| Background and Aim:Gastric cancer is a common malignant tumor. In recent years, with the changeof lifestyle and the improvement of diagnosis and treatment, the incidence andmortality have been significant lower, but is still the fourth most common malignanttumor. A number of studies have demonstrated that Helicobacter pylori infection andgastric cancer are closely related. In1994H. pylori has been classifed as classifiedas I carcinogen by WHO. H. pylori infection rate of natural population worldwidehas been more than half of the total population. However, the pathogenesis of H.pylori induced gastric cancer remains unclear. Therefore, it is clinically important toclarity the mechanisms of gastric cancer，to explore the early diagnosis of gastriccancer and gene therapy is a new method has important significance.PTEN is one of the most valuable tumor suppressor genes, only to p53, andencodes a dually functional phosphatase with lipid and protein phosphatase activities.Phosphorylation is the most significant method of post-translational modification ofPTEN. CK2phosphorylation on S370and S385causes stabilization of PTEN. AndPTEN plays an important role in cell migration, invasion, cellular actin cytoskeletonand growth by regulating downstream target factor FAK. Helicobacter pyloriinfection plays the originating and leading roles in the development of gastriccarcinoma, but how influences the gastric carcinogenesis mechanism by PTEN/FAKsignaling pathway is not clear. Here we investigate the roles of PTEN/FAK signalingpathway in different stages of gastric lesions in vivo or in vitro, and its relation to H.pylori infection.Materials and Methods:1. Immunohistochemical analysis the PTEN, p-PTEN, FAK and p-FAKproteins in different gastric mucosal injury tissues.2. We performed immunohistochemical analysis to determine the expression of PTEN, p-PTEN, FAK and p-FAK proteins in gastric mucosa of Mongolian gerbilsat6or12months.3. After incubation with H. pylori at a MOI of50for0,0.5,1,3,6or12h inGES-1, Western blot were used to detect expression of PTEN/FAK related proteins.4. Build different gastric epithelial cell lines with overexpression of PTENprotein, GES-1, GES-1-Empty, GES-1-PTEN-WT or GES-1-PTEN-MT, weperformed the incubation1h with H. pylori at a MOI of50, Western blot were usedto detect expression of PTEN/FAK related proteins.5. Standard Transwell assays were used in GES-1, GES-1-Empty,GES-1-PTEN-WT or GES-1-PTEN-MT, we performed the incubation1h with H.pylori at a MOI of50. After incubation for20h, invasion cells were counted afterCrystal violet staining.Results:1. Differential expression of PTEN, p-PTEN, FAK and p-FAK in different stagesof gastric lesions.(1) Immunohistochemical analysis showed that PTEN, p-PTEN, FAK andp-FAK proteins were mainly located in the cytoplasm of epithelial cells. Theexpression of PTEN protein was reduced from CNAG to GC (p <0.001).Theexpression of PTEN was also showed a similar trend (p <0.001and p <0.05). Thep-PTEN protein expression was increased from CNAG to IM, and considering PTENprotein was high expression in Dys and GC (p <0.001). In control group, p-PTENexpression was also presented a similar result (p <0.001). In the control group,expression of p-PTEN was no significant difference at all stages (p>0.05). Theexpression levels of FAK in the study were significantly increased as pathologicalstages progressed from CNAG to GC. In CNAG group compared with Dys or GCgroups, there were significant differences. Moreover, in control group, FAKexpression was significantly higher in patients with CNAG compared with GC(30.38and52%). The results showed that the expression ratio of p-FAK was47.91%in the CNAG group,100%in the IM group,86.55%in the Dys group and87.34%inthe GC group. There were no significant differences in p-FAK expression between IM and Dys or CG, and among the other stages of disease were significantlydifferent (p <0.001). The p-FAK expression level was significantly decreased inCNAG compared with IM, Dys and GC.(2) Among all groups, PTEN protein expression was no significant differencebetween H. pylori infection and control group (p>0.05). However, in patients withCNAG, p-PTEN protein expression was significantly increased in H. pylori infectiongroup than control group (p <0.05). Besides, there was no significant difference inthe expression of FAK and p-FAK between patients with and those without H. pyloriinfection in CNAG, IM and Dys (p>0.05). However, in patients with GC, theexpression of FAK was significantly lower in the presence of H. pylori infection thanthat control group (p <0.05), while there was no significant difference in theexpression of p-FAK.2. The effect of H. pylori infection on the expression of PTEN/FAK pathwayrelated proteins in gastric mucosa of Mongolian gerbils(1) We performed immunohistochemical analysis to determine the expressionof PTEN, p-PTEN, FAK and p-FAK proteins in gastric mucosa of Mongolian gerbils.There was no significant difference in the expression of PTEN between6and12months in H. pylori and control group (p>0.05), and between H. pylori and controlgroup at6and12months (p>0.05). The p-PTEN protein expression wassignificantly increased at12months compared with6months (p<0.001). In addition,the expression of p-PTEN was no obvious difference between H. pylori and controlgroup at6months (p>0.05). However, the expression of p-PTEN was significantlyhigher in Mongolian gerbils challenged with H. pylori than that without H. pylori at12months (p <0.001).(2) Compared to6months, FAK expression in Mongolian gerbils wassignificantly higher at12months (p <0.001, p=0.001). FAK expression was noobvious difference between H. pylori and control group at6or12months (p>0.05).The expression of p-FAK was significantly higher at12months with H. pyloricompared with6months (p <0.001). p-FAK expression was no obvious differencebetween H. pylori with and control group at6months (p>0.05), however, theexpression of p-FAK was significantly higher in Mongolian gerbils challenged with H. pylori than the control group at12months (p <0.001).3. The effect of H. pylori in the expression of PTEN/FAK pathway relatedproteins in gastric cell linesAfter incubation with H. pylori at a MOI of50, there was no significantdifference in the expression of PTEN (p>0.05), however, the expression of p-PTENincreased0.5h after incubation, and reached the peak at1h and kept increasingduring12h (p <0.001). After incubation with medium alone, there was no significantdifference in the expression of PTEN and p-PTEN (p>0.05). After incubation withH. pylori at a MOI of50, there was no significant difference in the expression ofFAK (p>0.05), however, the expression of p-FAK increased0.5h after incubation,and reached the peak at3h and kept increasing during12h (p <0.001). Afterincubation with medium alone, there was no significant difference in the expressionof FAK and p-FAK (p>0.05).4. The role of PTEN in activation of PTEN/FAK pathway by H. pylori(1) We performed the incubation with H. pylori1h (MOI=50:1) in GES-1,GES-1-Empty, GES-1-PTEN-WT and GES-1-PTEN-MT, PTEN expression was noobvious difference (p>0.05), however, p-PTEN (p <0.001, p <0.001, p=0.001and p <0.05) was significantly higher with H. pylori than that of incubated withmedium alone.(2) After incubation1h with H. pylori at a MOI of50in GES-1, GES-1-Empty,GES-1-PTEN-WT and GES-1-PTEN-MT, FAK expression was no obviousdifference (p>0.05). The expression of p-FAK (p <0.001, p <0.001and p <0.05)was significantly higher with H. pylori than that of incubated with medium alone inGES-1, GES-1-Empty and GES-1-PTEN-WT, however, there was no significantdifference in GES-1-PTEN-MT (p>0.05). The expression of p-FAK (p <0.05) inGES-1-PTEN-WTwas significantly lower than GES-1,5. Effect of cell invasion induced after H. pylori infection by PTEN/FAKpathwayGiven that wPTEN expression was negatively correlated with p-FAK, wepostulated that wPTEN might influence cell invasion by H. pylori infection. Asshown in Figure3C, standard Transwell assays were next performed that the number of cell invasion showed a significant increase with H. pylori compared to that ofincubated with low serum medium alone (113.67±7.23versus118.10±9.00,85.06±10versus150.32±7.57,159.03±15.96versus179.67±6.02,158.97±5.00versus185.87±4.93, p <0.05). The cells in GES-1-PTEN-WT group (p <0.001)were significantly decreased compared to GES-1group, but increased inGES-1-PTEN-MT (p <0.001).Conclusions:1. In vivo, reduced expression of PTEN, increased PTEN phosphorylation andhigh expression of FAK and p-FAK could contribute to gastric carcinogenesis.2. H. pylori could induce a significant increase of PTEN phosphorylation anddecrease of phosphatase activity, activating its downstream effector FAK to.Therefore, we speculate that p-PTEN may be a novel inactivation of PTEN and theprocess is triggered by H. pylori infection.3. H. pylori infection improved the cell invasive ability by decreasing PTENphosphatase activity. |
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