chikusetsusaponin iva butyl ester (cs-iva-be), a novel il6r … · cs-iva-be (fig. 1a) is a...

Small Molecule Therapeutics

Chikusetsusaponin IVa Butyl Ester (CS-IVa-Be),a Novel IL6R Antagonist, Inhibits IL6/STAT3Signaling Pathway and Induces CancerCell ApoptosisJie 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 thepathogenesis of many cancers. Agents that suppress IL6/STAT3signaling have cancer-therapeutic potential. In this study, wefound that chikusetsusaponin IVa butyl ester (CS-IVa-Be), atriterpenoid saponin extracted from Acanthopanas gracilistylusW.W.Smith, induced cancer cell apoptosis. CS-IVa-Be inhibitedconstitutive and IL6-induced STAT3 activation, repressed STAT3DNA-binding activity, STAT3 nuclear translocation, IL6-inducedSTAT3 luciferase reporter activity, IL6-induced STAT3-regulatedantiapoptosis gene expression in MDA-MB-231 cells, and IL6-induced TF-1 cell proliferation. Surprisingly, CS-IVa-Be inhibitedIL6 family cytokines rather than other cytokines induced STAT3activation. Further studies indicated that CS-IVa-Be is an antag-

onist of IL6 receptor via directly binding to the IL6Rawith a Kd of663�74nmol/L and theGP130 (IL6Rb)with aKd of 1,660� 243nmol/L, interfering with the binding of IL6 to IL6R (IL6Ra andGP130) in vitro and in cancer cells. The inhibitory effect of CS-IVa-Be on the IL6–IL6Ra–GP130 interaction was relatively specific asCS-IVa-Be showed higher affinity to IL6Ra than to LIFR (Kd: 4,910� 1,240 nmol/L) and LeptinR (Kd: 4,990� 915 nmol/L). We nextdemonstrated that CS-IVa-Be not only directly induced cancer cellapoptosis but also sensitized MDA-MB-231 cells to TRAIL-induced apoptosis via upregulating DR5. Our findings suggestthat CS-IVa-Be as a novel IL6R antagonist inhibits IL6/STAT3signaling pathway and sensitizes theMDA-MB-231 cells to TRAIL-induced cell death. Mol Cancer Ther; 15(6); 1190–200. �2016 AACR.

IntroductionIL6 is a pleiotropic cytokine-mediating cancer associated

chronic inflammation and inflammatory response, playsimportant roles in the tumorigenesis (1). IL6 possesses threetopologically distinct receptor binding sites: site 1 for bindingto the 80 kDa chain IL6Ra, and sites 2 and 3 for interacting withthe two subunits of the signaling chain glycoprotein 130(GP130). The IL6 signaling transduces after the homodimer-ization of GP130, which becomes associated with IL6-bind-ing chain (IL6Ra) in the presence of IL6. (2). As a commonreceptor, GP130 also transduces signals delivered by the leu-

kemia inhibitory factor (LIF), the IL11, the oncostatin M(OSM), and the others (3).

STAT3, the key factor mediating the IL6-induced signalingbecomes tyrosine phosphorylated by IL6-induced cytokine recep-tor-associated kinases, the Janus kinase (JAK) family proteins (4).The IL6/STAT3 signaling pathway mediates tumor immune sup-pression (5), tumor cell survival, premetastatic niche formation,and chemotherapy resistance (6).

Breast cancer is the most common malignancy and theprimary cause of cancer-related death in women worldwide(7). IL6/JAK/STAT3 signaling plays a critical and pharmacolog-ically targetable role in orchestrating the composition of thebreast tumor microenvironment that promotes cancer cellgrowth, invasion, and metastasis (8, 9). Increasing clinical datahave indicated that the circulating levels of IL6 in breast cancerpatients correlate with clinical tumor stage and poor prognosis.IL6 plays two distinct roles in breast cancer: the systemic IL6,reflecting whole body metabolic and inflammatory status, iscorrelated with poor prognosis, advanced disease, and metas-tasis; the paracrine and autocrine IL6 signaling controls cancercell growth, cancer stem cell renewal, and metastasis (10, 11).In summary, the IL6-mediated excessive STAT3 activation playsimportant roles in breast cancer.

Inhibitors of the IL6/STAT3 signaling pathway should beeffective against the cancers with high levels of STAT3 activa-tion. As a matter of fact, JAK3 inhibitor tofacitinib (12, 13) andIL6Ra mAb tocilizumab (14) have been used in clinical trial.However, no natural IL6/STAT3 inhibitory compound has been

1Affiliated Hospital of Integrated Traditional Chinese and WesternMedicine, Nanjing University of Chinese Medicine, Nanjing, China.2LaboratoryofCellular andMolecularBiology, JiangsuProvinceAcad-emy of Traditional Chinese Medicine, Nanjing, China. 3Shanghai Insti-tute of Materia Medica, Chinese Academy of Sciences, Shanghai,China. 4Human Oncology and Pathogenesis Program, MemorialSloan-Kettering Cancer Center, New York, New York.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

J. Yang and S. Qian are the co-first authors of this article.

Corresponding Author: Peng Cao, Jiangsu Province Academy of TraditionalChinese Medicine, 100#, Shizi Street, Hongshan Road, Nanjing, 210028, China.Phone: 8625-8560-8666; Fax: 8625-8560-8666; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-15-0551

�2016 American Association for Cancer Research.

MolecularCancerTherapeutics

Mol Cancer Ther; 15(6) June 20161190

on June 8, 2020. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

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

http://mct.aacrjournals.org/

used in cancer clinical trial. Identifying novel compounds thatsuppress the JAKs signaling or antagonize IL6R is a pressingissue to prevent and treat the cancers with aberrant activatedIL6/STAT3 signaling.

TRAIL is a promising target for cancer therapy because itpreferentially induces cancer cell apoptosis with little or no effectson normal cells (15). However, some highly malignant cancers,including breast cancer (16), are resistant to TRAIL-inducedapoptosis. The resistance of cancer cells to TRAIL occur at variouspoints of the TRAIL signaling pathways, dysfunctions of the deathreceptors DR4 and DR5, overexpression of cellular FLICE-likeinhibitory protein (c-FLIP), B-cell lymphoma extra-large (Bcl-xL),myeloid cell leukemia 1 (Mcl-1), and the inhibitor of apoptosisproteins (IAP), all lead to TRAIL resistance (17). TRAIL resistancecan occur through activating STAT3 in cancer cells (18). Therefore,a combination of TRAIL with STAT3 inhibitor may prove to be asound strategy to overcome the resistance. Identifying novelagents that inhibit STAT3 activation not only can directly induceapoptosis but also sensitize cancer cells to TRAIL-mediatedapoptosis.

CS-IVa-Be (Fig. 1A) is a triterpenoid saponin extracted from atraditional Chinese medicine (TCM) herb Acanthopanas gracilis-tylus W.W.Smith that exhibits immunomodulation (19) andantithrombotic effects (20). The pharmacologic effects of CS-IVa-Be have not been reported yet. In this study, we demonstratedthat as an IL6R antagonist, CS-IVa-Be inhibited IL6/STAT3 sig-naling, repressed cancer cell growth, and synergizedwith TRAIL toinduce breast cancer cell apoptosis.

Materials and MethodsCell lines and cultures

Human breast cancer MDA-MB-231 and MCF-7 cells, humanhepatocellular cancer HepG2 andMHCC97L cells, cervical cancerHeLa cells, leukemic U937 and TF-1 cells, and gastric cancer SGC-7901 cells were purchased from Cell Bank of the ShanghaiInstitute of Biochemistry and Cell Biology in June 2013. Humanfetal liver cell line LO2 was purchased from Kunming Institute ofZoology, Chinese Academy of Sciences in June 2013. All cell lineshad been authenticated by the provider through short tandemrepeat (STR) profiling. All cell lines were preserved in liquid N2

after receipt and passaged for fewer than 6 months when we usethem to do experiments. The cell lines were last tested in June2013 by above cell bank through STR profiling. MDA-MB-231,MCF-7, and HepG2 cells were cultured in MEM supplementedwith 10%FBS.HeLa andMHCC97L cells were cultured inDMEM(Invitrogen) supplemented with 10% FBS(Invitrogen). U937,SGC-7901, and LO2 cells were cultured in RPMI1640 mediasupplemented with 10% FBS, and TF-1 cells were cultured inRPMI1640 media supplemented with 10% FBS, and 2 ng/mLgranulocyte-macrophage colony-stimulating factor (GM-CSF).All cell lines were cultured in a humidified atmosphere with a 5%CO2 incubator at 37�C.

Agents and antibodiesCS-IVa-Be was identified and purified from the fruit of

the Acanthopanas gracilistylus W.W.Smith. The purity of CS-IVa-Be is 98.8 %, which was determined by high-performanceliquid chromatography-evaporative light scattering detector(HPLC-ELSD) method. A 20 mmol/L solution of CS-IVa-Bewas prepared in DMSO, stored as small aliquots at 4�C.

STAT3, phospho-STAT3 (Tyr705), phospho-STAT1(Tyr701),STAT1, phosphor-STAT5(Tyr694), STAT5, phosphor-ERK1/2(Thr201/Tyr204), ERK1/2, phosphor-AKT(Ser473), AKT, phos-phor-JAK1(Tyr1022/1023), phosphor-JAK2(Tyr1007/1008), phos-phor-Src(Tyr416), Bcl-xL, Mcl-1, Survivin, XIAP, caspase-3,caspase-8, caspase-9, PARP, DR5, DR4, c-FLIP, IL6Ra, GAPDHantibodies were purchased from Cell Signaling Technology.Anti-IL6 and anti-GP130 antibodies were purchased fromAbcam. IL6 ELISA Kit was purchased from R&D Systems.His-probe, NE-PER Nuclear and Cytoplasmic Extraction, andLightShift Chemiluminescent EMSA Kit were obtained fromSanta Cruz Biotechnology. Methyl thiazolyl tetrazolium (MTT),N-acetyl-L-cysteine (NAC), SP600125, PD98059, SB203580, andZ-VAD-FMK were purchased from Sigma-Aldrich. Recombinanthuman TNFa, IL6, EGF, IFNg were obtained from Tocris Bio-science. Recombinant human IL6Ra, GP130, LIFR, and Leptinreceptor were purchased from Sino Biological Inc. Pierce ClassicMagnetic IP/Co-IP Kit were purchased from Thermo FisherScientific.

Immunoblot assayCells were plated in 6-well plate at a density of 5 � 106

cells/mL overnight before being treated with CS-IVa-Be. Thetotal cell lysates were prepared as described previously (21).The equal amounts of proteins from each sample were sub-jected to SDS-PAGE followed by transfer to polyvinylidenedifluoride (PVDF) membranes. After being blocked with 1%(w/v) BSA in TBST for 2 hours, the membranes were incu-bated with primary antibody overnight at 4�C then washedand incubated with secondary antibody conjugated with IgGDyLight for 1 hour at room temperature. Afterward, mem-branes were washed and scanned with an Odyssey infraredfluorescent scanner (LI-COR) and analyzed with Odysseysoftware version 3.

Cytotoxicity assayLO2, HepG2, MHCC97L, SGC-7901, TF-1, U937, MDA-MB-

231, andMCF-7 cells were plated in triplicate in a 96-well plate ata density of 1� 104 cells with 100 mL culture medium per well inthe absence or presence of indicated concentrations of CS-IVa-Befor 24 hours. Cell viability was then determined by the MTT assayas described previously (22).

Apoptosis assayApoptotic cells were determined by using an FITC Annexin V

Apoptosis Detection Kit (BD Biosciences) according to the man-ufacturer's protocol. The cells were washed and incubated withbinding buffer containing Annexin V-FITC and PI in the dark atroom temperature for 15 minutes. Afterward, the degree ofapoptosis was analyzed by FACScan laser flow cytometer (FACSCalibur; Becton Dickinson). The data were analyzed using thesoftware CELLQuest.

JC-1 stainingMitochondrial membrane potential was examined via JC-1

staining (Beyotime Institute of Biotechnology, China). MDA-MB-231 cells were incubated with JC-1 working solution at 37�Cin the dark for 20 minutes and observed by fluorescence micros-copy. In healthy cells, JC-1 forms complexes of J-aggregatesshowing punctate red fluorescence at 590 nm emission

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

www.aacrjournals.org Mol Cancer Ther; 15(6) June 2016 1191




wavelength; however, in apoptotic cells, JC-1 remains in themonomeric form showing diffused green fluorescence at 530 nmemission wavelength.

Immunofluorescence stainingMDA-MB-231 cells were seeded at the density of 70% to 80 %

confluence per well into 24-well chamber slides. After beingtreated with test drugs for the indicated times, cells were fixedwith cold 4% (w/v) paraformaldehyde for 20minutes, rehydratedin PBS for 15minutes, and permeabilized in 0.1% (w/v) TritonX-100 at room temperature for 10minutes. After being washedwithPBS, the cells were blocked with 3% BSA for 1 hour and thenincubated with primary antibody at 4�C overnight followed byFITC-conjugated secondary antibody for 1 hour at room temper-ature. The images of green fluorescence–stained pSTAT3 werecaptured using a fluorescence microscope.

Luciferase reporter gene activity assayWe used the luciferase reporter assay (the Dual-Luciferase

Reporter Assay System; Promega) to investigate the IL6-inducedtranscriptional activity of STAT3. Transient transfection was per-formed in 96-well plates at a cell density of 50% to 70% conflu-ence per well. The STAT3 luciferase reporter plasmid was cotrans-fected with the pRL-TK plasmid (which encodes Renilla luciferaseas an internal control for transfection efficiency) using Lipofecta-mine 2000 (Invitrogen) according to the manufacturer's instruc-tions. The transfected cells were treated with CS-IVa-Be for variousperiods of time, and the cell lysates were prepared for assessmentof luciferase activity. Firefly and Renilla luciferase activities weremeasured using a luminometer (Centro XS3 LB960) according tothe manufacturer's instructions. Relative firefly luciferase activitynormalized by Renilla luciferase activity was expressed as foldinduction after treatment with CS-IVa-Be compared with vehiclecontrol (DMSO).

Electrophoretic mobility shift assaySTAT3–DNA-binding activity was analyzed by electrophoretic

mobility shift assay (EMSA) using biotin-labeled, double-strand-ed STAT3 consensus-bindingmotif probe. STAT3 consensus oligoprimers were as follows: forward, 50-GATCCTTCTGGGAATTCC-TAGATC-30; reverse, 30-CTAGGAAGACCCTTAAGGATCTAG-50.Nuclear protein extracts were prepared from CS-IVa-Be–treatedMDA-MB-231 cells and incubated with biotin-labeled probe.STAT3–DNA complex was separated from free oligonucleotideon 5% nondenaturing polyacrylamide gels, then transferred tonylonmembranes, and cross-linked for 15minutes under a hand-held UV lamp. Cross-linked, biotin-labeled DNA was detectedusing the Chemiluminescent Nucleic Acid Detection Module(Pierce Biotechnology, Inc.).

IL6 inhibitory bioassayAfter being starved in media without GM-CSF for 24 hours,

various concentrations of CS-IVa-Be (2.5, 5, 7.5, 10, 12.5) mmol/Lin the presence of IL6 (25 ng/mL) were added to the TF-1 cells andincubated for 24or 48hours; cell viabilitywas thendeterminedbythe MTT assay.

In vitro kinase assayIn vitro JAKs kinase (JAK2, JAK3, TYK2) assay for a series of CS-

IVa-Be concentrations was performed by BPS Bioscience Service

using Kinase-Glo Plus Luminescence Kinase Assay Kit (Promega).In vitro panel kinases (JAK1, JAK2, JAK3, TYK2, EGFR, IGF1R,PDGFRa, FGFR1) assay with two concentrations (25 and50 mmol/L) of CS-IVa-Be was performed by Eurofins' KinaseProfiler service according to Cerep's validation Standard Operat-ing Procedure.

IL6 receptor binding assay in cell levelThe cell-based IL6-receptor binding was performed using

the kit of Fluorokine Biotinylated Human Interleukin 6 (R&DSystems) followed by the manufacturer's protocol. First, U937,MDA-MB-231, HeLa, andHepG2 cells were treated with the testcompound for various periods of time and the cells wereharvested and washed twice with PBS to remove any residualgrowth factors that may be present in the culture media. Next,the cells were resuspended in PBS to a final concentration of 4�106 cells/mL, 10 mL of biotinylated IL6 was added to a 25 mLaliquot of the cell suspension for a total reaction volume of35 mL. As a negative staining control, an aliquot of cells werestained with 10 mL of negative control reagent (a soybeantrypsin inhibitor protein biotinylated as the IL6). The cellswere then incubated for 30 minutes at 4�C followed by theaddition of 10 mL Avidin-FITC reagent and another 30-minuteincubation at 4�C in the dark before being analyzed by flowcytometry.

Analysis of IL6Ra, DR4, and DR5 surface expressionCells were treated with CS-IVa-Be for various periods of times

before being detached with trypsin containing EDTA andwashed twice with staining buffer. Cells were then incubatedwith mouse monoclonal anti-human DR5, DR4, or IL6Raantibodies for 45 minutes at 4�C, washed twice, incubated withFITC-conjugated secondary antibody for 30 minutes at 4�C andwashed again, then resuspended in staining buffer for flowcytometric analysis.

IL6 receptor binding assay in vitroIL6 receptor binding was performed following the method

described previously (23). Briefly, 96-well plates (Nunc) werecoated with 500 ng/mL of human recombinant soluble IL6receptor (sIL6R), washed, and blocked. The sIL6R-coated 96-well plates were incubated with 6.2 nmol/L of recombinanthuman IL6, or 6.2 nmol/L of IL6 plus one of six concentra-tions of CS-IVa-Be, in triplicates, and washed to removeunbound IL6, the wells then were incubated with a mono-clonal rabbit anti-human IL6 antibody (200 ng/mL), washed,and incubated with goat anti-rabbit IgG-HRP (40 ng/mL;Santa Cruz Biotechnology). Sample wells were washed anddeveloped with tetramethylbenzidine (100 mL/well, 10 min-utes at room temperature and stopped with 2 mol/L H2SO4).Optical density in each well was determined using a micro-plate reader (Multiskan Ascent, Thermo Scientific) 450 nmwith a correction wavelength of 630 nm. Nonspecific bindingof IL6 is determined by the reading of wells without sIL6R.Data were analyzed by nonlinear regression analysis usingGraphPad Prism4 (GraphPad). Maximum binding of IL6(100% bound) is defined as the absorbance in the absenceof CS-IVa-Be. The absorbance in the presence of variousconcentrations of CS-IVa-Be is calculated as percent of max-imum binding of IL6.

Yang et al.

Mol Cancer Ther; 15(6) June 2016 Molecular Cancer Therapeutics1192




Microscale thermophoresis assayBinding assay of labeled recombinant human IL6Ra, GP130,

LIFR, and LeptinR to CS-IVa-Be was performed using the Mono-lith NT.115 Kit (NanoTemper Technologies). Briefly, 10 mmol/Lrecombinant human IL6Ra, GP130, LIFR, and LeptinR werelabeled in PBS buffer using the Protein Labeling Kit RED-NHSfollowed by the manufacturer's protocol (NanoTemper Technol-ogies). A 16-point titration series of CS-IVa-Be were added to thelabeled protein (200 nmol/L) in PBST buffer (PBS þ 0.05%Tween-20), the total volume of mixed sample was 20 mL and thevolume ratio of CS-IVa-Be to labeled protein is 1:1, the finalDMSO concentration for each protein-compound sample was2%. The protein-compound samples were equilibrated for 5minutes at room temperature, loaded into the MST PremiumCoated capillaries (NanoTemper Technologies) and assayed bymicroscale thermophoresis (MST). The thermophoresis data wereanalyzed using NT Analysis 1.5.41 (NanoTemper Technologies)to compute Kd value according to the law of mass action. Theexperiment was performed in triplicate, and the fits are repre-sented as mean � SD. Data normalization and curve fitting wereperformed using GraphPad Prism 5.

Coimmunoprecipitation assayThe interaction of IL6Ra with GP130 in the presence of IL6

was assayed by coimmunoprecipitation (co-IP) assay accordingto the manufacturer's protocol of Pierce Classic MagneticIP/Co-IP Kit. At room temperature, 10 mg of recombinanthuman IL6Ra was preincubated with 0, 5, or 10 mmol/L ofCS-IVa-Be for 2 hours in IP wash buffer, 10 mg of recombinanthuman IL6 was added and incubated with GP130 for another 2hours, and then 2 mg of His antibody was added and incubatedfor 2 hours to form immune complex. Twenty-five microlitersof prewashed Pierce Protein A/G Magnetic Beads were placedinto the above immune complex and incubated for 1 hour withmixing. Then the beads were washed and the target antigen waseluted with Alternative Elution, and the target antigen and thebinding proteins were immunoblotted with the indicated anti-bodies by Western blot assay.

siRNA-mediated gene silencingDR5-specific siRNAwas obtained fromRiboBio. Transient trans-

fection was performed using Lipofectamine 2000. Briefly, cellswere transfected with indicated concentration of siRNAs for

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Figure 1.CS-IVa-Be induces cancer cellapoptosis. A, the chemical structure ofCS-IVa-Be. B, LO2, HepG2, MHCC97L,SGC-7901, TF-1, U937, MDA-MB-231,and MCF-7 cells were treated withvarious concentrations of CS-IVa-Befor 24 hours and then subjected toMTT assay; the value of IC50 wascalculated. C, cell lysates from MDA-MB-231–treated with variousconcentrations (0, 5, 7.5, 10mmol/L) ofCS-IVa-Be for 24 hours were analyzedfor apoptosis proteins. MDA-MB-231cells were treated with variousconcentrations (0, 7.5, 10 mmol/L) ofCS-IVa-Be for 24 hours followingtreatment by Z-VAD-FMK for 30minutes, and subjected to MTT assay(D) and apoptosis assay (E). F, MDA-MB-231 cells treated with variousconcentrations (0, 5, 7.5, 10mmol/L) ofCS-IVa-Be for 24 hours were stainedwith JC-1 and then subjected tocytometry assay. This is arepresentative result of threerepetitive experiments with similarresults and error bars mark SDs(� , P < 0.05).






24 hours, followed by treatment with CS-IVa-Be for indicatedtime, and the cells were then harvested for flow cytometry analysis.

Statistical analysisThe experimental results presented in the figures are represen-

tative of three or more independent experiments. The data arepresented as the mean values � SD. Statistical comparisonsbetween the groups were done using one-way ANOVA. Valuesof P < 0.05 were considered to be statistically significant.

ResultsCS-IVa-Be induces cancer cell apoptosis

CS-IVa-Be (Fig. 1A), a triterpenoid saponin extracted fromAcanthopanas gracilistylus W.W.Smith, was identified as a celldeath inducer. We investigated the effects of CS-IVa-Be on theviability of cancer cells and found that CS-IVa-Be inhibits theviability of various kinds of cancer cells (including hepatocel-lular, gastric, leukemia, and breast cancers). Breast cancer MDA-MB-231 and MCF-7 cells were more sensitive to the CS-IVa-Be–induced cell viability attenuation than other cancer cells (Fig.

1B). We next investigated whether the CS-IVa-Be induced breastcancer cell inhibition due to apoptosis. We then found that CS-IVa-Be induced pro-caspase-3, -8, and -9 cleavage (Fig. 1C).Furthermore, Z-VAD-FMK, a pan-caspase inhibitor, significantlyoffsets the CS-IVa-Be–induced MDA-MB-231 cell viabilityattenuation and apoptosis (Fig. 1D and E). In addition, CS-IVa-Be induced a decrease of mitochondrial membrane poten-tial (Fig. 1F). The results indicate that CS-IVa-Be promotesbreast cancer cell apoptosis.

CS-IVa-Be selectively inhibits constitutive and IL6 familymember–induced STAT3 activation in MDA-MB-231 cells

To identify the signaling pathways that were involved inthe CS-IVa-Be–induced cell apoptosis, we investigated theeffects of CS-IVa-Be on STAT3, NF-kB, MAPK, and AKT signal-ing. CS-IVa-Be was found to inhibit the constitutive and IL6-induced STAT3 and JAK1, JAK2, Src activation rather thanconstitutive and IL6-induced ERK1/2 and AKT activation(Fig. 2A and B). Other IL6 family cytokines (IL11, LIF, andOSM, which share the same GP130 receptor to transducesignal) inducing STAT3 activation were also inhibited by

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Figure 2.CS-IVa-Be inhibits IL6/STAT3activation in MDA-MB-231 cells. A, celllysates fromMDA-MB-231 cells treatedwith various concentrations (0, 5, 7.5,10 mmol/L) of CS-IVa-Be for 24 hourswere analyzed for pSTAT3 (Tyr705),STAT3, pSTAT1(Tyr701), STAT1,pSTAT5 (Tyr694), STAT5, pAKT, AKT,pERK1/2, andERK1/2,withGAPDHasaloading control. B, cell lysates fromMDA-MB-231 cells treatedwith variousconcentrations (0, 2.5, 5, 7.5, 10 mmol/L) of CS-IVa-Be for 8 hours, followedby 15-minute IL6 (25 ng/mL)stimulation were analyzed for pSTAT3(Tyr705), STAT3, pJAK1, pJAK2, pSrc,pAKT, AKT, pERK1/2, and ERK1/2.GAPDH was used as a loading control.C, cell lysates from MDA-MB-231 cellstreated with 10 mmol/L of CS-IVa-Befor 8 hours, followed by 15-minutestimulation of 25 ng/mL IL11, LIF, OSM,respectively, were analyzed forpSTAT3 (Tyr705), STAT3. GAPDH wasused as a loading control. D, celllysates fromMDA-MB-231 cells treatedwith 10 mmol/L of CS-IVa-Be for 8hours, followed by 15-minutestimulation of 100 ng/mL EGF andIFNg , respectively, were analyzed forpSTAT3 (Tyr705), STAT3, pSTAT1(Tyr701), STAT1, pSTAT5 (Tyr694), andSTAT5. GAPDH was used as a loadingcontrol. This is a representative resultof three repetitive experimentswith similar results.

Yang et al.





CS-IVa-Be (Fig. 2C). But interestingly, constitutive and EGF-induced STAT3, STAT5 activation, or IFNg-induced STAT3,STAT1 activation were not inhibited by CS-IVa-Be (Fig. 2Aand D). In addition, ROS production was observed uponCS-IVa-Be treatment (Supplementary Fig. S1A), but treatmentwith ROS, JNK1/2/3, ERK1/2, or p38-specific inhibitor did notreverse the CS-IVa-Be–induced cell viability attenuation inMDA-MB-231 cells (Supplementary Fig. S1B). CS-IVa-Be hadno inhibitory effects on the TNFa-induced NF-kB transcriptionactivity (Supplementary Fig. S1C) and NF-kB DNA-bindingactivity (Supplementary Fig. S1D).

CS-IVa-Be abrogates IL6/STAT3 downstream signalingWe further investigated the effects of CS-IVa-Be on constitutive

and IL6-induced STAT3 downstream signaling in MDA-MB-231cells. CS-IVa-Be was found to inhibit the pSTAT3 nuclear trans-location (Fig. 3A), the STAT3–DNAbinding activity (Fig. 3B), andIL6-induced STAT3 luciferase activity (Fig. 3C) in MDA-MB-231cells. CS-IVa-Be also downregulated the constitutive and IL6-induced Survivin, XIAP, Bcl-xL, and Mcl-1 expression (Fig. 3Dand E). IL6-induced TF-1 cell proliferation was also inhibited byCS-IVa-Be (Fig. 3F). These findings suggest that CS-IVa-Be inhibitIL6/STAT3 downstream signaling.

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24 h48 h

A B

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D

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/LCS-IVa-Be for 8 hours and then pSTAT3(Tyr705) was labeled through immunofluorescence staining; scale bar, 20 mm. B, MDA-MB-231 cells weretreated 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 toEMSA with STAT3 probe. C, MDA-MB-231 cells were transfected with STAT3 luciferase reporter vector for 24 hours, and then treated with variousconcentrations (0, 2.5, 5, 7.5, 10 mmol/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 wereanalyzed 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 withvarious concentrations (0, 2.5, 5, 7.5, 10 mmol/L) of CS-IVa-Be for 24 hours, followed by IL6 (25 ng/mL) stimulation for 15 minutes, were analyzedfor 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 markSDs (�, P < 0.05).






CS-IVa-Be does not inhibit the enzyme activity of nonreceptoror receptor tyrosine kinases of STAT3 in vitro

As STAT3 phosphorylation was mediated by several non-receptor tyrosine kinases (24) and receptor kinases (25), weinvestigated whether CS-IVa-Be impact the activity of upstreamkinases. CS-IVa-Be has showed inhibitory effects on the IL6-induced phosphorylation of JAK1, JAK2, and Src (Fig. 2B) inMDA-MB-231 cells. The inhibitory effect of CS-IVa-Be on IL6-induced STAT3 activation might be through its repression onJAK1, JAK2, and Src kinase activity. However, by in vitro kinaseassay, we found no inhibitory effect of CS-IVa-Be on theenzyme activity of tyrosine kinases such as JAKs family kinases(JAK1, JAK2, JAK3, and TYK2; Fig. 4A and B), Src, EGFR, insulin-like growth factor 1 receptor (IGF1R), platelet-derived growthfactor receptor a (PDGFRa), and fibroblast growth factorreceptor 1 (FGFR1; Fig. 4B), even at 50 mmol/L, the dose ismuch higher than the IC50 of CS-IVa-Be in MDA-MB-231 cells.Therefore, we suggest that CS-IVa-Be is not a tyrosine kinaseinhibitor.

CS-IVa-Be blocks IL6 binding to its cell surface receptorWe had shown that CS-IVa-Be inhibited IL6-induced JAKs

and STAT3 activation in breast cancer cells, without affectingJAKs enzyme activity in vitro. These findings made us speculate

that CS-IVa-Be might disrupt the upstream signaling such asthe interaction of IL6 to its receptor. To test this hypothesis, weinvestigated whether CS-IVa-Be affect the binding of IL6 tocell surface IL6R in cancer cells using biotin-labeled IL6 byflow cytometry analysis. To rule out the possibility that CS-IVa-Be might downregulate IL6R protein levels, we also measuredthe cell surface IL6Ra expression upon CS-IVa-Be treatment.The results demonstrated that CS-IVa-Be indeed lowered bio-tin-labeled IL6 binding to IL6R in various cancer cells tested(U937, MDA-MB-231, Hela, HepG2) in a dose-dependentmanner, without decreasing IL6Ra cell surface expression(Fig. 5A and B and Supplementary Fig. S2).

To further confirm our hypothesis that CS-IVa-Be inhibitsIL6–IL6R binding, we investigated the effects of CS-IVa-Be onIL6–IL6Ra interaction using an in vitro ELISA assay. Our resultsshowed that CS-IVa-Be significantly inhibited the IL6–IL6Rainteraction with maximal inhibition rate less than 20%(Fig. 5C). As CS-IVa-Be cannot antagonize IL6–IL6Ra interac-tion completely, we considered CS-IVa-Be could be a noncom-petitive IL6Ra antagonist.

Biochemical characterization of CS-IVa-Be/IL6R interactionAs our results have illustrated that CS-IVa-Be inhibited the

IL6–IL6R interaction, we next studied the binding specificity

JAK2 JAK3 TYK2%

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% R

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–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)]

IC50 > 30 μmol/L IC50 > 30 μmol/L IC50 > 30 μmol/L

(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)

% Inhibition of control value (mol/L)

EGFR

FGFR1

IGF1RJAK1

JAK2

JAK3

PDGFRaSrc

Tyk2

−20 −10 10 20 30 40 50 60 70 80 90 1000

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

A

B

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) on JAK1, JAK2, JAK3, Tyk2, Src, EGFR, FGFR1, andPDGFRa kinase activity in vitro.

Yang et al.





of CS-IVa-Be to IL6 or IL6R. We first measured the bindingaffinity (Kd: 48 � 2 nmol/L) of IL6 to IL6Ra (SupplementaryFig. S3A) using MST analysis, which allows a sensitive detec-tion of small-molecule binding to a protein target. Next,we measured the binding characteristics of CS-IVa-Be to IL6or IL6R and found that CS-IVa-Be can bind to IL6Ra(Kd: 663 � 74 nmol/L) and IL6Rb (GP130; Kd: 1,660 �243 nmol/L; Fig. 5D and E) but had no binding affinity toIL6 (Supplementary Fig. S3B). The affinity of CS-IVa-Be toIL6R was relatively higher than other IL6 family receptors suchas LeptinR (Kd: 4,990 � 915 nmol/L) and LIFR (Kd: 4,910 �1,240 nmol/L; Fig. 5E and Supplementary Fig. S3). IL6Rapretreatment with CS-IVa-Be also inhibited IL6Ra/GP130interaction in the presence of IL6 (Fig. 5F). The expressionof IL6R, IL6, and pSTAT3 in different cancer cell lines werecompared (Supplementary Fig. S4A–S4C). As MDA-MB-231cells secrete the highest levels of IL6 (Supplementary Fig. S4B),we also considered whether CS-IVa-Be could inhibit IL6 secre-tion; however, CS-IVa-Be exhibited little inhibitory effect onIL6 secretion (Supplementary Fig. S4D). Overall, these resultssuggest that CS-IVa-Be binds to IL6R (IL6Ra and GP130) anddisrupts IL6/IL6Ra/GP130 interaction.

CS-IVa-Be synergized with TRAIL to induce apoptosis in breastcancer cells

Resistance to TRAIL-induced apoptosis in many cancerslimits its clinical use as an anticancer agent (26), and aberrantSTAT3 activation has been suggested to be a part of the causes

(27). As we have proved that CS-IVa-Be is an effective IL6/STAT3 inhibitor, we next determined whether CS-IVa-Be cansynergize with TRAIL to induce apoptosis in breast cancer cells.We treated the breast cancer MDA-MB-231 cells with CS-IVa-Bealone or combined with TRAIL and analyzed cell apoptosis. Thecells treated by TRAIL/CS-IVa-Be combination demonstratedobvious apoptotic morphology (Fig. 6A) and increased cellapoptosis (Fig. 6B and C).

CS-IVa-Be upregulates DR5 synergized with TRAILWith our data showing CS-IVa-Be activating caspase-8, we

hypothesized that the death receptor pathways could be involvedin the CS-IVa-Be–induced apoptosis. We then investigated theeffects of CS-IVa-Be on the expression of death receptors andfound thatCS-IVa-Be increasedDR5protein levels in breast cancerMDA-MB-231 and MCF-7 cells (Fig. 6D). We next analyzed thecell surface DR5 expression levels in CS-IVa-Be–treatedMDA-MB-231 cells, whichwere also increased uponCS-IVa-Be treatment. Incontrast, the levels ofDR4did not increase byCS-IVa-Be (Fig. 6D).It has previously been suggested that the upregulation of DR5synergizes with TRAIL to induce apoptosis. We then examinedwhether CS-IVa-Be induced upregulation of DR5 synergized withTRAIL. Transfection of MDA-MB-231 cells with DR5-specificsiRNA resulted in a marked inhibition of DR5 surface expression(data not shown). After the combined treatment with CS-IVa-Beand TRAIL, the apoptosis rate in the DR5 knocked down cells wassignificantly reduced compared with the control (Fig. 6E). Wesuggest that the synergized induction of apoptosis by CS-IVa-Be

IL6Ra expression IL6–IL6 receptor binding

IL6–

IL6R

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Isotype IsotypeCS-IVa-Be

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Kd = 663 ± 74 (nmol/L)

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.5 25 50 100 0 1 2 3 4 5 6

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IL6RaGP130 (IL6Rb)LeptinRLIFR

663 ± 741,660 ± 2434,990 ± 9154,910 ± 1,240

IgG His antibody

CS-IVa-Be(mmol/L)

His-IL6Ra

GP130

0 0 5 10

IP: Anti-His

IP: Anti-His

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A

C

E F

D

BFigure 5.CS-IVa-Be disrupts IL6/IL6Ra/GP130interaction. Cytometry analysis of theinhibitory effect of CS-IVa-Be onIL6–IL6R interaction in U937 cells.Biotinylated IL6 was used to label cellsurface IL6R.A, the levels of cell surfaceIL6Ra in cellswithCS-IVa-Be treatmentor not (left). B, cell surface IL6–IL6Rbinding levels in cells with CS-IVa-Betreatment or not (right). C, ELISAanalysis of the inhibitory effect of CS-IVa-Be on IL6–IL6Ra binding in vitro.The maximum binding of IL6 (100%bound) is defined as without CS-IVa-Be, whereas the effects of variousconcentrations of CS-IVa-Be werecalculated as the percentages ofmaximum binding of IL6. MST analysisof CS-IVa-Be binding to IL6Ra, GP130,LeptinR, and LIFR. D, CS-IVa-Be–binding affinity to IL6Ra. E, CS-IVa-Be–binding affinity to GP130, LeptinR, andLIFR, respectively. F, recombinanthuman IL6Ra with His tag wasincubated with 0, 5, or 10 mmol/LCS-IVa-Be for 2 hours, thenmixed withrecombinant human GP130 and IL6,and immunoprecipitated with anti-Hisantibody. The precipitates werewashed, suspended in reducing samplebuffer, and immunoblotted withanti-His or anti-GP130 antibody.These are representative results ofthree repetitive experiments withsimilar results.






and TRAIL was dependent on the DR5 upregulation, at leastpartially.

DiscussionCS-IVa-Be, a triterpenoid saponin from TCM Acanthopanas

gracilistylus W.W.Smith, has previously been shown to possesscytotoxic activity in many cancer cells (28), although the mech-

anism remains elusive. Herein, we uncovered that CS-IVa-Bedecrease cell viability in various cancer cells by inducing apopto-sis. Numerous signaling pathways, such as IL6/STAT3, ROS/MAPK, TNFa/NF-kB, and AKT, are involved in regulating cellproliferation and survival (29, 30). AlthoughCS-IVa-Be treatmentinduced ROSproduction, we demonstrated that oxidative stress isnot the primary cause for CS-IVa-Be–induced cell apoptosis.TNFa/NF-kB and AKT are also not involved in CS-IVa-Be–

ControlA

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MDA-MB-231 MCF-7

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0 5 7.5 10 0 5 7.5 10

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– 30 30

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CS-IVa-Be (7.5 μμmol/L)

CS-IVa-Be (μmol/L)

CS-IVa-Be (μmol/L)

DR5

7.5 μmol/L(130)7.5 μmol/L(22)

Control (22)

Isotype (28) Isotype (20)

Control (81)DR5 DR4

GAPDH

TRAIL (ng/mL)

TRAIL (ng/mL)

Ap

op

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te (

%)

PARP

GAPDH

CS-IVa-Be (μmol/L)

CS-IVa-Be (μmol/L)

Scramble siRNA (nmol/L)

DR5 siRNA (nmol/L)

TRAIL (60 ng/mL)

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 for24 hours, and the cell morphology was recorded (A), cell apoptosis was analyzed by Annexin V/PI double staining assay (B), and Western blot analysis wasdone 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 thensubjected 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 cytometryassaywithDR5orDR4antibodies, andmouse anti-human IgGwasused as an isotype control (bottom). E,MDA-MB-231 cellswere transfectedwith scramble siRNAorDR5 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 byAnnexin V/PI double staining assay. These are representative results of three repetitive experiments with similar results and error bars mark SDs (� , P < 0.05).

Yang et al.





mediated cell apoptosis. We next discovered CS-IVa-Be inhibitedIL6 family cytokines induced STAT3 activation. Especially, CS-IVa-Be showed more potential inhibitory effect on IL6-inducedSTAT3 activation than other family cytokines (Fig. 2B and C).Interestingly, CS-IVa-Be showed no inhibitory effect on EGF orIFNg-induced STAT3, STAT5, or STAT1 activation as well asconstitutive STAT5 or STAT1 activation.

Tyrosine kinases (JAKs, Src, EGFR, FGFR1, IGF1R, andPDGFRa)are not involved in the mechanism of CS-IVa-Be–mediatedIL6/STAT3 inhibition because CS-IVa-Be showed no inhibitoryeffect on the enzyme activity of any of the kinases. We furtherfound that CS-IVa-Be directly binds to IL6R (IL6Ra and GP130)and disrupts the interaction of IL6/IL6Ra/GP130 in vitro and incancer cells. As CS-IVa-Be showed higher affinity to IL6Ra thanother IL6 family receptors (LIFR and LeptinR), we suggest that CS-IVa-Be is a relatively specific IL6R antagonist, interfering IL6-induced STAT3 activation. In view of the fact that GP130 is acommon subunit of IL6 family receptors and CS-IVa-Be also candirectly bind GP130, CS-IVa-Be exhibits inhibitory effect on otherIL6 family cytokines (IL11, LIF, and OSM) inducing STAT3 acti-vation. Theevidence thatU937 cells express relativelyhigh levels ofcell surface IL6Ra than other cancer cells (Fig. 5A and Supple-mentary Fig. S4A) and IL6Ra knockdown partly reversed the CS-IVa-Be–induced U937 cell apoptosis (Supplementary Fig. S4E andS4F) also demonstrate the key role of IL6Ra in mediating CS-IVa-Be–induced cancer cell apoptosis.

Many kinds of natural compounds have been reported toabolish IL6/STAT3 signaling through different mechanisms(31) such as targeting IL6Ra (32) or GP130 (33), directly inhibit-ing JAKs (34), inducing PTPase expression (35), inhibiting STAT3translocation (36), andblocking JAK–STAT3 interaction (37). Thenatural antagonist of IL6R is rarely reported except for the ERBF,which is isolated from bufadienolide and sensitizes breast cancercells to TRAIL-induced apoptosis via inhibition of STAT3/Mcl-1pathway (38). In accordance with their results, our data suggestCS-IVa-Be is a novel natural IL6R antagonist and shows anticanceractivity.

To further confirm the inhibitory effect of CS-IVa-Be on cancercells was via IL6/STAT3 inhibition, we investigated the GP130,pSTAT3, STAT3, IL6 secretion levels, and cell surface IL6Raexpression in different cancer cells (Supplementary Fig. S4A–S4C) and compared the sensitivity of different cancer cells toCS-IVa-Be. MDA-MB-231 cells express the highest levels ofGP130, pSTAT3, and IL6 than other cancer cells determinedMDA-MB-231 cell line is sensitive to CS-IVa-Be. We found thatMCF-7 cell line expresses low level of IL6 and pSTAT3, but it isalso sensitive to CS-IVa-Be. It has been reported that low level ofIL6 is indispensible to maintain the growth of MCF-7 breastcancer cells (39). In addition, CS-IVa-Be also can bind GP130,and the expression of GP130 in MCF-7 cells is higher than othercancer cells except MDA-MB-231. We speculate that MCF-7 cellsare more dependent on GP130-mediated other IL6 family cyto-

kine–induced signaling to survive, and antagonizing GP130 byCS-IVa-Be can effectively inhibit MCF-7 viability.

TRAIL resistance in many cancers limits its therapeutic use inclinical scenarios. The STAT3 trans-regulated prosurvival genesSurvivin, c-FLIP, XIAP, Bcl-xL, and Mcl-1 are involved in regu-lating cell apoptosis and TRAIL sensitivity to cancer cells (40).The expression levels of these genes were attenuated by CS-IVa-Be, which exhibits enhanced apoptosis in breast cancer cellsvia upregulating DR5 expression, which is consistent with thereport that upregulation of the cell surface DR5 was dependenton the suppression of STAT3 activation (41). These studiessuggest that DR5 is negatively regulated by STAT3, and theinhibition of STAT3 phosphorylation by CS-IVa-Be accounts forDR5 induction and the apoptosis enhancing synergy withTRAIL.

In summary, we characterizedCS-IVa-Be as a novel natural IL6Rantagonist, which inhibits IL6/STAT3 signaling, induces cancercell apoptosis, and synergizes with TRAIL in breast cancer cells.Therefore, synergistic combination of CS-IVa-Be with TRAIL haspotential to be a more effective strategy to treat cancers withaberrant IL6/STAT3 activation.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J. Yang, Q. Yu, S.P. Gao, P. CaoDevelopment of methodology: Y. Yang, Q. YuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Yang, X. CaiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Yang, S. Qian, X. Sun, Q. Yu, S.P. GaoWriting, review, and/or revision of the manuscript: J. Yang, S. Qian, Q. Yu,S.P. Gao, P. CaoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Qian, W. Lu, C. HuStudy supervision: C. Hu, Q. Yu, P. Cao

AcknowledgmentsThe authors thank Xiaojun Xu and Ping Zhou in State Key Laboratory of

Natural Medicine (China Pharmaceutical University), for providing help withthe MST analysis.

Grant SupportP. Cao received Jiangsu Province Funds for Distinguished Young Scientists

(BK20140049) grant, J. Yang received National Natural Science Foundation ofChina (No. 81403151) grant, and X.Y. Sun received National Natural ScienceFoundation of China (No. 81202576) grant.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received July 1, 2015; revised February 15, 2016; accepted February 24, 2016;published OnlineFirst February 29, 2016.

References1. Rattigan Y, Hsu JM, Mishra PJ, Glod J, Banerjee D. Interleukin 6 mediated

recruitment of mesenchymal stem cells to the hypoxic tumor milieu. ExpCell Res 2010;316:3417–24.

2. Kishimoto T, Akira S, Taga T. Interleukin-6 and its receptor: a paradigm forcytokines. Science 1992;258:593–7.

3. Kishimoto T, Taga T, Akira S. Cytokine signal transduction. Cell 1994;76:253–62.

4. Schindler C, Levy DE, Decker T. JAK-STAT signaling: from interferons tocytokines. J Biol Chem 2007;282:20059–63.

5. Ferguson SD, Srinivasan VM, Heimberger AB. The role of STAT3 in tumor-mediated immune suppression. J Neurooncol 2015;123:385–94.

6. SpitznerM, Ebner R,Wolff HA, Ghadimi BM,Wienands J, GradeM. STAT3:a novel molecular mediator of resistance to chemoradiotherapy. Cancers2014;6:1986–2011.






7. Place AE, Jin Huh S, Polyak K. The microenvironment in breast cancerprogression: biology and implications for treatment. Breast Cancer Res2011;13:227.

8. Senst C, Nazari-Shafti T, Kruger S, Honer Zu Bentrup K, Dupin CL, ChaffinAE, et al. Prospective dual role of mesenchymal stem cells in breast tumormicroenvironment. Breast Cancer Res Treat 2013;137:69–79.

9. ChangQ, Bournazou E, Sansone P, BerishajM,Gao SP,Daly L, et al. The IL-6/JAK/Stat3 feed-forward loop drives tumorigenesis and metastasis. Neo-plasia 2013;15:848–62.

10. Fuksiewicz M, Kowalska M, Kotowicz B, Rubach M, Chechlinska M,Pienkowski T, et al. Serum soluble tumour necrosis factor receptor typeI concentrations independently predict prognosis in patients with breastcancer. Clin Chem Lab Med 2010;48:1481–6.

11. Dethlefsen C,Hojfeldt G,Hojman P. The role of intratumoral and systemicIL-6 in breast cancer. Breast Cancer Res Treat 2013;138:657–64.

12. Cutolo M, Meroni M. Clinical utility of the oral JAK inhibitor tofacitinib inthe treatment of rheumatoid arthritis. J Inflamm Res 2013;6:129–37.

13. Vyas D, O'Dell KM, Bandy JL, Boyce EG. Tofacitinib: the first janus kinase(JAK) inhibitor for the treatment of rheumatoid arthritis. Ann Pharmac-other 2013;47:1524–31.

14. Hashizume M, Tan SL, Takano J, Ohsawa K, Hasada I, Hanasaki A, et al.Tocilizumab, a humanized anti-IL-6R Antibody, as an emerging therapeu-tic option for rheumatoid arthritis: molecular and cellular mechanisticinsights. Int Rev Immunol 2015;34:265–79.

15. Walczak H, Miller RE, Ariail K, Gliniak B, Griffith TS, Kubin M, et al.Tumoricidal activity of tumor necrosis factor-related apoptosis-inducingligand invivo. Nat Med 1999;5:157–63.

16. Rahman M, Pumphrey JG, Lipkowitz S. The TRAIL to targeted therapyof breast cancer. Adv Cancer Res 2009;103:43–73.

17. Zhang LD, Fang BL. Mechanisms of resistance to TRAIL-induced apoptosisin cancer. Cancer Gene Ther 2005;12:228–37.

18. Chen KF, Tai WT, Liu TH, Huang HP, Lin YC, Shiau CW, et al. Sorafenibovercomes TRAIL resistance of hepatocellular carcinoma cells through theinhibition of STAT3. Clin Cancer Res 2010;16:5189–99.

19. Spelman K, Burns J, Nichols D, Winters N, Ottersberg S, Tenborg M.Modulation of cytokine expression by traditional medicines: a review ofherbal immunomodulators. Altern Med Rev 2006;11:128–50.

20. Chen XC, Xia L, Hu S, Huang G. Inhibitory effects of Acanthopanaxgraci-listylussaponins on human platelet aggregation and platelet factor 4 liber-ation invitro. Zhongguo Yao Li Xue Bao . 1996;17:523–6.

21. Isomoto H, Mott JL, Kobayashi S, Werneburg NW, Bronk SF, Haan S, et al.Sustained IL-6/STAT-3 signaling in cholangiocarcinoma cells due to SOCS-3 epigenetic silencing. Gastroenterology 2007;132:384–96.

22. Takada Y, Ichikawa H, Badmaev V, Aggarwal BB. Acetyl-11-keto-beta-boswellic acid potentiates apoptosis, inhibits invasion, and abolishesosteoclastogenesis by suppressing NF-kappa B and NF-kappa B-regulatedgene expression. J Immunol 2006;176:3127–40.

23. Vardanyan M, Melemedjian OK, Price TJ, Ossipov MH, Lai J, Roberts E,et al. Reversal of pancreatitis-induced pain by an orally available, smallmolecule interleukin-6 receptor antagonist. Pain 2010;151:257–65.

24. ImadaK, LeonardWJ. The Jak-STATpathway.Mol Immunol . 2000;37:1–11.25. Song LX, Turkson J, Karras JG, Jove R, Haura EB. Activation of Stat3

by receptor tyrosine kinases and cytokines regulates survival inhuman non-small cell carcinoma cells. Oncogene 2003;22:4150–65.

26. Dimberg LY, Anderson CK, Camidge R, Behbakht K, Thorburn A, FordHL. On the TRAIL to successful cancer therapy? Predicting and counter-acting resistance against TRAIL-based therapeutics. Oncogene 2013;32:1341–50.

27. Lanuti P, BertagnoloV, Pierdomenico L, Bascelli A, Santavenere E, Alinari L,et al. Enhancement of TRAIL cytotoxicity by AG-490 in human ALL cells ischaracterized by downregulation of cIAP-1 and cIAP-2 through inhibitionof Jak2/Stat3. Cell Res 2009;19:1079–89.

28. Wang J, Lu J, Lv C, Xu T, Jia L. Three new triterpenoid saponins from root ofGardeniajasminoidesEllis. Fitoterapia 2012;83:1396–401.

29. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell2011;144:646–74.

30. Sung B, Park B, Yadav VR, Aggarwal BB. Celastrol, a triterpene, enhancesTRAIL-induced apoptosis through the down-regulation of cell survivalproteins and up-regulation of death receptors. J Biol Chem 2010;285:11498–507.

31. Sansone P, Bromberg J. Targeting the interleukin-6/Jak/stat pathway inhuman malignancies. J Clin Oncol 2012;30:1005–14.

32. Hayashi M, Rho MC, Fukami A, Enomoto A, Nonaka S, Sekiguchi Y, et al.Biological activity of a novel nonpeptide antagonist to the interleukin-6receptor 20S,21-epoxy-resibufogenin-3-formate. J Pharmacol Exp Thera-peut 2002;303:104–9.

33. Hong SS, Choi JH, Lee SY, Park YH, Park KY, Lee JY, et al. A novel small-molecule inhibitor targeting the IL-6 receptor beta subunit, glycoprotein130. J Immunol 2015;195:237–45.

34. Wang Y, Ma X, Yan S, Shen S, Zhu H, Gu Y, et al. 17-hydroxy-jolkinolide B inhibits signal transducers and activators of transcrip-tion 3 signaling by covalently cross-linking Janus kinases andinduces apoptosis of human cancer cells. Cancer Res 2009;69:7302–10.

35. Yang J, Cai X, Lu W, Hu C, Xu X, Yu Q, et al. Evodiamine inhibits STAT3signaling by inducing phosphatase shatterproof 1 in hepatocellular carci-noma cells. Cancer Lett 2013;328:243–51.

36. Bharadwaj U, Eckols TK, Kolosov M, Kasembeli MM, Adam A, Torres D,et al. Drug-repositioning screening identified piperlongumine as a directSTAT3 inhibitor with potent activity against breast cancer. Oncogene2015;34:1341–53.

37. ShinDS, Jung SN, Yun J, Lee CW,HanDC, KimB, et al. Inhibition of STAT3activation by KT-18618 via the disruption of the interaction between JAK3and STAT3. Biochem Pharmacol 2014;89:62–73.

38. Dong Y, Yin S, Li J, Jiang C, Ye M, Hu H. Bufadienolide com-pounds sensitize human breast cancer cells to TRAIL-induced apo-ptosis via inhibition of STAT3/Mcl-1 pathway. Apoptosis 2011;16:394–403.

39. Jiang XP, Yang DC, Elliott RL, Head JF. Down-regulation of expression ofinterleukin-6 and its receptor results in growth inhibition of MCF-7 breastcancer cells. Anticancer Res 2011;31:2899–906.

40. Krueger A, Schmitz I, Baumann S, Krammer PH, Kirchhoff S. CellularFLICE-inhibitory protein splice variants inhibit different steps of caspase-8activation at the CD95 death-inducing signaling complex. J Biol Chem2001;276:20633–40.

41. Ivanov VN, Zhou H, Partridge MA, Hei TK. Inhibition of ataxia telangiec-tasia mutated kinase activity enhances TRAIL-mediated apoptosis inhuman melanoma cells. Cancer Res 2009;69:3510–9.


Yang et al.




2016;15:1190-1200. Published OnlineFirst February 29, 2016.Mol Cancer Ther Jie Yang, Shihui Qian, Xueting Cai, et al. Cancer Cell ApoptosisAntagonist, Inhibits IL6/STAT3 Signaling Pathway and Induces Chikusetsusaponin IVa Butyl Ester (CS-IVa-Be), a Novel IL6R

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chikusetsusaponin iva butyl ester (cs-iva-be), a novel il6r … · cs-iva-be (fig. 1a) is a...

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