Inhibitory Effects Of Synthetic Peptide PШ On High Peritoneal Metastasis Of Gastric Cancer

Gastric cancer is still the second most frequent type of malignancies among all tumors worldwide. The poor prognosis of gastric cancer is mostly caused by the extensive metastasis to the lymph nodes, liver, and peritoneal dissemination even if curative resection was performed. The main cause of recurrence after curative resection of advanced tumors is peritoneal metastasis, which is also the most common cause of noncurative surgery. Advanced gastric cancer which has invaded the gastric serosa carries a very poor prognosis, as special peritoneal dissemination frequently occurs even after various attempts have been made because the contributions of these therapies to patient survival were unsatisfactory. Therefore, it is urgently needed to develop effective therapies against peritoneal dissemination and improv the gloomy prognosis. In search of new strategies for the treatment of specific peritoneal dissemination, the potential application of tumor-targeted therapies attracts more and more attention. Phage display is an immensely powerful technique to isolate and identify peptide that can not only interact with target sites but also exert biological properties to the target cells. To isolate targeting ligands which are specific for high peritoneum metastasis of gastric cancer cells and can also inhibit metastasis ability to the target cells of the peritoneum, we performed phage display panning and obtained a specific peptide as promising for the development of therapy vectors or targeting drugs against peritoneum metastasis of gastric cancer.【Objectives】1. To identify the sequence similarities of PШpeptide with those of known proteins by bioinformatics methods and provide some clues for further studying the peptide bio-characteristics; 2. To study the affinity of PШpeptide with GC9811-P cells; 3. To evaluate the potential utility of PШpeptide in anti-peritoneum metastasis of gastric carcinoma cells; 4. To study gene expression profile of human gastric carcinoma cell line with peritoneal metastatic potential for screening peritoneal metastasis-related genes; 5. To identify the receptors responsible for peptide PШ-specific binding as promising for elucidating the cellular and molecular mechanisms of gastric cancer with peritoneal metastasis.【Methods】1. Study on the characteristics of PШpeptide using bioinformatics database Integrated bioinformatics database ExPASy was used to analyze and predict PШpeptide molecular weight (Mr), isoelectric point (pI), hydrophobicity and half-lives. BLASTP,PDB_ISL and SCOP database were employed to compare the sequence similarities of PШpeptide with those of other known proteins.2. Study on the affinity of PШpeptide with GC9811-P cells(1) Binding assays: Forty individual phages were randomly isolated and inally five candidate peptides with unique amino acid sequences were identified, using binding assays to test the binding efficiency of different phages to the target cells; (2) Cellular enzyme-linked immunosorbent assay (CELISA) : The binding effect of phage - displaying PШpeptide towards different gastric cancer cells was observed by CELISA, and the plates were read on an automated ELISA plate reader at the OD of 490 nm; (3) Competitive inhibition assay: After GC9811-P cells were preincubated with different concentrations of the peptide PШin culture medium, the phage18 were added into the medium, and the binding efficiency was tested. To make sure whether the phage binding to GC9811-P cells was mediated by the displayed peptide; (4) Using immunocytochemistry, flow cytometry analysis, and internalization assays to observe the binding between peptide PШand GC9811-P cells, and further observe whether internalization was active when the cells were incubated with phage - displaying peptide PШat 37°C; (4) Binding assay in vivo: BALB/cnu/nu mice were inoculated intraperitoneally with GC9811-P cells inoculated with the peptide PШ–FITC. Forty-eight hours later, the mice were killed and the peritoneal tumor was collected and observed under fluorescence microscopy.3. Peptide PШinhibit peritoneal dissemination of gastric cancer in vitro and in vivo(1) Cytotoxicity assays: The cytotoxicity of peptide PШto GC9811-P cells was performed by using the MTT assay; (2) migrate assay: to observe the effect of peptide PШon GC9811-P cells migration; (3) Adhesion assay: The peptide PШto interfere attachment of GC9811-P cells with extracellular matrix substrates was assessed by cell adhesion assay; (4) In vitro invasion assay: Invasion assays were performed by transwell plate pre-coated with Matrigel. To observe whether the synthetic peptide PШcould inhibit invasion ability of GC9811-P cells; (5) Tumor implantation and therapy: using orthotopic implantation animal experimental model to observe whether the peptide PШcould inhibit peritoneal dissemination of gastric cancer.4. Differential gene expression profiles of gastric cancer cells with high metastatic potential to peritoneumWe performed global analysis on differential gene expression of a gastric cancer cell line (GC9811) and its derivative sublines with high potential for metastasis to the peritoneal cavity (GC9811-P). By applying a high-density oligonucleotide array method, expression of approximately 11901 genes was analyzed, and selected some genes were confirmed by the RT-PCR method.5. Identification of peptide PШ-specific binding receptors(1) Extraction of GC9811-P cells total protein and membrane protein; (2) elution of binding protein: Ni-sepharose FF packing and 6-His coupled with peptide PШincubated with GC9811-P cells membrane protein, using different concentrations of imidazole solution to gradient elution, and eluting peak was collected; (3) SDS-PAGE; (4)the protein was analysis by MALDI-TOF-MS.【Results】1. General characteristics of peptide PШpredicted by bioinformatics The results of ExPASy data bank showed that Peptide PШwas weak acid, hydrophilic, and it's estimated half-live was 1.9h. Molecular formula was C57H92N12O19S1. Similar sequences were found between peptide PШand fission yeast, lipopolysaccharide biosynthesis protein and Histone acetyltransferase by BLASTP.2. Peptide PШhas specially affinity with GC9811-P cells(1) These selected phages were tested for their binding effect on GC9811-P. The amino acid sequence SMSIASPYIALE of the phage-displaying was the best for binding to the GC9811-P, and the peptide was named PШand the phage clone was named phage 18; (2) The binding effect of phage-displaying PШpeptide towards different cells was observed by CELISA. No significant binding of the phage was observed on the GES-1, SGC-7901, and GC9811, while the positive result was obtained on GC9811-P, AGS, and MKN45 by the hage-displaying peptide PШ. The phage-displaying PШpeptide was the best binder to GC9811-P cells for it showed up to 500-fold greater binding yields than the original library; (3) Internalization experiment: Phage-displaying peptide PШnot only special bind but also internalize to GC9811-P cells; (4) Competitive and inhibitory results between the synthetic peptide PШand the phage-displaying peptide PШon GC9811-P cells. There was a decreasing binding efficiencies value of the phage displaying peptide PШwith increasing amounts of peptide PШ. The assay indicated that the phage binding to GC9811-P cells was mediated by the displayed peptide; (5) After incubation with PШ-FITC conjugates, the GC9811-P cells were washed and examined using a fluorescence microscope. The result show PШ-FITC stained a significant fraction. We also found that the presence of excessive unlabeled PШabolished the PШ-FITC internalization into GC9811-P cells. These results indicated that peptide PШitself can bind to GC9811-P cells and that the binding is specific; (6) Flow cytometry analysis of phage displaying peptide PШbinding to GC9811-P cells. When GC9811-P cells were incubated with the peptide PШ-FITC, the percentage of positive cells marked by fluorescence was 90.2% and the fluorescence intensity was significantly higher than control group. The fluorescence intensity was increased along with peptide PШ-FITC incubated time and dose. The result indicated that peptide PШcan specially bind to GC9811-P cells; (7) Binding test in vivo: The peritoneal tumor of nude mice caused by the GC98l1-P cells showed strong fluorescence under fluorescence microscope. It's indicated that peptide PШalso can specially bind to GC9811-P cells in vivo.3. Peptide PШcould inhibit peritoneal dissemination of gastric cancer(1) Cytotoxicity assay: Exposure of the cells to various concentrations of peptide PШfor 24 and 48 h showed no obvious cell growth inhibition. Peptide PШshowed no apparent toxicity on GC9811-P cells by MTT assay; (2) migrate assay showed peptide PШcould significantly inhibit GC9811-P cells migration; (3) Our results from the adhesion assay indicated that peptide PШeffectively inhibited adhesiveness of GC9811-P cells compared with those from the control (50.9±2.1) % vs. (27±3.0) %). The difference was significant (p<0.01) ; (4) Using in vitro matrigel invasion assay, we observed that the invasive ability of GC9811-P cells incubated with peptide PШwas significantly decreased, and the number of traversed membrane cells were 43.5±4.3, whereas, the number of traversed membrane cells which GC9811-P cell incubated alone were 165.6±24.3, (p<0.01); (5) Significant decreases in the number of peritoneal disseminated macroscopic nodules were observed in animal models injected with synthetic peptide PШ. Tumor nodes per square centimeter at the peritoneum in GC9811-P control group were 11.5±4.3, while tumor nodes per square centimeter at the peritoneum in GC9811-P treatment group were 2.1±0.4 (p<0.001).4. Differential gene expression profiles of gastric cancer cells with high metastatic potential to peritoneumAmong the 11901 target genes, 248 genes (2.1%) were differentially expressed, and up-regulated genes and down-regulated genes were 218 and 30 respectively. Bioinformatical analysis revealed that these genes were closely associated with cell signal transduction, cell metabolize, cytoskeleton, motility, immunity, oncogene and tumor suppressor, cell cycle and cell apoptosis. Real-time PCR showed expression of PTEN, S100A4, CTNNB1 mRNA in concordance with the result of gene chips.5. Identification ofr peptide PШ-specific binding receptors(1) After peptide PШincubated with GC9811-P membrane protein, the proteins were eluted at 112 minutes by 300mmol/L imidazole; (2) Except for relative molecular mass 20-40Kd from SDS-Page gel, there was a strip which the relative molecular mass were 60 Kd in gel. The result suggested there exist multimer of peptide PШconjugated membrane protein in the eluting solution; (3) The proteins were treated by in-gel digestion, drying and then analyzed by MALDI-TOF-MS. After MASCOT database searching, the protein were identified. They were Heme oxygenase 1 and ST2.【Conclusion】1. The sequence of peptide PШwere similar to those of some domains of known proteins by bioinformatic protein databases, which provide some evidences for further study the biological functions of the antagonists; 2. Peptide PШhas high affinity with high peritoneal metastasis of gastric cancer GC9811-P cell; 3. Peptide PШcould inhibit peritoneal dissemination of gastric cancer, which ma be mediated by Heme oxygenase 1 and ST2.