Study On The Relationship Between INOS Expression And Cisplatin Chemosensitivity In Gastric Cancer Induced By Oxidative Stress

BackgroundAt present,the five-year survival rate of gastric cancer in China is low,mainly because most patients with gastric cancer are diagnosed at advanced stage,and clinical chemotherapy is prone to drug resistance.Tumor chemotherapy drug resistance significantly affects the survival time of patients,therefore,the research on chemotherapy and the mechanism of chemotherapy drug resistance is constantly being explored.Some studies have shown that metabolic reprogramming is one of the markers of cancer,and it also plays an important role in chemotherapy and drug resistance.Among them,the oxidative damage caused by reactive oxygen species produced by oxidative stress is an important event of cancer cell apoptosis induced by chemotherapy.However,the role and mechanism of active oxygen in chemotherapy of gastric cancer is not clear.It is not clear whether the changes of reactive oxygen species during chemotherapy are related to the effect of chemotherapy for gastric cancer.In addition,the results of related experiments show that reactive oxygen species produced by different chemotherapeutic drugs are involved in the formation of chemotherapeutic resistance.However,the mechanism of how reactive oxygen species participate in the formation of chemotherapy resistance in gastric cancer is still unclear.Objective:In this study,pathological immunohistochemistry,MTT and Western blot were used to detect the expression of inducible nitric oxide synthase((induced nitric oxide synthase,iNOS))and nuclear factor-κB(nuclear factor kappa-B,NF-kappa B(NF-κB)in different stages of gastric adenocarcinoma,and the expression of iNOS and NF--B in gastric cancer MKN-45 cells treated with H2O2,cisplatin and H2O2+cisplatin.To explore the effect of oxidative stress and cisplatin chemotherapy on the expression of iNOS in gastric cancer and their interaction,and to explore the related mechanism through in vitro cell test,in order to reveal the role of iNOS in chemotherapy and chemotherapy resistance,which is expected to provide a new target for reversing chemotherapy resistance of gastric cancer and improve the therapeutic effect of advanced gastric adenocarcinoma.Methods:1、Clinically collect 78 cancer tissue specimens from patients with gastric adenocarcinoma,and collect relevant clinical data such as stage,pathological results,chemotherapy regimen and so on.2、The expression of iNOS protein in gastric adenocarcinoma was analyze d by pathological immunohistochemistry in neoadjuvant chemotherapy+operati on group,simple operation group,early and advanced group by pathological i mmunohistochemistry.3、Chi-square test was used to analyze the positive expression rate of iNOS protein in gastric adenocarcinoma tissues of neoadjuvant chemotherapy+operation group,simple operation group,early stage and advanced stage group.4、A sufficient number of MKN-45 human gastric cancer cells were cultured in vitro and subcultured into 4 groups with the same number of cells.after the culture reached the target number,the experimental group was treated with t-BHP,cisplatin and cisplatin+t-BHP respectively,while the control group was treated with the same amount of complete culture medium.5、The MTT experiment was used to detect the optimal concentration of different concentrations of H2O2,cisplatin,and cisplatin+ H2O2 acting on MKN-45 cells,calculate the IC50 value,and detect the optimal time of action.6、Western blot method was used to detect the expression of iNOS protein in t-BHP,cisplatin,cisplatin+t-BHP and gastric cancer cells in the control group,and the gray level of iNOS and NF-κB protein was analyzed with Quantity one software(Bio-Rad)Value,and compared with the internal reference protein β-actin,calculate the expression of iNOS and NF-κB protein,and make a pairwise comparison.Results:1、The results of pathological immunohistochemistry showed that the expression of iNOS in advanced gastric cancer patients who underwent radical gastrectomy was significantly higher than that in early patients(P<0.05).2、Pathological immunohistochemistry results showed that for advanced patients,the expression of iNOS in the cancer tissue of gastric cancer patients in the direct surgery group was significantly higher than that in the neoadjuvant chemotherapy+surgery group,and the difference was statistically significant(P<0.05).3、The results of MTT detection showed that compared with the simple H 2O2 group,the inhibition rate of gastric cancer MKN-45 cells was significantly increased in the H2O2+CDDP group by adding different concentrations of H 2O2 to the culture holes of the same amount of CDDP.Among them,the inhib ition rate of 0.0001%H2O2+CDDP increased from 38.69%to 69.24%compared with 0.0001%H2O2 alone,an increase of about 2 times.IC50=1.523e-006(8.192e007to2.833e-006)of the H2O2+ CDDP group.The IC50 of H2O2+CDDP group was significantly lower than that of H2O2 group(P<0.001).In 0.60 μg/ml CDDP,1.523eMel 0.06%H2O2 was added to gastric cancer MKN-45 cells with an inhibition rate of 50%.4、Western-blot detection showed that the expression of iNOS in the CDDP group decreased significantly;the expression of iNOS protein in the H2O2 group was significantly different from that in the CDDP group(P=0.007);compared with the CDDP-treated gastric cancer group,the iNOS protein in the H2O2+CDDP intervention group The expression level increased significantly,and the difference between the two groups was significant(P=0.0138).5、Western-blot detection showed that the expression of NF-κB in the CDDP group decreased significantly;the expression of NF-κB protein in the H2O2 group was significantly different from that in the CDDP group(P=0.013);compared with the CDDP-treated gastric cancer group,the H2O2+CDDP intervention group The expression of NF-κB protein increased significantly,and there was a significant difference between the two groups(P=0.027)Conclusion:Oxidative stress induced by H2O2 significantly increased the expression of iNOS and NF-κB in gastric cancer.Cisplatin chemotherapy down-regulated the expression of iNOS and NF-κB protein in gastric cancer,which not only reduced the inflammatory state of gastric cancer lesions,but also induced drug resistance Oxidative stress in cancer cells induced by H2O2 can improve the sensitivity of gastric cancer cells to cisplatin chemotherapy and reverse drug resistance through increased expression of iNOS.The results are expected to provide a new treatment strategy for refractory gastric cancer.