Screening And Identification Of Ovarian Cancer Targeting Polypeptides

BackgroundOvarian cancer is just going up to third incidence following cervical cancer and uterine cancer. It is the most fatal gynecological malignancy and is characterized by insidious onset, rapid progression and clinical survival rate. Thus, it is no doubt that ovarian cancer has caused great threat to the health of women. Unfortunately, so far, a sensitive method for early diagnosis of ovarian cancer is not ideal, so that many patients have lost the opportunity to be found and the optimal surgical treatment. Even if the relapse rate of early stage ovarian cancer is up to 40%. The treatment of ovarian cancer includes surgery, chemotherapy, radiotherapy and immunotherapy, but as mentioned above, many patients have reached advanced stage and has obvious shift, so the use of conventional therapy, including chemotherapy for non operative treatment of the main. However, the current chemotherapy drugs are not only low targeting and poor curative effect but also have strong toxic and side effects.In summary, the research and development of molecular fragments that can be used for targeting diagnosis and targeting therapy is an urgent problem in the medical practice of ovarian cancer, whereas the targeting peptides are such molecules with great expectation.Currently, the phage displayed peptide library is the most effective technique to screen the targeting peptides. Since it was put forward in 1985, it has been widely used in various fields of biomedicine because of the high efficiency of the technology and the advantages of the peptide molecule itself. Due to molecular weight small, non-toxic, immunogenicity, easy synthesis, high affinity and sensitivity, targeting peptides can be served as targeting molecular probes for early imaging diagnosis. Furthermore, these peptides conjugated with other biological materials, nanoparticles, anti-cancer drugs can be used for targeting therapy, which can greatly improve the efficacy of the drugs and significantly reduce toxic and side effects. It is of great significance to the surveillance and treatment of ovarian cancer.ObjectiveWe aimed to screen a novel peptide that specifically and sensitively binds to ovarian cancer cell lines by modified procedure from 12 peptide library. The specificity and sensitivity have been identified at cell level. Moreover, it is the foundation to develop specific probes and targeting therapy of ovarian cancer in the years to come.MethodsWhole-cell subtractive screening from a phage display 12 peptide library was performed on ovarian cancer cell line, SKOV3, and human embryonic kidney cell line HEK293 used as controls. After five rounds of biopanning,60 phage clones were picked randomly, amplified, purified and titered. Positive phage clones were identified using Enzyme-Linked Immunosorbent Assay. To obtain the consensus peptide sequences, the ssDNA of positive phage clones were extracted, sequenced, and translated into protein. Subsequently, bioinformatics analysis of consensus sequences was performed by different software. Finally, the immunofluorescence assay in human ovarian cancer SKOV3 cells and normal cells and other cancer cells was studied to further assess the targeting specificity and sensitivity of the best positive phage clone.Results1. After five rounds of whole-cell subtractive screening, the number of phages recovered from SKOV3 cells was significant enrichment.2. A total of 60 phage clones were picked randomly, amplified, purified, tittered. After three ELISA assays,44 phage clones showed higher affinity to SKOV3 cells. Among these 44 phage clones, the R20 had the best targeting capacity.3. According to the results of DNA sequencing, nine different consensus sequences were obtained, including AWPXXXXXXKAL、ASPXXXXXXVGP、QPTXXXXX XRSK、KLHXXXXXXSLI、YDSXXXXXXPRA、KSGXXXXXXPNF、WSNXXX XXXTYG、ELIXXXXXXYLS and NIAXXXXXXGNR, named OSP1-OSP9 respectively.4. Homology analysis of the nine peptide sequences demonstrated that they were high similar to some immunoglobulins, tumor markers, and transmembrane receptors.5. Analysis of amino acid composition and characteristics of the 9 peptides, there were 19 kinds of amino acids, which are abundant in proline, alanine, and serine. The frequency was 12.96%,12.04% and 9.26%.6. Hydrophobicity analysis suggested that there were three hydrophobic peptides, OSP1, OSP7, OSP8 included. But the others were hydrophilic peptides. Furthermore, isoelectric point analysis showed that there were four acidic peptides, OSP2, OSP6, OSP7 and OSP8 included. However, the others were alkaline peptides.7. Both ELISA and immunofluorescence assay revealed that the R20 had the best targeting ability to SKOV3 cells with high specificity and sensitivity. However, it showed no binding activity to other cancer cell lines, including breast cancer, cervical cancer, gastric cancer, hepatocellular cancer and colorectal cancer. Thus, R20 was identified as the best positive clones.ConclusionsBiopanning by phage display technology, a best targeting ovarian cancer cells clone/peptide sequence, R20/OSP2, were obtained. Cellular level appraisal results indicated the targeting peptide had the developing potential to be used as a targeting molecular probe and a guide element targeting therapy drugs of ovarian cancer.