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[Cancer Research 65, 4827-4835, June 1, 2005]
© 2005 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

A Novel Peptide Specifically Binding to Interleukin-6 Receptor (gp80) Inhibits Angiogenesis and Tumor Growth

Jen-Liang Su1, Kuo-Pao Lai2, Chi-An Chen3, Ching-Yao Yang1,4,5, Pei-Sheng Chen1, Chiao-Chia Chang1, Chia-Hung Chou1, Chi-Lun Hu2, Min-Liang Kuo1, Chang-Yao Hsieh2 and Lin-Hung Wei2

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

Requests for reprints: Lin-Hung Wei, Department of Oncology, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei 100, Taiwan. Phone: 886-2-2312-3456, ext. 7140; Fax: 886-2-2371-1174; E-mail: weilh{at}ha.mc.ntu.edu.tw.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Experimental and clinical findings support the essential role of interleukin (IL)-6 in the pathogenesis of various human cancers and provide a rationale for targeted therapeutic investigations. A novel peptide, S7, which selectively binds to IL-6 receptor (IL-6R) {alpha} chain (gp80) and broadly inhibits IL-6-mediated events, was identified using phage display library screening. The synthetic S7 peptide specifically bound to soluble IL-6R as well as cognate human IL-6R{alpha}, resulting in a dose-dependent blockade of the interaction between IL-6 and IL-6R{alpha}. S7 peptide prevents IL-6–mediated survival signaling and sensitizes cervical cancer cells to chemotherapeutic compounds in vitro. The in vitro analysis of antiangiogenic activity showed that S7 peptide substantially inhibits IL-6–induced vascular endothelial growth factor-A expression and angiogenesis in different cancer cell lines. Furthermore, S7 peptide was bioavailable in vivo, leading to a significant suppression of IL-6–induced vascular endothelial growth factor–mediated cervical tumor growth in severe combined immunodeficient mice. These observations show the feasibility of targeting IL-6/IL-6R interaction using the small peptide and highlight its potential in the clinical applications.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin (IL)-6 is a secreted, pleiotropic cytokine that regulates the physiologic and pathologic responses to various disease processes, including inflammation, myocardial infarction, autoimmune disorder, Alzheimer's disease, osteoporosis, and hematologic and nonhematologic malignancies (1–5). The biological activities of IL-6 are mediated through binding to a membrane-bound glycoprotein IL-6 receptor (IL-6R) {alpha} chain (gp80) on target cells. Specifically, the IL-6/IL-6R complex initiates homodimerization of the ubiquitously expressed gp130 (ß chain), activates a cytoplasmic tyrosine kinase bound to gp130, and then triggers Janus-activated kinase/signal transducers and activators of transcription (STAT), Ras/mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K)/Akt signaling (6, 7). IL-6 signaling mediated via gp130 interferes with many cellular functions, such as cell growth and survival, differentiation, cell mobility, and angiogenesis, and is thereby critically involved in the pathogenesis of various human cancers (8–10). Therefore, blocking IL-6 activity may be of clinical benefit in the management of 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 with cervical cancer, and its production is related to the severity of cervical neoplasia (11). Cervical cancer cells, as well as lymphocytes, macrophages, and endothelial cells, produce substantially high microenvironmental IL-6 levels, which promote cervical tumor growth via autocrine or paracrine mechanisms or both (12, 13). Previously, we showed that, by inducing vascular endothelial growth factor (VEGF) via the STAT3 pathway, IL-6 is involved in the angiogenic switch that occurs during cervical oncogenesis (14, 15). Furthermore, overexpression of IL-6 in cervical cancer cells confers resistance to cisplatin cytotoxicity by up-regulating the apoptotic threshold through a PI3K/Akt signaling (16). Taken together, these observations suggest that blocking IL-6 activity would be a feasible strategy 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 in various applications, including epitope mapping and identification of peptide mimics of nonpeptide ligands. In fact, peptides that target tumor cells and tumor vasculature have been successfully identified using the phage display approach (17, 18). Here, we apply the recombinant system to the manufacture of identified peptides that react with the IL-6R. The experimental results showed that this selective interactive ligand is valid for inhibiting tumor angiogenesis and growth of human cervical cancer in vitro and in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies and reagents. Antihuman VEGF antibody, anti–ß-actin antibody, biotin-labeled anti–IL-6 antibody, anti–IL-6R{alpha} antibody, and human IL-6R{alpha} 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-ERK1/2 antibody, 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. Matrigel was 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 hepatoma cells, 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 µg/mL), L-glutamine (2 mmol/L), and sodium pyruvate (1 mmol/L, Invitrogen). Cell cultures were maintained at 37°C in a humidified 5% CO2 atmosphere. Human umbilical vein endothelial cells (HUVEC), endothelial cell growth medium, trypsin-EDTA, and trypsin-neutralizing solutions were purchased from Clonetics (San Diego, CA). All cell cultures were conducted according to the supplier's recommendations.

Screening a 7-mer phage display library with soluble KDR. The procedure for screening the phage display library was modified according to instructions of the manufacturer of the kit (New England Biolabs, Beverly, MA). The cell culture dishes (60 mm diameter) were coated with human IL-6R{alpha} proteins. IL-6R{alpha} protein was added at 100 µg/mL in 3 mL/dish and incubated at 4°C for 24 hours before blocking with phosphate buffer containing 1% bovine serum albumin (BSA) for 1 hour at 37°C. The phage library containing 1012 clones was sequentially added to non-bait-coated dishes (those coated with human IL-6Rß proteins) for preabsorption. In each case, the library was shaken gently at room temperature for 1 hour. Finally, the preabsorbed library was applied to soluble IL-6R{alpha} (sIL-6R{alpha})–coated dishes for specific screening. After thorough washing, plate-bound phage clones were eluted with elution buffer [0.22 mol/L glycine-HCl (pH 2.2)] and neutralized immediately. Four rounds of selection were done, after which individual plaques were picked at random and subjected to analysis by phage ELISA and DNA sequencing following amplification in Escherichia coli ER2537.

Phage ELISA using various types of cells. C33A, HeLa, and Siha cervical carcinoma cells, BCC cells, and HEK293 cells were grown in DMEM supplemented with 10% FBS. Human umbilical cords were digested with 0.1% collagenase II, and HUVECs were collected and grown in M199 medium supplemented with 10% FBS. For phage ELISA assays, cells were seeded in 96-well plates at 90% confluence. After an overnight incubation, cells were fixed with ice-cold glutaraldehyde (0.125%) in PBS for 10 minutes at room temperature and then washed with PBS. PBS containing 3% BSA was used to block the plates by overnight incubation at 4°C. After blocking, phages [5 x 1012 plaque-forming units (pfu)/mL] were added to the plates and incubated for 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 ELISA using a horseradish peroxidase (HRP)–conjugated anti-M13 monoclonal antibody (mAb).

Competition assay of positive phages and synthesized peptides. sIL-6R{alpha} (250 µg/mL) was immobilized on 96-well plates and blocked with phosphate buffer containing 1% BSA for 1 hour at 37°C. In competition experiments, seven different phage clones (1012 pfu/mL), which were selected by the above phage ELISA or various concentrations of S1 or S7 peptide, were then incubated with sIL-6R{alpha}-coated or cell-coated plates for 1 hour at room temperature. The bound IL-6 was detected by ELISA. After the preincubation, human IL-6 protein (50 ng/mL, 100 µL) was added directly to the wells without removal of the phage. After additional 2 hours of 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 added for 1 hour and HRP-conjugated streptavidin then was added for 1 hour. The free avidin conjugate was washed away and freshly prepared substrate solution (TMB) was then added to each well. The reaction was allowed to proceed for 10 minutes, after which the color development was stopped by the addition of H3PO4 (1.0 mol/L). The absorbance at 450 nm was read with a reference wavelength of 650 nm (A450/650).

Immunofluorescence staining. Suspensions of 2.5 x 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-6R{alpha} antibody and monoclonal anti-M13 antibody were applied to the slides at a dilution of 1:50 and incubated at 4°C 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 µm.

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 µg/mL), aprotinin (1 µg/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, immunoreactive 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 terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling assay. Cells were harvested and washed with PBS, and hypodiploid cells were analyzed by flow cytometry. Briefly, 1 x 106 cells were washed with PBS, resuspended in 500 µL buffer (0.5% Triton X-100, PBS, 0.05% RNase A), and incubated for 30 minutes. Finally, propidium iodide solution (50 µg/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, the extent 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 protocol provided with the kit.

RNA isolation and reverse transcription-PCR. Total RNA was isolated by using RNazol B reagent according to the manufacturer's instructions. Total RNA (3 µg) was reverse transcribed into single-stranded cDNA using a Moloney murine leukemia virus reverse transcriptase and random hexamers (Promega). Amplification of growth factor cDNA and ß-actin cDNA as an internal control in each reaction was carried out by PCR with the primers described as follows: VEGF-A: 5'-AGCTACTGCCATCCAATCGC-3' (forward) and 5'-GGGCGAATCCAATTCCAAGAG-3' (reverse); ß-actin: 5'-GATGATGATATCGCCGCGCT-3' (forward) and 5'-TGGGTCATCTTCTCGCGGTT-3' (reverse). Primers were used at a final concentration of 0.5 µmol/L. Reaction mixture was first denatured at 95°C for 10 minutes. The PCR conditions applied were 95°C for 1 minute, 52°C for 1 minute, and 72°C for 1 minute for 30 cycles followed by 72°C for 10 minutes. PCR products were visualized by ethidium bromide staining after agarose gel electrophoresis.

Human vascular endothelial growth factor-A immunoassay. Conditioned 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 µL) was incubated with 50 µL 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 room temperature. 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 standardization purposes, 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 µg/mL). At 90% confluence, cultured medium was changed 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 –80°C until use.

Determination of human umbilical vein endothelial cell proliferation. HUVECs were plated onto six-well dishes (Falcon, Becton Dickinson) at a concentration of 2.5 x 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 M199 medium. 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 enumerated using 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. Each individual 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 4°C and mixed to homogeneity using cooled pipette tips. The bottom of 96-well cell culture plates were coated with a thin layer of Matrigel (40 µL), which was left to polymerize at 37°C for 30 minutes. HUVECs were first resuspended in M199 medium containing 1% serum to 2.5 x 104 cells/100 µL, mixed with 100 µL M199 medium containing 1% serum and 100 µL CM from C33A/neo or C33A/IL-6 cells treated in various ways, and finally plated onto the Matrigel-coated surface. 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 µL) containing serum-free medium from cells treated in 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 and photographed immediately. Plugs were then dissolved in PBS and incubated at 4°C 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-6 cells 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 the National Taiwan University College of Medicine, Taipei, Taiwan) were inoculated s.c. with 1 x 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 excised and fixed in 10% neutral buffered formalin.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of peptide sequences that bind to human interleukin-6 receptor {alpha}. To identify peptides that target IL-6R{alpha} and inhibit IL-6 binding to IL-6R{alpha}, we used the phage display technique to screen a 7-mer random cyclic peptide phage library. A random 7-mer peptide phage library composed of 2 x 109 independent phage clones was obtained from a biopanning screen against plate-bound sIL-6R{alpha}. In one setting, the IL-6R{alpha}-bound phages were recovered by elution with acidic buffer [0.2 mol/L glycine-HCl (pH 2.2)]. For each biopanning, the number of phages (pfu) in the inputs and outputs were compared with determine the degree of selection. The total number of phages bound to IL-6R{alpha} was increased from 7.4 x 104 pfu in the first round to 4.6 x 106 pfu in the third round. After three rounds of selection, roughly 10.3% (31 of 300) of the phage clones analyzed exhibited IL-6R{alpha}-binding activity (data not shown). DNA sequencing of these 31 sIL-6R{alpha}-binding clones showed that seven independent peptide sequences were selected (Table 1). In light of the ability of the selected phage clones to bind sIL-6R{alpha}, we examined the ability of these phage clones to block interaction between IL-6R{alpha} and its ligand, IL-6. To this end, a fixed amount of phage particles (1011 pfu/mL) was added to sIL-6R{alpha}-coated wells, and their ability to block the binding of IL-6 to sIL-6R{alpha} was examined. This analysis revealed that phage S7 significantly inhibited the binding of IL-6 to sIL-6R{alpha} (Fig. 1A); phage S5 and phage S6 inhibited the interaction to a lesser extent, and other clones did not interfere with binding. Thus, phage S7 was chosen for further study. The binding affinity of phage S7 to different cell lines was determined by the in vitro binding assay. Phage S7 showed higher binding affinity than phage S1 in all membrane-type IL-6R{alpha}-expressing cell lines but not in IL-6R{alpha}-negative HUVEC cells (Fig. 1B). Immunofluorescence staining revealed that phage S7, but not phage S1, binds to the plasma membrane of C33A cervical cancer cells (Fig. 1C) and other IL-6R{alpha}-expressing cell lines (data not shown). Further confirmation of the specificity of phage S7 binding to IL-6R{alpha} was obtained by laser scan confocal microscopic observation of C33A cells double stained with anti–IL-6R{alpha} and anti-M13 antibodies, which showed that phage S7 tightly colocalized with IL-6R{alpha} on the cell membrane (Fig. 1D). The above data clearly show that phage S7 specifically binds to IL-6R{alpha} and blocks the interaction between IL-6 and IL-6R{alpha}.


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  		Table 1. Sequences of the peptides selected by binding to sIL-6R{alpha}

 


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  		Figure 1. Characterization of binding activity of selected phage display clones. A, individual sIL-6R{alpha}-binding phage clones were tested for their ability to compete with IL-6 protein for binding to immobilized sIL-6R{alpha}. Phages were added to sIL-6R{alpha}-coated wells in a 96-well plate at a concentration of 1012 pfu/mL. After 1 hour of incubation 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 increase compared 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-6 for binding to immobilized sIL-6R{alpha}) were added to various cell monolayers combined with IL-6 protein and incubated for 2 hours at room temperature. After washing, the cell-bound phages were detected using HRP-conjugated anti-M13 mAb and TMB substrate as described in Materials and Methods. Columns, means of at 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 C33A cervical carcinoma cells by immunofluorescence staining. C33A cells (4 x 105) were seeded onto coverslips. After treatment with phage clone 1 and phage clone 7 for 2 hours, immunostaining was done using anti-M13 antibody followed by FITC-conjugated antimouse IgG. Membrane localization of phage clone 7 was then observed by fluorescence microscopy. The position of the cell nucleus was confirmed by staining with Hoechst 33258 fluorescent dye. Original magnification, x400. Representative of three independent experiments. D, immunofluorescence staining of phage clone 7–treated C33A cell monolayer shows colocalization of IL-6R{alpha} and phage clone 7. Polyclonal goat antihuman IL-6R{alpha} antibody and monoclonal anti-M13 antibody were applied to the phage clone 7–treated C33A cells and incubated at 4°C overnight. After washes in PBS, the samples were treated with donkey antigoat IgG rhodamine-conjugated secondary antibody and FITC-conjugated goat antimouse secondary antibody for 1 hour at room temperature. The immunofluorescence-labeled cells were then analyzed by fluorescence microscopy as described in Materials and Methods.

 
S7 peptide blocks the interaction between interleukin-6 and interleukin-6 receptor {alpha}. Based on the above results, phage-encoded peptides (specifically S7) were synthesized. An in vitro competition assay revealed that peptide S7 could antagonize the binding of IL-6 to sIL-6R{alpha} in a dose-dependent manner (Fig. 2A) and significantly reduce the binding of IL-6 to IL-6R{alpha} in five different cell lines (Fig. 2B). These data clearly show that peptide S7 binds to IL-6R{alpha} and interferes with the interaction between IL-6 and IL-6R{alpha}.



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  		Figure 2. S7 peptide competes with IL-6 protein for binding to IL-6R{alpha}. A, various concentrations of the S7 peptide or the S1 control peptide were tested for ability to competitively inhibit the binding of IL-6 protein to immobilized sIL-6R{alpha}. Various concentrations of S1 or S7 peptide were incubated with sIL-6R{alpha}-coated plates for 1 hour at room temperature. After this preincubation, human IL-6 protein (50 ng/mL, 100 µL) was added directly to the wells without removal of the phage for 2 hours. The bound IL-6 was then measured using ELISA as described in Materials and Methods. Columns, means of three independent experiments; bars, SD. Competition ability of S7 peptide was significantly elevated at 25 to 250 µmol/L compared with control. *, P < 0.05, two-tailed Student's t test. Each treatment was done in three separate experiments and incubations were conducted in triplicate. B, S7 peptide (50 µmol/L) and S1 control peptide (50 µmol/L) were tested for ability to competitively inhibit the binding of IL-6 protein to IL-6R{alpha} in various cell lines. The bound IL-6 was then measured using ELISA as described in Materials and Methods. Each treatment was done in three separate experiments and incubations were conducted in triplicate. Columns, means of three independent experiments; bars, SD. *, P < 0.05, statistically significant increase compared with the corresponding value of S1 peptide.

 
S7 peptide decreased interleukin-6–induced Mcl-1 expression and antiapoptosis in cervical cancer cells. It has been reported that IL-6 acts as an antiapoptotic factor via up-regulation of Mcl-1 protein through the PI3K/Akt and MAPK signaling pathways in a variety of human malignancies, including cervical cancer (16, 22–25). To examine the effects of peptide S7 in IL-6-induced antiapoptotic signaling, the forms of Akt and MAPK [which are reported to function in the regulation of cervical cancer cell survival (16)] were examined. Western blot analysis revealed that the phosphorylated Akt and ERK1/2 MAPK levels were increased in IL-6-treated C33A cervical cancer cells and this activation could be significantly inhibited by treatment with S7 peptide but not with S1 control peptide (Fig. 3A). Furthermore, IL-6-induced Mcl-1 protein up-regulation was also abolished by S7 peptide (Fig. 3B, top). Whether S7 peptide can functionally block IL-6-induced antiapoptosis in cervical cancer cells was investigated by measuring the effect of various treatments on apoptotic cell death detected by flow cytometry. The data revealed that IL-6 can noticeably protect C33A cells from cisplatin-induced apoptosis and, more importantly, S7 peptide interferes with this protection (Fig. 3B, bottom). It has been reported that two IL-6 type cytokines, IL-11 and CNTF, induce STAT3 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 reveal any inhibitory effect on IL-11- or CNTF-induced phosphorylation of STAT3. Together, these data show that S7 peptide can block IL-6-mediated survival signaling and subsequent antiapoptosis in cervical cancer cells.



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  		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 treated with human IL-6 protein (50 ng/mL) in the presence or absence of S1 peptide (50 µmol/L) or S7 peptide (50 µmol/L). Cell lysates were obtained and subjected to SDS-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 blot analyses 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 µg) were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with specific anti–Mcl-1 antibodies; ß-actin served as the internal loading control. Representative of at least three independent experiments. Bottom, S7 peptide inhibits IL-6-induced protection against cisplatin-induced apoptosis in C33A cervical carcinoma cells. Cells were treated with IL-6 protein combined with S1 or S7 peptide and incubated with 5 µmol/L cisplatin for 24 hours. FACScan was done as an apoptosis assay as described in Materials and Methods. Columns, means of three independent experiments; bars, SD. *, P < 0.05, statistically significant decrease compared with values of cisplatin-treated control. Representative of at least three independent experiments. C, HT-29 and HepG2 cells were treated with human IL-11 protein (50 ng/mL) and human CNTF protein (1 nmol/L) in the presence or absence of S1 peptide (50 µmol/L) or S7 peptide (50 µmol/L). Cell lysates were obtained and subjected to SDS-PAGE followed by immunoblotting with various antibodies, such as anti–phospho-STAT3 and anti-STAT3. Numbers below lanes, level of protein expression compared with the control.

 
S7 peptide inhibits interleukin-6–induced vascular endothelial growth factor-A expression and angiogenesis in different cancer cell lines. Besides its role in antiapoptosis, IL-6 is important in angiogenesis via up-regulation of VEGF-A in cervical cancer (15). Thus, the effects of S7 peptide on the expression of VEGF-A protein and mRNA in C33A cells were examined by Western blot and reverse transcription-PCR (RT-PCR) analysis, respectively. VEGF-A protein and mRNA levels were increased in response to IL-6 treatment and this 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 C33A cells (Fig. 4B). In addition, IL-6 has been reported to induce VEGF-A expression in other cancer cells, such as multiple myeloma and BCC (28, 29). Thus, the ability of S7 peptide to inhibit IL-6-induced VEGF-A expression in these cells was examined. VEGF-A was dramatically induced by IL-6 treatment in RPMI 8226 multiple myeloma cells (Fig. 4C, left) and BCC cells (Fig. 4C, right). More important, addition of S7 peptide abolished IL-6-induced VEGF-A expression in both cell lines (Fig. 4C). Furthermore, by the application of a human VEGF-A immunoassay, we were also able to detect the production of secreted VEGF-A in response to IL-6 with or without S7 peptide. As shown in Fig. 4D, elevated levels of VEGF-A secretion due to IL-6 stimulation paralleled the increased expression of VEGF-A protein, indicating that a mature and functionally active VEGF-A protein was simultaneously being generated in IL-6-treated cells. The IL-6R{alpha} antagonist S7 peptide notably decreased IL-6-mediated VEGF-A secretion in all three cell lines (Fig. 4D). These data clearly show that S7 peptide inhibits IL-6-induced VEGF-A up-regulation in several different types of cancer cells.



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  		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 in IL-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 µmol/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). ß-actin served as the internal loading control. Representative of at least three independent experiments. B, IL-6-overexpressing C33A cells (C33A/IL-6) or vector control cells (C33A/neo) were treated with S1 peptide (50 µmol/L) or S7 peptide (50 µmol/L) for 16 hours followed by analysis the expression of VEGF-A protein and mRNA by Western blot and RT-PCR, respectively. ß-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 of S7 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 postcultured medium was collected and assayed for VEGF-A by immunoassay 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 vector control value.

 
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 angiogenic activity, 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, CM from IL-6-overexpressed cells (IL-6-CM) significantly increased the proliferation of HUVECs compared with the control CM (neo-CM), and IL-6-CM-induced HUVECs proliferation seemed to be greatly attenuated by S7 peptide but not by S1 peptide.



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  		Figure 5. S7 peptide abolishes IL-6-mediated angiogenic response in vitro and in vivo. A, HUVEC cell proliferation was measured in 24-well plates following treatment with CM from vector control cells (neo-CM) or that derived from IL-6-overexpressed cells (IL-6-CM) in the presence or absence of S7 peptide (50 µmol/L). Trypan blue exclusion method was done to determine the level of HUVEC 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 control value. 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 from C33A/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 6 hours). The experiment was conducted thrice with similar results. Bottom, quantitative results of tube formation assay. Briefly, cells were washed, fixed in methanol, and then stained in DifQuik solution some 2 hours before tube area measurement. Three replicate fields of triplicate wells were digitally photographed. Tube area was quantified using MetaMorph software (Universal Imaging Corp., West Chester, PA). Columns, means of three independent experiments; bars, SD. *, P < 0.05, statistically significant increase compared with the corresponding value of CTL-CM. C, top, S7 peptide inhibits angiogenesis 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 nude mice at sites lateral to the abdominal midline. Animals were sacrificed 7 days after injection. The mouse skin was detached and the Matrigel plug was recovered and photographed immediately. Bottom, the plugs were then minced and homogenized with a tissue homogenizer, and hemoglobin levels in the plugs were determined using Drabkin's solution according to the manufacturer's instructions as described in Materials and Methods. Columns, means of three independent experiments; bars, SD. *, P < 0.05, statistically significant decrease compared with values of neo-CM-treated control.

 
The effect of IL-6-CM on the morphologic differentiation of HUVECs was investigated by application of a tube-like structure formation assay. HUVECs were placed onto growth factor–reduced Matrigel with or without CM from IL-6-overexpressed C33A cells (C33A/IL-6-CM) for 6 hours. C33A/IL-6-CM stimulation led to the formation 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 completely prevented by S7 peptide but not by S1 peptide (Fig. 5B, top, III and IV). Quantitation of these cell cords, as described previously (31), revealed that C33A/IL-6-CM induced enhanced tube-like structure formation (Fig. 5B, bottom, 5.1-fold induction compared with control) and that this effect was almost completely inhibited by S7 peptide.

Next, we investigated whether S7 peptide inhibited IL-6-induced angiogenesis by the Matrigel plug assay. Matrigel plugs containing CM from vector control cells or IL-6-overexpressing C33A cells combined with or without S7 peptide (obtained by mixing the CM from C33A/IL-6 ± S7 with 0.5 mL cold Matrigel) were implanted into C57BL/6J mice and recovered 7 days later. Macroscopic analysis revealed 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 angiogenic activity was also reduced by S7 peptide but not by S1 peptide in this in vivo assay (Fig. 5C, top, III and IV). Similarly, hemoglobin level was clearly elevated in plugs containing C33A/IL-6-CM than in plugs containing C33A/neo-CM, and hemoglobin induction was almost totally inhibited by S7 peptide (Fig. 5C, bottom). The above data strongly suggest that IL-6-induced angiogenic effects can be blocked 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 could represent a therapeutic strategy to target IL-6-mediated tumor growth in vivo, SCID mice were injected s.c. with IL-6-overexpressed C33A human cervical carcinoma cells (C33A/IL-6) or C33A/neo vector 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 (but not of S1) peptide led to a significant reduction of IL-6-induced tumor 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 and ERK in tumors were significantly inhibited by S7 peptide (Fig. 6B). Furthermore, we investigated whether S7 peptide induced apoptotic cell death in tumor by TUNEL assay. As shown in Fig. 6B (right), the number of apoptotic cells was notably increased in S7 peptide–treated tumors but not in S1 peptide–treated tumors. Thus, these results show a vigorous antitumor effect of S7 peptide in IL-6-induced signaling and tumor growth in vivo.



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  		Figure 6. S7 peptide inhibits IL-6-mediated tumor growth in vivo. A, SCID mice were inoculated s.c. with 1 x 106 IL-6-overexpressing C33A cervical carcinoma cells (C33A/IL-6) or vector control cells (C33A/neo; 10 mice per group). After 3 days, treatment was initiated with i.p. injections of 50 mg/kg S1 or S7 peptide every 2 days in two other C33A/IL-6-inoculated groups (10 mice per group). The tumor size in SCID mice with various treatments was measured every 3 days, and the volume was calculated. Mean ± SE of 10 individual mice. B, animals were sacrificed 42 days after inoculation with tumor cells (39 days after injection with S1 or S7 peptide). The tumor was recovered and photographed immediately following the analysis of protein expression and apoptotic cell death by Western blot and TUNEL assay, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Data from experimental and clinical studies have shown a critical role of IL-6 in active cancers (5). IL-6 activities are mediated through specific low-affinity binding to {alpha} chain (gp80) and subsequently through the ability of this complex to recruit ß subunit (gp130) and thereby trigger signal transduction (6). Notably, in contrast to ubiquitious expression of the transmembrane spanning gp130, cellular distribution of the cognate IL-6R is limited and its expression is predominantly confined to hepatocytes and leukocyte subpopulations (32). However, gp80 could be cleaved from the cell membrane molecule by a transmembrane metalloproteinase or translated from an alternatively spliced mRNA (33). This soluble receptor (sIL-6R) binds IL-6 with an affinity similar to that of the cognate receptor (0.5-2 nmol/L), and more importantly, the sIL-6R/IL-6 complex is capable of activating cells via interaction with membrane-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 in numerous clinical conditions, indicating that its production is part of the disease process (34). By screening a random cyclic peptide phage display library, this study has identified a novel peptide that can specifically bind to IL-6R{alpha}, 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 shown by ELISA (Fig. 1A). Phage S7, expressing the peptide LSLITRL, was the most efficient phage with high affinity to sIL-6R. Cell based experiments have further shown that phage S7 had good binding to membrane-type IL-6R{alpha}-positive cell lines but not to membrane-type IL-6R{alpha}-negative HUVECs (Fig. 1B). Moreover, the addition of chemically synthesized phage S7 encoded peptide has led to a significant reduction in the binding of IL-6 to IL-6R{alpha} in various cell lines. All these specific inhibitory effects were verified by the colocalization of phage S7 and IL-6R{alpha} at the cell membrane using laser scan confocal microscopy (Fig. 1C and D).

Cervical cancer is a major health problem worldwide despite advances in screening programs. The progression of cervical cancer has been associated with increased levels of IL-6 in serum and cervicovaginal secretions (11). IL-6 is capable of promoting cervical tumor progression and dissemination by autocrine and/or paracrine mechanisms (13, 35). Platinum-based chemotherapy is the treatment of choice for the management of patients with metastatic and recurrent cervical cancer. We have shown previously that overexpression of IL-6 in cervical cancer cells caused a marked resistance to apoptosis induced by cisplatin or doxorubicin. This effect was primarily attributed to the up-regulation of Mcl-1 through a PI3K/Akt pathway (16). In the current study, S7 peptide was able to effectively inhibit IL-6-induced phosphorylation of Akt and ERK, resulting in down-regulation of Mcl-1 (Fig. 3A and B). Treatment with S7 peptide blocks IL-6 signaling mediated through gp130 and thus sensitizes cervical cancer cells to cisplatin in vitro. Endogenous IL-6 is a resistance factor for chemotherapeutic compounds used in the treatment of cancers (such as prostate cancer and renal cell carcinoma) that are poorly responsive to chemotherapy. Enhanced cytotoxicity could be achieved in these cases using a combination of cisplatin with anti–IL-6 or anti–IL-6R mAb (36, 37). The addition of agents with the ability to inhibit IL-6 activity in humans may thus improve combination therapy for cancer patients.

That IL-6 activates cervical tumor angiogenesis and induces rapid tumor growth has been shown in an animal model (15). The present study verified the in vivo efficacy of S7 IL-6R antagonist peptide by showing its vigorous suppression of IL-6-induced tumor growth in SCID mice (Fig. 6), which correlates with substantial antiangiogenic activity in vivo. Similarly, anti-VEGF antibody was shown to substantially inhibit IL-6-mediated angiogenesis and tumor growth in nude mice (15). Interestingly, recent studies show that IL-6 also induced basic fibroblast growth factor–dependent angiogenesis in BCC via Janus-activated kinase/STAT3 and PI3K/Akt signaling (10), suggesting that interruption of IL-6 signaling might be a more feasible molecular target for blocking IL-6-stimulated angiogenesis in diverse human cancers. Furthermore, during late stages of tumor growth, markedly increased serum level and tumor expression of IL-6 are responsible for the development of cancer cachexia (38). In agreement with animal studies demonstrating that specific IL-6 inhibitors can block tumor-induced cachexia in vivo (39, 40), many clinical trials have shown that anti–IL-6 mAb therapy decreased the incidence of cancer-related anorexia and cachexia in patients with malignant disease (5). Taken together, these results support the notion that inhibition of IL-6 activity may be useful in the management of cancer patients with advanced disease.

In summary, we found a novel small peptide that was a targeting ligand against IL-6R{alpha}, elucidated the molecular mechanism of its action, 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 in the vast majority of studies. Our findings highlight the potential application of peptide-S7 in the management of patients with malignant disease.


   Acknowledgments
 
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 accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Julie Yu for carefully editing the article.

Received 1/20/05. Revised 3/14/05. Accepted 3/22/05.


   References
Top
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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