| Research BackgroundGastric cancer is one of the demanding malignancy worldwide,with a very high tumor-related mortality rate,and the prognosis of gastric cancer is poor.Gastric cancer in China has become an important health burden,and the overall effect of gastric cancer treatment in China was still not satisfied,although great progress had been made both in the clinical and the translational research.The patient’s clinical symptoms are non-specific in the early stages of the disease,so in our country when the clinical diagnosis of gastric cancer had been made,the vast majority of patients were already in advanced stage,and the overall survival rate is far less dismal than the counterpart patients in Japan and Korea.Therefore,the caner signal pathway and kinase mechanism research,related to the early diagnosis and tumor progression,has been becoming an important foundation for the research and prevention of gastric cancer.Ca MKKβ plays an important role in the regulation of systematic energy balance and key cellular functions,and the emerging role as the metabolic regulation force us to link Ca MKKβ with the cancer metabolism.Recent studies have revealed that abnormalities of Ca MKKβ function participated in many type of malignancy including cancers arising from the alimentary tract,however less attention had been paid on the expression and function of Ca MKKβ in gastric cancer.The main downstream target of Ca MKKβ including AMPK,Ca MK and Akt,and there maybe many complicated crosstalk between these kinase.STO-609 is a selective inhibitor of Ca MKKβ,which had been found to exhibiting inhibitory effect on a variety type of tumors,especially on cell proliferation in vitro.However,only sporadic research concering the role of STO-609 on Ca MKKβ and its downstream molecular target had been reported,and the specific mechanism of STO-609 inhibition of tumor is still unknown.The mechanism study of inhibitory effect of STO-609,had become a prerequisite for it as a potential drugs for the treatment of gastric cancerPurpose1.To stydy the expression partten of Ca MKKβ in gastric cancer tissues and non-cancerous tissues,and to investigate whether theres is a corelation between the Ca MKKβ expression and the patients’ s clinical or pathological character and the prognosis of gastric cancer.2.To investigate the effect of STO-609 on the proliferation,invasion and apoptosis of gastric cancer cells,and to explore the effect of STO-609 on the downstream kinase of Ca MKKβ.3.To explore the possible mechanism STO-609 on gastric cancer cells,and its relationship between the Ca MKKβ/AMPK cascade or other downstream kinases pathway of Ca MKKβ.Methods1.Immunohistochemical method was used to detect the expression of Ca MKKβin gastric cancer tissues and adjacent tissues and normal tissues.2.Clinical analysis of the expression of Ca MKKβ in gastric cancer and the clinicopathological features and prognosis of gastric cancer were analyzed by statistical software.The histological typing,tumor diameter,Lauren type and lymph node metastasis were analyzed.3.The effect of STO-609 on the survival rate of gastric adenocarcinoma cells was investigated by trypan blue staining.4.Western blot was used to detect the expression of Ca MKKβ and its downstream pathway AMPK,Akt and ERK in the in gastric adenocarcinoma cells.5.Apoptosis-Necrosis quantitative assay kit and Western Blot were used to detect the effect of STO-609 on the apoptosis rate of gastric adenocarcinoma cells.6.Trans-well invasion chamber was used to detect the effect of STO-609 on the invasion of gastric adenocarcinoma cells.7.sh RNA inhibition of Ca MKKβ and AMPK were used to study the effect of STO-609 on the viability,apoptosis and invasion of gastric adenocarcinoma cells,and the relationship between the expression of AMPK,Akt and ERK.8.Data were analyzed statistically using IBM SPSS 24.0.Results1.The expression of Ca MKKβ was positive in 61 of 93 cases of gastric carcinoma,and in 15 of 38 cases in non-cancerous tissues.The expression of Ca MKKβ in gastric carcinoma was 1.56 ± 1.05,and the score of non-cancerous tissue was 0.65 ± 0.89,the difference was statistically significant.2.The expression of Ca MKKβ in gastric carcinoma was correlated with the depth of tumor invasion and the strong positive expression of Ca MKKβ could be a predictor of poor prognosis.3.The cell viability was inhibit by STO-609 both in gastric adenocarcinoma SUN-1 cells and N87 cells.4.The activation of AMPK,Akt and ERK in gastric adenocarcinoma SUN-1cells and N87 cells were inhibited by STO-6095.STO-609 had pro-apoptosis effect on both the SUN-1 cells and N87 cells of gastric adenocarcinoma.6.STO-609 inhibited the invasion of gastric adenocarcinoma SUN-1 cells and N87 cells.7.Compared with the control group,Ca MKKβ sh RNA inhibited the pro-apoptosis effect of STO-609 on SUN-1 cells,however AMPK sh RNA did not have any effect on the pro-apoptosis effect.8.Compared with the control group,AMPK sh RNA have no effect on the the viability inhibition and apoptosis induced by STO-609,also AMPK sh RNA have no effect on the activates Akt and ERK in SUN-1 cells.Conclusion1.The expression of Ca MKKβ in gastric cancer tissues was significantly higher than that in adjacent tissues and normal tissues.Expression of Ca MKKβ was correlated with the depth of tumor invasion and strong positive expression also was a indepent predictor of poorer prognosis,which could be a potential molecular marker.2.STO-609 inhibited the cell viability of gastric adenocarcinoma cells,which may be related to the induction of apoptosis,and the activation of Ca MKKβdownstream AMPK,Akt and ERK maybe involved in apoptosis induction.STO-609 inhibited the invasion of gastric adenocarcinoma cells,suggesting that Ca MKKβSTO-609 inhibitor may be used as a novel anticancer drug.3.We further found that,AMPK silencing had no effect on STO-609 induced apoptosis of gastric cancer cells,suggesting that AMPK,although as a key downstream molecular of Ca MKKβ,maybe not involved in the STO-609 induced apoptosis.4.Finally,base on our overall results,we hypothesized that: the pro-apoptosis effect of STO-609 on gastric cancer cells,may by the inhibition of the Ca MKKβdownstream Akt、ERK or the interaction between the regulation signal pathway. |
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