A Novel Peptide Specifically Binding to Interleukin-6 ...cancerres.aacrjournals.org/content/canres/65/11/4827.full.pdf · A Novel Peptide Specifically Binding to Interleukin-6 Receptor

A Novel Peptide Specifically Binding to Interleukin-6 Receptor

(gp80) Inhibits Angiogenesis and Tumor Growth

Jen-Liang Su,1Kuo-Pao Lai,

2Chi-An Chen,

3Ching-Yao Yang,

1,4,5Pei-Sheng Chen,

1

Chiao-Chia Chang,1Chia-Hung Chou,

1Chi-Lun Hu,

2Min-Liang Kuo,

1

Chang-Yao Hsieh,2and Lin-Hung Wei

2

1Laboratory of Molecular and Cellular Toxicology, Institute of Toxicology, National Taiwan University College of Medicine andDepartments of 2Oncology, 3Obstetrics and Gynecology, 4Surgery, and 5Traumatology, National Taiwan University Hospital andNational Taiwan University College of Medicine, Taipei, Taiwan

Abstract

Experimental and clinical findings support the essential roleof interleukin (IL)-6 in the pathogenesis of various humancancers and provide a rationale for targeted therapeuticinvestigations. A novel peptide, S7, which selectively bindsto IL-6 receptor (IL-6R) A chain (gp80) and broadly inhibitsIL-6-mediated events, was identified using phage displaylibrary screening. The synthetic S7 peptide specifically boundto soluble IL-6R as well as cognate human IL-6RA, resultingin a dose-dependent blockade of the interaction between IL-6and IL-6RA. S7 peptide prevents IL-6–mediated survivalsignaling and sensitizes cervical cancer cells to chemother-apeutic compounds in vitro . The in vitro analysis ofantiangiogenic activity showed that S7 peptide substantiallyinhibits IL-6–induced vascular endothelial growth factor-Aexpression and angiogenesis in different cancer cell lines.Furthermore, S7 peptide was bioavailable in vivo , leading to asignificant suppression of IL-6–induced vascular endothelialgrowth factor–mediated cervical tumor growth in severecombined immunodeficient mice. These observations showthe feasibility of targeting IL-6/IL-6R interaction using thesmall peptide and highlight its potential in the clinicalapplications. (Cancer Res 2005; 65(11): 4827-35)

Introduction

Interleukin (IL)-6 is a secreted, pleiotropic cytokine thatregulates the physiologic and pathologic responses to variousdisease processes, including inflammation, myocardial infarction,autoimmune disorder, Alzheimer’s disease, osteoporosis, andhematologic and nonhematologic malignancies (1–5). The biolo-gical activities of IL-6 are mediated through binding to amembrane-bound glycoprotein IL-6 receptor (IL-6R) a chain(gp80) on target cells. Specifically, the IL-6/IL-6R complex initiateshomodimerization of the ubiquitously expressed gp130 (h chain),activates a cytoplasmic tyrosine kinase bound to gp130, and thentriggers Janus-activated kinase/signal transducers and activators oftranscription (STAT), Ras/mitogen-activated protein kinase(MAPK), and phosphatidylinositol 3-kinase (PI3K)/Akt signaling(6, 7). IL-6 signaling mediated via gp130 interferes with manycellular functions, such as cell growth and survival, differentiation,cell mobility, and angiogenesis, and is thereby critically involved inthe pathogenesis of various human cancers (8–10). Therefore,

blocking IL-6 activity may be of clinical benefit in the managementof patients with malignant diseases.IL-6 plays a pivotal role in the human cervical oncogenesis. IL-6

levels are increased in cervicovaginal secretions of patients withcervical cancer, and its production is related to the severity ofcervical neoplasia (11). Cervical cancer cells, as well as lymphocytes,macrophages, and endothelial cells, produce substantially highmicroenvironmental IL-6 levels, which promote cervical tumorgrowth via autocrine or paracrine mechanisms or both (12, 13).Previously, we showed that, by inducing vascular endothelial growthfactor (VEGF) via the STAT3 pathway, IL-6 is involved in theangiogenic switch that occurs during cervical oncogenesis (14, 15).Furthermore, overexpression of IL-6 in cervical cancer cells confersresistance to cisplatin cytotoxicity by up-regulating the apoptoticthreshold through a PI3K/Akt signaling (16). Taken together, theseobservations suggest that blocking IL-6 activity would be a feasiblestrategy in the management of cervical cancer.To validate IL-6R as a therapeutic target, we screened a phage

display peptide library for peptide ligands reactive with IL-6R.Random peptide libraries displayed on phage have been used invarious applications, including epitope mapping and identificationof peptide mimics of nonpeptide ligands. In fact, peptides thattarget tumor cells and tumor vasculature have been successfullyidentified using the phage display approach (17, 18). Here, we applythe recombinant system to the manufacture of identified peptidesthat react with the IL-6R. The experimental results showed that thisselective interactive ligand is valid for inhibiting tumor angiogen-esis and growth of human cervical cancer in vitro and in vivo .

Materials and Methods

Antibodies and reagents. Antihuman VEGF antibody, anti–h-actinantibody, biotin-labeled anti–IL-6 antibody, anti–IL-6Ra antibody, and

human IL-6Ra protein were purchased from R&D Systems (Minneapolis,

MN). Anti–phospho-Akt1 (Ser473) antibody, anti-Akt1 antibody, anti–phospho-

extracellular signal-regulated kinase 1/2 (ERK1/2) antibody, anti-ERK1/2antibody, anti–Mcl-1 antibody, donkey antigoat IgG rhodamine-conjugated

antibody, and goat antimouse IgG FITC-conjugated antibody were all

purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Human VEGF-A

ELISA kit and recombinant IL-6 were purchased from R&D Systems. Matrigelwas acquired from Collaborative Research (Bedford, MA). Tetramethyl

benzidine (TMB) was obtained from Sigma Chemical Co. (St. Louis, MO).

Cell culture. C33A, HeLa, and Siha (cervical carcinoma) cells, basal

cell carcinoma (BCC) cells, HT-29 colon cancer cells, HepG2 hepatomacells, and HEK293 fibroblast cells were cultured in DMEM (Invitrogen,

Carlsbad, CA) containing 10% fetal bovine serum (FBS; Bioserum, Victoria,

Australia), penicillin (100 units/mL), streptomycin (100 Ag/mL), L-glutamine(2 mmol/L), and sodium pyruvate (1 mmol/L, Invitrogen). Cell cultures

were maintained at 37jC in a humidified 5% CO2 atmosphere. Human

umbilical vein endothelial cells (HUVEC), endothelial cell growth medium,

Requests for reprints: Lin-Hung Wei, Department of Oncology, National TaiwanUniversity Hospital, No. 7, Chung-Shan South Road, Taipei 100, Taiwan. Phone: 886-2-2312-3456, ext. 7140; Fax: 886-2-2371-1174; E-mail: [email protected].

I2005 American Association for Cancer Research.

www.aacrjournals.org 4827 Cancer Res 2005; 65: (11). June 1, 2005

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trypsin-EDTA, and trypsin-neutralizing solutions were purchased from

Clonetics (San Diego, CA). All cell cultures were conducted according to thesupplier’s recommendations.

Screening a 7-mer phage display library with soluble KDR. Theprocedure for screening the phage display library was modified according toinstructions of the manufacturer of the kit (New England Biolabs, Beverly,MA). The cell culture dishes (60 mm diameter) were coated with human IL-6Ra proteins. IL-6Ra protein was added at 100 Ag/mL in 3 mL/dish andincubated at 4jC for 24 hours before blocking with phosphate buffer con-taining 1% bovine serum albumin (BSA) for 1 hour at 37jC. The phage librarycontaining 1012 clones was sequentially added to non-bait-coated dishes(those coated with human IL-6Rh proteins) for preabsorption. In each case,the library was shaken gently at room temperature for 1 hour. Finally, thepreabsorbed librarywas applied to soluble IL-6Ra (sIL-6Ra)–coateddishes forspecific screening. After thorough washing, plate-bound phage clones wereeluted with elution buffer [0.22 mol/L glycine-HCl (pH 2.2)] and neutralizedimmediately. Four rounds of selection were done, after which individualplaques were picked at random and subjected to analysis by phage ELISAand DNA sequencing following amplification in Escherichia coli ER2537.

Phage ELISA using various types of cells. C33A, HeLa, and Siha cervicalcarcinoma cells, BCC cells, and HEK293 cells were grown in DMEMsupplementedwith 10% FBS. Human umbilical cords were digestedwith 0.1%collagenase II, and HUVECs were collected and grown in M199 mediumsupplemented with 10% FBS. For phage ELISA assays, cells were seeded in 96-well plates at 90% confluence. After an overnight incubation, cells were fixedwith ice-cold glutaraldehyde (0.125%) in PBS for 10 minutes at roomtemperature and then washed with PBS. PBS containing 3% BSA was used toblock the plates by overnight incubation at 4jC. After blocking, phages [5 �1012 plaque-forming units (pfu)/mL] were added to the plates and incubatedfor 2 hours at room temperature. Wells were then washed six times with TBS(pH 7.5) containing 0.1% Tween 20, and bound phage was detected by ELISAusing a horseradish peroxidase (HRP)–conjugated anti-M13 monoclonalantibody (mAb).

Competition assay of positive phages and synthesized peptides. sIL-6Ra (250 Ag/mL) was immobilized on 96-well plates and blocked withphosphate buffer containing 1% BSA for 1 hour at 37jC. In competitionexperiments, seven different phage clones (1012 pfu/mL), which wereselected by the above phage ELISA or various concentrations of S1 or S7peptide, were then incubated with sIL-6Ra-coated or cell-coated plates for1 hour at room temperature. The bound IL-6 was detected by ELISA. Afterthe preincubation, human IL-6 protein (50 ng/mL, 100 AL) was addeddirectly to the wells without removal of the phage. After additional 2 hoursof incubation at room temperature, the plates were thoroughly washed with

0.1% BSA/PBS buffer (pH 8.5); biotin-conjugated anti–IL-6 mAb was addedfor 1 hour and HRP-conjugated streptavidin then was added for 1 hour. Thefree avidin conjugate was washed away and freshly prepared substratesolution (TMB) was then added to each well. The reaction was allowed toproceed for 10 minutes, after which the color development was stopped bythe addition of H3PO4 (1.0 mol/L). The absorbance at 450 nm was read witha reference wavelength of 650 nm (A450/650).

Immunofluorescence staining. Suspensions of 2.5� 103 cells in medium

were allowed to adhere to glass Nunc chamber slides for 16 hours. Phage was

added to the cells for 2 hours at room temperature. Cells to be analyzed by

staining were washed twice with PBS, fixed for 25 minutes at room

temperature in 3% paraformaldehyde, and then blocked by incubation in

2.5% BSA in PBS. Polyclonal goat antihuman IL-6Ra antibody andmonoclonal

anti-M13 antibodywere applied to the slides at a dilution of 1:50 and incubated

at 4jC overnight. After washes in PBS, the samples were treated with donkey

antigoat IgG rhodamine-conjugated secondary antibody (R&D Systems) and

FITC-conjugated goat antimouse secondary antibody (R&D Systems) at a

dilution of 1:200 for 1 hour at room temperature. The immunofluorescence-

labeled cells were then analyzed by fluorescence microscopy.

Laser scan confocal microscopy. Immunofluorescence-labeled cells

were analyzed using an inverted laser scanning microscope (Zeiss LSM 410

invert, Carl Zeiss, Oberkochen, Germany) equipped with both argon ion

(488 nm) and HeNe (543 nm) lasers. For double labeling, the confocal overlay

mode was used, and images from two different channels, one green and one

red, collected simultaneously on the same focal plane. Colocalization of two

labeled antigens was detected as a single yellow image when the images

from both channels were overlaid. For each image, the cells were optically

sectioned from the ventral to the dorsal surface at intervals of 1 Am.

Western blot analysis. Cells were incubated in serum-free DMEM for

24 hours before treatment with or without IL-6 (50 ng/mL) and cells were

lysed in radioimmunoprecipitation assay buffer [Tris-HCl (50 mmol/L;

pH 7.5), NaCl (120 mmol/L), NP40 (0.5%), NaF (100 mmol/L), Na3VO4

(200 mmol/L), phenylmethylsulfonyl fluoride (1 mmol/L), leupeptin (1 Ag/mL), aprotinin (1 Ag/mL)] for 15 minutes on ice. The cell lysates were

prepared as described previously (19). An equal quantity of protein from the

cell lysates was resuspended in gel sample buffer, resolved by 10% SDS-PAGE,

and transferred to nitrocellulose membranes (Millipore Corp., Milford, MA).

After blocking, blots were incubated with specific primary antibodies, and

following washing and incubation with secondary antibodies, immunoreac-

tive proteins were visualized by an enhanced chemiluminescence detection

system (Amersham, Arlington Heights, IL). Where indicated, the membranes

were stripped and reprobed with another antibody.

Quantification of apoptosis by flow cytometry and terminaldeoxynucleotidyl transferase–mediated dUTP nick end labeling assay.Cells were harvested and washed with PBS, and hypodiploid cells wereanalyzed by flow cytometry. Briefly, 1 � 106 cells were washed with PBS,

resuspended in 500 AL buffer (0.5% Triton X-100, PBS, 0.05% RNase A), and

incubated for 30 minutes. Finally, propidium iodide solution (50 Ag/mL,

0.5 mL) was added. Cells were then left on ice for 15 to 30 minutes.Fluorescence emitted from propidium iodide-DNA complexes was quantified

after laser excitation of the fluorescent dye by fluorescence-activated cell

sorting flow cytometry (Becton Dickinson, Mountain View, CA). Finally, theextent of apoptosis was determined by measuring DNA content of the cells

below the G0-G1 peak. Terminal deoxynucleotidyl transferase–mediated

dUTP nick end labeling (TUNEL) assay was done in tissue sections using

apoptosis detection kit (Promega, Madison, WI) as described in the protocolprovided with the kit.

RNA isolation and reverse transcription-PCR. Total RNA was isolated

by using RNazol B reagent according to themanufacturer’s instructions. Total

RNA (3 Ag) was reverse transcribed into single-stranded cDNA using aMoloney murine leukemia virus reverse transcriptase and random hexamers

(Promega). Amplification of growth factor cDNA and h-actin cDNA as an

internal control in each reaction was carried out by PCR with the primers

described as follows: VEGF-A: 5V-AGCTACTGCCATCCAATCGC-3V ( forward)and 5V-GGGCGAATCCAATTCCAAGAG-3V (reverse); h-actin: 5V-GATGATGA-TATCGCCGCGCT-3V ( forward) and 5V-TGGGTCATCTTCTCGCGGTT-3V(reverse). Primers were used at a final concentration of 0.5 Amol/L. Reaction

Table 1. Sequences of the peptides selected by binding tosIL-6Ra

Phage clone Encoded insert

S1 LSLMPRL

S2 NPMMRPL

S3 QMRTTIR

S4 RLMMLQQS5 MLLQNRQ

S6 TLQASIL

S7 LSLITRL

NOTE: The phage display peptide library was subjected to four rounds

of biopanning against plate-immobilized recombinant human IL-6Ra

protein (sIL-6Ra). Individual phage clones selected by this procedure

were then analyzed for their ability to bind to immobilized sIL-6Ra ina phage ELISA assay. This resulted in the identification of seven

individual phage clones, which scored positively in this assay. The

sequences of encoded inserts from these clones are shown above.

Cancer Research

Cancer Res 2005; 65: (11). June 1, 2005 4828 www.aacrjournals.org



mixture was first denatured at 95jC for 10 minutes. The PCR conditionsapplied were 95jC for 1 minute, 52jC for 1 minute, and 72jC for 1 minute for

30 cycles followed by 72jC for 10 minutes. PCR products were visualized by

ethidium bromide staining after agarose gel electrophoresis.

Human vascular endothelial growth factor-A immunoassay. Condi-tioned medium (CM) was concentrated by Amicon Ultra Centrifugal Filter

Devices (Millipore). VEGF-A levels in culture supernatants were assayed

using a quantitative sandwich ELISA assay (R&D Systems) according to the

manufacturer’s instructions. In brief, cell supernatant (50 AL) was incubatedwith 50 AL of assay diluents for 2 hours at room temperature in a 96-well

tissue culture plate coated with a mAb against VEGF-A. After five

consecutive washes, a conjugate consisting of a polyclonal VEGF-A antibody

and HRP was added, and the mixture was incubated for 2 hours at roomtemperature. Following the subsequent addition of a color reagent,

absorbance was measured at 450 nm using a Thermo-Max microplate

reader (Molecular Devices Co., Menlo Park, CA). For standardizationpurposes, serial dilutions of recombinant human VEGF-A were assayed at

the same time. All experiments were carried out in triplicate.

Collection of conditioned medium. C33A/neo and C33A/IL-6 cells

were grown in DMEM containing 10% FBS, penicillin (100 units/mL), and

streptomycin (100 Ag/mL). At 90% confluence, cultured medium waschanged to serum-free medium, and C33A/IL-6 cells were incubated with S1

or S7 peptide for a further 24 hours. CM was collected, centrifuged to

remove any cellular contaminants, and then stored at �80jC until use.

Determination of human umbilical vein endothelial cell prolifera-tion. HUVECs were plated onto six-well dishes (Falcon, Becton Dickinson)

at a concentration of 2.5 � 105 cells per well in M199 supplemented with

10% FBS. One day after seeding, HUVECs were stimulated with CM from

cells treated in various ways, the CM having been mixed with M199medium. Twenty-four hours later, the viable cells were counted using a

trypan blue exclusion method. For the trypan blue exclusion assay, the cells

were first washed with PBS, trypsinized, and then resuspended in 1 mL PBS.

The numbers of clear and trypan blue–stained cells were then enumeratedusing a hemocytometer (improved Neubauer ruling) and a phase-contrast

light microscope, and the cell number was multiplied by the dilution factor

to obtain the corresponding total number of the cells in the sample. Eachindividual experiment was repeated thrice.

Tube formation assay. Assessment of in vitro capillary tube–like

formation used a growth factor–reduced basement membrane Matrigel

matrix. The Matrigel was thawed at 4jC and mixed to homogeneity using

Figure 1. Characterization of binding activity of selected phage display clones. A, individual sIL-6Ra-binding phage clones were tested for their ability to compete withIL-6 protein for binding to immobilized sIL-6Ra. Phages were added to sIL-6Ra-coated wells in a 96-well plate at a concentration of 1012 pfu/mL. After 1 hour ofincubation with phage, IL-6 protein was added to the wells, and bound IL-6 was then measured using biotin-conjugated anti–IL-6 mAb, HRP-conjugated streptavidin,and TMB substrate as described in Materials and Methods. Columns, mean of three independent experiments; bars, SD. *, P < 0.05, statistically significant increasecompared with the corresponding control value. B, phage clone 1 and phage clone 7 (1012 pfu/mL; which had been selected based on their ability to compete with IL-6for binding to immobilized sIL-6Ra) were added to various cell monolayers combined with IL-6 protein and incubated for 2 hours at room temperature. Afterwashing, the cell-bound phages were detected using HRP-conjugated anti-M13 mAb and TMB substrate as described in Materials and Methods. Columns, means ofat least three independent experiments in triplicate; bars, SD. C, phage clone 1 and phage clone 7 were tested for their binding activity and location in C33Acervical carcinoma cells by immunofluorescence staining. C33A cells (4 � 105) were seeded onto coverslips. After treatment with phage clone 1 and phage clone 7 for2 hours, immunostaining was done using anti-M13 antibody followed by FITC-conjugated antimouse IgG. Membrane localization of phage clone 7 was thenobserved by fluorescence microscopy. The position of the cell nucleus was confirmed by staining with Hoechst 33258 fluorescent dye. Original magnification, �400.Representative of three independent experiments. D, immunofluorescence staining of phage clone 7–treated C33A cell monolayer shows colocalization of IL-6Raand phage clone 7. Polyclonal goat antihuman IL-6Ra antibody and monoclonal anti-M13 antibody were applied to the phage clone 7–treated C33A cells and incubatedat 4jC overnight. After washes in PBS, the samples were treated with donkey antigoat IgG rhodamine-conjugated secondary antibody and FITC-conjugated goatantimouse secondary antibody for 1 hour at room temperature. The immunofluorescence-labeled cells were then analyzed by fluorescence microscopy asdescribed in Materials and Methods.

IL-6R–Specific Peptide Inhibits Tumor Growth




cooled pipette tips. The bottom of 96-well cell culture plates were coated

with a thin layer of Matrigel (40 AL), which was left to polymerize at 37jCfor 30 minutes. HUVECs were first resuspended in M199 medium

containing 1% serum to 2.5 � 104 cells/100 AL, mixed with 100 AL M199

medium containing 1% serum and 100 AL CM from C33A/neo or C33A/IL-6

cells treated in various ways, and finally plated onto the Matrigel-coatedsurface. Six hours later, the cells were fixed in 4% paraformaldehyde and

stained with 0.1% crystal violet. Three microscopic fields were selected at

random and photographed, and the number of tube-like structures per field

was measured as described previously (20).In vivo study of angiogenesis using Matrigel plug assay. This assay

was done as described previously (21). Angiogenesis was measured in

Matrigel. Matrigel (500 AL) containing serum-free medium from cells treatedin various ways was injected s.c. into 4- to 8-week-old female BALB/c nude

mice at sites lateral to the abdominal midline, two injections per mouse. All

measurements were made at least in triplicate. Animals were sacrificed

7 days after Matrigel injection. The Matrigel plugs were recovered andphotographed immediately. Plugs were then dissolved in PBS and incubated

at 4jC overnight. Hemoglobin levels were determined using Drabkin’s

solution (Sigma Chemical) according to the manufacturer’s instructions.

Tumor growth inhibition experiment. The C33A/neo and C33A/IL-6cells were well established as described previously (16). For the tumor

growth inhibition experiments, 6- to 8-week-old female severe combined

immunodeficient (SCID) mice (supplied by the animal center in theNational Taiwan University College of Medicine, Taipei, Taiwan) were

inoculated s.c. with 1 � 106 C33A/neo or C33A/IL-6 cells. Beginning 3 days

later, S1 or S7 peptide (dissolved in PBS) was injected i.p. every 2 days.Tumor development was followed in individual animals (10 per group) by

twice weekly sequential caliper measurements of length (L) and width (W).

Tumor volume was calculated by the formula LW2/2. After 42 days, the mice

were killed; the tumors were removed and weighed. A segment was excisedand fixed in 10% neutral buffered formalin.

Results

Identification of peptide sequences that bind to humaninterleukin-6 receptor A. To identify peptides that target IL-6Raand inhibit IL-6 binding to IL-6Ra, we used the phage displaytechnique to screen a 7-mer random cyclic peptide phage library. Arandom 7-mer peptide phage library composed of 2 � 109

independent phage clones was obtained from a biopanning screenagainst plate-bound sIL-6Ra. In one setting, the IL-6Ra-boundphages were recovered by elution with acidic buffer [0.2 mol/Lglycine-HCl (pH 2.2)]. For each biopanning, the number ofphages (pfu) in the inputs and outputs were compared withdetermine the degree of selection. The total number of phagesbound to IL-6Ra was increased from 7.4 � 104 pfu in the first roundto 4.6 � 106 pfu in the third round. After three rounds of selection,roughly 10.3% (31 of 300) of the phage clones analyzed exhibited IL-6Ra-binding activity (data not shown). DNA sequencing of these 31sIL-6Ra-binding clones showed that seven independent peptidesequences were selected (Table 1). In light of the ability of theselected phage clones to bind sIL-6Ra, we examined the ability ofthese phage clones to block interaction between IL-6Ra and itsligand, IL-6. To this end, a fixed amount of phage particles (1011 pfu/mL) was added to sIL-6Ra-coated wells, and their ability to blockthe binding of IL-6 to sIL-6Ra was examined. This analysis revealedthat phage S7 significantly inhibited the binding of IL-6 to sIL-6Ra(Fig. 1A); phage S5 and phage S6 inhibited the interaction to a lesserextent, and other clones did not interfere with binding. Thus, phageS7 was chosen for further study. The binding affinity of phage S7 todifferent cell lines was determined by the in vitro binding assay.Phage S7 showed higher binding affinity than phage S1 in allmembrane-type IL-6Ra-expressing cell lines but not in IL-6Ra-negative HUVEC cells (Fig. 1B). Immunofluorescence stainingrevealed that phage S7, but not phage S1, binds to the plasmamembrane of C33A cervical cancer cells (Fig. 1C) and other IL-6Ra-expressing cell lines (data not shown). Further confirmation of thespecificity of phage S7 binding to IL-6Ra was obtained by laser scanconfocal microscopic observation of C33A cells double stained withanti–IL-6Ra and anti-M13 antibodies, which showed that phage S7tightly colocalized with IL-6Ra on the cell membrane (Fig. 1D). Theabove data clearly show that phage S7 specifically binds to IL-6Raand blocks the interaction between IL-6 and IL-6Ra.S7 peptide blocks the interaction between interleukin-6 and

interleukin-6 receptor A. Based on the above results, phage-encoded peptides (specifically S7) were synthesized. An in vitrocompetition assay revealed that peptide S7 could antagonize thebinding of IL-6 to sIL-6Ra in a dose-dependent manner (Fig. 2A) andsignificantly reduce the binding of IL-6 to IL-6Ra in five different celllines (Fig. 2B). These data clearly show that peptide S7 binds to IL-6Ra and interferes with the interaction between IL-6 and IL-6Ra.S7 peptide decreased interleukin-6–induced Mcl-1 expres-

sion and antiapoptosis in cervical cancer cells. It has beenreported that IL-6 acts as an antiapoptotic factor via up-regulation ofMcl-1 protein through the PI3K/Akt and MAPK signaling pathwaysin a variety of human malignancies, including cervical cancer (16,22–25). To examine the effects of peptide S7 in IL-6-induced

Figure 2. S7 peptide competes with IL-6 protein for binding to IL-6Ra.A, various concentrations of the S7 peptide or the S1 control peptide weretested for ability to competitively inhibit the binding of IL-6 protein to immobilizedsIL-6Ra. Various concentrations of S1 or S7 peptide were incubated withsIL-6Ra-coated plates for 1 hour at room temperature. After this preincubation,human IL-6 protein (50 ng/mL, 100 AL) was added directly to the wells withoutremoval of the phage for 2 hours. The bound IL-6 was then measured using ELISAas described in Materials and Methods. Columns, means of three independentexperiments; bars, SD. Competition ability of S7 peptide was significantly elevatedat 25 to 250 Amol/L compared with control. *, P < 0.05, two-tailed Student’st test. Each treatment was done in three separate experiments and incubationswere conducted in triplicate. B, S7 peptide (50 Amol/L) and S1 control peptide(50 Amol/L) were tested for ability to competitively inhibit the binding of IL-6 proteinto IL-6Ra in various cell lines. The bound IL-6 was then measured using ELISAas described in Materials and Methods. Each treatment was done in threeseparate experiments and incubations were conducted in triplicate. Columns,means of three independent experiments; bars, SD. *, P < 0.05, statisticallysignificant increase compared with the corresponding value of S1 peptide.

Cancer Research




antiapoptotic signaling, the forms of Akt and MAPK [which arereported to function in the regulation of cervical cancer cell survival(16)] were examined. Western blot analysis revealed that thephosphorylated Akt and ERK1/2 MAPK levels were increased in IL-6-treated C33A cervical cancer cells and this activation could besignificantly inhibited by treatment with S7 peptide but not with S1control peptide (Fig. 3A). Furthermore, IL-6-induced Mcl-1 proteinup-regulation was also abolished by S7 peptide (Fig. 3B , top).Whether S7 peptide can functionally block IL-6-induced antiapop-tosis in cervical cancer cells was investigated by measuring theeffect of various treatments on apoptotic cell death detected by flowcytometry. The data revealed that IL-6 can noticeably protect C33Acells from cisplatin-induced apoptosis and, more importantly, S7peptide interferes with this protection (Fig. 3B , bottom). It has beenreported that two IL-6 type cytokines, IL-11 and CNTF, induceSTAT3 phosphorylation in HT-29 and HepG2 cells, respectively(26, 27). In a parallel experiment to test the specificity of S7 peptide,

we analyzed the inhibitory effect of S7 peptide on IL-11- and CNTF-mediated signaling. As shown in Fig. 3C , S7 peptide did not revealany inhibitory effect on IL-11- or CNTF-induced phosphorylation ofSTAT3. Together, these data show that S7 peptide can block IL-6-mediated survival signaling and subsequent antiapoptosis incervical cancer cells.S7 peptide inhibits interleukin-6–induced vascular endothe-

lial growth factor-A expression and angiogenesis in differentcancer cell lines. Besides its role in antiapoptosis, IL-6 is importantin angiogenesis via up-regulation of VEGF-A in cervical cancer (15).Thus, the effects of S7 peptide on the expression of VEGF-A proteinand mRNA in C33A cells were examined byWestern blot and reversetranscription-PCR (RT-PCR) analysis, respectively. VEGF-A proteinand mRNA levels were increased in response to IL-6 treatment andthis induction was almost totally blocked by S7 peptide (Fig. 4A).Likewise, treatment with S7 peptide also inhibited VEGF-A up-regulation in response to transfection of human IL-6 cDNA into

Figure 3. S7 peptide inhibits IL-6-mediated survival signaling and subsequent antiapoptosis. A, 80% confluent C33A cells were starved for 24 hours and then treatedwith human IL-6 protein (50 ng/mL) in the presence or absence of S1 peptide (50 Amol/L) or S7 peptide (50 Amol/L). Cell lysates were obtained and subjected toSDS-PAGE followed by immunoblotting with various antibodies, such as anti–phospho-ERK1/2, anti–phospho-Akt, anti-ERK1/2, and anti-Akt. B, top, Western blotanalyses of the antiapoptotic protein Mcl-1 expression in the presence or absence of IL-6 protein with or without S7 peptide. Equal amounts of cell lysates (75 Ag) wereresolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with specific anti–Mcl-1 antibodies; h-actin served as the internal loadingcontrol. Representative of at least three independent experiments. Bottom, S7 peptide inhibits IL-6-induced protection against cisplatin-induced apoptosis in C33Acervical carcinoma cells. Cells were treated with IL-6 protein combined with S1 or S7 peptide and incubated with 5 Amol/L cisplatin for 24 hours. FACScan was done asan apoptosis assay as described in Materials and Methods. Columns, means of three independent experiments; bars, SD. *, P < 0.05, statistically significant decreasecompared with values of cisplatin-treated control. Representative of at least three independent experiments. C, HT-29 and HepG2 cells were treated with humanIL-11 protein (50 ng/mL) and human CNTF protein (1 nmol/L) in the presence or absence of S1 peptide (50 Amol/L) or S7 peptide (50 Amol/L). Cell lysates were obtainedand subjected to SDS-PAGE followed by immunoblotting with various antibodies, such as anti–phospho-STAT3 and anti-STAT3. Numbers below lanes, level ofprotein expression compared with the control.





C33A cells (Fig. 4B). In addition, IL-6 has been reported to induceVEGF-A expression in other cancer cells, such as multiple myelomaand BCC (28, 29). Thus, the ability of S7 peptide to inhibit IL-6-induced VEGF-A expression in these cells was examined. VEGF-Awas dramatically induced by IL-6 treatment in RPMI 8226 multiplemyeloma cells (Fig. 4C , left) and BCC cells (Fig. 4C , right). Moreimportant, addition of S7 peptide abolished IL-6-induced VEGF-Aexpression in both cell lines (Fig. 4C). Furthermore, by theapplication of a human VEGF-A immunoassay, we were also ableto detect the production of secreted VEGF-A in response to IL-6 withor without S7 peptide. As shown in Fig. 4D , elevated levels of VEGF-Asecretion due to IL-6 stimulation paralleled the increased expressionof VEGF-A protein, indicating that a mature and functionally activeVEGF-A protein was simultaneously being generated in IL-6-treatedcells. The IL-6Ra antagonist S7 peptide notably decreased IL-6-mediated VEGF-A secretion in all three cell lines (Fig. 4D). Thesedata clearly show that S7 peptide inhibits IL-6-induced VEGF-A up-regulation in several different types of cancer cells.The crucial role of VEGF-A in angiogenesis (30) also prompted us

to examine whether S7 peptide inhibited IL-6-induced angiogenesis.To examine whether S7 peptide inhibited IL-6-induced angiogenicactivity, angiogenesis assays (such as proliferation and capillary-like tubule formation by HUVECs in vitro) were done with CM

collected from cells treated as indicated. As shown in Fig. 5A , CMfrom IL-6-overexpressed cells (IL-6-CM) significantly increased theproliferation of HUVECs compared with the control CM (neo-CM),and IL-6-CM-induced HUVECs proliferation seemed to be greatlyattenuated by S7 peptide but not by S1 peptide.The effect of IL-6-CM on the morphologic differentiation of

HUVECs was investigated by application of a tube-like structureformation assay. HUVECs were placed onto growth factor–reducedMatrigel with or without CM from IL-6-overexpressed C33A cells(C33A/IL-6-CM) for 6 hours. C33A/IL-6-CM stimulation led to theformation of elongated and robust tube-like structures (Fig. 5B , top ,II) composed of many more cells than was the case for the control(Fig. 5B , top , I). In addition, we also found that C33A/IL-6-CM-induced tube-like cell cord formation was almost completelyprevented by S7 peptide but not by S1 peptide (Fig. 5B , top , IIIand IV ). Quantitation of these cell cords, as described previously(31), revealed that C33A/IL-6-CM induced enhanced tube-likestructure formation (Fig. 5B , bottom , 5.1-fold induction comparedwith control) and that this effect was almost completely inhibitedby S7 peptide.Next, we investigated whether S7 peptide inhibited IL-6-induced

angiogenesis by the Matrigel plug assay. Matrigel plugs containingCM from vector control cells or IL-6-overexpressing C33A cells

Figure 4. S7 peptide inhibits IL-6-induced VEGF-A expression in different cell types. A, determination of the protein (top ) and mRNA (bottom ) levels of VEGF-A inIL-6-treated C33A cells in the presence or absence of S7 peptide. C33A cells were treated with IL-6 (50 ng/mL) combined with or without S7 peptide (50 Amol/L),following which Western blot analysis was used to determine VEGF-A (top ). Total RNA was isolated and subjected to RT-PCR using specific primers for VEGF-A(bottom ). h-actin served as the internal loading control. Representative of at least three independent experiments. B, IL-6-overexpressing C33A cells (C33A/IL-6 ) orvector control cells (C33A/neo ) were treated with S1 peptide (50 Amol/L) or S7 peptide (50 Amol/L) for 16 hours followed by analysis the expression of VEGF-A proteinand mRNA by Western blot and RT-PCR, respectively. h-actin served as the internal loading control. Representative of at least three independent experiments.C, determination of the protein and mRNA levels of VEGF-A in IL-6-treated RPMI 8226 multiple myeloma cells (left ) and BCC cells (right ) in the presence or absence ofS7 peptide by Western blot analysis and RT-PCR, respectively. Representative of at least three independent experiments. D, production of VEGF-A in C33A cells,BCC cells, and RPMI 8226 cells stably transfected with human IL-6 cDNA or control vector in the presence or absence of S7 peptide for 24 hours. The postculturedmedium was collected and assayed for VEGF-A by immunoassay as described in Materials and Methods. Columns, means of at least three independentexperiments in triplicate; bars, SD. *, P < 0.05, statistically significant increase compared with the corresponding vector control value.

Cancer Research




combined with or without S7 peptide (obtained by mixing the CMfromC33A/IL-6F S7 with 0.5mL coldMatrigel) were implanted intoC57BL/6J mice and recovered 7 days later. Macroscopic analysisrevealed that the plugs containing the C33A/neo-CM (Fig. 5C ,top , I) were much paler than those containing C33A/IL-6-CM(Fig. 5C , top , II). Supportively, the IL-6-mediated angiogenicactivity was also reduced by S7 peptide but not by S1 peptide inthis in vivo assay (Fig. 5C , top , III and IV ). Similarly, hemoglobinlevel was clearly elevated in plugs containing C33A/IL-6-CM thanin plugs containing C33A/neo-CM, and hemoglobin induction wasalmost totally inhibited by S7 peptide (Fig. 5C , bottom). The abovedata strongly suggest that IL-6-induced angiogenic effects can beblocked by S7 peptide both in vitro and in vivo .S7 peptide suppressed interleukin-6–induced tumor growth

in an animal model. Finally, to explore whether S7 peptide couldrepresent a therapeutic strategy to target IL-6-mediated tumorgrowth in vivo , SCID mice were injected s.c. with IL-6-overexpressedC33A human cervical carcinoma cells (C33A/IL-6) or C33A/neovector control cells. S7 or S1 peptide (50 mg/kg) was injected i.p.every 2 days, and the growth of the tumors was assessed over time.The results of this analysis showed that administration of S7 (butnot of S1) peptide led to a significant reduction of IL-6-inducedtumor growth, and S7 peptide inhibited tumor growth by 76%(P < 0.05) at the end of the experiment (Fig. 6A). In addition, IL-6-mediated VEGF-A expression and phosphorylation of Akt andERK in tumors were significantly inhibited by S7 peptide (Fig. 6B).Furthermore, we investigated whether S7 peptide induced apoptoticcell death in tumor by TUNEL assay. As shown in Fig. 6B (right), thenumber of apoptotic cells was notably increased in S7 peptide–treated tumors but not in S1 peptide–treated tumors. Thus, theseresults show a vigorous antitumor effect of S7 peptide in IL-6-induced signaling and tumor growth in vivo .

Discussion

Data from experimental and clinical studies have shown a criticalrole of IL-6 in active cancers (5). IL-6 activities are mediated through

specific low-affinity binding to a chain (gp80) and subsequentlythrough the ability of this complex to recruit h subunit (gp130) andthereby trigger signal transduction (6). Notably, in contrast toubiquitious expression of the transmembrane spanning gp130,cellular distribution of the cognate IL-6R is limited and itsexpression is predominantly confined to hepatocytes and leukocytesubpopulations (32). However, gp80 could be cleaved from the cellmembrane molecule by a transmembrane metalloproteinase ortranslated from an alternatively spliced mRNA (33). This soluble

Figure 5. S7 peptide abolishes IL-6-mediated angiogenic response in vitro andin vivo . A, HUVEC cell proliferation was measured in 24-well plates followingtreatment with CM from vector control cells (neo-CM ) or that derived fromIL-6-overexpressed cells (IL-6-CM ) in the presence or absence of S7 peptide(50 Amol/L). Trypan blue exclusion method was done to determine the level ofHUVEC cell proliferation as described in Materials and Methods. Columns,means of at least three independent experiments in triplicate; bars, SD. *, P <0.05, statistically significant increase compared with the corresponding controlvalue. B, top, HUVECs were seeded onto the Matrigel layer in 24-well plates.The assay was done in the presence of neo-CM (I ) and IL-6-CM (II ; CM fromC33A/neo or C33A/IL-6 cells, respectively) or IL-6/S1-CM (III ) and IL-6/S7-CM(IV ; CM from C33A/IL-6 cells treated with S1 or S7 peptide, respectively, for 6hours). The experiment was conducted thrice with similar results. Bottom,quantitative results of tube formation assay. Briefly, cells were washed, fixed inmethanol, and then stained in DifQuik solution some 2 hours before tube areameasurement. Three replicate fields of triplicate wells were digitallyphotographed. Tube area was quantified using MetaMorph software (UniversalImaging Corp., West Chester, PA). Columns, means of three independentexperiments; bars, SD. *, P < 0.05, statistically significant increase comparedwith the corresponding value of CTL-CM. C, top, S7 peptide inhibitsangiogenesis in the Matrigel plug assay. Matrigel mixture containing neo-CM (I ),IL-6-CM (II), IL-6/S1-CM (III ), or IL-6/S7-CM (IV ) was injected s.c. into nudemice at sites lateral to the abdominal midline. Animals were sacrificed 7 daysafter injection. The mouse skin was detached and the Matrigel plug wasrecovered and photographed immediately. Bottom, the plugs were then mincedand homogenized with a tissue homogenizer, and hemoglobin levels in the plugswere determined using Drabkin’s solution according to the manufacturer’sinstructions as described in Materials and Methods. Columns, means of threeindependent experiments; bars, SD. *, P < 0.05, statistically significant decreasecompared with values of neo-CM-treated control.





receptor (sIL-6R) binds IL-6 with an affinity similar to that of thecognate receptor (0.5-2 nmol/L), and more importantly, the sIL-6R/IL-6 complex is capable of activating cells via interaction withmembrane-bound gp130 (32). This unique feature makes the sIL-6R/IL-6 complex an agonist rather than an antagonist for target cells.Consequently, elevated sIL-6R levels have been documented innumerous clinical conditions, indicating that its production is partof the disease process (34). By screening a random cyclic peptidephage display library, this study has identified a novel peptide thatcan specifically bind to IL-6Ra, negatively regulate IL-6 signaling,and diminish IL-6-mediated cervical tumorigenesis in vivo .To date, we have identified three different phage clones that can

bind to sIL-6R and compete with IL-6 for binding to IL-6R as shownby ELISA (Fig. 1A). Phage S7, expressing the peptide LSLITRL, wasthe most efficient phage with high affinity to sIL-6R. Cell basedexperiments have further shown that phage S7 had good binding tomembrane-type IL-6Ra-positive cell lines but not to membrane-type IL-6Ra-negative HUVECs (Fig. 1B). Moreover, the addition ofchemically synthesized phage S7 encoded peptide has led to asignificant reduction in the binding of IL-6 to IL-6Ra in various celllines. All these specific inhibitory effects were verified by thecolocalization of phage S7 and IL-6Ra at the cell membrane usinglaser scan confocal microscopy (Fig. 1C and D).

Cervical cancer is a major health problem worldwide despiteadvances in screening programs. The progression of cervical cancerhas been associated with increased levels of IL-6 in serum andcervicovaginal secretions (11). IL-6 is capable of promoting cervicaltumor progression and dissemination by autocrine and/or paracrinemechanisms (13, 35). Platinum-based chemotherapy is the treat-ment of choice for the management of patients with metastatic andrecurrent cervical cancer. We have shown previously that over-expression of IL-6 in cervical cancer cells caused amarked resistanceto apoptosis induced by cisplatin or doxorubicin. This effect wasprimarily attributed to the up-regulation of Mcl-1 through a PI3K/Akt pathway (16). In the current study, S7 peptide was able toeffectively inhibit IL-6-induced phosphorylation of Akt and ERK,resulting in down-regulation of Mcl-1 (Fig. 3A and B). Treatmentwith S7 peptide blocks IL-6 signaling mediated through gp130 andthus sensitizes cervical cancer cells to cisplatin in vitro . EndogenousIL-6 is a resistance factor for chemotherapeutic compounds used inthe treatment of cancers (such as prostate cancer and renal cellcarcinoma) that are poorly responsive to chemotherapy. Enhancedcytotoxicity could be achieved in these cases using a combination ofcisplatin with anti–IL-6 or anti–IL-6R mAb (36, 37). The addition ofagents with the ability to inhibit IL-6 activity in humans may thusimprove combination therapy for cancer patients.

Figure 6. S7 peptide inhibits IL-6-mediated tumorgrowth in vivo . A, SCID mice were inoculateds.c. with 1 � 106 IL-6-overexpressing C33Acervical carcinoma cells (C33A/IL-6) or vectorcontrol cells (C33A/neo; 10 mice per group). After3 days, treatment was initiated with i.p. injectionsof 50 mg/kg S1 or S7 peptide every 2 days intwo other C33A/IL-6-inoculated groups (10 miceper group). The tumor size in SCID mice withvarious treatments was measured every 3 days,and the volume was calculated. Mean F SE of10 individual mice. B, animals were sacrificed 42days after inoculation with tumor cells (39 daysafter injection with S1 or S7 peptide). The tumorwas recovered and photographed immediatelyfollowing the analysis of protein expression andapoptotic cell death by Western blot and TUNELassay, respectively.

Cancer Research




That IL-6 activates cervical tumor angiogenesis and induces rapidtumor growth has been shown in an animal model (15). The presentstudy verified the in vivo efficacy of S7 IL-6R antagonist peptide byshowing its vigorous suppression of IL-6-induced tumor growth inSCID mice (Fig. 6), which correlates with substantial antiangiogenicactivity in vivo . Similarly, anti-VEGF antibody was shown tosubstantially inhibit IL-6-mediated angiogenesis and tumor growthin nude mice (15). Interestingly, recent studies show that IL-6 alsoinduced basic fibroblast growth factor–dependent angiogenesis inBCC via Janus-activated kinase/STAT3 and PI3K/Akt signaling (10),suggesting that interruption of IL-6 signaling might be a morefeasible molecular target for blocking IL-6-stimulated angiogenesisin diverse human cancers. Furthermore, during late stages of tumorgrowth, markedly increased serum level and tumor expression of IL-6 are responsible for the development of cancer cachexia (38). Inagreement with animal studies demonstrating that specific IL-6inhibitors can block tumor-induced cachexia in vivo (39, 40), manyclinical trials have shown that anti–IL-6 mAb therapy decreased theincidence of cancer-related anorexia and cachexia in patients with

malignant disease (5). Taken together, these results support thenotion that inhibition of IL-6 activity may be useful in themanagement of cancer patients with advanced disease.In summary, we found a novel small peptide that was a targeting

ligand against IL-6Ra, elucidated the molecular mechanism of itsaction, and showed its antitumor efficacy both in vitro and in vivo .Numerous clinical studies have been reported using targeted anti–IL-6 mAb therapy for cancer. The antibodies were well tolerated inthe vast majority of studies. Our findings highlight the potentialapplication of peptide-S7 in the management of patients withmalignant disease.

Acknowledgments

Received 1/20/2005; revised 3/14/2005; accepted 3/22/2005.Grant support: National Science Council, Taiwan grants NSC-92-2314-B002-329

and NSC-2314-B002-172.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Julie Yu for carefully editing the article.

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Editor's Note

Editor's Note: A Novel Peptide SpecificallyBinding to Interleukin-6 Receptor (gp80)Inhibits Angiogenesis and Tumor GrowthJen-Liang Su, Kuo-Pao Lai, Chi-An Chen, Ching-Yao Yang,Pei-Sheng Chen, Chiao-Chia Chang, Chia-Hung Chou,Chi-Lun Hu, Min-Liang Kuo, Chang-Yao Hsieh, andLin-Hung Wei

The editors weremade aware of concerns regarding potentialmanipulation of data inthis article (1). An internal review by the editors determined that in Fig. 3A, the P-ERKWestern blot bands in lanes 3 and 4 appear to be spliced together. In addition, thesame Western blot image appears to have been used in Fig. 3C (STAT3) and Fig. 6B(b-actin). Because satisfactorily corrected figures could not be provided, the editorsare publishing this note to alert readers to these concerns.

Reference1. Su JL, Lai KP, Chen CA, Yang CY, Chen PS, Chang CC, et al. A novel peptide specifically binding to

interleukin-6 receptor (gp80) inhibits angiogenesis and tumor growth. Cancer Res 2005;65:4827–35.

Published online July 15, 2019.Cancer Res 2019;79:3791doi: 10.1158/0008-5472.CAN-19-1712�2019 American Association for Cancer Research.

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http://crossmark.crossref.org/dialog/?doi=10.1158/0008-5472.CAN-19-1712&domain=pdf&date_stamp=2019-6-28

2005;65:4827-4835. Cancer Res Jen-Liang Su, Kuo-Pao Lai, Chi-An Chen, et al. Receptor (gp80) Inhibits Angiogenesis and Tumor GrowthA Novel Peptide Specifically Binding to Interleukin-6

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