| Gastric cancer is one of the most common cancers of digestive system with the second rank high incidence rate among all of the cancers and the third leading cause of cancer death. Adenocarcinoma makes up of approximately 90% of gastric cancers, which is also the most frequent type in China. Data from previous findings have demonstrated that interleukin-1β(IL-1β) is closely associated with the gastric carcinogenesis and gastric cancer progression. However, the molecular mechanisms involved in IL-1β promoted gastric cancer remain to be determined. Non-receptor tyrosine protein kinase(c-Src) is the product of proto-oncogene SRC, which is the earliest discovered Src family kinase(SFKs) member. IL-1β has been identified to be able to activate c-Src in lung adenocarcinoma cells, leading to increased cell migration; and both IL-1β and c-Src are capable of promoting gastric adenocarcinoma(GA) cell survival. However, whether IL-1β is able to activate c-Src in GA cells, leading to the biological changes of GA cells has not been reported yet. Therefore, we chose to preliminarily investigate this topic in this study.In this study, gastric adenocarcinoma cell of MKN-45 was chosen for the study. The aim of this study is to preliminarily elucidate whether IL-1β is able to activate c-Src in MKN-45 cells and whether the activation of c-Src by IL-1β is able to increase GA cell survival, and preliminarily understand the mechanism which regulates IL-1β-promoted GA cell survival whether involved in STAT3 with the purposes to provide a good foundation of experiment for finding a novel therapeutic target for patients with GA. The whole paper includes three sections.The content of the first section is “Preliminary investigation of IL-1β-increased gastric adenocarcinoma(GA) cell survival via c-Src”. The methods used in this section included: 1. Use of Annexin V plus PI staining, and then flow cytometry analysis to detect the effect of IL-1β stimulation on the survival of MKN-45 cells. Followed, IL-1β neutralizing antibody was used to pre-treat MKN-45 cells, and then the inhibition effect of IL-1β neutralizing antibody on IL-1β-promoted MKN-45 cell survival was checked. 2. The expression of phosphorylated c-Src induced by IL-1β and the inhibition effect of IL-1β neutralizing antibody and the c-Src specific inhibitor PP1 on the activation of c-Src induced by IL-1β were examined by western blot. 3. The inhibition effect of the c-Src specific inhibitor PP1 on IL-1-promoted MKN-45 cell survival was investigated by pre-treated MKN-45 cells with PP1 and then analyzed by Annexin V plus PI staining, and flow cytometry detection of the apoptotic cells.The content of the second section is “Obtaining the low c-Src expression of MKN-45 cell lines and investigation of the effect of inhibition of the c-Src expression on IL-1-promoted GA cell survival”. The methods used in this section included: 1. Establishment of MKN-45 cell clones with stable low c-Src expression by transfected two c-Src sh RNA targeted on two different c DNA of human c-Src. 2. Annexin V plus PI staining and then flow cytometry analysis to detect the inhibition effect of silence of c-Src expression on IL-1β-promoted MKN-45 cell survival.The content of the third section is “Preliminary investigation of the mechanism involving IL-1β-promoted GA cell survival via activation of c-Src”. The methods used in this section included: 1. Preliminary investigation of the activation of c-Src by IL-1β whether involving IL-1β induced interaction of TRAF6 with c-Src by co-immunoprecipitation assay. 2. Western-blot analysis of the effect of c-Src and Stat3 specific inhibitors PP1 and WP1066 on the activation of Stat3 induced by IL-1β-promoted Stat3 activation 3. Further investigation of IL-1β-promoted GA cell survival whether involving Stat3 by use of Stat3 inhibitor WP1066 to pre-treat MKN-45 cells, and then performance apoptosis assay.The results of first section: 1. IL-1β stimulation was able to reduce MKN-45 cell apoptosis induced by serum-withdrawalThe results of Annexin V plus PI staining and then flow cytometry analysis exhibited that IL-1β stimulation significantly reduced MKN-45 cell apoptosis induced by serum-withdrawal, compared with that of MKN-45 cells without IL-1β stimulation p <0.05; which was inhibited by pre-treated MKN-45 cells with IL-1β neutralizing antibody, indicating that IL-1β was able to increase MKN-45 cell survival. 2. IL-1β was able to activate c-Src in MKN-45 cellsWestern blot results exhibited that IL-1β stimulation was able to activate c-Src in MKN-45 cells. The expression of phosphorylated c-Src was increased in response to IL-1β stimulation, which was inhibited by IL-1β neutralizing antibody and the c-Src specific inhibitor PP1, indicating that IL-1β was able to activate c-Src. 3. The results from Annexin V plus PI staining and then flow cytometry analysis exhibited that the effect of IL-1β-promoted MKN-45 cell survival was inhibited by c-Src specific inhibitors PP1,indicating that IL-1β-promoted GA cell survival was potential through activation of c-Src.The results of second section: 1. Successfully obtaining MKN-45 cell clones with stable low expression of c-Src through stably transfected p GPU6/GFP/Neo-c-SRC sh RNA1 and p GPU6/GFP/Neo-c-SRC sh RNA2, and MKN-45 cell clones with stable expression of si RNA negative control(scramble sh RNA) through stably transfected p GPU6/GFP/Neo-Scramble sh RNA. 2. Annexin V plus PI staining and then flow cytometry analysis results demonstrated that inhibition of the expression of c-Src by c-Src sh RNA significantly decreased IL-1β-promoted MKN-45 cell survival, further indicating IL-1β-promoted GA cell survival was through c-Src.The results of the third section: 1. The results of co-immunoprecipitation assay indicated that the interaction of TRAF6 with c-SRC was detected in MKN-45 cells in response to IL-1β, leading to the activation of c-SRC, which was blocked by IL-1β neutralizing antibody. 2. Western blot results exhibited that IL-1β-increased GA cell survival was associated with IL-1β-promoted the activation of c-Src, then in turn activated the c-Src downstream signaling molecular Stat3. The expression of phosphorylated Stat3 was significantly increased in response to IL-1β, which was inhibited by c-Src inhibitor PP1 and Stat3 inhibitor WP1066. 3. Annexin V plus PI staining and then flow cytometry analysis results exhibited that pre-treated MKN-45 cells with Stat3 inhibitor WP1066 was able to significantly inhibit IL-β-promoted GA cell survival.Conclusions: From the results of this study, we got preliminary conclusions: IL-1β is able to activate c-Src, the latter in turn activates Stat3, resulting in the increased MKN-45 cell survival. |
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