| Backgroud andAim:Gastric carcinoma is a worldwide malignant disease with high incidence andmortality, and it is characterized by genome instability through severe DNA damagecaused by various factors, including Helicobacter pylori (H.pylori) infection, heredityand living habits. It is well-established that H.pylori infection is a definite etiologicalfactor in gastric carcinogenesis. However, the mechanism through which H.pyloriinfection contributes to the development of gastric cancer has not been fullyelucidated. The latest study showed an increased rate of DNA double strand breaks(DSBs) in H.pylori infected gastric mucosa cells. The catalytic subunit of theDNA-dependent protein kinase (DNA-PKcs) and the Ku70/Ku80heterodimer(Ku70/80)are crucial promoters of the non-homologous end joining (NHEJ) pathway.Accumulating evidence has shown that dysregulation of DNA-PKcs and Ku70/80isassociated with pathological processes in various tumors. The expression changes andbiological function of DNA-PKcs and Ku70/80in gastric cancer are still unclear. Wedetermined a possible pathological role of DNA-PKcs and Ku70/80-mediated DNArepair pathways in H.pylori related human gastric pathology processes.Materials and Methods：1. A total of146gastric biopsies were obtained from gastric carcinoma patients[H.pylori-negative:89and H.pylori-positive:57] and34from normal subjects[H.pylori-negative:16and H.pylori-positive:18] via surgery and endoscopic detection.The expression of DNA-PKcs and the Ku70/80protein was detected byimmunohistochemistry.2. GES-1and AGS cells were co-cultured with H.pylori (ATCC43504, Cag A+Vac A+) at a cell/bacteria ratio of1:100from1h,3h,6h,12h to24h, the whole celllysated were harvested and subjected to western blot using specific antibodies,western blot for β-actin served as loading control.3. The Mongolian gerbils were divided into two groups:(1) control (n=30)housed normally according to the feeding standard, and randomly sacrificed fifteen gerbils at6,12months of experiment;(2) H.pylori infected group (n=60) receivingintragastric administration of living H.pylori levitation liquid in concentration of109CFU/ml five times in each other day,0.5ml/per time and randomly sacrificed thirtyof them at6,12months of the experiment for analysis.Immunohistochemistry assaywere used to estimate DNA-PKcs and Ku70/80protein expression in gerbils.Results:1. Expression of DNA-PKcs and Ku70/80in human gastric mucosa tissues andthe correlation between the expression of DNA-PKcs and Ku70/80and differentclinical parameters in gastric carcinoma patientsOverall, the positive rates of both DNA-PKcs and Ku70/80were significantlyincreased in gastric cancer (χ2=133.04, P <0.001for DNA-PKcs and χ2=13.06, P <0.01for Ku) compared with normal gastric mucosa (NGM). There was hardly anydetectable expression of DNA-PKcs in NGM, and the positive rate of DNA-PKcsprotein expression in patients with NGM was0%(0/34), whereas the rate in gastriccancer (GC) was93.8%(137/146). The difference between the two groups wasstatistically significant. Additionally, the positive rate of Ku70/80was79.4%(27/34)in NGM and96.6%(141/146) in GC. The DNA-PKcs protein level was significantlyincreased in GC (Mann-Whitney U=39.00, P <0.001), compared with NGM. Inaddition, there was a significant difference in the expression of Ku70/80(Mann-Whitney U=1117.00, P <0.001) between GC and NGM. There was also asignificant difference in Ku70/80protein expression between GC patients with andwithout H.pylori infection (P <0.05). Spearman analysis showed a negativecorrelation between tumor differentiation and DNA-PKcs expression (r=-0.447, P <0.05). Moreover, Ku70/80expression was negatively correlated with both clinicalstage (r=-0.189, P <0.05) and H.pylori colonization (r=-0.168, P <0.05).2.The effect of H.pylori on the expression of DNA-PKcs and Ku70/80in gastriccell linesAfter incubation with Hp at a MOI of100, the expression of DNA-PKcs was1.16±0.08in GES-1cells co-cultured with H.pylori for1hour, while it’s1.04±0.32inthe control, the difference was statistically significant (t1h=4.67，P<0.05). With theextended response time, the expression of Ku70/80were1.58±0.33,1.84±0.41, 1.97±0.34,3.72±1.43and3.74±1.55in GES-1cells co-cultured with H.pylori for1,3,6,12and24hours respectively, and it’s1.24±0.42in GES-1cells without H.pylori,the difference were statistically significant (t=3.57,4.20,5.03,8.11and8.14,P<0.05). Ku70/80protein expression in AGS cells were4.69±0.86,3.67±0.66,2.41±0.25,1.35±0.36and1.32±0.11after co-cultured with H.pylori for1,3,6,12and24hours respectively, all decreased significantly than that in AGS cells withoutH.pylori, which was4.84±0.77(t=34.13,27.68,19.81,4.47and5.68，P<0.05).3. Effects of H.pylori on the expression of DNA-PKcs and Ku70/80inMongolian gerbilsAt six and twelve months post-challenge, the total positive rate of DNA-PKcs ininfected gerbils was98.1%(53/54), while it’s all negative in the controls (0/25), thedifference between the two groups was statistically significant (χ2=74.55,P<0.01).However, the total positive rate of Ku70/80heterodimer was68.5%(37/54) and92%(23/25) in infected gerbils and control gerbils respectively, the differencebetween the two groups was statistically significant (χ2=5.16,P<0.05).Conclusions:1. Enhanced DNA-PKcs and Ku70/80expression may be closely associated withgastric carcinoma.2. In Vitro, H.pylori could increase the expression of Ku70/80in GES-1cells, aswell as inhibit Ku70/80heterodimer expression inAGS cells.3. In vivo, H.pylori infection could promote the expression of DNA-PKcs anddown-regulate Ku70/80expression in gerbil gastric mucosa, suggested that theimbalanced expression of DNA-PKcs and Ku70/80exacerbated H.pylori inducedDNAdamage in gastric mucosa cells.4. The abnormal DNA repairing activities mediated by DNA-PKcs and Ku70/80heterodimer may be associated closely with pathological process in gastric mucosacaused by H.pylori. |
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