Mechanism Investigation Of Oroxin A Suppressing The Esophageal Squamous Cell Carcinoma Growth In Vivo And In Vitro

Esophageal cancer(EC)is a prevalent malignant tumor of digestive tract in modern clinic,and its morbidity and mortality rank the seventh and sixth in the world respectively.China ranks first in both morbidity and mortality of the new cases of esophageal cancer in the world.Esophageal cancers are classified as esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma(EAC)based on their histopathological subtypes,esophageal squamous cell carcinoma is the main histological and epidemiological types of esophageal cancer in China.The occurrence and development of esophageal cancer are multi-factor and multi-stage,and the pathological types and risk factors of each country and region are also different.It is very difficult to detect esophageal cancer in the early clinical stage,and more than 90%of patients with esophageal cancer are in the middle and late stage.At present,the main treatment manner of esophageal cancer in clinic including chemotherapy,radiotherapy,surgery and chemotherapy combined surgery,although the diagnosis and treatment of esophageal cancer in recent years has made some progress,latest immunotherapies have made a significant contribution to the treatment of cancer,and some immunocheckpoint blockers may be approved by FDA,the quality of life of the esophageal cancer patients and survival time has improved,but the 5 years of survival rate is still less than 30%of the advanced patients and some issues such as immunity requirements and biological dosages of drugs remain unresolved.The poor prognosis of esophageal cancer,extremely high recurrence and metastasis,and chemotherapy resistance are still a major problem which we are faced with.Therefore,it is necessary to search for effective and low toxicity side effect of anti-tumor drugs and specific molecular targets for the treatment of esophageal cancer.In recent decades,screening anti-tumor active ingredients from natural products has become an important direction in cancer research.Flavonoids belong to polyphenolic natural compounds that widely exist in many plants.They have the rich pharmacological activity and significant anti-tumor effect,but have little toxic effect on normal cells.Therefore,in the previous experiment study,we screened more than 50 compounds,and the experimental results showed that Oroxin A could significantly inhibit the growth of esophageal squamous cell carcinoma cells KYAE150 and KYSE450.Oroxin A is an effective flavonoid component isolated from the the traditional Chinese medicinal herb named Oroxylum indicum.Previous research results have shown that Oroxin A has a certain inhibitory effect on breast cancer cells and lung cancer cells,but the effect and mechanism of Oroxin A on esophageal squamous cell carcinoma cells have not been studied.In this study,we used MTT assay,Foci assay and soft Agar assay to detect the effect of Oroxin A on the growth of esophageal squamous cell carcinoma.In order to further investigate the mechanism of Oroxin A that inhibit esophageal squamous carcinoma cell proliferation,we tested proteomics and phosphorylation of omics changes by biology mass after treated KYSE150 cell with Oroixn A.Through bioinformatics analysis,we have processed the phosphorylation of differentially expressed proteins in KYSE150 cells compared with the control cells.With analysising the pathways,we got the important differential expression phosphorylation protein.In the study,we also used a serious of experiment to inform the results,and detected that Oroxin A could bind to the target protein by binding assay to reveal the mechanism of inhibiting ESCC growth by Oroxin A.Methods1.Cell toxicity experiment:KYSE150,KYSE 450 cells and SHEE cells were treated with different concentrations of Oroxin A,the survival rate were calculated and draw the curve.The concentration of Oroxin A was selected at the survival rate which is IC50 value to do the cell proliferation assay and has no toxicity on normal cell.2.Cell proliferation assay:Different concentrations of Oroxin A treated KYSE140,KYSE150,KYSE410 and KYSE450 cells to explore the ESCC cell proliferation after Oroxin A treatment.3.Anchoragein-dependent cell growth:To determine the inhibitory effect of Oroxin A on anchoragein-dependent cell growth,KYSE150 and KYSE450 cells were treated with different concentrations of Oroxin A.4.Foci Formation Assay:To explore effect of Oroxin A on ESCC cell growth,KYSE150 and KYSE450 cells were treated with different concentrations of Oroxin A.5.Proteomic and phosphoproteomic analysis:Oroxin A treated the KYSE150 cells with the concentration of 10 μM for 24 h,performed proteomic and phosphoproteomic analysis;based on mass spectrum to find potential targets and mechanism of suppressing the proliferation of ESCC after Oroxin A treatment.6.Western blotting assay:confirmed expression level of phosphorylated proteins including STAT3Tyr705,STAT3Ser727,NFκBser907 after Oroxin A treatment.7.Patient-derived xenograft(PDX)mouse model:We established PDX model using ESCC tissue fragments and transplanted on back of mice,and then treated different dose of Oroxin A(50 mg/kg or 100 mg/kg)by intraperitoneal injection to evaluated the inhibiting effect of Oroxin A on ESCC growth in vivo.8.Immunohistochemical(IHC)analysis:To evaluated the expression level of phosphorylated proteins STAT3Tyr705,STAT3ser727,NFkBSer907 in the PDX tumor tissues with Oroxin A treated.9.Binding assay:To determine the potential molecular target proteins of aiming at Oroxin A,sepharose 4B-pull down assay was investigated.10.Cell cycle:To detect the cell cycle change with Oroxin A treatment.Results1.Cell toxicity experiment:The results of IC50 value against Oroxin A indicated that 20 μM Oroxin A has no cytotoxicity on normal esophageal epithelial cells(SHEE).2.Cell proliferation assay:The results showed that KYSE140,KYSE150,KYSE410 and KYSE450 cells after different concentrations treatment with Oroxin A could inhibit the cell proliferation of KYSE140,KYSE150,KYSE410,KYSE450 significantly.3.Anchorage in-dependent cell growth:Compared with the control group,Oroxin A could restrained the cell colony formation ability of KYSE150 and KYSE450 significantly with the increasing concentration.4.Foci Formation Assay:Compared with the control group,Oroxin A could inhibite the cell growth and cell colony formation ability of KYSE150 and KYSE450 cells strongly with the increasing concentration.5.Phosphoproteomic analysis:Phosphoproteomic profile revealed the phosphorylation proteins and sites of STAT3TYr705,STAT3ser727,NFKBSer907 were dramatically down regulated.6.Western blotting assay:The western blotting results can confirmed the reduction of the expression of the phosphorylated proteins,and we found that phosphorylation of STAT3Tyr705,STAT3Ser727,NFkBSer907.7.Patient-derived xenograft(PDX)mouse model:The experiment results showed that Oroxin A could suppress the tumor growth without decreasing the mice body weight compared with the control group.8.Immunohistochemical(IHC)analysis:The IHC results showed that the expression of STAT3Tyr705,STAT3Ser727,NFκBSer907 were significantly reduced in the PDX mice tissues with Oroxin A treatment.9.Binding assay:The results of IC50 against Oroxin A can bind with JAK2 obviously and it may be the potential target.10.Cell cycle:The results indicated that Oroxin A could induce the cell cycle arrest at G0/G1 phase.ConclusionOroxin A can significantly suppress the proliferation of esophageal squamous carcinoma cells by binding to JAK2 in JAK/STAT signaling pathway and inhibiting the phosphorylation level of STAT3Tyr705,STAT3Ser727,NFκBSer907,also colud induce cell cycle arrest at G0/G1 phase,then inhibited the growth of ESCC in vivo and in vitro.