| Esophageal cancer(EC)is recognized as one of the major cancers that threaten the safety of human life in the world.In some countries like Asia and some low-income countries such as South Africa,esophageal cancer has seriously affected human life.The main pathological types of esophageal cancer were esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma(EAC).In China,the incidence of esophageal squamous cell carcinoma accounts for more than 90%of the incidence of esophageal cancer,especially in some plain areas.Due to the lack of adequate early diagnosis and treatment,esophageal cancer is often diagnosed in the middle and advanced stage of development,and the clinical situation is not optimistic.Up to now,there are many treatments,and surgery is still the main method to treat esophageal cancer,but the postoperative recurrence rate is still very high.In recent years,some studies have shown that the use of chemoprophylaxis after surgery could prevent tumor recurrence effectively.Therefore,some low-toxic and efficient drugs is urgently needed especially some target drugs to delay the recurrence of esophageal cancer and improve the 5-year survival rate of patients.Our group screened some common natural compounds extracted from traditional Chinese medicine and found that pectolinarigenin can effectively inhibit the proliferation of esophageal squamous cell carcinoma.Pectolinarigenin,a flavonoid with many biological characteristics,has been shown to inhibit the growth of a variety of tumors,including lung cancer,gastric cancer and so on,but its mechanism is still unclear.In our study,we confirmed the inhibitory effect of pectolinarigenin on the proliferation of esophageal squamous cell carcinoma by cell proliferation experiment,soft agar cloning and plate cloning.In order to better study the mechanism of its inhibitory effect on esophageal squamous cell carcinoma,the data analysis methods of proteomics and phosphorylated proteomics were used in this study.Pectolinarigenin can significantly inhibit the expression of pYAPS109,pSTAT3Y705,pNF?BS907,pMAPK3T202,pGSK-3BY216 and pRB1T826 in esophageal squamous cell carcinoma cells.These results are consistent with some of the previous studies.We also found a decrease in the expression of pRB1T826 in the CDK6-RB1 pathway.We use protein pull-down experiments to confirm that pectolinarigenin could directly bind to CDK6 protein kinase.The kinase experiment in vitro showed that CDK6 could activate RB1 and increase the expression of pRB1T826,while pectolinarigenin could directly bind to CDK6 to inhibit the activation of RB1 protein by CDK6,thus reducing the expression of pRB1T826.In order to accurately evaluate the inhibitory effect of pectolinarigenin on the proliferation of esophageal squamous cell carcinoma,we established a humanized transplanted tumor model of esophageal squamous cell carcinoma(Patient-derived xenograft,PDX).It was found that pectolinarigenin had a significant inhibitory effect on the proliferation of esophageal squamous cell carcinoma in vivo,and the results of immunohistochemical staining showed that pectolinarigenin could significantly reduce the expression of pRB1T826 in vivo.To sum up,this study found that pectolinarigenin could down regulate expression of pYAPS109?pSTAT3Y705?pNF?cBS907?pMAPK3T202?pGSK-3BY216 and pRB1T826 involved in ten KEGG pathways like viral carcinogenesis pathway?Vibrio cholerae infection?Prolactin signaling pathway and T cell receptor signaling pathway and so on.The results of further studies indicated that pectolinarigenin can bind to CDK6 directly to inhibit the activation of CDK6 protein kinase on RBI,reducing the expression of pRB1T826,and playing its inhibitory effect on esophageal squamous cell carcinoma in vivo and in vitro.Therefore,the results show that,pectolinarigenin could target for esophageal squamous cell carcinoma by inhibiting the activation of CDK6 to pRB1T826.Methods and purpose1.Cytotoxicity assay:This experiment was used to study the toxicity of pectolinarigenin on esophageal squamous cell carcinoma.2.Cell proliferation assay:To detect the inhibitory effect of pectolinarigenin on the proliferation of esophageal squamous cell carcinoma.3.Soft agar assay:This experiment was used to simulate and study the effect of pectolinarigenin on the clone formation ability of esophageal squamous cell carcinoma cells4.Plate clone formation experiment:To further confirm the ability of pectolinarigenin to inhibit the cloning of esophageal squamous cell carcinoma cells.5.Proteomics and phospho-proteomics experiments:To analyze the changes of intracellular protein and phosphorylated protein level after pectolinarigenin treated.6.Western blotting assay:In order to verify whether the expression of related proteins were down-regulated in the results of phospho-proteomics,as well as the expression level of the total related targets in the pathway.7.Molecular pull-down experiment and computer molecular simulation docking experiment:In order to verify whether pectolinarigenin can bind to CDK6 directly.8.In vitro kinase experiment:To verify whether CDK6 can phosphorylate and activate RB1 protein at Tyr826 site and whether pectolinarigenin can inhibit the phosphorylation of RB1 by CDK6.9.PDX model:The inhibitory effect of pectolinarigenin on ESCC was detected in vivo.10.Immunohistochemical staining:To study the expression of pRB1T826 and proliferation marker Ki67 in tumor tissue treated with pectolinarigenin.Results1.Cytotoxicity test:The results showed that pectolinarigenin with 20 ?M has obvious toxic effect on esophageal squamous cell carcinoma.2.Cell proliferation experiment:Pectolinarigenin could inhibit the proliferation of esophageal squamous cell carcinoma in a dose-dependent manner.3.Soft Agar clone formation experiment:Pectolinarigenin could significantly inhibit the soft Agar clone formation ability of esophageal squamous cell carcinoma cells at 10 ?M.4.Plate clone formation experiment:This experiment further confirmed that pectolinarigenin could significantly inhibit the cloning ability of esophageal squamous cell carcinoma cells at 10 ?M.5.Proteomic and phosphorylated proteomic experiments:Pectolinarigenin can significantly down-regulate the expression levels of pYAPS109,pSTAT3Y705,pNF?BS907,pMAPK3T202,pGSK-3BY216 and pRB1T826.6.Western blotting showed that pectolinarigenin could significantly inhibit the expression of pYAPS109,pSTAT3Y705,pNF?BS907,pMAPK3T202,pGSK-3BY216 and pRB1T826 in esophageal squamous cell carcinoma.7.The results of molecular pull-down experiment and computer molecular simulation docking experiment show that pectolinarigenin can bind to CDK6 molecule directly.8.In vitro kinase experiment:CDK6 can phosphorylate and activate RBI protein at Tyr826 site,while pectolinarigenin can directly act on CDK6 protein and inhibit the activation of RB1 by CDK6,thus reducing the expression of pRB1T826.9.PDX model:Pectolinarigenin had no obvious side effects on mice,and it could significantly inhibit the proliferation of human esophageal squamous cell carcinoma,and the weight and volume of tumor decreased with the increase of concentration.10.Immunohistochemical staining:Pectolinarigenin could significantly reduce the expression of pRB1T826 and cell proliferation marker Ki67 in esophageal squamous cell carcinoma.ConclusionPectolinarigenin can inhibit the proliferation of esophageal squamous cell carcinoma by reducing the expression of pYAPS109,pSTAT3Y705,pNF?BS907,pMAPK3T202 and pGSK-3BY216.In addition,it can also inhibit the proliferation of esophageal squamous cell carcinoma by binding to CDK6 protein kinase and reducing the phosphorylation activation of RB1 at Tyr826 site,thus reducing the expression of pRB1T826. |
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