Phage Display Screen Identifies A Novel Peptide That Controls Ovarian Cancer

BackgroundOvarian cancer is the most lethal gynecological malignancy and characterizedby insidious onset, rapid progression and poor survival.Indeed,seventy-five percentof ovarian cancer patients were with evidence of metastatic spreading beyond theovaries.There was not a reagent that could effectively inhibit metastasis of ovariancancer. So, it’s urgent to find new treatment of ovarian cancer and new methods ofmetastatic interference in clinical. The phage display technique is a useful tool toidentify short peptides or antibodies that can specifically bind to and regulatefunctions of target proteins and the latter could be used for tumor early diagnosis,vaccines and as therapeutic targets. If such proteins are cell surface proteins, theycould also be useful for delivery of bioactive agents, such as small-molecule drugsand radionucleotides into cells.In addition, to investigate the efficacy of selectedpeptide on ovarian cancer, we carried out a series of experiments both in vitro and invivo. To the best of our knowledge, this was the first study reporting the use of phagedisplay technology to target the ovarian tumor.MethodsPrimary culture of ovarian epithelial cells was made by cell brush innovatively，Purified by erythrocyte spallation and differential adherence, and adding epidermalgrowth factor into serum-free DMEM-F12medium. The cells were identified by HEstaining and immunocytochemical staining.To establish a method for primary culturing human normal ovarian epithelial cells so as to used for further in vitroexperiments.With the HO8910cells as the target cells and human normal ovarian epithelialcells as the absorber cells,4rounds of panning from a PH.D-C7C phage-displaypeptide library were carried out. Individual phage clones were selected and identifiedby ELISA assay. Positive phage clones were characterized by DNA sequencing andbioinformatics analysis. The positive phage clones specifically bound to HO8910were identified by immunofluorescence detection.3.The candidate peptide1(SWQIGGN, translated from the selected phage DNAsequence) was synthesized and labeled with biotin.We have set three groups:thepeptide1-HO8910(K1), control peptide15-HO8910(K15), and HO8910cells(balnk).The in vitro effects of the selected peptides on cells were investigated for theircancer-related functions, including the cell adhesion, spreading, motility, andinvasion.To further investigate whether the peptide had influences on ovarian cancer invivo, we established peptide1incubation with HO8910cells mouse alartransplantation tumor models.The effects of peptide1on the gene expression ofVEGF in HO8910cells was meatured to understand the effects of peptide1on cellinvasion and migration.And a tunnel assay was performed to detect the effect ofpeptide on cell apoptosis.ResultsHigh purity epithelial cells were obtained(＞95%).The epthelial cells becameadherent after24hours ’culture, form edmonolayer cell colony after culturing7to12days, polygonal or flat, well transmission of light and refractivity. The cells could bepassaged6to8generation and grew well in the early days. Cytokeratin19wasalmost expressed and cell growth curve showed “S” type.The method for culturingovarian surface epithelial from human ovarian is successfully established. And it is asimple、efficient and practical way of primary cell culture.After4rounds of biopanning, Compared with the number of the first roundphages, the number of the last round phages were244times higher.12positive phageclones showed specific binding to HO8910cells. The sequence of the peptide was read as “SWQIGGN”. The results of immunofluorescence detection indicated thatphage1could be specifically bound to HO8910.3.The difference in cell growth activity among the three groups was statisticallysignificant from the3nd to6th day, cell growth ability of HO8910-peptide1wasweakened physically (p<0.05).These results of the transwell chamber migration andMatrigel invasion assay indicated that the peptide1might alter the metastaticpotentials of HO8910cells,The decrease in the number of invading and migratingcells in the KD cells, compared to those of the parental and mock cells wasstatistically significant.All resultsindicated that the selected peptide1not onlysignificantly inhibited cell growth but also could inhibit cell metastasis in vitro.4. The growth rate of abdominal circumference,the number of tumor position, totalnumber of disseminated tumor nudolesthe tumor weight were all significantlydecreased，compared with the other two groups(p<0.05).Furthermore, There was nostatistically significance in the tumor growth between Control and HO8910group(p>0.05). These results suggested that the peptide was able to inhibit the growth anddissemination capacities of ovarian cancer cell line HO8910in the transplantationmode.ConclusionA method for culturing ovarian surface epithelial from human ovarian issuccessfully established. And it is a simple、efficient and practical way of primarycell culture.Whole-cell subtraction biopanning for HO8910cells through random phagepeptide library was established successfully, which may provides a potential vectorfor targeting therapy of ovarian cell carcinoma patients.the phage display-identified tumor cell-binding peptide(SWQIGGN) can controlovarian cancer cell viability, migration, invasion, and adhesion capacity in vitro aswell as tumor growth and metastasis in vivo.The effect of this novel peptide (SWQIGGN) which could control ovarian cancerin vitro and in vivo may be connected with the download of VEGF. It will make greatprogress in developing innovative agents to effectively control ovarian cancer metastasis and progression.