Development Of Peptides From HepG2 Cells As The Candidate Targets For The Therapy And Diagnosis Of Hepatocarcinoma

Cellular hepatocarcinoma is a common malignant tumor generated from liver cells with high mortality. Although surgery operation, chemical and radiation therapies are available in current clinical practice, drawbacks such as delayed diagnosis, missed chance for surgery therapy, and the strong side effects of chemical and radiation therapy are the important difficulties to save the life of patient. The development of modern targeting therapy and molecular imaging diagnosis for cancers are the hopeful ways to overcome the difficulties. For the needs of these new developments, the screening for specific and sensitive biomarkers from the cancer cell surface has been becoming important emergent lab task for the investigators in the world. The biopanning of peptide library via phage display method is an important approach to develop such biomarker targeting cancer cells specifically.Phage display technology has been developed to be a powerful technique for selecting specific polypeptides or proteins, which was developed as a molecular mimicking method in 1985. This technique allows fragments of foreign peptides or proteins to be expressed as fusion proteins displayed on the phage surface. These fusion proteins can keep their relatively independent spatial structure and biological activity, establish direct contact between random peptides and DNA coding sequences, and make all kinds of polypeptide ligands to some molecular targets (antibody, enzyme, cell surface receptor, etc) by in vitro affinity panning procedures to be rapidly identified. Phage display technology has been widely exploited to construct tumor-associated antibody and peptide libraries, identify molecule markers for cancer diagnosis and leading peptides with anti-tumor activities, and has been used for antitumor agents targeted transportation research.In this research paper, based on the new research progress both at home and abroad, phage display peptide library was used to screen the colorectal cancer cells specific peptides. Liver cancer HepG2 cell line was used as target cells, for four rounds subtraction biopanning from phage-displayed random dodecapeptide library, in order to identify peptide motif sequences that can bind to liver cancer cells with high affinity and specificity. This paper was for further research on liver cancer early diagnosis agents and targeted drugs.Objective: Phage display-12 random peptide library was used to screen the peptides binding specifically to human liver cancer HepG2 cells, identify their affinity and specificity to HepG2 cells, analyze amino acid sequence constitution and hydrophobic characteristics, search for the ligands specifically binding to HepG2 cancer cells, analyze amino acids clusters sites of these proteins binding to cells, to provide experimental basis for the phage-displayed peptides in tumor early diagnosis and targeted drugs research.Methods:HepG2 cells were used as the target cells, and human embryo kidney HEK293 cells as negative absorber cells for 4 rounds whole-cell subtraction biopanning from phage display-12 random peptide library. Thirty phage clones were picked up randomly, amplified and titered. Positive phage clones with high affinity and specificity were identified by Enzyme-Linked Immunosorbent Assay (ELISA). The affinity of positive phage clones with HepG2 cells was determined in order to exclude false positive clones. The ssDNA of positive phage clones, unrelated phage and some negative phage clones were extracted and sequenced full automatic to identify the conservative sequence. The characteristic of amino acid sequence was analyzed by BioEdit software. The homology between peptides was analysed by Alignment, searching for the similar amino acid sites contained in several peptides and motif sequences. Searching for proteins data bank, homologous sequence and potentially binding ligands or cell surface receptor were identified by BLAST method. The specificity of positive phage clones which can target to HepG2 cells was detected by immunofluorescence assay.Results:After four rounds of subtraction biopanning in vitro, phage clones that could bind to colorectal cancer cells were enriched and 30 phage clones were picked up from tittered LB/IPTG/Xgal plate with about 100 plaques. The affinity of 30 phage clones was identified by ELISA. Whole Cell-ELISA results suggested that 10 positive clones, including PC6, PC11, PC16, PC18, PC26, PC27 and PC28 could bind to HepG2 cells with higher affinity. Compared to other 7 positive clones, PC23 phage clone had the highest affinity to HepG2 cells. The ssDNA sequencing of positive phage clones showed four peptide sequences:LLADTTHHRPWT, LLADTPHHRPWT, FGWVTPHHELRS, SLSDTTHHGPWP. Four peptides had certain homology and conservative sequence XXXD(V)TT(P)HHXP(L)W(R)X (Motif Sequence). Analysis of the amino acid composition suggested that amino acid sequences are abundant serine, proline, arginine, valine and aspartate. The analysis of peptides'hydrophobicity showed that these four peptides were hydrophilic polypeptide. Homology analysis of four peptide sequences by BLAST in protein data bank showed that they had high similarity with some cell surface receptors or ligands. Immunofluorescence detection of positive phage clones targeting to HepG2 cells indicated that those positive clones, which displayed four sequenced peptides, could specificially bind to HepG2 cells rather than HEK293 cells.Conclusions:Subtractive bio-panning from phage display peptide library against liver cancer cells, then identified by whole Cell-ELISA,8 positive phage clones binding specifically to HepG2 cancer cells were screened out in this study. Four peptide sequences were obtained by DNA sequencing, and they have common sequence: XXXD(V)TT(P)HHXP(L)W(R)X by amino acid sequences characteristic analysis. These obtained HepG2 cell specific peptides and motif sequence may be the epitopes in ligand protein, which may have the ability to bind specially to some tumor cell surface receptors. Immunofluorescence assay showed that those positive clones, which displayed four sequenced peptides, were HepG2 cells specific phage clones. The peptides may be developed to be anti-cancer drugs targeted delivery agents or novel .molecular imaging probes that can be used for tumor early diagnosis. This research paper does provide an experimental basis for targeted tumor therapy.