A Novel IL6R Antagonist, Inhibits IL6/STAT3 Signaling Pathway And

Published OnlineFirst February 29, 2016; DOI: 10.1158/1535-7163.MCT-15-0551

Small Molecule Therapeutics Molecular Cancer Therapeutics Chikusetsusaponin IVa Butyl Ester (CS-IVa-Be), a Novel IL6R Antagonist, Inhibits IL6/STAT3 Signaling Pathway and Induces Cancer Cell Apoptosis Jie Yang1,2, Shihui Qian2, Xueting Cai1,2, Wuguang Lu1,2, Chunping Hu1,2, Xiaoyan Sun1,2, Yang Yang1,2, Qiang Yu3, S. Paul Gao4, and Peng Cao1,2

Abstract

The activation of IL6/STAT3 signaling is associated with the onist of IL6 receptor via directly binding to the IL6Ra with a Kd of pathogenesis of many cancers. Agents that suppress IL6/STAT3 663 74 nmol/L and the GP130 (IL6Rb) with a Kd of 1,660 243 signaling have cancer-therapeutic potential. In this study, we nmol/L, interfering with the binding of IL6 to IL6R (IL6Ra and found that chikusetsusaponin IVa butyl ester (CS-IVa-Be), a GP130) in vitro and in cancer cells. The inhibitory effect of CS-IVa- triterpenoid saponin extracted from Acanthopanas gracilistylus Be on the IL6–IL6Ra–GP130 interaction was relatively specificas W.W.Smith, induced cancer cell apoptosis. CS-IVa-Be inhibited CS-IVa-Be showed higher affinity to IL6Ra than to LIFR (Kd: 4,910 constitutive and IL6-induced STAT3 activation, repressed STAT3 1,240 nmol/L) and LeptinR (Kd: 4,990 915 nmol/L). We next DNA-binding activity, STAT3 nuclear translocation, IL6-induced demonstrated that CS-IVa-Be not only directly induced cancer cell STAT3 luciferase reporter activity, IL6-induced STAT3-regulated apoptosis but also sensitized MDA-MB-231 cells to TRAIL- antiapoptosis gene expression in MDA-MB-231 cells, and IL6- induced apoptosis via upregulating DR5. Our findings suggest induced TF-1 cell proliferation. Surprisingly, CS-IVa-Be inhibited that CS-IVa-Be as a novel IL6R antagonist inhibits IL6/STAT3 IL6 family cytokines rather than other cytokines induced STAT3 signaling pathway and sensitizes the MDA-MB-231 cells to TRAIL- activation. Further studies indicated that CS-IVa-Be is an antag- induced cell death. Mol Cancer Ther; 15(6); 1190–200. 2016 AACR.

Introduction kemia inhibitory factor (LIF), the IL11, the oncostatin M (OSM), and the others (3). IL6 is a pleiotropic cytokine-mediating cancer associated STAT3, the key factor mediating the IL6-induced signaling chronic inflammation and inflammatory response, plays becomes tyrosine phosphorylated by IL6-induced cytokine recep- important roles in the tumorigenesis (1). IL6 possesses three tor-associated kinases, the Janus kinase (JAK) family proteins (4). topologically distinct receptor binding sites: site 1 for binding The IL6/STAT3 signaling pathway mediates tumor immune sup- to the 80 kDa chain IL6Ra, and sites 2 and 3 for interacting with pression (5), tumor cell survival, premetastatic niche formation, the two subunits of the signaling chain glycoprotein 130 and chemotherapy resistance (6). (GP130). The IL6 signaling transduces after the homodimer- Breast cancer is the most common malignancy and the ization of GP130, which becomes associated with IL6-bind- primary cause of cancer-related death in women worldwide ing chain (IL6Ra) in the presence of IL6. (2). As a common (7). IL6/JAK/STAT3 signaling plays a critical and pharmacolog- receptor, GP130 also transduces signals delivered by the leu- ically targetable role in orchestrating the composition of the breast tumor microenvironment that promotes cancer cell growth, invasion, and metastasis (8, 9). Increasing clinical data 1 Affiliated Hospital of Integrated Traditional Chinese and Western have indicated that the circulating levels of IL6 in breast cancer Medicine, Nanjing University of Chinese Medicine, Nanjing, China. 2Laboratory of Cellular and Molecular Biology, Jiangsu Province Acad- patients correlate with clinical tumor stage and poor prognosis. emy of Traditional Chinese Medicine, Nanjing, China. 3Shanghai Insti- IL6 plays two distinct roles in breast cancer: the systemic IL6, tute of Materia Medica, Chinese Academy of Sciences, Shanghai, reflecting whole body metabolic and inflammatory status, is China. 4Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, New York. correlated with poor prognosis, advanced disease, and metastasis; the paracrine and autocrine IL6 signaling controls cancer Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). cell growth, cancer stem cell renewal, and metastasis (10, 11). In summary, the IL6-mediated excessive STAT3 activation plays fi J. Yang and S. Qian are the co- rst authors of this article. important roles in breast cancer. Corresponding Author: Peng Cao, Jiangsu Province Academy of Traditional Inhibitors of the IL6/STAT3 signaling pathway should be Chinese Medicine, 100#, Shizi Street, Hongshan Road, Nanjing, 210028, China. effective against the cancers with high levels of STAT3 activa- Phone: 8625-8560-8666; Fax: 8625-8560-8666; E-mail: [email protected] tion. As a matter of fact, JAK3 inhibitor tofacitinib (12, 13) and doi: 10.1158/1535-7163.MCT-15-0551 IL6Ra mAb tocilizumab (14) have been used in clinical trial. 2016 American Association for Cancer Research. However, no natural IL6/STAT3 inhibitory compound has been

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CS-IVa-Be, a Novel IL6R Antagonist, Inhibits IL6/STAT3

used in cancer clinical trial. Identifying novel compounds that STAT3, phospho-STAT3 (Tyr705), phospho-STAT1(Tyr701), suppress the JAKs signaling or antagonize IL6R is a pressing STAT1, phosphor-STAT5(Tyr694), STAT5, phosphor-ERK1/2 issue to prevent and treat the cancers with aberrant activated (Thr201/Tyr204), ERK1/2, phosphor-AKT(Ser473), AKT, phos- IL6/STAT3 signaling. phor-JAK1(Tyr1022/1023), phosphor-JAK2(Tyr1007/1008), phos- TRAIL is a promising target for cancer therapy because it phor-Src(Tyr416), Bcl-xL, Mcl-1, Survivin, XIAP, caspase-3, preferentially induces cancer cell apoptosis with little or no effects caspase-8,caspase-9,PARP,DR5,DR4,c-FLIP,IL6Ra,GAPDH on normal cells (15). However, some highly malignant cancers, antibodies were purchased from Cell Signaling Technology. including breast cancer (16), are resistant to TRAIL-induced Anti-IL6 and anti-GP130 antibodies were purchased from apoptosis. The resistance of cancer cells to TRAIL occur at various Abcam. IL6 ELISA Kit was purchased from R&D Systems. points of the TRAIL signaling pathways, dysfunctions of the death His-probe, NE-PER Nuclear and Cytoplasmic Extraction, and receptors DR4 and DR5, overexpression of cellular FLICE-like LightShift Chemiluminescent EMSA Kit were obtained from inhibitory protein (c-FLIP), B-cell lymphoma extra-large (Bcl-xL), Santa Cruz Biotechnology. Methyl thiazolyl tetrazolium (MTT), myeloid cell leukemia 1 (Mcl-1), and the inhibitor of apoptosis N-acetyl-L-cysteine (NAC), SP600125, PD98059, SB203580, and proteins (IAP), all lead to TRAIL resistance (17). TRAIL resistance Z-VAD-FMK were purchased from Sigma-Aldrich. Recombinant can occur through activating STAT3 in cancer cells (18). Therefore, human TNFa, IL6, EGF, IFNg were obtained from Tocris Bio- a combination of TRAIL with STAT3 inhibitor may prove to be a science. Recombinant human IL6Ra, GP130, LIFR, and Leptin sound strategy to overcome the resistance. Identifying novel receptor were purchased from Sino Biological Inc. Pierce Classic agents that inhibit STAT3 activation not only can directly induce Magnetic IP/Co-IP Kit were purchased from Thermo Fisher apoptosis but also sensitize cancer cells to TRAIL-mediated Scientific. apoptosis. CS-IVa-Be (Fig. 1A) is a triterpenoid saponin extracted from a Immunoblot assay traditional Chinese medicine (TCM) herb Acanthopanas gracilis- Cells were plated in 6-well plate at a density of 5 106 tylus W.W.Smith that exhibits immunomodulation (19) and cells/mL overnight before being treated with CS-IVa-Be. The antithrombotic effects (20). The pharmacologic effects of CS- total cell lysates were prepared as described previously (21). IVa-Be have not been reported yet. In this study, we demonstrated The equal amounts of proteins from each sample were sub- that as an IL6R antagonist, CS-IVa-Be inhibited IL6/STAT3 sig- jected to SDS-PAGE followed by transfer to polyvinylidene naling, repressed cancer cell growth, and synergized with TRAIL to difluoride (PVDF) membranes. After being blocked with 1% induce breast cancer cell apoptosis. (w/v) BSA in TBST for 2 hours, the membranes were incubated with primary antibody overnight at 4C then washed Materials and Methods and incubated with secondary antibody conjugated with IgG DyLight for 1 hour at room temperature. Afterward, mem- Cell lines and cultures branes were washed and scanned with an Odyssey infrared Human breast cancer MDA-MB-231 and MCF-7 cells, human fluorescent scanner (LI-COR) and analyzed with Odyssey hepatocellular cancer HepG2 and MHCC97L cells, cervical cancer software version 3. HeLa cells, leukemic U937 and TF-1 cells, and gastric cancer SGC- 7901 cells were purchased from Cell Bank of the Shanghai Institute of Biochemistry and Cell Biology in June 2013. Human Cytotoxicity assay fetal liver cell line LO2 was purchased from Kunming Institute of LO2, HepG2, MHCC97L, SGC-7901, TF-1, U937, MDA-MB- Zoology, Chinese Academy of Sciences in June 2013. All cell lines 231, and MCF-7 cells were plated in triplicate in a 96-well plate at 4 had been authenticated by the provider through short tandem a density of 1 10 cells with 100 mL culture medium per well in the absence or presence of indicated concentrations of CS-IVa-Be repeat (STR) profiling. All cell lines were preserved in liquid N2 after receipt and passaged for fewer than 6 months when we use for 24 hours. Cell viability was then determined by the MTT assay them to do experiments. The cell lines were last tested in June as described previously (22). 2013 by above cell bank through STR profiling. MDA-MB-231, MCF-7, and HepG2 cells were cultured in MEM supplemented Apoptosis assay with 10 % FBS. HeLa and MHCC97L cells were cultured in DMEM Apoptotic cells were determined by using an FITC Annexin V (Invitrogen) supplemented with 10% FBS(Invitrogen). U937, Apoptosis Detection Kit (BD Biosciences) according to the man- SGC-7901, and LO2 cells were cultured in RPMI1640 media ufacturer's protocol. The cells were washed and incubated with supplemented with 10% FBS, and TF-1 cells were cultured in binding buffer containing Annexin V-FITC and PI in the dark at RPMI1640 media supplemented with 10% FBS, and 2 ng/mL room temperature for 15 minutes. Afterward, the degree of granulocyte-macrophage colony-stimulating factor (GM-CSF). apoptosis was analyzed by FACScan laser flow cytometer (FACS All cell lines were cultured in a humidified atmosphere with a 5% Calibur; Becton Dickinson). The data were analyzed using the CO2 incubator at 37 C. software CELLQuest.

Agents and antibodies JC-1 staining CS-IVa-Be was identified and purified from the fruit of Mitochondrial membrane potential was examined via JC-1 the Acanthopanas gracilistylus W.W.Smith. The purity of CS- staining (Beyotime Institute of Biotechnology, China). MDA- IVa-Be is 98.8 %, which was determined by high-performance MB-231 cells were incubated with JC-1 working solution at 37C liquid chromatography-evaporative light scattering detector in the dark for 20 minutes and observed by fluorescence micros- (HPLC-ELSD) method. A 20 mmol/L solution of CS-IVa-Be copy. In healthy cells, JC-1 forms complexes of J-aggregates was prepared in DMSO, stored as small aliquots at 4C. showing punctate red fluorescence at 590 nm emission

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wavelength; however, in apoptotic cells, JC-1 remains in the using Kinase-Glo Plus Luminescence Kinase Assay Kit (Promega). monomeric form showing diffused green fluorescence at 530 nm In vitro panel kinases (JAK1, JAK2, JAK3, TYK2, EGFR, IGF1R, emission wavelength. PDGFRa, FGFR1) assay with two concentrations (25 and 50 mmol/L) of CS-IVa-Be was performed by Eurofins' Kinase Immunofluorescence staining Profiler service according to Cerep's validation Standard Operat- MDA-MB-231 cells were seeded at the density of 70% to 80 % ing Procedure. confluence per well into 24-well chamber slides. After being fi treated with test drugs for the indicated times, cells were xed IL6 receptor binding assay in cell level with cold 4% (w/v) paraformaldehyde for 20 minutes, rehydrated The cell-based IL6-receptor binding was performed using in PBS for 15 minutes, and permeabilized in 0.1% (w/v) TritonX- the kit of Fluorokine Biotinylated Human Interleukin 6 (R&D 100 at room temperature for 10 minutes. After being washed with Systems) followed by the manufacturer's protocol. First, U937, PBS, the cells were blocked with 3% BSA for 1 hour and then MDA-MB-231, HeLa, and HepG2 cells were treated with the test incubated with primary antibody at 4 C overnight followed by compound for various periods of time and the cells were FITC-conjugated secondary antibody for 1 hour at room temper- harvested and washed twice with PBS to remove any residual fl – ature. The images of green uorescence stained pSTAT3 were growth factors that may be present in the culture media. Next, fl captured using a uorescence microscope. the cells were resuspended in PBS to a final concentration of 4 106 cells/mL, 10 mLofbiotinylatedIL6wasaddedtoa25mL Luciferase reporter gene activity assay aliquot of the cell suspension for a total reaction volume of We used the luciferase reporter assay (the Dual-Luciferase 35 mL. As a negative staining control, an aliquot of cells were Reporter Assay System; Promega) to investigate the IL6-induced stained with 10 mL of negative control reagent (a soybean transcriptional activity of STAT3. Transient transfection was per- trypsin inhibitor protein biotinylated as the IL6). The cells formed in 96-well plates at a cell density of 50% to 70% conflu- were then incubated for 30 minutes at 4C followed by the ence per well. The STAT3 luciferase reporter plasmid was cotrans- addition of 10 mL Avidin-FITC reagent and another 30-minute fected with the pRL-TK plasmid (which encodes Renilla luciferase incubation at 4C in the dark before being analyzed by flow as an internal control for transfection efficiency) using Lipofecta- cytometry. mine 2000 (Invitrogen) according to the manufacturer's instructions. The transfected cells were treated with CS-IVa-Be for various Analysis of IL6Ra, DR4, and DR5 surface expression periods of time, and the cell lysates were prepared for assessment Cells were treated with CS-IVa-Be for various periods of times of luciferase activity. Firefly and Renilla luciferase activities were before being detached with trypsin containing EDTA and measured using a luminometer (Centro XS3 LB960) according to washed twice with staining buffer. Cells were then incubated the manufacturer's instructions. Relative firefly luciferase activity with mouse monoclonal anti-human DR5, DR4, or IL6Ra normalized by Renilla luciferase activity was expressed as fold antibodies for 45 minutes at 4 C, washed twice, incubated with induction after treatment with CS-IVa-Be compared with vehicle FITC-conjugated secondary antibody for 30 minutes at 4 Cand control (DMSO). washed again, then resuspended in staining buffer for flow cytometric analysis. Electrophoretic mobility shift assay STAT3–DNA-binding activity was analyzed by electrophoretic IL6 receptor binding assay in vitro mobility shift assay (EMSA) using biotin-labeled, double-strand- IL6 receptor binding was performed following the method ed STAT3 consensus-binding motif probe. STAT3 consensus oligo 0 described previously (23). Briefly, 96-well plates (Nunc) were primers were as follows: forward, 5 -GATCCTTCTGGGAATTCC- 0 0 0 coated with 500 ng/mL of human recombinant soluble IL6 TAGATC-3 ; reverse, 3 -CTAGGAAGACCCTTAAGGATCTAG-5 . receptor (sIL6R), washed, and blocked. The sIL6R-coated 96- Nuclear protein extracts were prepared from CS-IVa-Be–treated well plates were incubated with 6.2 nmol/L of recombinant MDA-MB-231 cells and incubated with biotin-labeled probe. human IL6, or 6.2 nmol/L of IL6 plus one of six concentra- STAT3–DNA complex was separated from free oligonucleotide tions of CS-IVa-Be, in triplicates, and washed to remove on 5% nondenaturing polyacrylamide gels, then transferred to unbound IL6, the wells then were incubated with a mono- nylon membranes, and cross-linked for 15 minutes under a hand- clonal rabbit anti-human IL6 antibody (200 ng/mL), washed, held UV lamp. Cross-linked, biotin-labeled DNA was detected and incubated with goat anti-rabbit IgG-HRP (40 ng/mL; using the Chemiluminescent Nucleic Acid Detection Module Santa Cruz Biotechnology). Sample wells were washed and (Pierce Biotechnology, Inc.). developed with tetramethylbenzidine (100 mL/well, 10 minutes at room temperature and stopped with 2 mol/L H2SO4). IL6 inhibitory bioassay Optical density in each well was determined using a micro- After being starved in media without GM-CSF for 24 hours, plate reader (Multiskan Ascent, Thermo Scientific) 450 nm various concentrations of CS-IVa-Be (2.5, 5, 7.5, 10, 12.5) mmol/L with a correction wavelength of 630 nm. Nonspecificbinding in the presence of IL6 (25 ng/mL) were added to the TF-1 cells and of IL6 is determined by the reading of wells without sIL6R. incubated for 24 or 48 hours; cell viability was then determined by Data were analyzed by nonlinear regression analysis using the MTT assay. GraphPad Prism4 (GraphPad). Maximum binding of IL6 (100% bound) is defined as the absorbance in the absence In vitro kinase assay of CS-IVa-Be. The absorbance in the presence of various In vitro JAKs kinase (JAK2, JAK3, TYK2) assay for a series of CS- concentrations of CS-IVa-Be is calculated as percent of max- IVa-Be concentrations was performed by BPS Bioscience Service imum binding of IL6.

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Microscale thermophoresis assay Coimmunoprecipitation assay Binding assay of labeled recombinant human IL6Ra, GP130, The interaction of IL6Ra with GP130 in the presence of IL6 LIFR, and LeptinR to CS-IVa-Be was performed using the Mono- was assayed by coimmunoprecipitation (co-IP) assay according lith NT.115 Kit (NanoTemper Technologies). Briefly, 10 mmol/L to the manufacturer's protocol of Pierce Classic Magnetic recombinant human IL6Ra, GP130, LIFR, and LeptinR were IP/Co-IP Kit. At room temperature, 10 mg of recombinant labeled in PBS buffer using the Protein Labeling Kit RED-NHS human IL6Ra was preincubated with 0, 5, or 10 mmol/L of followed by the manufacturer's protocol (NanoTemper Technol- CS-IVa-Be for 2 hours in IP wash buffer, 10 mg of recombinant ogies). A 16-point titration series of CS-IVa-Be were added to the human IL6 was added and incubated with GP130 for another 2 labeled protein (200 nmol/L) in PBST buffer (PBS þ 0.05% hours, and then 2 mg of His antibody was added and incubated Tween-20), the total volume of mixed sample was 20 mL and the for 2 hours to form immune complex. Twenty-five microliters volume ratio of CS-IVa-Be to labeled protein is 1:1, the final of prewashed Pierce Protein A/G Magnetic Beads were placed DMSO concentration for each protein-compound sample was into the above immune complex and incubated for 1 hour with 2%. The protein-compound samples were equilibrated for 5 mixing. Then the beads were washed and the target antigen was minutes at room temperature, loaded into the MST Premium eluted with Alternative Elution, and the target antigen and the Coated capillaries (NanoTemper Technologies) and assayed by binding proteins were immunoblotted with the indicated anti- microscale thermophoresis (MST). The thermophoresis data were bodies by Western blot assay. analyzed using NT Analysis 1.5.41 (NanoTemper Technologies) to compute Kd value according to the law of mass action. The siRNA-mediated gene silencing experiment was performed in triplicate, and the fits are repre- DR5-specific siRNA was obtained from RiboBio. Transient trans- sented as mean SD. Data normalization and curve fitting were fection was performed using Lipofectamine 2000. Briefly, cells performed using GraphPad Prism 5. were transfected with indicated concentration of siRNAs for

A B 30 1 2 3 4 R =R =H, R =Bu, R =Glc 25

( μ mol/L) 10

CO 50

4 IC R3OOC OR 5 O O Figure 1. HO 1 0 R2O OR CS-IVa-Be induces cancer cell CS-IVa-Be LO2 TF-1 U937 apoptosis. A, the chemical structure of HepG2 MCF-7

CS-IVa-Be. B, LO2, HepG2, MHCC97L, MHCC97LSGC-7901 SGC-7901, TF-1, U937, MDA-MB-231, MDA-MB-231 and MCF-7 cells were treated with various concentrations of CS-IVa-Be for 24 hours and then subjected to C D CS-IVa-Be (μmol/L) 0 5 7.5 10 MTT assay; the value of IC50 was calculated. C, cell lysates from MDA- 120 Pro-PARP – MB-231 treated with various Cleaved-PARP 100 concentrations (0, 5, 7.5, 10 mmol/L) of Pro-Caspase-8 80 CS-IVa-Be for 24 hours were analyzed for apoptosis proteins. MDA-MB-231 60 cells were treated with various 40 Cell viability concentrations (0, 7.5, 10 mmol/L) of Cleaved-caspase-8 (% of control) 20 CS-IVa-Be for 24 hours following Cleaved-caspase-3 treatment by Z-VAD-FMK for 30 0 minutes, and subjected to MTT assay Cleaved-caspase-9 CS-IVa-Be (μmol/L) – ––7.5 7.5 7.5 10 10 10 (D) and apoptosis assay (E). F, MDA- GAPDH MB-231 cells treated with various Z-VAD-FMK (μmol/L) – 2040–– 20 40 20 40 concentrations (0, 5, 7.5, 10 mmol/L) of CS-IVa-Be for 24 hours were stained with JC-1 and then subjected to EF cytometry assay. This is a 30 30 representative result of three repetitive experiments with similar 25 25 results and error bars mark SDs 20 (, P < 0.05). 15 20 JC-1 10 15 5 (Green/red ratio) Apoptosis rate (%) Apoptosis rate 0 10 CS-IVa-Be (μmol/L) 7.5 7.5 10 10 –– CS-IVa-Be (μmol/L) 0157.50 Z-VAD-FMK (μmol/L) –––20 20 20

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24 hours, followed by treatment with CS-IVa-Be for indicated 1B). We next investigated whether the CS-IVa-Be induced breast time, and the cells were then harvested for flow cytometry analysis. cancer cell inhibition due to apoptosis. We then found that CS- IVa-Be induced pro-caspase-3, -8, and -9 cleavage (Fig. 1C). Statistical analysis Furthermore, Z-VAD-FMK, a pan-caspase inhibitor, significantly The experimental results presented in the figures are represen- offsets the CS-IVa-Be–induced MDA-MB-231 cell viability tative of three or more independent experiments. The data are attenuation and apoptosis (Fig. 1D and E). In addition, CS- presented as the mean values SD. Statistical comparisons IVa-Be induced a decrease of mitochondrial membrane poten- between the groups were done using one-way ANOVA. Values tial (Fig. 1F). The results indicate that CS-IVa-Be promotes of P < 0.05 were considered to be statistically significant. breast cancer cell apoptosis.

CS-IVa-Be selectively inhibits constitutive and IL6 family Results member–induced STAT3 activation in MDA-MB-231 cells CS-IVa-Be induces cancer cell apoptosis To identify the signaling pathways that were involved in CS-IVa-Be (Fig. 1A), a triterpenoid saponin extracted from the CS-IVa-Be–induced cell apoptosis, we investigated the Acanthopanas gracilistylus W.W.Smith, was identiﬁed as a cell effects of CS-IVa-Be on STAT3, NF-kB, MAPK, and AKT signal- death inducer. We investigated the effects of CS-IVa-Be on the ing. CS-IVa-Be was found to inhibit the constitutive and IL6- viability of cancer cells and found that CS-IVa-Be inhibits the induced STAT3 and JAK1, JAK2, Src activation rather than viability of various kinds of cancer cells (including hepatocel- constitutive and IL6-induced ERK1/2 and AKT activation lular, gastric, leukemia, and breast cancers). Breast cancer MDA- (Fig. 2A and B). Other IL6 family cytokines (IL11, LIF, and MB-231 and MCF-7 cells were more sensitive to the CS-IVa-Be– OSM, which share the same GP130 receptor to transduce induced cell viability attenuation than other cancer cells (Fig. signal) inducing STAT3 activation were also inhibited by

A B IL6 (25 ng/mL) CS-IVa-Be (μmol/L) 0 5 7.5 10 CS-IVa-Be (μmol/L) 002.5 5 7.5 10 pSTAT3 pSTAT3 Figure 2. STAT3 CS-IVa-Be inhibits IL6/STAT3 STAT3 activation in MDA-MB-231 cells. A, cell pSTAT1 pJAK1 lysates from MDA-MB-231 cells treated STAT1 with various concentrations (0, 5, 7.5, pSTAT5 pJAK2 10 mmol/L) of CS-IVa-Be for 24 hours 705 STAT5 pSrc were analyzed for pSTAT3 (Tyr ), STAT3, pSTAT1(Tyr701), STAT1, pAKT pAKT pSTAT5 (Tyr694), STAT5, pAKT, AKT, AKT AKT pERK1/2, and ERK1/2, with GAPDH as a loading control. B, cell lysates from pERK1/2 pERK1/2 MDA-MB-231 cells treated with various ERK1/2 ERK1/2 concentrations (0, 2.5, 5, 7.5, 10 mmol/ L) of CS-IVa-Be for 8 hours, followed GAPDH GAPDH by 15-minute IL6 (25 ng/mL) stimulation were analyzed for pSTAT3 (Tyr705), STAT3, pJAK1, pJAK2, pSrc, C IL11 LIF OSM pAKT, AKT, pERK1/2, and ERK1/2. GAPDH was used as a loading control. CS-IVa-Be (μmol/L) 0 0 10 0 0 10 0 0 10 C, cell lysates from MDA-MB-231 cells pSTAT3 treated with 10 mmol/L of CS-IVa-Be for 8 hours, followed by 15-minute STAT3 stimulation of 25 ng/mL IL11, LIF, OSM, respectively, were analyzed for GAPDH pSTAT3 (Tyr705), STAT3. GAPDH was used as a loading control. D, cell lysates from MDA-MB-231 cells treated D EGF IFNγ with 10 mmol/L of CS-IVa-Be for 8 hours, followed by 15-minute CS-IVa-Be (μmol/L) 0010 0010 stimulation of 100 ng/mL EGF and pSTAT3 pSTAT3 IFNg, respectively, were analyzed for pSTAT3 (Tyr705), STAT3, pSTAT1 STAT3 STAT3 (Tyr701), STAT1, pSTAT5 (Tyr694), and STAT5. GAPDH was used as a loading pSTAT5 pSTAT1 control. This is a representative result of three repetitive experiments STAT5 STAT1 with similar results.

GAPDH GAPDH

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CS-IVa-Be (Fig. 2C). But interestingly, constitutive and EGF- CS-IVa-Be abrogates IL6/STAT3 downstream signaling induced STAT3, STAT5 activation, or IFNg-induced STAT3, We further investigated the effects of CS-IVa-Be on constitutive STAT1 activation were not inhibited by CS-IVa-Be (Fig. 2A and IL6-induced STAT3 downstream signaling in MDA-MB-231 and D). In addition, ROS production was observed upon cells. CS-IVa-Be was found to inhibit the pSTAT3 nuclear trans- CS-IVa-Be treatment (Supplementary Fig. S1A), but treatment location (Fig. 3A), the STAT3–DNA binding activity (Fig. 3B), and with ROS, JNK1/2/3, ERK1/2, or p38-speciﬁc inhibitor did not IL6-induced STAT3 luciferase activity (Fig. 3C) in MDA-MB-231 reverse the CS-IVa-Be–induced cell viability attenuation in cells. CS-IVa-Be also downregulated the constitutive and IL6- MDA-MB-231 cells (Supplementary Fig. S1B). CS-IVa-Be had induced Survivin, XIAP, Bcl-xL, and Mcl-1 expression (Fig. 3D no inhibitory effects on the TNFa-induced NF-kBtranscription and E). IL6-induced TF-1 cell proliferation was also inhibited by activity (Supplementary Fig. S1C) and NF-kB DNA-binding CS-IVa-Be (Fig. 3F). These ﬁndings suggest that CS-IVa-Be inhibit activity (Supplementary Fig. S1D). IL6/STAT3 downstream signaling.

AB CS-IVa-Be (0 μmol/L) CS-IVa-Be (5 μmol/L)

CS-IVa-Be (μmol/L)

0 2.5 5 7.5 10 pSTAT3 (Tyr705) STAT3

C D 120 CS-IVa-Be (μmol/L) 0 5 7.5 10 100 Survivin 80 c-FLIP 60

40 XIAP

(% of IL6 control) 20 Mcl-1

STAT3 luciferase activity luciferase STAT3 0 Bcl-xL CS-IVa-Be (μmol/L) 0 0 2.5 5 7.5 10 GAPDH IL6 (25 ng/mL)

EF IL6 (25 ng/mL) 120 CS-IVa-Be (μmol/L) 0 0 2.5 5 7.5 10 24 h 100 48 h Survivin 80 c-FLIP 60 XIAP 40 Mcl-1 20 Bcl-xL Cell viability (% of control) 0 GAPDH CS-IVa-Be (μmol/L) 0 0 2.5 5 7.5 10 12.5

IL6 (25 ng/mL)

Figure 3. CS-IVa-Be inhibits IL6/STAT3 downstream signaling. A, immunoﬂuorescence staining of pSTAT3 (Tyr705). MDA-MB-231 cells were treated with 5 mmol/L CS-IVa-Be for 8 hours and then pSTAT3(Tyr705) was labeled through immunoﬂuorescence staining; scale bar, 20 mm. B, MDA-MB-231 cells were treated with various concentrations (0, 2.5, 5, 7.5, 10 mmol/L) of CS-IVa-Be for 24 hours, after which nuclear proteins were extracted and subjected to EMSA with STAT3 probe. C, MDA-MB-231 cells were transfected with STAT3 luciferase reporter vector for 24 hours, and then treated with various concentrations(0,2.5,5,7.5,10mmol/L) of CS-IVa-Be for 8 hours. The STAT3 luciferase activity was measured following stimulation with IL6 (25 ng/mL) for 8 hours. D, cell lysates from MDA-MB-231 cells treated with various concentrations (0, 5, 7.5, 10 mmol/L) of CS-IVa-Be for 24 hours were analyzed for Survivin, c-FLIP, XIAP, Mcl-1, and Bcl-xL. GAPDH was used as a loading control. E, cell lysates from MDA-MB-231 cells treated with variousconcentrations(0,2.5,5,7.5,10mmol/L) of CS-IVa-Be for 24 hours, followed by IL6 (25 ng/mL) stimulation for 15 minutes, were analyzed for Survivin, c-FLIP, XIAP, Mcl-1, and Bcl-xL. GAPDH was used as a loading control. F, inhibition of IL6-induced proliferation of TF-1 cells by CS-IVa-Be. TF-1 cells were incubated with IL6 (25 ng/mL) in the presence of various concentrations (0, 2.5, 5, 7.5, 10, 12.5 mmol/L) of CS-IVa-Be for 24 or 48 hours, and cell viability was measured by MTT assay. This is a representative result of three repetitive experiments with similar results and error bars mark SDs (, P < 0.05).

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A JAK2 JAK3 TYK2 (10 μmol/L ATP/0.2 mg/mL poly-Glu-Tyr) (10 μmol/L ATP/0.2 mg/mL poly-Glu-Tyr) (10 μmol/L ATP/0.1 mg/mL IRS-1 peptide)

120 120 120 110 110 110 100 100 100 90 90 90 80 80 80 70 70 70 60 60 60 50 50 50 40 40 40 30 μ 30 μ 30 μ 20 IC50 > 30 mol/L 20 IC50 > 30 mol/L 20 IC50 > 30 mol/L 10 10 10 % Remaining activity 0 % Remaining activity 0 % Remaining activity 0 –10 –10 –10 −1 0 1 2 3 4 5 −1 0 1 2 3 4 5 −1 0 1 2 3 4 5 CS-IVa-Be [Log (nmol/L)] CS-IVa-Be [Log (nmol/L)] CS-IVa-Be [Log (nmol/L)]

B % Inhibition of control value (mol/L) −20 −10 0 10 20 30 40 50 60 70 80 90 100 EGFR FGFR1 IGF1R JAK1 JAK2 JAK3 PDGFRa Src Tyk2

CS-IVa-Be Test concentration:5.0E-06 M Test concentration:2.0E-05 M

Figure 4. The effect of CS-IVa-Be on JAKs and other growth factor receptors in vitro kinase enzymatic activity. A, the effect of CS-IVa-Be on JAK2, JAK3, TYK2 kinase enzymatic activity in vitro. B, the effect of CS-IVa-Be (25 and 50 mmol/L)onJAK1,JAK2,JAK3,Tyk2,Src,EGFR,FGFR1,and PDGFRa kinase activity in vitro.

CS-IVa-Be does not inhibit the enzyme activity of nonreceptor that CS-IVa-Be might disrupt the upstream signaling such as or receptor tyrosine kinases of STAT3 in vitro the interaction of IL6 to its receptor. To test this hypothesis, we As STAT3 phosphorylation was mediated by several non- investigated whether CS-IVa-Be affect the binding of IL6 to receptor tyrosine kinases (24) and receptor kinases (25), we cell surface IL6R in cancer cells using biotin-labeled IL6 by investigated whether CS-IVa-Be impact the activity of upstream flow cytometry analysis. To rule out the possibility that CS-IVa- kinases. CS-IVa-Be has showed inhibitory effects on the IL6- Be might downregulate IL6R protein levels, we also measured induced phosphorylation of JAK1, JAK2, and Src (Fig. 2B) in the cell surface IL6Ra expression upon CS-IVa-Be treatment. MDA-MB-231 cells. The inhibitory effect of CS-IVa-Be on IL6- The results demonstrated that CS-IVa-Be indeed lowered bio- induced STAT3 activation might be through its repression on tin-labeled IL6 binding to IL6R in various cancer cells tested JAK1, JAK2, and Src kinase activity. However, by in vitro kinase (U937, MDA-MB-231, Hela, HepG2) in a dose-dependent assay, we found no inhibitory effect of CS-IVa-Be on the manner, without decreasing IL6Ra cell surface expression enzyme activity of tyrosine kinasessuchasJAKsfamilykinases (Fig. 5A and B and Supplementary Fig. S2). (JAK1,JAK2,JAK3,andTYK2;Fig.4AandB),Src,EGFR,insulin- To further confirm our hypothesis that CS-IVa-Be inhibits like growth factor 1 receptor (IGF1R), platelet-derived growth IL6–IL6R binding, we investigated the effects of CS-IVa-Be on factor receptor a (PDGFRa), and fibroblast growth factor IL6–IL6Ra interaction using an in vitro ELISA assay. Our results receptor 1 (FGFR1; Fig. 4B), even at 50 mmol/L, the dose is showed that CS-IVa-Be significantly inhibited the IL6–IL6Ra much higher than the IC50 of CS-IVa-Be in MDA-MB-231 cells. interaction with maximal inhibition rate less than 20% Therefore, we suggest that CS-IVa-Be is not a tyrosine kinase (Fig. 5C). As CS-IVa-Be cannot antagonize IL6–IL6Ra interac- inhibitor. tion completely, we considered CS-IVa-Be could be a noncom- petitive IL6Ra antagonist. CS-IVa-Be blocks IL6 binding to its cell surface receptor We had shown that CS-IVa-Be inhibited IL6-induced JAKs Biochemical characterization of CS-IVa-Be/IL6R interaction and STAT3 activation in breast cancer cells, without affecting As our results have illustrated that CS-IVa-Be inhibited the JAKs enzyme activity in vitro.Thesefindings made us speculate IL6–IL6R interaction, we next studied the binding specificity

1196 Mol Cancer Ther; 15(6) June 2016 Molecular Cancer Therapeutics

CS-IVa-Be, a Novel IL6R Antagonist, Inhibits IL6/STAT3

a Figure 5. A IL6R expression B IL6–IL6 receptor binding CS-IVa-Be disrupts IL6/IL6Ra/GP130 CS-IVa-Be interaction. Cytometry analysis of the m Isotype Isotype (5 mol/L) inhibitory effect of CS-IVa-Be on CS-IVa-Be (0 mmol/L) CS-IVa-Be IL6–IL6R interaction in U937 cells. (0 mmol/L) Biotinylated IL6 was used to label cell CS-IVa-Be surfaceIL6R.A,thelevelsofcellsurface (5 mmol/L) IL6Ra in cells with CS-IVa-Be treatment or not (left). B, cell surface IL6–IL6R binding levels in cells with CS-IVa-Be treatmentornot(right).C,ELISA analysis of the inhibitory effect of CS- C D IVa-Be on IL6–IL6Ra binding in vitro. 120 120 Kd = 663 ± 74 (nmol/L) The maximum binding of IL6 (100% 100 100 bound) is defined as without CS-IVa- 80 Be, whereas the effects of various 80 IL6Ra concentrations of CS-IVa-Be were 60 60 calculated as the percentages of 40 40 maximum binding of IL6. MST analysis of CS-IVa-Be binding to IL6Ra,GP130, 20 20 Bound fraction (%) – IL6–IL6R a binding (%) LeptinR, and LIFR. D, CS-IVa-Be 0 0 0123456 binding affinity to IL6Ra.E,CS-IVa-Be– 0 25 50 6.25 12.5 100 binding affinity to GP130, LeptinR, and 1.625 3.125 CS-IVa-Be [Log (nmol/L)] LIFR, respectively. F, recombinant CS-IVa-Be (mmol/L) human IL6Ra with His tag was incubated with 0, 5, or 10 mmol/L CS-IVa-Be for 2 hours, then mixed with EFIgG His antibody recombinant human GP130 and IL6, and immunoprecipitated with anti-His CS-IVa-Be 00 510 (mmol/L) antibody. The precipitates were IL6 family receptors Kd (nmol/L) washed, suspended in reducing sample IP: Anti-His His-IL6Ra buffer, and immunoblotted with IL6Ra 663 ± 74 IB: Anti-His anti-His or anti-GP130 antibody. GP130 (IL6Rb) 1,660 ± 243 These are representative results of LeptinR 4,990 ± 915 IP: Anti-His GP130 three repetitive experiments with LIFR 4,910 ± 1,240 IB: Anti-GP130 similar results. of CS-IVa-Be to IL6 or IL6R. We first measured the binding (27).AswehaveprovedthatCS-IVa-BeisaneffectiveIL6/ affinity (Kd:48 2nmol/L)ofIL6toIL6Ra (Supplementary STAT3 inhibitor, we next determined whether CS-IVa-Be can Fig. S3A) using MST analysis, which allows a sensitive detec- synergize with TRAIL to induce apoptosis in breast cancer cells. tion of small-molecule binding to a protein target. Next, We treated the breast cancer MDA-MB-231 cells with CS-IVa-Be we measured the binding characteristics of CS-IVa-Be to IL6 alone or combined with TRAIL and analyzed cell apoptosis. The or IL6R and found that CS-IVa-Be can bind to IL6Ra cells treated by TRAIL/CS-IVa-Be combination demonstrated (Kd:663 74 nmol/L) and IL6Rb (GP130; Kd:1,660 obvious apoptotic morphology (Fig. 6A) and increased cell 243 nmol/L; Fig. 5D and E) but had no binding affinity to apoptosis (Fig. 6B and C). IL6 (Supplementary Fig. S3B). The affinity of CS-IVa-Be to IL6R was relatively higher than other IL6 family receptors such CS-IVa-Be upregulates DR5 synergized with TRAIL as LeptinR (Kd: 4,990 915 nmol/L) and LIFR (Kd: 4,910 With our data showing CS-IVa-Be activating caspase-8, we 1,240 nmol/L; Fig. 5E and Supplementary Fig. S3). IL6Ra hypothesized that the death receptor pathways could be involved pretreatment with CS-IVa-Be also inhibited IL6Ra/GP130 in the CS-IVa-Be–induced apoptosis. We then investigated the interaction in the presence of IL6 (Fig. 5F). The expression effects of CS-IVa-Be on the expression of death receptors and of IL6R, IL6, and pSTAT3 in different cancer cell lines were found that CS-IVa-Be increased DR5 protein levels in breast cancer compared (Supplementary Fig. S4A–S4C). As MDA-MB-231 MDA-MB-231 and MCF-7 cells (Fig. 6D). We next analyzed the cells secrete the highest levels of IL6 (Supplementary Fig. S4B), cell surface DR5 expression levels in CS-IVa-Be–treated MDA-MB- we also considered whether CS-IVa-Be could inhibit IL6 secre- 231 cells, which were also increased upon CS-IVa-Be treatment. In tion; however, CS-IVa-Be exhibited little inhibitory effect on contrast, the levels of DR4 did not increase by CS-IVa-Be (Fig. 6D). IL6 secretion (Supplementary Fig. S4D). Overall, these results It has previously been suggested that the upregulation of DR5 suggest that CS-IVa-Be binds to IL6R (IL6Ra and GP130) and synergizes with TRAIL to induce apoptosis. We then examined disrupts IL6/IL6Ra/GP130 interaction. whether CS-IVa-Be induced upregulation of DR5 synergized with TRAIL. Transfection of MDA-MB-231 cells with DR5-specific CS-IVa-Be synergized with TRAIL to induce apoptosis in breast siRNA resulted in a marked inhibition of DR5 surface expression cancer cells (data not shown). After the combined treatment with CS-IVa-Be Resistance to TRAIL-induced apoptosis in many cancers and TRAIL, the apoptosis rate in the DR5 knocked down cells was limits its clinical use as an anticancer agent (26), and aberrant significantly reduced compared with the control (Fig. 6E). We STAT3 activation has been suggested to be a part of the causes suggest that the synergized induction of apoptosis by CS-IVa-Be

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CS-IVa-Be (7.5 μmol/L) A Control TRAIL (60 ng/mL) TRAIL (0 ng/mL) TRAIL (60 ng/mL)

B C

60 50

40 PARP 30 20 10 GAPDH Apoptosis rate (%) 0

TRAIL (ng/mL) – – 60 60 TRAIL (ng/mL) – – 60 60 CS-IVa-Be (μmol/L) – 7.5 7.5– CS-IVa-Be (μmol/L) – 7.5 – 7.5

DE 35 MDA-MB-231 MCF-7 30 μ CS-IVa-Be ( mol/L) 0 5 7.5 10 0 5 7.5 10 25 DR5 20 GAPDH 15 10

Apoptosis rate (%) 5

DR5 DR4 0 Control (81) 7.5 μmol/L(22) 7.5 μmol/L(130) Isotype (20) μ Isotype (28) CS-IVa-Be ( mol/L) – 7.5––––– 7.5 7.5 7.5 7.5 7.5

Control (22) TRAIL (ng/mL) – –3030––30 30 –– 30 30 Scramble siRNA (nmol/L) – – – ––50 50 50 50 ––– DR5 siRNA (nmol/L) – – – –––––50 50 50 50

Figure 6. CS-IVa-Be sensitized to TRAIL-induced cancer cell apoptosis. MDA-MB-231 cells were treated with 7.5 mmol/L CS-IVa-Be, TRAIL (60 ng/mL), or a combination for 24 hours, and the cell morphology was recorded (A), cell apoptosis was analyzed by Annexin V/PI double staining assay (B), and Western blot analysis was done with PARP antibody (C). D, MDA-MB-231 and MCF-7 cells were treated with different concentrations (0, 5, 7.5, 10 mmol/L) of CS-IVa-Be for 24 hours, and then subjected to Western blot assay with DR5 antibody (top). MDA-MB-231 cells treated with 7.5 mmol/L of CS-IVa-Be for 24 hours were subjected to cytometry assay with DR5 or DR4 antibodies, and mouse anti-human IgG was used as an isotype control (bottom). E, MDA-MB-231 cells were transfected with scramble siRNA or DR5 siRNA for 24 hours, treated with 7.5 mmol/L CS-IVa-Be, 30 ng/mL TRAIL, or in combination for 24 hours, and the cell apoptosis was analyzed by Annexin V/PI double staining assay. These are representative results of three repetitive experiments with similar results and error bars mark SDs (, P < 0.05).

and TRAIL was dependent on the DR5 upregulation, at least anism remains elusive. Herein, we uncovered that CS-IVa-Be partially. decrease cell viability in various cancer cells by inducing apoptosis. Numerous signaling pathways, such as IL6/STAT3, ROS/ Discussion MAPK, TNFa/NF-kB, and AKT, are involved in regulating cell proliferation and survival (29, 30). Although CS-IVa-Be treatment CS-IVa-Be, a triterpenoid saponin from TCM Acanthopanas induced ROS production, we demonstrated that oxidative stress is gracilistylus W.W.Smith, has previously been shown to possess not the primary cause for CS-IVa-Be–induced cell apoptosis. cytotoxic activity in many cancer cells (28), although the mech- TNFa/NF-kB and AKT are also not involved in CS-IVa-Be–

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CS-IVa-Be, a Novel IL6R Antagonist, Inhibits IL6/STAT3

mediated cell apoptosis. We next discovered CS-IVa-Be inhibited kine–induced signaling to survive, and antagonizing GP130 by IL6 family cytokines induced STAT3 activation. Especially, CS- CS-IVa-Be can effectively inhibit MCF-7 viability. IVa-Be showed more potential inhibitory effect on IL6-induced TRAIL resistance in many cancers limits its therapeutic use in STAT3 activation than other family cytokines (Fig. 2B and C). clinical scenarios. The STAT3 trans-regulated prosurvival genes Interestingly, CS-IVa-Be showed no inhibitory effect on EGF or Survivin, c-FLIP, XIAP, Bcl-xL,andMcl-1 are involved in regu- IFNg-induced STAT3, STAT5, or STAT1 activation as well as lating cell apoptosis and TRAIL sensitivity to cancer cells (40). constitutive STAT5 or STAT1 activation. The expression levels of these genes were attenuated by CS-IVa- Tyrosine kinases (JAKs, Src, EGFR, FGFR1, IGF1R, and PDGFRa) Be, which exhibits enhanced apoptosis in breast cancer cells are not involved in the mechanism of CS-IVa-Be–mediated via upregulating DR5 expression, which is consistent with the IL6/STAT3 inhibition because CS-IVa-Be showed no inhibitory report that upregulation of the cell surface DR5 was dependent effect on the enzyme activity of any of the kinases. We further on the suppression of STAT3 activation (41). These studies found that CS-IVa-Be directly binds to IL6R (IL6Ra and GP130) suggest that DR5 is negatively regulated by STAT3, and the and disrupts the interaction of IL6/IL6Ra/GP130 in vitro and in inhibition of STAT3 phosphorylation by CS-IVa-Be accounts for cancer cells. As CS-IVa-Be showed higher affinity to IL6Ra than DR5 induction and the apoptosis enhancing synergy with other IL6 family receptors (LIFR and LeptinR), we suggest that CS- TRAIL. IVa-Be is a relatively specific IL6R antagonist, interfering IL6- In summary, we characterized CS-IVa-Be as a novel natural IL6R induced STAT3 activation. In view of the fact that GP130 is a antagonist, which inhibits IL6/STAT3 signaling, induces cancer common subunit of IL6 family receptors and CS-IVa-Be also can cell apoptosis, and synergizes with TRAIL in breast cancer cells. directly bind GP130, CS-IVa-Be exhibits inhibitory effect on other Therefore, synergistic combination of CS-IVa-Be with TRAIL has IL6 family cytokines (IL11, LIF, and OSM) inducing STAT3 acti- potential to be a more effective strategy to treat cancers with vation. The evidence that U937 cells express relatively high levels of aberrant IL6/STAT3 activation. cell surface IL6Ra than other cancer cells (Fig. 5A and Supple- mentary Fig. S4A) and IL6Ra knockdown partly reversed the CS- Disclosure of Potential Conflicts of Interest IVa-Be–induced U937 cell apoptosis (Supplementary Fig. S4E and No potential conflicts of interest were disclosed. S4F) also demonstrate the key role of IL6Ra in mediating CS-IVa- Be–induced cancer cell apoptosis. Authors' Contributions Many kinds of natural compounds have been reported to Conception and design: J. Yang, Q. Yu, S.P. Gao, P. Cao abolish IL6/STAT3 signaling through different mechanisms Development of methodology: Y. Yang, Q. Yu Acquisition of data (provided animals, acquired and managed patients, (31) such as targeting IL6Ra (32) or GP130 (33), directly inhibit- provided facilities, etc.): J. Yang, X. Cai ing JAKs (34), inducing PTPase expression (35), inhibiting STAT3 Analysis and interpretation of data (e.g., statistical analysis, biostatistics, translocation (36), and blocking JAK–STAT3 interaction (37). The computational analysis): J. Yang, S. Qian, X. Sun, Q. Yu, S.P. Gao natural antagonist of IL6R is rarely reported except for the ERBF, Writing, review, and/or revision of the manuscript: J. Yang, S. Qian, Q. Yu, which is isolated from bufadienolide and sensitizes breast cancer S.P. Gao, P. Cao cells to TRAIL-induced apoptosis via inhibition of STAT3/Mcl-1 Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Qian, W. Lu, C. Hu pathway (38). In accordance with their results, our data suggest Study supervision: C. Hu, Q. Yu, P. Cao CS-IVa-Be is a novel natural IL6R antagonist and shows anticancer activity. Acknowledgments To further confirm the inhibitory effect of CS-IVa-Be on cancer The authors thank Xiaojun Xu and Ping Zhou in State Key Laboratory of cells was via IL6/STAT3 inhibition, we investigated the GP130, Natural Medicine (China Pharmaceutical University), for providing help with pSTAT3, STAT3, IL6 secretion levels, and cell surface IL6Ra the MST analysis. expression in different cancer cells (Supplementary Fig. S4A– S4C) and compared the sensitivity of different cancer cells to Grant Support CS-IVa-Be. MDA-MB-231 cells express the highest levels of P. Cao received Jiangsu Province Funds for Distinguished Young Scientists GP130, pSTAT3, and IL6 than other cancer cells determined (BK20140049) grant, J. Yang received National Natural Science Foundation of MDA-MB-231 cell line is sensitive to CS-IVa-Be. We found that China (No. 81403151) grant, and X.Y. Sun received National Natural Science MCF-7 cell line expresses low level of IL6 and pSTAT3, but it is Foundation of China (No. 81202576) grant. also sensitive to CS-IVa-Be. It has been reported that low level of The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked IL6 is indispensible to maintain the growth of MCF-7 breast advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate cancer cells (39). In addition, CS-IVa-Be also can bind GP130, this fact. and the expression of GP130 in MCF-7 cells is higher than other cancer cells except MDA-MB-231. We speculate that MCF-7 cells Received July 1, 2015; revised February 15, 2016; accepted February 24, 2016; are more dependent on GP130-mediated other IL6 family cyto- published OnlineFirst February 29, 2016.

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1200 Mol Cancer Ther; 15(6) June 2016 Molecular Cancer Therapeutics

Chikusetsusaponin IVa Butyl Ester (CS-IVa-Be), a Novel IL6R Antagonist, Inhibits IL6/STAT3 Signaling Pathway and Induces Cancer Cell Apoptosis

Jie Yang, Shihui Qian, Xueting Cai, et al.

Mol Cancer Ther 2016;15:1190-1200. Published OnlineFirst February 29, 2016.

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