The Study On Tagging Certain Proteins From PC3M Binded To The Specific Peptide Concerning The Metastases Of Human Prostatic Cancer By Western Blotting

Prostate carcinoma is one of the most common tumors in genitourinary system. With the change of life style, the incidence rate of PC is increasing gradually in our country and most of the PCs are at progression stage when discovered. Metastasis to the distant internal organs is the leading cause of death, with 30% prostate cancer patients undergoing metastasis at first diagnosis and more than 80% suffering bone metastasis during the whole disease course. Thus, investigating the mechanisms of the invasion and metastasis of tumor cells has been and will be one of the momentous tasks for fundamental and clinical researches.The highly metastatic human prostate carcinoma cell-binding peptides can be obtained from the phage-displayed random peptide library using subtraction method. Human prostate cell, human prostate carcinoma cell with low metastatic potential PC3, were first used as the target cells for subtractive biopanning from a 7-mer peptide phage-displayed library. The short peptides binding specifically to the human prostate cell and PC3 were gotten rid of from the library. Then the human prostate carcinoma cell with high metastatic potential PC3M was usedas panning target to select target binders so the short peptides binding specifically to PC-3M can be obtained. It might help design new peptide targeted therapy for metastatic human prostate carcinoma that cuts down the adverse side reaction with improving effectiveness.In this paper, immunoblotting was used to investigate the specific binding site on PC-3M cell surface of the peptides correlated with highly metastatic human prostate carcinoma, and accordingly develop novel specific marker proteins in diagnosis and treatment of PC. The phage-displayed short peptides correlated with highly metastatic human prostate carcinoma were co-incubated with plasmic and membranous proteins extracted from PC3M, and the proteins from PC-3M specifically binding to the phage-displayed short peptides were detected using the antibody against the phage-displayed short peptides; protein mobility curve and standard protein marker curve were developed to measure the molecular weight of the target proteins, and the correlation of the expression of the proteins with metastasis was also investigated.We selected three short peptide clones correlated with highly metastatic human prostate carcinoma B08,B10,B11 and carried out BLASTP using as query B08,B10,B11. The result showed no full match sequence for B08 but two full match sequences for B10,B11. The correlation of these two sequences with metastasis of PC was not mentioned. So these three short peptides are the potential highly metastatic human prostate carcinoma related peptides, and thus play important roles in metastasis.The immunoblotting and the protein mobility curve and standard proteinmarker curve showed that the B08 peptide could bind to the membranous proteins with molecular weight at 112KDa,92KDa and plasmic proteins with molecular weight at 112KDa,92KDa and 62KDa respectively of PC-3M; B10 peptide could bind to the membranous proteins with molecular weight at 112KDa,92KDa respectively while it could bind to the plasmic proteins with molecular weight at 112KDa,55KDa,40KDa; for B11 short peptide, it could bind to the 112KDa,92KDa membranous protein while binding to the plasmic proteins with molecular weight at112KDa,32.5KDa respectively.Above all, we successfully obtained the membranous and plasmic proteins of PC-3M that could specifically bind to the highly metastatic human prostate carcinoma correlated- peptides clones B08,B10,B11 using immunoblotting. These proteins might be the potential novel specific marker proteins in diagnosis and treatment of PC.