The Inhibition Effect Of ARHI On Pancreatic Cancer And Its Possible Mechanism Of Celullar Transduction Pathway

Pancreatic cancer is a highly malignancy tumor with poor prognosis and symptoms obscurity, the incidence and mortality rate of pancreatic cancer is steadily increased in recent years. Mechanism of pancreatic cancer pathogenesis is still not fully understood which makes the early dignosis and treatment diffficult, it spurs us to reveal the internal secret of pancreatic cancer and searches the available pathway in tumor treatment.ARHI is a maternally imprinted tumor suppressor gene, the prime work from our laboratory suggested that lost expression of ARHI protein was in some pancreatic carcinomas tissue, and meanwhile it presented hyper-methylation of CpG islands (CpGâ… 45.5%; CpGâ…¡27.3%%) in its gene promoter region. In this study, we constructed eukaryotic expressing vector with wild-type ARHI which was transfected into the Panc-1 pancreatic cancer cell line without ARHI gene. So that the aim was to explore the association and possible mechanisms of gene ARHI and methylation with pancreatic carcinoma pathogenesis.It is well known that pathogenesis of carcinoma is a process involving in multistage and multiple genes, and in which the cellular transduction pathway is absolutely necessarily to tumor pathogenesis. This study approaches the relationship between ARHI gene or demethylation., and cellular transduction pathway. The aims of the study to explore the mechanism of ARHI, tumor suppressor gene, to regulate the carcinoma pathogenesis. For this popure, the reseach work will introduce in three partsThe effect of the ARHI, as tumor suppressing gene, on pancreatic cancerThe plasmid plRES2-EGFP-ARHI with wild-type ARHI was constructed, and then which transfected into Panc-1 (pancreatic cancer cell line without ARHI gene). The Panc-1 alone and empty plasmid were as double controls: 1. Westernblot was used to examine the expression of protein ARHI, MAPK/ERK1/2 and JAK-STAT3 in Panc-1. The changes were evaluated by the ratio of functional protein to its total protein; 2. Flow cytometry was used to analyze the effect of protein ARHI to apoptosis and cell cycle of Panc-1; 3. The proliferation of Panc-1 cell is analysed by MTT; 4. Morphology was observed through methylene blue and Hoechst33258 stain in the proliferation and aoptosis.The results: 1. The eukaryotic vector pIRES2-EGFP-ARHI was constructed which tranfected into Panc-1. The transfected Panc-1 expressed the protein of ARHI again; 2. ARHI can significantly slow down the speed of proliferation of Pane-1; 3 ARHI could increase the percentage of apoptosis of Panc-1 (P＜0.05), but it had little effect on cell cycle; 4. Compared with the double control, ARHI significantly decrease expression of protein P-ERK1/2 at 120h (P＜0.05); 5. The Similar change could be seen in P-STAT3 at 96h, 120h (P＜0.05).Conclusions: 1. The eukaryotic expressing vector pIRES2-EGFP-ARHI successfully constructed; 2. ARHI gene could inhibited the proliferation of Panc-1 without change of morphology; 3. Gene ARHI could raise percentage of apoptosis to Panc-1, but had little effect to cell cycle; 4. ARHI could decrease the expression of function protein in cellular transduction pathway such as P-ERK and P-STAT3.The effect of tumor suppress gene, ARHI, on growth of pancreatic carcinoma xenografted in Nude MiceIn order to study the inhibition effect of ARHI on the pancreatic cancer, the ARHI gene stable transfected Panc-1 cell line was established by G418 selection, the empty plasmid transfected cell line as a control. The ARHI mRNA and protein expression were detected by RT-PCR and Westernblot, which suggested that stable transfection was successful. The ARHI gene stable transfected Panc-1 cell line and control group was injected subcutaneously into nude mice to establish the pancreatic carcinoma xenografted model. ARHI protein expression in xenografted model was studied by immunohistochemistry, meanwhile the tumor growth, size and mice body weight were recorded.Results: 1. The ARHI stable transfected Panc-1 cells expressed the ARHI protein and ARHI mRNA, but that did not in non-transfected Panc-1 cells; 2. In pancreatic carcinoma xenograffed model, the ARHI protein expressed in the higher level than that in the control group; 3. The growth of tumor in nude mice was much slower in ARHI transfected Panc-1 cells than that in control group (P＜0.05). In the meantime, the size of tumors by ARHI transfected Panc-1 cells were also smaller than control group. (P＜0.05), but the body weight of mice had no difference between these two groups. Conclusions: In this part of study, the ARHI gene stable transfected Panc-1 cell line and pancreatic carcinoma xenografted nude mice model were successfully established, the data in the animal level showed that the ARHI gene had an inhibition effect on pancreatic cancinoma growth. These supplied the evidence and establish the well basis in the study of the relationship of ARHI and pancreatic cancer.The study theI relationship between methylation and cellular transduction signaling pathway in panerearic tumorIn this study, specimens of human pancreatic cancer, pancreatic carcinoma xenografted of Nude Mice and pancreatic cancer cell lines (Panc-l&P3) were used. 1. MAPK/ERK1/2 and JAK-STAT3 protein were measured by Westemblot; 2. The growth of the two kind of cells, who were treated with either different concentrations of PD98059 (MAPK/ERK1/2 specific inhibitor, 50 and 20umol/L), 5Aza-dC (drug of de-methylation, 1umol/L ), combination of the two drugs or not, were measured by MTT assay; 3. Flow cytometry was used to analyze the effect of different concentration of PD98059 on apoptosis and cell cycle of the two cell lines; 4. The morphology of cells apoptosis was observed through Hoechst33258 stain; 5. The effect of PD98059 on the growth of pancreatic carcinoma xenografted of Nude Mice were evaluated according to record the size of tumor and of body weight of mice.The results: 1. Among the 6 tissues of human pancreatic cancer, 3 of them were up-regulation on the expression of MAPK/ERK1/2 proteins, 4 of them were up-regulation on the expression of JAK-STAT3; 2. In two cell lines treated by 5Aza-dC, the ratio of P-STAT3/T-STAT3 protein in P3 at 120h and in Panc-1 at 96h and 120h expressed decrease significantly (P＜0.05) comparing of the control, And also the simlar results were obtained in the change of ratio of MAPK/ERK1/2 protein in the same cell lines and same time; 3. Comparing of control, proliferations of Pane-1 and P3, who were treated by PD98059, PD98059 50umol/L+ 5Aza-dC lumol/L, were significantly decreased (P＜0.05). Among the different agents groups and two cell lines, The inhibition in group of the combination(PD98059+SAza-dC) was the most strong in Panc-1 cells, but it was in the group of PD98059 50umol/L in P3 cells. If combination of PD98059 and 5Aza-dC to treat either Panc-1 or P3 cells, the inhibition effect of proliferation on Panc-1 was additive, however, it hadnot similar additive effect in P3 cells; 4. PD98059 50umol/L enhances the apoptosis of both Panc-1 and P3 (P＜0.05) cells, the cells in G₁ phase were significantly increase (P＜0.05) and those in S phase were decreased in P3 cells. but it did not have same change seen in Panc-1; 5. PD98059 could inhibited the growth of pancreatic carcinoma xenografted of Nude Mice by the MAPK/ERK1/2 cellular transduction pathyway.Conclusions: 1. The expression of MAPKUERK1/2 and JAK-STAT3 proteins were abnormal in the tissue of human pancreatic cancar; 2. The protein of P-ERK/1/2 could promote proliferation of Panc-1 and P3 cells which could be blocked by PD98059. in time and concentration dependent; 3. The P-ERK/1/2 can induce apoptosis of Panc-1 and P3 cells; 4. The effect of de-methylation agent on apoptosis and proliferation were through the cellular transduction MAPK/ERK1/2 pathway; 5 No matter PD98059 or de-methylation agent could change the expression of the protein of MAPK/ERK1/2 and JAK-STAT3. It suggested the two cellular transduction proteins may have relationship to promote the pancreatic cancer.