Helicobacter Pylori Induces Epithelial-mesenchymal Transition Of Gastric Epithelial Cells By Activation Of Akt Signaling Pathway

Backgroud and Aim:Gastric cancer is the fourth most common malignancy worldwide, and thesecond most common cancer and the first leading cause of cancer death in China.Although the incidence and mortality of gastric cancer have declined, the morbidityof gastric cancer in China is still high, about17million people died of gastric cancerevery year. Now it’s widely recognized that Hp as a chronic gastritis pathogens, playsan important role in the occurrence and recurrence of peptic ulcer. H. pylori infectionhas been classified as a definite carcinogen for the development of gastric carcinomain1994WHO/.IARC. During development and pathological contexts such as fibrosisand cancer progression,epithelial cells can initiate a complex transcriptionalreprogramming, accompanied by dramatically morphological changes, which named“epithelial-mesenchymal transition”(EMT)．In this transition, epithelial cells losetheir epithelial characteristics to acquire mesenchymal properties and increasedmotile and invasive behavior. EMT plays an important role in apoptosis andproliferation,migration and adhesion. The study has confirmed that Hp couldpromote invasion and metastasis of gastric epithelial cells by activating PI3K/Aktsignaling pathway through inducing EMT. Therefore, in our study, we exploredwhether Hp infection of normal gastric mucosa would induce EMT by activating thePI3K/Akt signaling pathway, resulting in the proliferation and apoptosis disorder,which could develop new theoretical basis for Hp infection-related disease controlstrategies.Materials and Methods:1. A total of165tissue specimens of chronic non-atrophic gastritis (19H.pylori-positive and20H. pylori-negative), intestinal metaplasia (19H. pylori-positiveand21H. pylori-negative), dysplasia (19H. pylori-positive and20H.pylori-negative), and gastric cancer (22H. pylori-positive and25H. pylori-negative)were recruited for immunohistochemical analysis of Akt,p-Akt,GSK-3β,p-GSK-3β,E-cadherin and N-cadherin expression. 2. 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 todetect expression of Akt-GSK-3β-EMT related proteins using Western blot.3. GES-1cells were pretreated with or without Akt Inhibitor VIII or DMSO for1h at the indicated concentrations prior to incubation with H. pylori strainATCC43504at a MOI of50or medium alone. After incubation for1h, cell lysateswere used to detect expression of Akt-GSK-3β-EMT related proteins using Westernblot.4,. Cytoactivetest：MTT methodResults:1. Differential expression of Akt-GSK-3β-EMT signaling pathway protein indifferent stages of gastric lesions in relation to H. pylori infection（1）Overall, there was no significant difference in the expression ofAkt amongall groups (p>0.05). In the presence or absence of H. pylori infection, the expressionof Akt was also no significant difference（p>0.05）. There was no significantdifference in the expression of p-Akt among all groups (p>0.05). In the presence ofH. pylori infection, there was also no significant difference in the expression of p-Aktamong all groups (p>0.05). In the absence of H. pylori infection, the expression ofp-Akt was progressively increased from chronic non-atrophic gastritis to gastriccancer (p <0.05). There was no significant difference in the expression of Aktbetween patients with and those without H. pylori infection among all groups (p>0.05) and in the expression of p-Akt 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-Akt wassignificantly higher in the presence of H. pylori infection than that in the absence ofthe infection (p <0.05).(2) Overall, the expression of GSK-3β was progressively increased from chronicnon-atrophic gastritis to intestinal metaplasia,but sustained low expression in gastriccancer (p <0.001). In the presence or absence of H. pylori infection, the expressionof GSK-3β was also the same expression trend (p <0.001and p <0.05). Theexpression of p-GSK-3βwas progressively increased from chronic non-atrophic gastritis to gastric cancer (p <0.05). In the presence of H. pylori infection, theexpression of p-GSK-3βwas also progressively increased from chronic non-atrophicgastritis to intestinal metaplasia, dysplasia and sustained high expression in gastriccancer (p <0.05). In the absence of H. pylori infection, there was no significantdifference in the expression of p-GSK-3βamong all groups (p>0.05). There was nosignificant difference in the expression of GSK-3βand p-GSK-3β between patientswith and those without H. pylori infection in chronic non-atrophic gastritis, intestinalmetaplasia and dysplasia(p>0.05). However, in patients with gastric cancer, theexpression of GSK-3βwas significantly lower in the presence of H. pylori infectionthan in the absence of the infection, the expression of p-GSK-3β was significantlyhigher in the presence of H. pylori infection than in the absence of the infection (p <0.05).(3) The expression of E-cadherin was progressively decreased from chronicnon-atrophic gastritis to gastric cancer (p <0.001). In the presence or absence of H.pylori infection, the expression of E-cadherin was also progressively decreased fromchronic non-atrophic gastritis to intestinal metaplasia, dysplasia and sustained lowexpression in gastric cancer (p<0.001and p<0.05). The expression of N-cadherin wasprogressively increased from chronic non-atrophic gastritis to gastric cancer (p <0.05). In the presence of H. pylori infection, the expression of N-cadherin was alsoprogressively increased from chronic non-atrophic gastritis to gastric cancer (p <0.05). In the absence of H. pylori infection, the expression of N-cadherin wassustained high expression in dysplasia and gastric cancer (p <0.05). There was nosignificant difference in the expression of E-cadherin between patients with and thosewithout H. pylori infection in intestinal metaplasia and dysplasia(p>0.05), and in theexpression of N-cadherin between patients with and those without H. pylori infectionin chronic non-atrophic gastritis, intestinal metaplasia and dysplasia (p>0.05).However, in patients with chronic non-atrophic gastritis and gastric cancer, theexpression of E-cadherin was significantly lower in the presence of H. pyloriinfection than in the absence of the infection(p <0.05), and in patients with gastriccancer, the expression of N-cadherin was significantly higher in the presence of H.pylori infection than in the absence of the infection (p <0.05). 2.The effect of H. pylori in the expression of Akt-GSK-3β-EMT pathway relatedproteins in gastric cell linesAfter incubation with H. pylori at a MOI of50, there was no significantdifference in the expression of Akt and GSK-3β (p>0.05), however, the expressionof p-Akt increased0.5h after incubation, and reached the peak at3h and keptincreasing during12h after incubation (p <0.001), and the expression of p-GSK-3βincreased0.5h after incubation, and reached the peak at12h after incubation (p <0.001).The expression of E-cadherin decreased0.5h after incubation, and reached thebottom at6h and kept decreasing during12h after incubatio（np<0.05）, the expressionof N-cadherin increased0.5h after incubation, and reached the peak at3h and keptincreasing during12h after incubation (p <0.001). After incubation with mediumalone, there was no significant difference in the expression of Akt, p-Akt, GSK-3β,p-GSK-3β, E-cadherin and N-cadherin (p>0.05).3.The role of Akt in activation of Akt-GSK-3β-EM T pathway by H. pylori(1) After pretreated with Akt InhibitorVIII for1h, there was no significantdifference in the expression of Akt, GSK-3β, E-cadherin and N-cadherin (p>0.05),however, the expression of p-Akt (p <0.001) and p-GSK-3β (p <0.001) wassignificantly decreased. After pretreated with DMSO for1h, there was no significantdifference in the expression of Akt, p-Akt, GSK-3β, p-GSK-3β, E-Cadherin andN-Cadherin (p>0.05).(2)After these cell lines, which were pretreated with or without Akt InhibitorVIII or DMSO for1h, incubated with H. pylori at a MOI of50or medium alone for1h, there were no significant differences in the expression of Akt and GSK-3β amongall groups (p>0.05). However, the expression of p-Akt (p <0.05and p <0.01),p-GSK-3β (p <0.01and p <0.05), N-Cadherin（p<0.05、p<0.05）were significantlyhigher and the expression of E-cadherin were lowe（rp>0.05、p>0.05）in the cell linesincubated with H. pylori than that of incubated with medium alone in control andDMSO. However there was no significant difference in the expression of p-Akt,p-GSK-3β, E-Cadherin and N-Cadherin in the cell lines incubated with H. pylori inAkt Inhibitor VIII groups.4.The function of Akt-GSK-3β-EMT signaling pathway in the effect of H.pylori on the biology behavior in gastric epithelial cell lines(1) The results of MTT analysis of GES-1cells co-cultured with H.pylori atdifferent MOI rates. The result showed that there was no significant difference insurvival rate at MOI of5:1and10:1for12h (p>0.05), whereas our results indicatedan increase of survival rate in cells incubation with H. pylori at a MOI of50:1and100:1whith6h, reached the peak at12h(P<0.001). Furthermore, the survival rate ofcells for6h and12h was significantly higher than that of0.5h、1h and3h（p<0.001）,similar results were obtained between6h and12h（p<0.001）. GES-1cells wereco-cultured with H.pylori at indicated range of mulitiplicity of infection (MOI)between5-100:1.The survival rate was not different from that of control group for0.5、1、3h despite of its mulitiplicity of infection (MOI)（p>0.05）, while the survivalrate of groups whose MOI were50:1and100:1were higher than that of control groupand lower MOI groups when the action time lasted6h and12h（p<0.001）, the cellsurvival rate of MOI of100:1was significantly higher than that of MOI of50:1group(p <0.001).(2) The results of MTT analysis of GES-1cells pretreated with DMSO or AktInhibitor VIII were co-cultured with H.pylori at a MOI of50:1for different times.The survival rate increased at6h after incubation without Akt Inhibitor VIII andDMSO pretreatment, reached the peak at12h (p <0.001), and the rate at6h and12hwas significantly higher than that at0.5,1,3h (p <0.001), the12h cell survival ratewas significantly higher than that at6h (p <0.01); while the survival rate showed nodifference when H.pylori was co-cultured with GES-1cells with Akt Inhibitor VIIIpretreatment（p>0.05）. The survival rate was not different from each control groupswhen H.pylori was co-cultured with GES-1cells without Akt Inhibitor VIII andDMSO pretreatment for0.5,1,3h（p>0.05）, while the results became significantlydifferent when the incubation time lasted6h and12h（p>0.05）. Otherwise, thesurvival rate showed no difference when H.pylori was co-cultured with GES-1cellswith Akt Inhibitor VIII pretreatment within12h（p>0.05）.The cells survival rate hasno obvious difference between groups when H.pylori was co-cultured with GES-1cells without Akt Inhibitor VIII and DMSO pretreatment for0.5,1,3h（p>0.05）,while the survival rate were significantly higher when H.pylori was co-cultured with GES-1cells without Akt Inhibitor VIII and DMSO pretreatment thanthat of Akt Inhibitor VIII pretreatment of GES-1(p <0.001). There was nosignificant difference between cells without Akt Inhibitor VIII pretreatment andDSMO pretreatment (p>0.05).Conclusions:1. The results of vivo study showed that Akt-GSK-3β-EMT signaling pathwayswere involved in the occurrence and development of gastric mucosal lesions relatedto Hp infection and may induce EMT in the early stage of gastric mucosa lesions andpromote carcinogenesis of gastric cancer.2The results of vitro study showed that Hp could activate Akt signaling pathwayand act on its downstream effectors GSK-3β in the early stage of infection whichinduced the occurance of EMT in normal gastric mucosa cells, thereby causingproliferation and apoptosis disorders of gastric mucosal cell which participated in theincidence of gastric cancer.3. Helicobacter pylori could induce epithelial-mesenchymal transition of gastricepithelial cells by activation of Akt signaling pathway.4. Helicobacter pylori could increase viability of gastric epithelial cells byactivation Akt-GSK-3β-EMT signaling pathway.