| Gastric cancer is one of the most popular digestive tumors in the world that kills a lot of people in china. Improvement of the diagnosis and treatment of gastric cancer results in better prognosis and patients quality of life. During the past decades, the prognosis of gastric cancer was improved especially in early gastric cancer based on early diagnosis and treatment, which confers the five-year survival rate above 90%. However, there still are tremendous numbers of patients who are diagnosed with advanced and/or late stage gastric cancer in the clinic. The prognosis of these patients is quite unsatisfied even after intergrated therapy which including surgery, chemotherapy, radiation as well as traditional medicines. A total of 50% to 90% patients will die because of tumor recurrence and/or metastasis.Numerous reports have demonstrated that the host-tumor interactions play a significant role in the tumor development, process as well as metastasis. The mesenchymal cell adjacent to the tumor may not only stimulate tumor cell growth by supplying nutrition for tumor cells, but also afford tissue space and cell skeleton, which help tumor cell infiltrate to the extracelluar matrix membrane. Several investigations have been focused on the role of mesenchymal cell in tumor development and process.Hepatocyte growth factor is a multifunctional factor, which generates from mesenchymal cell including fibroblast cell, macrophage, epithelium, endothelium, lipid-storage cell as well as several cancer cells. HGF may function as a mitogen, motogen and morphogen after being combined with the tyrosine kinase receptor, c-Met, therefore, it play an important role in cell growth, migration, invasion as well as angiogenesis.Previous studies have reported that HGF may inhibit normal cells such as hepatic cell, kidney cell, corneal cell from apoptosis, its anti-apoptic effect can also be observed in several cancer cells including lung, breast cancer, glioma cells. Recently, HGF was found to suppress DNA-damage agent induced tumor apoptosis, which may result in tumor chemoresistance. However, this anti-apoptotic effect is cell selective. Furthmore, it was reported that the unsatisfied effects of chemotherapy in gastric cancer was possibly due to the chemoresistance and/or drug insensitivation to gastric cancer. It is rarely studied whether HGF may inhibit gastric cancer cell apoptosis that is induced by DNA damage agents, and whether the anti-apoptotic effect of HGF may be associated with chemoresistance in gastric cancer is still unclear.HGF may also stimulate cell motility, migration as well as invasion, which play a significant role in tumor invasion and metastasis. It is reported that HGF can stimulate cancer cell such as breast cancer, lung cancer, prostate cancer, and glioma cell invasion. However, this biological behavior is organ specific, few studies have concentrated on the influence of HGF on cancer invasion in gastric cancer.Based on the previous studies, we selected a gastric cancer cell line, MKN-45, which does not autonomously express HGF to investigate the influence of HGF on cell apoptosis and cancer invasion. Our study tried to evaluate the possible role of HGF on chemoresistance in gastric cancer as well as to demonstrate the possible mechanism of HGF on cancer invasion.This study was composed of four sections.Section I: pIRES2-EGFP-HGF plasmid constructionPUC-SRot/HGF cDNA was bestowed from Prof. Nakamura of Osaka University College of Medicine, Japan. A 2.3kb target of HGF cDNA was inserted using Not I site of thePUC-SRa plasmid. pIRES2-EGFP was purchased from B&D company. PUC-SRct/HGF and pIRES2-EGFP were both digested by Bam HI, after purification and proliferation of HGF cDNA and pIRES2-EGFP, the targeted band were ligated by T4 DNA ligase, the ligation product were then transformed into DH5ot sensitive E coli, the anti-kanamycin E coli clone were selected out. Among them, HGF inserted clone was confirmed by Bam HI digestion. The order of HGF insertion was confirmed by both Kpn I, Pst I digestion and PCR. Our results found three HGF insertion recombinant, both enzyme digestion and PCR reaction confirmed that HGF cDNA was inserted in a correct order. One of the recombinant was amplified and purified for further experiment.Section II: Establish a gastric cancer cell linethat may stably express HGFWe, firstly, selected several gastric cancer cell line including MKN-45, YCC-3, NCI-N87, SNU-16 and AGS to investigate HGF expression by using RT-PCR, we found that MKN-45 itself can not express HGF. Futhermore, MKN-45 is a cell line that expresses high level of tyrosine kinase receptor, c-Met. We then selected MKN-45 as a cell line for HGF transfection. HGF recombinant and null plasmid were transfected and selected by G418. HGF expression was detected by RT-PCR and western blot. The morphology and cell growth and proliferation was compared between HGF transfectant, control vector transfectant and wild type MKN-45 cell, and no significant difference was observed among them. We also check the HGF function in the stably expressed HGF transfectant cells using MDCK as a target cell, we found MDCK was scattered significantly when cultured with the medium of HGF transfectant. Therefore, we established a cell line that may stably and functionally express HGF.Section III, HGF suppresses adriamycin induced apoptosis in gastric cancer ceU line, MKN-45We investigated the influence of HGF on adriamycin induced apoptosis in MKN-45 cell line using MTT proliferation assay, DAPI stain, DNA defragmentation assay and flowcytometry. Our results found that cell proliferation was inhibited more significantly by adriamycin in control vector transfectant and wild type cells than that in HGF transfectant. We also count the apoptosiscell by staining with DAPI, and observed that HGF transfectant showed less cell apoptosis than that of nontransfectant. When cells treated with 0. lug/ml adriamycin, DNA radder was observed in wild type and control vector transfectant cells, However, it is not clear in HGF transfectant cells. Finally, we confirmed the above results by using flowcytometry with PI staining. Therefore, HGF may suppress adriamycin induced apoptosis in gastric cancer cell line, MKN-45.Section IV. Influence of HGF on cell adhesion, migration and invasion in gastric cancer cell line, MKN-45We investigated the influence of HGF on cell adhesion, migration and invasion in MKN-45 cell line using cell adhesion assay, boyden chamber assay, wound assay as well as detecting the tumor metastasis associated genes expression between three group of cells. There were no significant differences of the adhesion and migration rate between the HGF transfectant and control vector transfectant, wild type MKN-45 cell. No difference of cell invasion was found between the three types of cells by using wound assay. We finally assessed the tumor metastatic associated genes expression between the three type of cells, and no different expression was found between them regarding E-cadherin, itergrin, MMP2, MMP-9 as well as MT1-MMP. We therefore may conclude that HGF does not affect cell adhesion, migration and invasion in MKN-45 cell line, implying that HGF is not associated with tumor metastasis for MKN-45 cell.We successfully constructed a GFP expressing plasmid pIRES2-EGFP-HGF and established a gastric cancer cell line that can stably express HGF. Our results observed that HGF could suppress MKN-45 apoptosis that was induced by adriamycin. However, HGF could not enhance MKN-45 cell adhesion, migration and invasion. Our study implicate that HGF may be involved in chemoresitance in gastric cancer, nevertheless, the aggressive and invasive characteristics of MKN-45 may not be dependent on HGF/Met signaling, some other cytokines may play a role in MKN-45 cancer cell invasion.
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