Study On The Effect Of Guiqi Baizhu Prescription On The Growth And Metastasis Of Gastric Cancer Cells Through VEGFR-2 And C-Met Based On Traditional Chinese Medicine Chemical Bioinformatics

ObjectiveBased on the research of our group,the potential targets of Guiqi Baizhu Prescription（GQBZP）for gastric cancer（GC）treatment were determined.Cheminformatics-Molecular Docking-Microscale Thermophoresis（MST）-Molecular Dynamics（MD）simulation and cell biology experiments revealed the material basis and molecular mechanism of"multi-point effect and synergistic effect"of Guiqi Baizhu Recipe in GC treatment.Methods1.Based on vascular epidermal growth factor receptor 2（VEGFR-2）and cellular mesenchymal-epithelial transition factor（c-Met）targets,the potential material basis of GQBZP for GC treatment was revealed.1.1 The material basis of GC treated by GQBZP was revealed based on molecular docking technology.The crystal structure and active sites of relevant target proteins were determined according to literature.The small molecules effectively bound to targets proteins in GQBZP were screened by molecular docking technology.Representative small molecules were screened based on molecular docking results and molecular characteristics,and in vitro MST assay was used to detecte the affinity between representative small molecules and target proteins.Molecular toxicity predictions,CCK8 assay and MD simulation were used to verify the toxicity and in vitro activity of representative small molecules,and to determine the representative small molecule species of GQBZP.1.2 The activity of representative small molecules of GQBZP was verified by methods in cell biology.GC cell lines with high expression of target proteins were screened by western blot（WB）.CCK8 assay,clone formation assay,3D tumor microsphere assay and WB assay were used to verify the inhibitory ability of representative small molecules of GQBZP on GC cell proliferation.Flow cytometry（FCM）,Hoechst 33342 staining,mitochondrial membrane potential（MMP）assay and WB assay were used to verify the effects of representative small molecules of GQBZP on GC cell cycle and apoptosis.The effects of representative small molecules on GC cell migration,invasion and epithelial interstitial transformation（EMT）were detected by Transwell migration and invasion assay and WB assay.Enzyme activity assay was used to detect the inhibitory effect of representative small molecules of GQBZP on target protein kinase.RT-q PCR and WB experiments were used to reveal the possible mechanism of GQBZP representative small molecules inhibiting the proliferation of GC cells.2.Explore the effect of representative small molecule combination of GQBZP based on Transwell co-culture system.Human umbilical vein endothelial cells（HUVEC）were isolated,extracted and identified in vitro.The co-culture system was established with GC cell line with high expression of target protein and HUVEC.CCK8,RT-q PCR and WB assays were used to evaluate the co-culture model.After single or combined intervention of representative small molecules of GQBZP at appropriate concentration in co-cultured GC cells,FCM,Transwell assays and WB assays were used to detect the cell cycle,migration and invasion of co-cultured GC cells.The expression of PI3K/AKT signaling pathway related proteins was detected by WB assays.CCK8 assays,FCM,Transwell assays and WB assay were used to verify the inhibitory effect and the possible mechanism of LA and NOB combined with apatinib on co-cultured GC cells.Results:1.Based on VEGFR-2 and c-Met targets,the potential material basis of Guiqi Baizhu Recipe for GC treatment was revealed.1.1 Molecular docking technique was used to reveal the material basis of GC treated by GQBZP.Molecular docking results showed that there were 156 small molecules of GQBZP with VEGFR-2 protein docking score≤-7.36,mainly from licorice,rhubarb,angelica and astragalus.And there are 131 small molecules of GQBZP with c-Met protein docking score≤8.20,mainly from licorice,rhubarb and astragalus.We selected 7 small molecules targeting VEGFR-2 and 4 small molecules targeting c-Met from the top 20 rated molecules for MST validation,respectively,and found that K_d value of rhein was 15.88μM,and that of licoflavone A（LA）was 25.99μM.K_d value of rutin was 0.28μM,and that of nobiletin（NOB）was 0.71μM.The prediction result of small molecule toxicity showed that all 11 molecules were not hepatotoxicity.CCK8results showed that LA and aloe emodin had the best inhibitory effect on three GC cell lines in a time and dose dependent manner among the seven VEGFR-2 targeting molecules,while NOB had the best inhibitory effect on three GC cell lines in a time and dose dependent manner among the four c-Met targeting molecules.Combined with the MST results,we selected LA and NOB to continue the MD simulation,and the results showed that LA has a good affinity and stable binding with VEGFR-2,while NOB has a good affinity and stable binding with c-Met.1.2 Cell biology experiments were used to verify the in vitro antitumor activity of representative small molecules of GQBZP.Since the antitumor effect of NOB has been widely reported,but whether LA has antitumor effect has not been reported before,we further verified the antitumor effect of LA basing on cell biology experiments.Therefore,we used 25,50 and 100μM LA to intervene 20 ng/m L VEGF-stimulated MKN-45 cells,and the results showed that LA inhibited the proliferation,cycle,migration,invasion and EMT and promoteed cell apoptosis of VEGF-stimulated MKN-45 cells in a dose-dependent manner.Tyrosine kinase assay demonstrated that LA is a potential VEGFR-2 inhibitor with IC₅₀ of 14.36μM.GQBZP inhibited GC cell proliferation by decreasing the expression of phosphatidylinositol 3 kinase（PI3K）m RNA and protein kinase B（AKT）m RNA and protein phosphorylation levels of mitogen-activated extracellular signal regulated kinase（MEK）and extracellular regulated protein kinase（ERK）.2.Effect of representative molecules of GQBZP on proliferation of GC cells in co-culture system.We evaluated the co-culture system and found that VEGFR-2 and c-Met interaction existed in the co-culture system.Compared with MKN-45 cells,VEGF m RNA expression was increased in the co-culture MKN-45 cells,with statistically significant differences（P<0.05）.HGF m RNA expression in co-cultured HUVEC cells was higher than that in HUVEC cells,with statistically significant differences（P<0.05）.Compared with MKN-45 cells and HUVEC,the phosphorylation levels of VEGFR-2and c-Met proteins were increased in co-cultured MKN-45 cells and co-HUVEC（P<0.01）.The mechanism was related to activation of PI3K/AKT and MEK/ERK signaling pathways.We treated co-cultured MKN-45 cells with 25μM Apatinib,25μM LA and 25μM NOB,respectively.The results showed that compared with control group,Apatinib,LA and NOB significantly inhibited the proliferation,cycle,migration and invasion of co-cultured MKN-45 cells.The effect of the combination of the two or three drugs was better than that of the monotherapy group.The mechanism is related to targeting VEGFR-2 and c-Met and inhibition of downstream PI3K/AKT and MEK/ERK signaling pathways.Conclusion1.Molecular docking method analysis found that the main components that play a therapeutic role in GQBZP are mainly concentrated in mainly from licorice,rhubarb,angelica and astragalus.These Chinese herbal medicinal ingredients correspond to the sovereign,minister,assistant and envoy of GQBZP and accord with main affect of GQBZP to strengthen the spleen and hormonize stomach,tonify qi and blood,and promote blood circulation and transform stasis.The point of view of big data reveals preliminarily the material basis of GQBZP for the treatment of GC and fully embodies the side of"multi-point effect,synergistic effect".2.Chemical informatics method,literature research and cell biology experiments reveal that the antitumor effect and possible mechanism of LA and NOB and confirm the potential anti-GC effect of GQBZP.This study revealed that the scientific connotation of GQBZP in treating GC by strengthen the spleen and transform stasis,providing basis for the development and transformation of GQBZP,and at the same time,it can provide methodological reference for the modernization of TCM compound material research.