Screening And Identification Of Cervical Cancer Targeting Peptides

BackgroundCervical cancer is a popular malignant neoplasm for women in worldwide.It is evaluated with the third incidence in developed countries just following breast cancer and endometrial cancer.However,it is the leading course to threaten the health of women in developing countries.Cervical cancer is associated with many factors,such as early marriage and childbearing,smoking,low immunity,induced pregnancy abortion.The chronic persistent infection of high-risk human papilloma virus(HPV)is the most important oncogenic initiator.Although surgery operation,radiotherapy and chemotherapy are available in current clinical treatments,drawbacks such as delayed/missed diagnosis,dose-dependent toxicities,drug-resistance and strong side effects are the important difficulties to save the life of patients.Therefore,the development of more sensitive,early and safe methods are necessary for screening of cervical cancer.Phage displayed peptide library is well known as a peptide high-throughput screening method that was invented in 1985.Here,genotype and phenotype of exogenous peptides are linked by fusing the corresponding gene fragments to the minor coat protein III gene of the filamentous bacteriophage M13.The biopanning procedure is more likely to keep native structure and functional conformation of proteins,and requires no previous knowledge of the molecular composition at the site of interest.The technology provides the rapid,simple and economic identification of peptide ligands for various target molecules or structures.Compared with monoclonal antibody,targeted peptides may offer fundamental advantages because of small molecular weight,low immunogenicity,high affinity,rapid tissue penetration,and easy of synthesis.Furthermore,peptides can be labeled with the appropriate moieties or conjugated with nanoparticles,liposomes,anti-cancer drugs for early imaging diagnosis,targeting therapy,evaluating disease status and monitoring development of cervical cancer.ObjectiveEarly detection,diagnosis and early therapy are important to improve survival and reduce mortality of patients.To select a novel peptide binding cervical cancer specifically and sensitively,a 12-mer peptide library was used to make biopanning with the modified protocols on SiHa cells.The targeted peptide may be a promising lead candidate for molecular imaging and targeted drug delivery in the diagnosis and therapy of cervical cancer.MethodsA Ph.D-12 peptide library was used to select peptides that bind specifically to cervical cancer cell line SiHa and human embryonic kidney cell HEK293 was used as negative cells.After four rounds of whole-cell subtraction biopanning,57 phage clones were selected randomly,amplified,purified and titered.Positive phage clones with high specificity/sensitivity were characterized using ELISA.The ssDNA of positive phage clones,negative phage and unrelated phage clones were extracted and sequenced to obtain the consensus peptide sequences.Meanwhile,the homology,hydrophobicity and composition of amino acids of peptides were analyzed by different softwares.Moreover,the characterization and validation of the best clone that target SiHa cells were performed by immunofluorescence assay.Results1.After four rounds of screening,the number of phages recovered from SiHa cells was 21-fold higher than the first round.2.ELISA was performed to identify the cellular affinity.Of the 57 phage clones,36 clones showed higher binding to SiHa cells compared with UPRs and PBS control groups.Furthermore,the S7 clone appeared to bind most effectively to SiHa cells.3.After DNA sequencing,six consensus sequences were obtained,including KNLXXXXXXPLV,ETLXXXXXXIPW,QQLXXXXXXTPY,VTAXXXXXXTPL,SLSXXXXX)XTTS,TITXXXXXXSRK.4.Six peptide sequences existed certain degree of homology,which CSP2 and CSP3 had the highest similarity,approximately 50.0%.5.Homology analysis of six peptide sequences by BLAST in protein data bank showed that they had high similarity with some cell surface receptors,transcription factors,immunoglobulin,or transporters.6.Peptide sequences contained 17 kinds of amino acids,serine,theronine,and proline occupying the first 3 positions.The frequencies of occurrence were 16.67%,15.28%and 13.89%,respectively.7.According to pI values,CSP1,CSP5,CSP6 were alkaline polypeptides and others were acidic peptides.8.Excepting CSP1,other peptides were all hydrophilic.CSP6 showed the strongest hydropathy.9.S7 clone specifically/sensitively binding to SiHa cells was identified by immunofluorescence assay.Conclusions1.The phage clone S7 was identified as the best positive candidate that specifically bind to SiHa cells by ELISA and immunofluorescence assay.2.Peptides labeled with the appropriate moieties or conjugated with nanoparticles,liposomes,anti-cancer drugs provide new potential for early imaging diagnosis,targeting therapy,evaluating disease status and monitoring development of cervical cancer.