| Background and Aim:Gastric cancer is the fourth most common cancer worldwide, the third leading cause of cancer-related death in males and the fifth among females, indicating that it is still one of the most significant health burdens in the world, although the incidence and mortality of gastric cancer have declined. To date, surgical resection remains as the only curative means to improve survival of patients with gastric cancer, while chemotherapy has limited effects and is usually served to palliatively relieve patients’ symptoms and increase survival time. Therefore, it is necessary to investigate the mechanisms of gastric carcinogenesis and to develop novel strategies to prevent gastric cancer.For this purpose, our research objective focuses on PTEN, a tumor suppressor gene identified in1997. PTEN encodes a dually functional phosphatase with lipid and protein phosphatase activities and is involved in regulation of a variety of signaling transduction pathways, such as PI3K/Akt pathway, which are critical in cell apoptosis, adhesion, and mobility as well as regulation of chromosomal stability. Recent studies have indicated that PTEN was frequently inactivated in gastric cancers due to genetic or epigenetic changes, such as mutation, loss of heterozygosity, promoter hypermethylation and regulation of microRNA. However, it remains to be defined whether other mechanisms account for inactivation of PTEN and how PTEN inactivation promotes carcinogenesis. Indeed, phosphorylation is the most important mechanism of post-translational modification of PTEN, which leads to a loss of phosphatase activities or a gain of PTEN stability, and in turn results in loss of tumor suppressor function and increased cancer susceptibility.Helicobacter pylori infection plays a critical role in the development of gastric carcinoma through a multistep process from chronic gastritis to intestinal metaplasia, dysplasia and finally gastric carcinoma. Therefore, the aim of the present study was to determine the expression of PTEN and p-PTEN in different stages of gastric lesions with or without H. pylori infection, and the expression of PTEN/PI3K/Akt pathway related proteins with or without H. pylori infection in vivo or in vitro, in order to gain an insight into the role of PTEN phosphorylation in gastric cancer development in relation to H. pylori infection.Materials and Methods:1.15tissue specimens of gastric cancer and paired adjacent mucosa,4gastric cancer MKN-28, MKN-45, SGC-7901, and AGS cell lines and1non-cancerous gastric GES-1cell line were used to detect expression of PTEN and p-PTEN using Western blot.2. A total of160tissue specimens of chronic non-atrophic gastritis (20H. pylori-positive and20H. pylori-negative), intestinal metaplasia (20H.pylori-positive and20H. pylori-negative), dysplasia (20H.pylori-positive and20H. pylori-negative), and gastric cancer (20H.pylori-positive and20H. pylori-negative) were recruited for immunohistochemical analysis of PTEN and p-PTEN expression.3. Mongolian gerbils were orogastrically challenged with either sterile H. pylori strain ATCC43504or Brucella broth. The animals were euthanized at6or12months after last challenge and gastric tissues were collected and used to detect expression of PTEN/PI3K/Akt related proteins using immunohistochemistry.4. GES-1cells were incubated with H. pylori strain ATCC43504at a MOI of50or medium alone for0,0.5,1,3,6or12h. After incubation, cell lysates were used to detect expression of PTEN/PI3K/Akt related proteins using Western blot.5. GES-1cells were pretreated with or without LY294002or DMSO for1h at the indicated concentrations prior to incubation with H. pylori strain ATCC43504at a MOI of50or medium alone. After incubation for1h, cell lysates were used to detect expression of PTEN/PI3K/Akt related proteins using Western blot.6. GES-1cells were pretreated with or without Akt iVIII or DMSO for1h at the indicated concentrations prior to incubation with H. pylori strain ATCC43504at a MOI of50or medium alone. After incubation for1h, cell lysates were used to detect expression of PTEN/PI3K/Akt related proteins using Western blot.7. GES-1cells were incubated with H. pylori strain ATCC43504at a MOI of5,10,50or100or medium alone, after incubation for0,0.5,1,3,6or12h, cell viability were detected using MTT assay; GES-1, GES-1-Empty, GES-1-PTEN-WT or GES-1-PTEN-MT cells were incubated with H. pylori strain ATCC43504at a MOI of50or medium alone, after incubation for0,0.5,1,3,6or12h, cell viability were detected using MTT assay; GES-1cells were pretreated with or without Akt iVIII or DMSO for1h at the indicated concentrations prior to incubation with H. pylori strain ATCC43504at a MOI of50or medium alone, after incubation for0,0.5,1,3,6or12h, cell viability were detected using MTT assay.Results:1. Differential expression of PTEN and p-PTEN in gastric cancer tissues and cell lines(1)13cases (13/15,86.7%) showed a reduction in PTEN expression in gastric cancer tissues compared with the paired adjacent mucosa (p<0.001).10cases (10/15,66.7%) showed a reduction in p-PTEN expression in gastric cancer tissues compared with the paired adjacent mucosa (p>0.05).(2)10cases (10/15,66.7%) showed a increasing in the p-PTEN and PTEN ratio in gastric cancer tissues compared with the paired adjacent mucosa (p<0.05).(3) The levels of both PTEN and p-PTEN protein were higher in gastric cancer cell lines than in non-malignant GES-1cells, e.g.,3.43±0.41(p<0.001),3.45±0.25(p<0.01),3.25±0.39(p<0.01), and2.22±0.21(p<0.01) folds of MKN-28, MKN-45, SGC-7901, and AGS, respectively to the level of PTEN protein of GES-1, and4.60±0.36(p<0.01),19.5±1.39(p<0.001),18.9±1.68(p<0.01), and7.96±0.32(p<0.001) folds of MKN-28, MKN-45, SGC-7901, and AGS, respectively to the level of p-PTEN protein of GES-1.(4) The p-PTEN and PTEN ratio was higher in gastric cancer cell lines than that of non-malignant GES-1cells, e.g.,1.35±0.17(p>0.05),5.66±0.58(p<0.01),5.84±0.50(p<0.01), and3.60±0.31(p<0.01) folds of MKN-28, MKN-45, SGC-7901, and AGS, respectively to the ratio of GES-1.2. Differential expression of PTEN and p-PTEN in different stages of gastric lesions in relation to H. pylori infection(1) Overall, the expression of PTEN was progressively decreased from chronic non-atrophic gastritis to gastric cancer (p<0.001). In the presence or absence of H. pylori infection, the expression of PTEN was also progressively decreased from chronic non-atrophic gastritis to gastric cancer (p<0.001and p<0.05).(2) Overall, the expression of p-PTEN was progressively increased from chronic non-atrophic gastritis to intestinal metaplasia, and taking into account total PTEN reduction, was sustained high expression in dysplasia and gastric cancer (p<0.001). In the absence of H. pylori infection, the expression of p-PTEN was also progressively increased from chronic non-atrophic gastritis to intestinal metaplasia, and sustained high expression in dysplasia and gastric cancer (p<0.001). In the presence of H. pylori infection, there was no significant difference in the expression of p-PTEN among all groups (p>0.05).(3) There was no significant difference in the expression of PTEN between patients with and those without H. pylori infection among all groups (p>0.05), and in the expression of p-PTEN between patients with and those without H. pylori infection in intestinal metaplasia, dysplasia, and gastric cancer (p>0.05). However, in patients with chronic non-atrophic gastritis, the expression of p-PTEN was significantly higher in the presence of H. pylori infection than in the absence of the infection (p<0.05).3. The effect of H. pylori infection on the expression of PTEN/PI3K/Akt pathway related proteins in gastric mucosa of Mongolian gerbils(1) There was no significant difference in the expression of PTEN between6and12months after last challenge in Mongolian gerbils challenged with or without H. pylori (p>0.05), and between the groups challenged with and without H. pylori at6or12months after last challenge (p>0.05).(2) The expression of p-PTEN was significantly higher in Mongolian gerbils having12months challenge compared with those having6months challenge (p<0.001). There was no significant difference in the expression of p-PTEN between challenged with and without H. pylori at6months after last challenge (p>0.05), however, the expression of p-PTEN was significantly higher in Mongolian gerbils challenged with H. pylori than that without H. pylori at12months after last challenge (p<0.001).(3) The expression of PI3K was significantly higher in Mongolian gerbils having12months challenge compared with those having6months challenge (p<0.01). There was no significant difference in the expression of PI3K between challenged with and without H. pylori at6or12months after last challenge (p>0.05).(4) The expression of Akt was significantly higher in Mongolian gerbils having12months challenge compared with those having6months challenge (p<0.001). There was no significant difference in the expression of Akt between challenged with and without H. pylori at6or12months after last challenge (p=0.05, and p>0.05).(5) The expression of p-Akt (Ser473) was significantly higher in Mongolian gerbils having12months challenge compared with those having6months challenge (p<0.05). The expression of p-Akt (Ser473) was significantly higher in Mongolian gerbils challenged with H. pylori than that without H. pylori at6or12months after last challenge (p<0.05).(6) The expression of p-Akt (Thr308) was significantly higher in Mongolian gerbils having12months challenge compared with those having6months challenge (p<0.05). The expression of p-Akt (Thr308) was significantly higher in Mongolian gerbils challenged with H. pylori than that without H. pylori at6or12months after last challenge (p<0.05).(7) The expression of Bad was significantly higher in Mongolian gerbils having12months challenge with H. pylori compared with those having6months challenge with H. pylori (p<0.05), however, there was no significant difference in the expression of Bad between6and12months after last challenge in Mongolian gerbils challenged without H. pylori (p>0.05). There was no significant difference in the expression of Bad between challenged with and without H. pylori at6or12months after last challenge (p>0.05).(8) The expression of p-Bad was significantly higher in Mongolian gerbils having12months challenge without H. pylori compared with those having6months challenge without H. pylori (p<0.01), however, there was no significant difference in the expression of p-Bad between6and12months after last challenge in Mongolian gerbils challenged with H. pylori (p>0.05). The expression of p-Bad was significantly higher in Mongolian gerbils challenged with H. pylori than that without H, pylori at6months after last challenge (p<0.05), however, there was no significant difference in the expression of p-Bad between challenged with and without H. pylori at12months after last challenge (p>0.05).(9) There was no significant difference in the expression of GSK3β between6and12months after last challenge in Mongolian gerbils challenged with or without H. pylori (p>0.05), and between the groups challenged with and without H. pylori at6or12months after last challenge (p>0.05).(10) The expression of p-GSK3β was significantly higher in Mongolian gerbils having12months challenge with H. pylori compared with those having6months challenge with H. pylori (p<0.05), however, there was no significant difference in the expression of p-GSK3β between6and12months after last challenge in Mongolian gerbils challenged without H. pylori (p>0.05). There was no significant difference in the expression of p-GSK3β between challenged with and without H. pylori at6or12months after last challenge (p>0.05).4. The effect of H. pylori on the expression of PTEN/PI3K/Akt pathway related proteins in gastric cell lines(1) After incubation with H. pylori at a MOI of50, there was no significant difference in the expression of PTEN (p>0.05), however, the expression of p-PTEN increased0.5h after incubation, and reached the peak at3h and kept increasing during12h after incubation (p<0.001). After incubation with medium alone, there was no significant difference in the expression of PTEN and p-PTEN (p>0.05).(2) After incubation with H. pylori at a MOI of50, there was no significant difference in the expression of Akt, Bad, and GSK3β (p>0.05), however, the expression of p-Akt (Ser473) and p-Akt (Thr308) increased0.5h after incubation, and reached the peak at3h and kept increasing during12h after incubation (p<0.001), and the expression of p-Bad and p-GSK3β increased0.5h after incubation, and reached the peak at12h after incubation (p<0.001). After incubation with medium alone, there was no significant difference in the expression of Akt, p-Akt (Ser473), p-Akt (Thr308), Bad, p-Bad, GSK3β, and p-GSK3β (p>0.05).5. The role of PI3K in activation of PI3K/Akt pathway by H. pylori(1) After incubation with H. pylori at a MOI of50or with medium alone, there was no significant difference in the expression of PI3K (p>0.05).(2) After pretreated with LY294002for1h, there was no significant difference in the expression of PTEN, p-PTEN, PI3K, Akt, Bad, and GSK3β (p>0.05), however, the expression of p-Akt (Ser473)(p<0.001), p-Akt (Thr308)(p<0.001), p-Bad (p<0.001), and p-GSK3β (p<0.01) was significantly decreased. After pretreated with DMSO for1h, there was no significant difference in the expression of PTEN, p-PTEN, PI3K, Akt, p-Akt (Ser473), p-Akt (Thr308), Bad, p-Bad, GSK3β, and p-GSK3β(p>0.05).After these cell lines, which were pretreated with or without LY294002or DMSO for1h, incubated with H. pylori at a MOI of50or medium alone for1h, there were no significant differences in the expression of PTEN, PI3K, Akt, Bad, and GSK3β among all groups (p>0.05). However, the expression of p-PTEN (p<0.05, p <0.001, and p<0.01), p-Akt (Ser473)(p<0.001, p<0.01, and p<0.01), p-Akt (Thr308)(p<0.001,p<0.01, and p<0.01), p-Bad (p<0.05,p<0.05, and p<0.01), and p-GSK3β (p<0.05, p<0.05, and p<0.05) were significantly higher in the cell lines incubated with H. pylori than that of incubated with medium alone in control, DMSO, and LY294002groups.6. The role of Akt in activation of PI3K/Akt pathway by H. pyloriAfter pretreated with Akt iⅧ for1h, there was no significant difference in the expression of PTEN, p-PTEN, PI3K, Akt, Bad, and GSK3β (p>0.05), however, the expression of p-Akt (Ser473)(p<0.001), p-Akt (Thr308)(p<0.001), p-Bad (p<0.001), and p-GSK3β (p<0.01) was significantly decreased. After pretreated with DMSO for1h, there was no significant difference in the expression of PTEN, p-PTEN, PI3K, Akt, p-Akt (Ser473), p-Akt (Thr308), Bad, p-Bad, GSK3β, and P-GSK3β (p>0.05).After these cell lines, which were pretreated with or without Akt iVIII or DMSO for1h, incubated with H. pylori at a MOI of50or medium alone for1h, there were no significant differences in the expression of PTEN, PI3K, Akt, Bad, and GSK3β among all groups (p>0.05). However, the expression of p-PTEN (p<0.01, and p<0.01), p-Akt (Ser473)(p<0.05, and p<0.01), p-Akt (Thr308)(p<0.05, and p<0.01), p-Bad (p<0.05, and p<0.05), and p-GSK3β (p<0.01, and p<0.05) were significantly higher in the cell lines incubated with H. pylori than that of incubated with medium alone in control, and DMSO groups. The expression of p-PTEN (p< 0.01) was significantly higher in the cell lines incubated with H. pylori than that of incubated with medium alone in Akt iVIII group, however, there were no significant differences in the expression of p-Akt (Ser473), p-Akt (Thr308), p-Bad, and p-GSK3β (p>0.05) between the cell lines incubated with H. pylori and incubated with medium alone in Akt iVIII group.7. The role of PTEN/PI3K/Akt pathway in effect of cell viability induced by H. pylori(1) After incubation with H. pylori at a MOI of5or10in12h, there was no significant difference in cell viability of GES-1(p>0.05), however, the cell viability of GES-1increased6h after incubation, and reached the peak at12h after incubation with H. pylori at a MOI of50or100(p<0.001), and the cell viability was significantly higher in GES-1having12h incubation with H. pylori compared with those having6h incubation with H. pylori (p<0.001).(2) The cell viability of GES-1, GES-1-Empty, and GES-1-PTEN-MT increased6h after incubation, and reached the peak at12h after incubation with H. pylori at a MOI of50(p<0.001). The cell viability was significantly higher in GES-1, GES-1-Empty, and GES-1-PTEN-MT having6or12h incubation with H. pylori compared with those having0.5,1, or3h incubation with H. pylori (p<0.001), and the cell viability was significantly higher in GES-1, GES-1-Empty, and GES-1-PTEN-MT having12h incubation with H. pylori compared with those having6h incubation with H. pylori (p<0.01). However, after incubation with H. pylori at a MOI of50in12h, there was no significant difference in cell viability of GES-1-PTEN-MT (p>0.05).(3) The cell viability of GES-1, which were pretreated with or without DMSO for1h, increased6h after incubation, and reached the peak at12h after incubation with H. pylori at a MOI of50(p<0.001), the cell viability was significantly higher in these cell lines having6or12h incubation with H. pylori compared with those having0.5,1, or3h incubation with H. pylori (p<0.001), and the cell viability was significantly higher in these cell lines having12h incubation with H. pylori compared with those having6h incubation with H. pylori (p<0.01). However, after incubation with H. pylori at a MOI of50in12h, there was no significant difference in cell viability of GES-1, which was pretreated with Akt iⅧ (p>0.05).Conclusions:1. Reduced expression of PTEN and increased PTEN phosphorylation could contribute to gastric carcinogenesis, and that PTEN phosphorylation could be a novel mechanism of PTEN inactivation in gastric carcinogenesis.2. H. pylori phosphorylates and inactivates PTEN at the early stage, resulting in activation of PI3K/Akt pathway and subsequently promoting cell survival. |
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