Bufalin Targets BFAR To Regulate PI3K/AKT/mTOR Signal Pathway To Inhibit The Occurrence And Progression Of Gastric Cancer

Gastric cancer(GC)is the most common malignant tumor of the digestive system.Although the treatment has been continuously improved in recent years,the prognosis of patients is still poor due to the characteristics of high metastasis and easy recurrence of GC.Venenum Bufonis(VB)is a valuable traditional Chinese medicine,which is often used for analgesia,cardiotonic and anti-inflammation.Modern pharmacological studies have shown that Bufo bufonis has good anti-tumor effect.Bufalin is one of the most effective anticancer active components of VB.It has been proved to have good anticancer effect in many cancers,such as liver cancer,lung cancer,ovarian cancer,prostate cancer,neuroblastoma and so on.However,there are few studies on bufalin in GC.Previous studies in our group have shown that bufalin has a significant inhibitory effect on the proliferation of GC cells,but the clear target of direct interaction with bufalin is not clear.Therefore,finding the anti-tumor target of bufalin and its downstream signal pathway is very important to further clarify the anticancer mechanism of bufalin.Objective:The purpose of this study is to explore the inhibitory effect of bufalin on the occurrence and progression of GC and to find the interaction target and downstream signal pathway of bufalin,so as to further clarify the anti-tumor mechanism of bufalin and to provide a basis for bufalin as a new clinical drug for the treatment of GC.Methods:1.Bufalin inhibits the proliferation and migration of gastric cancer cells through PI3K/AKT/mTOR signal pathway.The effect of bufalin on the activity of GC cells(HGC-27 and MKN-45)and normal fibroblasts(L929)was detected by CCK-8,and the 50%inhibitory concentration was calculated to determine the working concentration of bufalin in the follow-up experiment.Scratch healing assay and Transwell assay were used to detect the effect of bufalin on the migration ability of two kinds of GC cells.Western blot was used to detect the effect of bufalin on the expression of migration-related proteins Ncadherin,E-cadherin,Vimentin and PI3K/AKT/mTOR proliferation signal pathway in two kinds of GC cells.2.BFAR is the direct interaction protein of bufalin.The proteins that can directly bind to bufalin were screened by human proteome microarray.Bioinformatics analysis was used to further evaluate the expression of candidate protein BFAR in GC,its significance in diagnosis and prognosis,and its participation in biological processes and signal pathways.The binding site of bufalin to the interacting protein BFAR was predicted by molecular docking technique.The effect of bufalin on the expression of BFAR in HGC-27 and MKN-45 GC cells was verified by Western blot.3.Bufalin targets BFAR and regulates PI3K/AKT/mTOR signal pathway to inhibit the migration ability of gastric cancer cells.BFAR was overexpressed in HGC-27 and MKN-45 GC cells by plasmid transfection,and the transfection efficiency was verified by Western blot.Scratch healing assay and Transwell assay were used to detect the effect of bufalin on the migration of GC cells through BFAR.Westernblot was used to detect the effect of bufalin on the expression of migration-related proteins N-cadherin,E-cadherin,Vimentin and PI3K/AKT/mTOR signal pathway proteins in GC cells through BFAR.4.HGC-27 and MKN-45 GC cell lines with stable BFAR overexpression were constructed by lentivirus infection.The cells were inoculated subcutaneously in nude mice to establish the subcutaneous transplanted tumor model of nude mice.After tumorigenesis,bufalin was injected intraperitoneally into nude mice to explore whether bufalin could affect the occurrence and development of GC through BFAR.The fluorescence intensity and range of subcutaneous transplanted tumor in nude mice were detected by threedimensional fluorescence imaging system in vivo,so as to detect the size of transplanted tumor.After treatment,the nude mice were killed,and the transplanted tumors and other major organs were removed and evaluated by HE staining.The expression of metastasisrelated proteins in the transplanted tumor was detected by Western blot.Results:1.Bufalin inhibited the viability of HGC-27 and MKN-45 GC cells in a time-and concentration-dependent manner,but had no significant effect on the viability of L929 cells.The 50% inhibitory concentration(IC50)of bufalin for 24,48,72 h was MKN-45: 24 h41.73 nmol/mL,48 h 18.59 nmol/mL,72 h 6.391 nmol/mL;HGC-27 and 24 h 80.68nmol/mL,48 h 21.78 nmol/mL,72 h 10.48 nmol/mL.Scratch healing assay and Transwell migration assay showed that bufalin significantly inhibited the migration ability of two kinds of GC cells in a concentration-dependent manner(p < 0.01).Western blot results showed that the expression of N-cadherin and Vimentin decreased significantly,while the expression of E-cadherin increased significantly in the two kinds of GC cells treated with bufalin for 48 h(p < 0.01).At the same time,bufalin significantly inhibited the phosphorylation of PI3 K,AKT and mTOR in HGC-27 and MKN-45 cells in a dosedependent manner(p < 0.01).2.Proteins with signal-to-noise ratio(SNR)greater than 3 in human proteome microarray results were selected as candidate proteins.The candidate protein was intersected with the results of GC biomarkers screened in previous studies,and BFAR was selected as the target protein for further study.The results of bioinformatics analysis showed that the expression of BFAR in GC was significantly higher than that in normal gastric tissue.The mutation rate of BFAR gene in GC patients is 2.3%,which indicates that the increased expression of BFAR in GC is mainly due to changes in expression rather than gene mutation.ROC curve showed that BFAR had high AUC,sensitivity and specificity in GC,which showed good diagnostic significance.In terms of prognosis,time-dependent ROC curve and K-M curve showed that the expression of BFAR was related to poor prognosis.The results of enrichment analysis show that BFAR is related to biological processes such as proliferation,invasion and metastasis,and may affect PI3K-related signal pathways.The results of molecular docking show that there are many potential binding modes between bufalin and BFAR,and the binding energies are all less than 0.Western blot results showed that the expression of BFAR in HGC-27 and MKN-45 GC cells decreased significantly after treatment with bufalin for 48 hours(p < 0.001).3.Western blot results showed that BFAR was significantly overexpressed in HGC-27 and MKN-45 cells after transfection.The results of scratch healing test and Transwell migration test showed that there was no significant difference in the migration ability of GC cells between the control group and the no-load group.Compared with the control group,the overexpression of BFAR could significantly promote the migration ability of the two kinds of GC cells,while bufalin could significantly reverse the migration promotion induced by BFAR.The results of Western blot showed that the overexpression of BFAR increased the expression of N-cadherin and Vimentin and decreased the expression of Ecadherin in two kinds of GC cells.Bufalin could reverse the change of expression induced by BFAR.In addition,the overexpression of BFAR increased the phosphorylation level of PI3 K,AKT and mTOR proteins in two kinds of GC cells.Bufalin could inhibit the phosphorylation of proteins related to PI3K/AKT/mTOR signal pathway induced by BFAR(p < 0.01).4.Bufalin targets BFAR and regulates PI3K/AKT/mTOR signaling pathway to inhibit the occurrence and progression of gastric cancer subcutaneous xenografts.In the field of fluorescence microscope,the infected HGC-27 and MKN-45 cells emit green fluorescence,and compared with the bright field in the same field,the infection efficiency is more than90%.Western blot results showed that BFAR was significantly overexpressed in HGC-27 and MKN-45 cells after lentivirus infection,and stable cell lines with over-expression of BFAR were successfully constructed.The results of three-dimensional fluorescence imaging in vivo showed that the fluorescence intensity and fluorescence range of the transplanted tumor in the BFAR overexpression group were significantly larger than those in the control group.After treatment with bufalin,the fluorescence intensity and fluorescence range of the transplanted tumor in the treatment group decreased significantly.The transplanted tumor was taken out and the tumor volume was measured in each group.The results showed that the average tumor volume of nude mice in the BFAR overexpression group was significantly larger than that in the control group,but the tumor volume in the treatment group decreased significantly after treatment with bufalin.The results of HE staining showed that all the tumors were solid tumors,and the tumor tissue in the treatment group showed large area necrosis.Bufalin had no obvious side effects on heart,liver,spleen,lung and kidney of nude mice.Western blot results showed that overexpression of BFAR increased the expression of N-cadherin and Vimentin and decreased the expression of E-cadherin.Bufalin could reverse the changes of protein expression induced by BFAR after treatment(p<0.01).In addition,overexpression of BFAR can increase the phosphorylation level of PI3 K,AKT and mTOR proteins in transplanted tumors,and bufalin treatment can inhibit the increase of phosphorylation level(p < 0.01).Conclusion:This study confirmed the inhibitory effect of bufalin on the occurrence and progression of GC.Bufalin inhibits the proliferation and migration of GC cells through PI3K/AKT/mTOR signal pathway in vitro.BFAR is a direct interacting protein of bufalin and has many potential binding sites with bufalin.BFAR is highly expressed in GC,which plays an important role in the diagnosis and prognosis of GC.Bufalin reverses the occurrence and progression of GC induced by BFAR in vivo and in vitro.Bufalin inhibits the carcinogenesis and progression of GC induced by BFAR by reversing the activation of PI3K/AKT/mTOR signal pathway induced by BFAR in vivo and in vitro.This study will provide theoretical evidence for bufalin as a clinical treatment of GC,and provide a new idea for BFAR as an early diagnosis and prognostic marker of GC.