| Objective: Endothelial nitric oxide synthase(e NOS,NOS3)is a member of the nitric oxide synthase family,acting as a catalytic enzyme involved in the production of nitric oxide in cells.In recent years research has confirmed that NOS3 participates in inhibiting tumor cell apoptosis,promoting angiogenesis,tumor cell proliferation,invasion and immunosuppression in breast cancer,bowel cancer,pancreatic cancer and prostate cancer.However,there are still few studies on the expression of NOS3 in malignant tumors,and the research on the mechanism that NOS3 affects the occurrence and development of tumors is not comprehensive and needs to be further explored.Gastric cancer is currently one of the most common malignant tumors worldwide.In China,most patients with gastric cancer are already in the advanced stage when they are diagnosed.Although radical gastric cancer resection is the core treatment program,which improves the patient's prognosis to a certain extent,about 1/3 of gastric cancer patients still relapse after surgery.Peritoneal dissemination accounts for almost 50% of gastric cancer recurrence.Although the treatment of gastric cancer has made great progress in recent years,the median survival time of gastric cancer patients is still only 7-16 months.Therefore,further exploration of the prevention and treatment of gastric cancer peritoneal metastasis is of great significance to improve the prognosis of gastric cancer patients and prolong the survival of gastric cancer patients.NOS3 as a prognostic marker in patients with gastric cancer and as a therapeutic target for peritoneal metastasis of gastric cancer has not been reported.Bufalin is one of the effective components of the traditional Chinese medicine toad venom.As the main effective component of Cinobufacin,bufalin is widely used to treat a variety of malignant tumors.Bufalin can promote tumor cell apoptosis,inhibit the proliferation,invasion and other malignant biological processes of tumor cells.However,the role of bufalin in peritoneal metastasis of gastric cancer and the role of NOS3 in this process are unknown.Methods: 1.NOS3 mRNA expression data in tumor tissues,normal tissues and human cancer cell lines and phenotype data of tumor patients were downloaded from UCSC Xena(https://xena.ucsc.edu/),Gene Expression Omnibus(GEO)(http://www.ncbi.nlm.nih.gov/geo/)database and CCLE database(Https://portals.broadinstitute.org/ccle/);2.The expression levels of NOS3 mRNA in tumor tissues,normal tissues and cancer cell lines were compared;3.t-test was used to compare the NOS3 expression level of tumor tissue and normal tissue in each tumor type;4.t-test was used to compare the NOS3 expression level of early tumor and advanced tumor stage in each tumor type;5.Kaplan-Meier analysis,Log-rank test and COX regression analysis was used to determine the relationship between NOS3 expression and the prognosis of patients in each tumor type;6.Immunohistochemical stain was used to detect the expression level of NOS3 protein in 90 gastric cancer tissues,and analyze its relationship with the clinicopathological parameters and overall survival time;7.siRNA or NOS3 inhibitor(L-NAME)were used to interfere with the expression of NOS3 in gastric cancer cells,and bufalin was also added to treat gastric cancer cells;8.MTT assay was used to detect the proliferation ability of gastric cancer cells;9 Adhesion assay was used to detect the ability of gastric cancer cells to adhere to Matrigel;10.Wound healing and Transwell assay was used to detect the invasion and migration ability of gastric cancer cells;11.Western blot experiment was used to detect the expression of NOS3,p-NOS3(Ser-1177),key proteins in the EMT process,and key proteins in the MAPK pathway in gastric cancer cells;12.MKN-45 P cells was used to construct a nude mice model of gastric cancer peritoneal metastasis.At the end of the experiment,the number and quality of peritoneal metastases and the ascites volume in nude mice were counted,and immunohistochemical stain was used to detect the expression of NOS3,E-cadherin and vimentin.Results:Part 1.Research on the expression and clinical significance of NOS3 in gastric cancer based on pan-cancer analysis.1.1 The expression level of NOS3 mRNA is highly tumor dependent,among which NOS3 expression level is the highest in gastric cancer tissue;1.2 The top three cell lines with high NOS3 mRNA expression levels are gastric adenocarcinoma,colon adenocarcinoma,and nervous system tumor cell lines;1.3 The expression level of NOS3 mRNA in rectal adenocarcinoma,gastric adenocarcinoma,pancreatic cancer,ovarian serous cystic adenocarcinoma,skin melanoma,and head and neck squamous cell carcinoma was significantly higher than that in normal tissues;1.4 In skin melanoma,NOS3 expressed higher in patients with advanced tumor stage.In gastric adenocarcinoma,NOS3 expression and tumor stage did not show a significant correlation;1.5 The overall survival time of gastric cancer patients with higher NOS3 mRNA expression was significantly shortened;1.6 Online database analysis found that NOS3 was an independent risk factor for the prognosis of gastric cancer patients;1.7 Immunohistochemical stain of 90 gastric cancer patients tissues showed that NOS3 is an independent risk factor for the prognosis of patients with gastric cancer;Part 2.The role and mechanism of NOS3 in the peritoneal metastasis of gastric cancer.2.1 Gastric cancer cell line MGC-803 and gastric cancer cell line with high potential for peritoneal metastasis,MKN-45 P,were selected for cell experiments.After knocking down the expression of NOS3 by siRNA,gastric cancer cells showed significant decrease in adhesion,invasion and migration capabilities;2.2 After L-NAME inhibiting the expression of NOS3,the adhesion,invasion and migration ability of gastric cancer cells is significantly reduced;2.3 Bioinformatics analysis found that NOS3 in gastric cancer may regulate the malignant biological behavior of gastric cancer through the MAPK pathway;2.4 After intervention in NOS3 expression in gastric cancer cells,the phosphorylation levels of ERK and p38 were significantly reduced,indicating that the activation of the MAPK pathway was downregulated;Part 3.Bufalin inhibits NOS3 activity on gastric cancer peritoneal metastasis.3.1 Bufalin reduces NOS3 phosphorylation(Ser1177)level by binding to NOS3 protein;3.2 After treatment of gastric cancer cells MGC-803 and MKN-45 P with bufalin,the adhesion,invasion and migration ability of gastric cancer cells were significantly inhibited;3.3 The gastric cancer cell line with high potential for peritoneal metastasis,MKN-45 P,was used to construct a nude mouse model of gastric cancer peritoneal metastasis.We found that the number and quality of peritoneal metastases and ascites volume in the bufalin single-agent,L-NAME single-agent and dual-agent groups were significantly reduced;3.4 In the experimental group,the expression of NOS3 protein in peritoneal metastases decreased,and the expression of E-cadherin increased while the expression of vimentin decreased.Conclusions: By analyzing public datasets and the pathological tissues of gastric cancer patients,we confirmed that the high expression of NOS3 is related to the poor prognosis of gastric cancer patients,and NOS3 is an independent risk factor for the prognosis of gastric cancer patients.NOS3 regulates the invasion,migration and colonization ability of gastric cancer cells through the MAPK signaling pathway.NOS3 may positively regulate the peritoneal metastasis of gastric cancer through the MAPK pathway,leading to a poor prognosis for gastric cancer patients.Bufalin is a clinical therapeutic drug that targets NOS3.It can reduce the phosphorylation level of NOS3 to inhibit the activity of NOS3.Bufalin might prevent the process of gastric cancer peritoneal metastasis by inhibiting the NOS3-MAPK signaling pathway.These results suggest that NOS3 may be a biomarker for predicting the overall survival time of gastric cancer patients and a potential target for gastric cancer treatment.Bufalin may inhibit the peritoneal metastasis of gastric cancer by down-regulating the activation of MAPK pathway through NOS3.This study provides more evidence to support the treatment of gastric cancer peritoneal metastasis by intraperitoneal infusion of bufalin. |
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