Screening Of Peptide Binding To Hepatocellular Carcinoma Cells And The Antitumor Activity Of The Fusion Expression Product With Antimicrobial Peptide

Human hepatocellular carcinoma (HCC) is the third leading worldwide cause ofdeath from cancer following lung cancer and gastric cancer. Now methods for earlydetection and the development of effective treatments are an eminent priority. Forthis reason, isolation of tumor-specific ligands is a growing area of research.Tumor-specific binding agents can be used as in vivo imaging agents that allow forearlier detection of tumors and micrometastasis. Additionally, these targeting agentscan be used to create guided drugs that can target the tumor while sparing healthytissues by conjugating the appropriate cell-targeting ligand to an anticancer drug.Phage display is a powerful technique for the isolation of peptides that bind to aparticular target with high affinity and specificity. The biopanning of intact cancercells can be used to isolate peptides that bind to cancer-specific cell surfacebiomarkers. The aim of this study was screening and identifying the peptide whichcan bind to HCC cells specifically, and linking this peptide with Melittin to make afusion peptide with targeted antitumor activity.In this research, we screened and identified a novel peptide that could bind toHepG2cells specifically by subtractive panning of a Ph.D.-12phage display libraryin vitro. The subtractive effect made by the negative molecular prior to biopanningreduced the binding quanties of non-specific phage clone, so as to get the specificbinding phages rapidly. After4rounds of biopanning,100clones were selected forfurther characterization. The results demonstrated that this biopanning strategy isfeasible. A cell-based ELISA assay was used to confirm the affinity of the phageclones to HepG2cells in vitro. In this part, the31obtained positive clones with highaffinty were getted.Clustal X1.86was used to do multiple alignment analysis for the read amino acidsequence. There was no general consensus sequence revealed, but Arg and Leu/Ile were encountered in all the sequences at least once; Pro also had a frequency ofoccurrence. UniProtKB/SWISS-Prot and NCBI/BLAST were used for homologyanalysis; the result showed that the similarity between24peptide sequences andknown protein sequence is not high. ExPASy, HeliQuest and The AntimicrobialPeptide Database were used to predict the biochemical properties of peptides and theprobable functions. AM-2,13,22were determined for further verification ultimately.AM-2,13,22and nonspecific control peptides were synthesized and coupledwith FITC and Biotin, and then verified by peptide-phage competitive inhibitionassay, immunocytochemistry, immunohistochemistry and immunofluorescence. Theresults of peptide-phage competitive inhibition assay showed that peptidesAM-2,13,22still have specific affinity to HepG2cell after isolated from phagevectors, when the concentration of AM-2was5M, the inhibition rate reached82.67%, as the most significant inhibitionfor, AM-2was used for identification ofbinding activity. The results of immunocytochemical stain and immunofluorescenceimaging supported that AM-2could bind to liver cancer cells specifically, while notnormal live cells. The results of immunohistochemistry stain andimmunofluorescence imaging supported that AM-2could bind to liver tissues withtumors specifically; while not liver tissues without tumors. The above-mentionedfindings strongly suggested that AM-2is potential to be used for early diagnosis andantitumor agents of liver cancer.A rigid linker A (EAAAK)2A was used to link AM-2and antimicrobial peptideMelittin while maintaining their respective structural and functional independence.Fusion peptide AM-2-A (EAAAK)2A-Melittin was synthesized to verify itsantitumor activity in vitro. MTT colorimetric detection was applied to verify theeffect of fusion peptide on cell proliferation and apoptosis, the results showed thatthe fusion peptides have selective killing effect; the design of target part did notreduce the antitumor activity of Melittin. Flow cytometry assay was used to identifythe combination rate of fusion peptide to HCC cells, the results showed that theAM-2improved the targeting of fusion peptide; and then was used to detect the cellapoptosis after treated by fusion peptide in different times, the results indicated thatthe fusion peptide can inhibite proliferation of HCC cells by induction of cell apoptosis. The changes on membrance of HepG2and SMMC-7721were found bySEM, the results showed there were lethal damages on the cell membrances. Theresults identified fusion peptide has a specific antitumor effect in vitro.In this study, we screened and identified a noval peptide which could bind toHCC cells specifivally using a phage display peptide library techniques. Specificityand affinity of this peptide were confirmed. Then the targeting peptide was linkedwith Melittin to formate a fusion peptide. Its antitumor activity in vitro was thenidentified. In the end we obtained a specific antitumor peptide, which may provideexperimental basis for further development of targeted drugs for cancer therapy.