Inhibition Of STAT3 Signaling Pathway By Terphenyllin Suppresses Growth And Metastasis Of Gastric Cancer

Objective Gastric cancer is a common type of malignant tumor with a relatively poor prognosis and presents a serious threat to global health.Even after radical resection,many patients will still suffer from local recurrence or distant metastasis.The five-year survival rate in patients with gastric cancer is often less than 35%.A common cure for advanced gastric cancer is chemoradiotherapy.Most of the existing chemotherapy drugs have severe adverse reactions and high drug resistance,and it is often difficult for gastric cancer patients who fail the first-line and second-line chemotherapy to benefit from radiotherapy since radiotherapy is only a local treatment.Therefore,finding safe and effective drugs to inhibit the migration of gastric cancer and improve the prognosis of patients is the focus and difficulty of current research.This paper aims to take STAT3 which over-expressed in many tumors as the research object,verify the specific binding between compound Terphenyllin and STAT3,study the effect of Terphenyllin on the downstream protein of STAT3 including c-Myc,Cyclin D1 and futher explore the activity of Terphenyllin in vitro and in vivo through a series of cell surface experiments and MKN1 orthotopic model.Methods To verify the specific binding of Terphenyllin to STAT3,we simulated the docking diagram of the specific binding of Terphenyllin to STAT3 using the chemical structure of Terphenyllin and the X-ray diffraction crystal structure of STAT3 protein and found a potential binding site on the surface of STAT3 protein.To study the effect of Terphenyllin on STAT3 signaling pathway,Western blot was used to detect the effect of the compound on STAT3,p-STAT3 and two downstream proteins c-Myc,Cyclin D1 under the intervention of Terphenyllin at different concentrations.To test the activity of Terphenyllin in vitro,CCK8 experiment,colony formation experiment,wound healing experiment and transwell invasion experiment were involved.Then,the effects of Terphenyllin on cell cycle synthesis and apoptosis of gastric cancer cells were detected by Propidium Idide/RNase staining buffer and FITC Annexin V Apoptosis Detection Kit I.In order to further explore the anti gastric cancer activity of Terphenyllin in vivo,we established an orthotopic gastric cancer mouse model.We do intraperitoneal injection every day,measure the weight of mice every three days,do fluorescence imaging on mice every seven days and finally do H&E stain with organs.So we can detect the effect of drugs on tumor growth in vivo and if there are obvious toxic and side effects on mouse body and other organs.Finally,in order to explore whether Terphenyllin can inhibit tumor metastasis,we performed fluorescence imaging on the peritoneum,liver and spleen of dissected mice,and compared the difference of the number of metastatic rice between the vehicle group and the terphenyllin group.Results Using the X-ray diffraction crystal structure of STAT3 protein,the docking diagram of the specific binding of Terphenyllin to STAT3 was simulated.A potential binding site was found on the surface of STAT3 protein,which preliminarily indicated that Terphenyllin specifically binds to STAT3.Western Blot experiment showed that Terphenyllin could inhibit the phosphorylation of STAT3 protein and reduce the expression levels of its downstream proteins c-Myc and Cyclin D1 in a concentration-dependent manner.In addition,the in vitro experiments showed that Terphenyllin had obvious cytotoxicity to both gastric cancer cell lines.And Terphenyllin can dose-dependently inhibit the colony formation,migration and invasion of two gastric cancer cell lines.Flow cytometry results showed that Terphenyllin also had obvious cell cycle arrest and pro-apoptotic effects on both MKN1 and BGC823 cell lines.Finally,the results of the in vivo experiment showed that the tumor volume and the number of metastatic mice in the Terphenyllin-treated group were significantly lower than those in the vehicle group,but the body weight of the mice,as well as the heart,liver,spleen,lung,kidney,brain were not affected after treatment.The results above all indicate that Terphenyllin can significantly inhibit the growth and metastasis of gastric cancer but did not show obvious toxic and side effects.Conclusion Terphenyllin directly binds to STAT3 protein through hydrogen bond,thus inhibiting the phosphorylation of STAT3 protein and reduces the expression levels of its downstream proteins c-Myc and Cyclin D1.In addition,Terphenyllin can inhibit the growth,proliferation,colony formation,migration and invasion of gastric cancer cells in a concentration-dependent manner,and promote apoptosis and cycle arrest.Finally,in the MKN1 orthotopic gastric cancer model,Terphenyllin can significantly inhibit the growth and metastasis of tumor in mice,but does not show obvious toxic and side effects.