After Capan-1 tumor development, which was 1 week after initial implantation, mice were divided into two treatment groups consisting of four mice (tumors : n=8): DMSO vehicle control and gavage injection of Bazedoxifene (5mg/kg/d). Mice bearing HPAF-II tumor were irrigated with Bazedoxifene (5mg/kg/d) and/or injected via abdomen with paclitaxel (15mg/kg, 2/w). Tumor growth was determined by measured the length (L) and width (W) of the tumor every other day with a caliper, and tumor volume was calculated on the basis of the following formula: volume = 0.52×LW2. After 21 days of treatment, tumors were harvested, snapfrozen in dry ice, and stored at −80 °C. Tumors tissue homogenates were lysed and
Mol Cancer Ther. Author manuscript; available in PMC 2017 November 01.
Wu et al.
Page 6
Author Manuscript
separated by SDS-PAGE to examine the expression of STAT3 phosphorylation, P-ERK1/2, P-AKT (Ser473), and Cleaved Caspase-3. Statistical Analysis Significance of correlations was done using GraphPad Prism software. Unpaired t tests were used for analyses assuming Gaussian populations with a 95% confidence interval. Data are presented as mean ± SE. Differences were analyzed with the Student t test, and significance was set at P
Mol Cancer Ther. Author manuscript; available in PMC 2017 November 01. Published in final edited form as: Mol Cancer Ther. 2016 November ; 15(11): 2609–2619. doi:10.1158/1535-7163.MCT-15-0921.
Bazedoxifene as a Novel GP130 Inhibitor for Pancreatic Cancer Therapy Xiaojuan Wu1,2, Yang Cao2,3, Hui Xiao2, Chenglong Li4, and Jiayuh Lin5,6 1Department
of Pediatric Surgery, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China
Author Manuscript
2Center
for Childhood Cancer and Blood Diseases, the Research Institute at Nationwide Children's Hospital, Department of Pediatrics, College of Medicine, the Ohio State University, Columbus OH, USA
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 OH, USA
5Department
of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, USA.
Abstract Author Manuscript Author Manuscript
The IL-6/GP130/STAT3 pathway is crucial for tumorigenesis in multiple cancer types, including pancreatic cancer and presents as a viable target for cancer therapy. We reported Bazedoxifene, which is approved as a selective estrogen modulator by FDA, as a novel inhibitor of IL-6/GP130 protein−protein interactions using multiple ligand simultaneous docking and drug repositioning approaches. STAT3 is one of the major downstream effectors of IL-6/GP130. Here, we observed Bazedoxifene inhibited STAT3 phosphorylation and STAT3 DNA binding, induced apoptosis, and suppressed tumor growth in pancreatic cancer cells with persistent IL-6/GP130/STAT3 signaling in vitro and in vivo. In addition, IL-6 but not INF-γ rescued Bazedoxifene-mediated reduction of cell viability. Bazedoxifene also inhibited STAT3 phosphorylation induced by IL-6 and IL-11, but not by OSM or STAT1 phosphorylation induced by INF-γ in pancreatic cancer cells, suggesting that Bazedoxifene inhibits GP130/STAT3 pathway mediated by IL-6 and IL-11. Furthermore, Bazedoxifene combined with paclitaxel or gemcitabine synergistically inhibited cell viability and cell migration in pancreatic cancer cells. These results indicate that Bazedoxifene is a potential agent and can generate synergism when combined with conventional chemotherapy in human pancreatic cancer cells and tumor Xenograft in mice. Therefore, our results support that Bazedoxifene as a novel inhibitor of GP130 signaling and may be a potential and safe therapeutic agent for human pancreatic cancer therapy.
6
Correspondence should be sent to Jiayuh Lin, Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Green Street, Baltimore, MD 21201, Tel:410-706-7469, [email protected]. Conflict of interest: The authors declare no conflict of interest.
Wu et al.
Page 2
Author Manuscript
Keywords Pancreatic Cancer; IL-6/GP130/STAT3; Bazedoxifene; STAT3 phosphylation; Drug resistance
Introduction
Author Manuscript
Human pancreatic cancer is one of the deadly malignant diseases with very poor clinical outcome. The median overall survival is approximately six months after surgical and chemoradiation therapies for locally advanced and metastatic stages of pancreatic cancer and the five-years overall survive rate is less than five percent. D ue to the absence of specific symptoms, the lack of early detection techniques, pancreatic cancer is usually diagnosed at advanced and metastatic stages and is not resected by surgery (1, 2). To date, chemo- and radiation-therapy has only limited success because of high resistance (3). Unfortunately, only less than 15% of all pancreatic cancer patients have a chance for surgical resection, after which 5-year survival rarely succeed to 20-25% (4, 5).
Author Manuscript
IL-6/GP130/STAT3 signaling pathway is frequently activated in many human cancer and contributes to oncogenesis and cancer progression (6,7). The present review is to highlight the role of IL-6 in pancreatic cancer development and progression (8). It is well-established that IL-6 is elevated in the serum of pancreatic cancer patients compared with healthy controls and those with chronic pancreatitis (9-13). Several studies raised strong evidence that elevated levels of IL-6 protein and mRNA in serum and tumor samples of patients with pancreatic cancer is associated with increased tumor size and poor prognosis (14). IL-6 binds a nonsignaling α-receptor IL-6R to form a binary complex (IL-6/IL-6Rα), which, after dimerization with GP130, leads to activation of receptor-associated JAKs. In turn, these several phosphorylate downstream targets including cytoplasmic STAT3, which after dimerization rapidly translocate to the nucleus and promotes pancreatic cancer-progression through transcriptional regulation of anti-apoptotic and pro-proliferative genes (14). STAT3 has been identified as a key oncogenic factor in a number of human cancers and is required for oncogenesis in mouse model of cancers (15, 16). In pancreatic cancers, constitutive activation of STAT3 by phosphorylation of Tyr705 has been reported in 30% to 100% of human tumor specimens, as well as in many pancreatic cancer cell lines (17, 18). In contrast, this pathway is inactive in normal pancreas, and correspondingly STAT3 is not required for pancreatic development or homeostasis (19). These studies suggest STAT3 activation by IL-6/GP130 signaling pathway play an important role in human pancreatic cancer development and progression.
Author Manuscript
Inhibiting IL-6/GP130 signaling might be a new therapeutic option for pancreatic cancer. One possibility would be the treatment with the humanized monoclonal anti-IL-6R antibodies, which is already approved for the treatment of some inflammation disease (20). However, its potential therapeutic effect on pancreatic cancer has not yet examined. Selective inhibitors of IL-6/GP130/STAT3 are more effective options for treatment of pancreatic cancer. We previous study explored a small molecular inhibitor of STAT3, LLL12, was proposed selectively blocking exogenous IL-6-induced STAT3 phosphorylation and nuclear translocation in two human pancreatic cancer cell lines (21). Due to the
Mol Cancer Ther. Author manuscript; available in PMC 2017 November 01.
Wu et al.
Page 3
Author Manuscript
importance of small molecules in drug discovery, and as there are no any examples of small molecules inhibiting the IL-6/GP130 pathway associated in pancreatic cancer treatment, it would be a good strategy to use small molecular inhibitor for the same. Bazedoxifene is known as a selective estrogen modulator and commonly used for the prevention for osteoporosis. Recently, we have discovered Bazedoxifene as a novel small molecular GP130 inhibitor, which binds to GP130 D1 domain (22). It may be expected to speed up the development of clinical therapies for the IL-6/GP130/STAT3 dependent cancers. In this study, we report the new role of Bazedoxifene as a GP130 inhibitor to inhibit GP130/STAT3 signaling pathway mediated by IL-6 and IL-11, induce apoptosis in pancreatic cancer cells, and suppress the tumor growth in human pancreatic cancer xenograft, suggesting that Bazedoxifene may serve as a novel therapeutic drug for pancreatic cancer by targeting GP130/STAT3 signaling pathway.
Author Manuscript
Materials and Methods Cell Lines and Reagents
Author Manuscript
Human pancreatic cancer cell lines (AsPC-1, PANC-1, HPAF-II, BxPC-3, HPAC, Capan-1) were purchased from ATCC (the American Type Culture Collection, Manassas, VA, USA). HPAF-II cells were cultured in Eagle's Minimum Essential Medium (DMEM), Capan-1 cells were maintained in Iscove's Modified Dubecco's Medium (IMDM) supplemented with 20% FBS and the others in Dulbecco's Modified Eagle Medium (DMEM) supplemented 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin. HPAF-II was purchased within two months before the experiments about HPAF-II was performed. AsPC-1, PANC-1, BxPC-3, Capan-1, and HPAC cell lines were frozen within two months of receipt and were resuscitated from early passage liquid nitrogen stocks as needed. Cells were cultured for less than 3 months before reinitiating cultures and were routinely inspected microscopically for stable phenotype. All cell lines were cultured in a humidified 37 °C incubator with 5% CO2. IL-6, IL-11, OSM and IFN-γ were purchased from Cell Signaling. The powder was dissolved in sterile PBS to make a 100ng/μl stock solution. Bazedoxifene was purchased from Acesys Pharmatech (USA) and paclitaxel and gemcitabine were bought from LC Laboratories (Woburn, MA, USA). These three drugs were dissolved in sterile dimethyl sulfoxide (DMSO) to make a 20mM stock solution. Aliquots of the stock solution were stored at −20°C. Western Blotting Assay
Author Manuscript
Human pancreatic cancer cell lines (HPAF-II, BxPC-3, Capan-1, HPAC) were harvested after treatment with Bazedoxifene or DMSO at 50-60% confluence overnight, then lysed in cold RIPA lysis buffer containing protease inhibitors cocktail and phosphatase inhibitor cocktail. The lysates were subjected to 10% or 12% SDS-PAGE gel and transferred to a PVDF membrane. Membranes were probed with a 1:1000 dilution of specific primary antibody and 1:10000 HRP conjugated secondary antibody. Primary antibodies against phosphorylated STAT3 (Tyr705, p-STAT3Y705), STAT3, phosphorylated STAT1 (Tyr701), STAT1, cleaved caspase-3, phospho-specific extracellular signal-regulated kinase (ERK) 1/2
Mol Cancer Ther. Author manuscript; available in PMC 2017 November 01.
Wu et al.
Page 4
Author Manuscript
(Threonine 202/Tyrosine 204), P-AKT (Ser473), GAPDH and secondary antibody are all from Cell Signaling Technology (Beverly, MA, USA). Membranes were analyzed using enhanced chemiluminescence plus reagents and scanned with the Storm Scanner (Amersham Pharmacia Biotech Inc, Piscataway, NJ). STATs Phosphorylation Induced by Cytokines or Growth Factors PANC-1, AsPC-1, and HPAF-II pancreatic cancer cells were seeded in 10cm plates and allowed to adhere overnight. The following night, the cells were serum-starved. The cells were then left untreated or were treated with Bazedoxifene (5-20μM) or DMSO. After 2 hours, the untreated and Bazedoxifene-treated cells were stimulated by IL-6 (50ng/ml), IL-11 (50ng/ml), OSM (50ng/ml), or INF-γ (50ng/ml) for 30 minutes. The cells were harvested and analyzed by western blot for p-STAT3Y705 or p-STAT1Y701.
Author Manuscript
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Author Manuscript
Cells were treated with Bazedoxifene (5-20μM) or DMSO at 50-60% confluence in the presence of 10% FBS for 24 hours. RNA from the cells was then extracted using RNeasy Kits (Qiagen) according to the manufacturer's instruction. Reverse transcription was done using an Omniscript reverse transcription kit (Qiagen). Polymerase chain reaction (PCR) amplification was performed under the following conditions : 5 min at 94 °C followed by 30 cycles of 30 seconds at 94 °C, 30 sec at 48-55 °C, and 60 seconds at 72 °C with a final extension of 10 min at 72 °C. The following primers were used: Cyclin D1, annealing at 52 °C, (For): 5’-GCTGGAGCCCGTGAAAAAGA-3’, (Rev): 5’CTCCGCCTCTGGCATTTTG-3’; Bcl-Xl , annealing at 48 °C, (For): 5’TTGGACAATGGACTGGTTGA-3’, (Rev): 5’-GTAGAGTGGATGGTCAGTG-3’; Survivin, annealing at 52 °C, (For): 5’-ACCAGGTGAGAAGTGAGGGA-3’, (Rev): 5’AACAGTAGAGGAGCCAGGGA-3’; GAPDH, annealing at 52 °C, (For): 5’TGATGACATCAAGAAGGTGGTGAAG-3’, (Rev): 5’TCCTTGGAGGCCATGTGGGCAT-3’ (integrated DNA Technologies, USA). MTT Cell Viability Assay
Author Manuscript
Human pancreatic cancer cell lines (HPAC, PANC-1, HPAF-II, BxPC-3, and Capan-1), were seeded in 96-well plates at a density of 3000 cells per well. The next day, IL-6 (50ng/ml), INF-γ (50ng/ml), or Bazedoxifene (10μM) alone, or combination of IL-6 or INF-γ with Bazedoxifene were added in triplicate to the plates in the presence of 0% FBS in HPAF-II cells for 24 hours. Different concentrations of Bazedoxifene (5-10μM), paclitaxel (1-2.5 μM), or gemcitabine (5 μM) alone, or Bazedoxifene plus paclitaxel or gemcitabine were add in triplicate to the plates in the presence of 10% FBS in BxPC-3 or Capan-1 cells or paclitaxel plus gemcitabine were add in the plates in PANC-1, HPAC, BxPC-3 and Capan-1 cells. The cells were incubated at 37°C for a period of 24-48 hours. BxPC-3 and Capan-1 cells were seeded in 96-well plates at a density of 3000 cells per well and cultured at 37 °C. The next day, GP130 siRNA (100 nM) or negative control siRNA was transfected into cells in triplicate using lipofectamine 2000 for 72 hours. 25μl of 3-(4,5-Dimethylthiazolyl)-2,5diphenyltetrazolium bromide (MTT, Sigma) was added to each sample in a volume of 100 μl and incubated for 4 hours. Then 150 μl of N, N-dimethylformamide (Sigma) solubilization solution was added to each well. The absorbance was read at 595 nm. Combination index Mol Cancer Ther. Author manuscript; available in PMC 2017 November 01.
Wu et al.
Page 5
Author Manuscript
(CI) was performed using data obtained from MTT assay with CompuSyn software. The CI values indicate a synergistic effect when 1, and an additive effect when equal to 1 (23). STAT3 DNA Binding Assay BxPC-3 cells were seeded in a 10-cm plate and treated with Bazedoxifene (5, 10μM) or DMSO for 24 hours. The nuclear extract kit (Clontech Inc., Mountain View, CA) was used to prepare cell nuclear extracts following the manufacturer's protocol. Nuclear extracts were analyzed for STAT3 DNA binding activity using a STAT3 DNA binding ELISA kit (Active Motif, Carlsbad, CA, USA) with an ELISA-based method. Absorbance was read at 450 nm. Wound Healing/Cell Migration Assay
Author Manuscript
When HPAC cells were 100% confluent, the monolayer was scratched in same width using a pipette tip. After washing, HPAC cells were then treated with different concentrations of Bazedoxifene or DMSO. In addition, we treated HPAC cells with Bazedoxifene, paclitaxel alone, or combination of them. After 24 hours culture, when the wound in the DMSO control was closed, images were captured by Leica Microsystems. Immunofluorescence
Author Manuscript
HPAF-II cells were seeded on glass coverslips in 6-well plate. The next day, the cells were cultured in serum free medium for 24 hours and pretreated with Bazedoxifene (20 μM) for 2 hours, followed by induction with 50ng/mL IL-6 for 30 minutes. Cells were fixed with cold methanol for 15 minutes and blocked with 5% normal goat serum and 0.3% Triton X-100 in PBS for 1 hour. The cells were incubated with primary antibodies of p-STAT3 Y705 (Cell Signaling, 1:100) overnight at 4 °C. After incubation with anti-rabbit FIFC-conjugated secondary antibody (Invitrogen, 1: 200), the cells were mounted with Vectashield Hardset mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Photomicrographs were captured by Leica Microsystems (Bannockburn, IL, USA). Mouse Xenograft Tumor Model
Author Manuscript
All animal studies were conducted in accordance with the principles and standard procedures approved by IACUC of the Research Institute at Nationwide Children's Hospital. Capan-1 (3 × 106) and HPAF-II (3 × 106) cells in Matrigel (BD Science Franklin Lakes, NJ) were injected subcutaneously into the both side of flank area of 6- week-old female athymic nude mice which were purchased from Harlan (Indianapo lis, IN, USA). After Capan-1 tumor development, which was 1 week after initial implantation, mice were divided into two treatment groups consisting of four mice (tumors : n=8): DMSO vehicle control and gavage injection of Bazedoxifene (5mg/kg/d). Mice bearing HPAF-II tumor were irrigated with Bazedoxifene (5mg/kg/d) and/or injected via abdomen with paclitaxel (15mg/kg, 2/w). Tumor growth was determined by measured the length (L) and width (W) of the tumor every other day with a caliper, and tumor volume was calculated on the basis of the following formula: volume = 0.52×LW2. After 21 days of treatment, tumors were harvested, snapfrozen in dry ice, and stored at −80 °C. Tumors tissue homogenates were lysed and
Mol Cancer Ther. Author manuscript; available in PMC 2017 November 01.
Wu et al.
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Author Manuscript
separated by SDS-PAGE to examine the expression of STAT3 phosphorylation, P-ERK1/2, P-AKT (Ser473), and Cleaved Caspase-3. Statistical Analysis Significance of correlations was done using GraphPad Prism software. Unpaired t tests were used for analyses assuming Gaussian populations with a 95% confidence interval. Data are presented as mean ± SE. Differences were analyzed with the Student t test, and significance was set at P
