Mechanism Of Yi Fei San Jie Pill Inhibiting EMT And Enhancing The Sensitivity Of Gefitinib In The Treatment Of Non-small Cell Lung Cancer

ObjectiveYi Fei San Jie PIL(YFSJP)is a Chinese patent medicine used in clinical treatment of non-small cell lung cancer(NSCLC),the formula is based on the experiential prescription from Dr.Lin Lizhu who is a famous traditional Chinese Medicine professor in Guangdong Province.As of today,as an in-hospital preparation of the First Affiliated Hospital of Guangzhou University of TCM,it is widely used in clinic and has achieved remarkable curative effect.In this study,we intend to explore and study the molecular mechanism of its antitumor effect through network pharmacology,conduct cell experiments and animal experiments,so as to further verify the antitumor effect of YFSJP,and lastly conduct RNASEQ sequencing to explore the molecular mechanism of YFSJP in the treatment of NSCLC.Methods1.Explore and verify the possible molecular mechanism of YFSJP in the treatment of NSCLC through network pharmacologyFirstly,to seek out the potential therapeutic targets of YFSJP through TCMSP and Batman database,and then collect the relevant potential therapeutic targets of NSCLC level through the data of Disgenet,OMIM and GeneCards.After that we combine the potential therapeutic targets,we found from both sides to obtain the intersection,and try to analyze the relevant pathways and mechanisms of the antitumor effect of YFSJP in the treatment of NSCLC.Based on the R software package cluster profiler,we have analyzed the pathway of both Gene Ontology(go)and Kyoto Encyclopedia of genes and genomes(KEGG),to further explore the molecular mechanism of YFSJP’s antitumor effect through intersection of the enrichment analysis results of both YFSJP and NSCLC.2.Efficacy of YFSJP and Gefitinib on the therapeutic Trial of Human NSCLC cell PC9 xenograft in nude miceAfter the xenograft model was successfully established,we divide the mice into four groups randomly:control group(0.9%NaCl),YFSJP treatment group(9.6g/kg),Gefitinib treatment group(50mg/kg)and combined administration group(YFSJP:9.6/kg+Gefitinib:50mg/kg),record and measure the size of tumor and the weight of the nude mouse every other day.After 21 days of administration,execute the mouse and isolate the subcutaneous transplanted tumors,also measure their volume and weight.Furthermore,verify the EMT related protein expression level from tumor examples through the WB test and run RNA SEQ sequencing examination.3.Make and prepare the YFSJP medicated serum solution and Gefitinib solution,explore and compare their effects on the functions of NSCLC cells A549 and PC9.Firstly,New Zealand rabbits were selected as the carrier and divided into two groups,one group with normal saline and the other group with YFSJP aqueous solution,given regularly and quantitatively,gavage administration.After 7 days,carotid blood was taken to obtain raw serum and YFSJP medicated serum.Secondly,use MTT method to measure the effects of YFSJP containing serum and Gefitinib solution on the growth and proliferation of A549 and PC9,also calculate the half inhibition rate(IC50).Thirdly,run the wound healing assay and Transwell cell migration test on A549 and PC9 with YFSJP medicated serum and Gefitinib solution,so as to detect the effects of their respective and combined effects on the migration ability of A549 and PC9.Furthermore,run the Transwell cell invasion test on A549 and PC9 with YFSJP medicated serum and Gefitinib solution,so as to detect the effects of their respective and combined effects on the invasion ability of A549 and PC9.Lastly,divide A549/PC9 into 4 groups:control group(raw rabbit serum),YFSJP medicated serum group,Gefitinib group and combined administration group(YFSJP medicated serum+Gefitinib),then run the Western blot(WB)test to detect the EMT related protein expression level in A549 and PC9 after intervention.After 24 hours of intervention,extract the proteins from A549 and PC9 in each group,and detect the protein expression level such as EGFR,p-EGFR1068,p38,p-p38,E-cadherin,Vimentin,etc.Results1.Efficacy of YFSJP and Gefitinib on the therapeutic Trial of Human NSCLC cell PC9 xenograft in nude mice1.1 tumor sizeThe average tumor size in Gefitinib group was smaller than that in control group;The mean tumor size of YFSJP group was smaller than that of control group;Compared with the control group,the average tumor size in the combined administration group was always smaller;Compared with Gefitinib group,the tumor size of the combined group was smaller.1.2 WB validation study on the mechanism of YFSJP affecting tumor growthThe expression level of Vimentin was significantly reduced in Gefitinib group and combined administration group.In addition,the phosphorylation level of EGFR1068 also decreased slightly,and the expression level of p-p38 in each group increased in turn,but the latter two had no statistical significance.2.Effects of YFSJP medicated serum solution and Gefitinib solution on the functions of NSCLC cells A549 and PC9.2.1 MTT method:(1)The results showed that raw rabbit serum could promote the proliferation of A549 and PC9,but the proliferation effect decreased with the decreasing of serum concentration,and the proliferation effect was not obvious at low concentration level.(2)After administration of YFSJP for 24h,there was no significant Inhibition of proliferation in PC9,but inhibition has increased step by step at 48h and 72h;On the other hand,significant Inhibition was observed after 72h in A549.It is considered that the protective components of serum to cells were decreased while the increasing of administration time.(3)Gefitinib can effectively inhibit the proliferation of A549 and PC9 cells in a time and dose-dependent manner.(4)After combined administration,there was significant Inhibition of proliferation in PC9 at 48h and 72h.On the other hand,there was significant Inhibition of proliferation in A549 at all of 24h,48h,and 72h.However,there was no statistical difference when we compare with the case of Gefitinib.The combination was not superior to Gefitinib alone.2.2 Wound healing assay and Transwell cell migration test2.2.1 Wound healing assay(1)In A549,the healing of combined administration group was slower than that of the control group at 36h,but the wound healing rate of Gefitinib group was significantly lower than that of other groups at 36h.(2)In PC9,the combined administration group and Gefitinib Group have limited and reduced the healing speed at 48h and 36h,combined administration was more effective than that of Gefitinib alone.2.2.2 Transwell cell migration test(1)In A549 cells,after 48 hours of administration,Gefitinib group significantly inhibited its migration ability,but YFSJP group and combined administration group had no significant inhibitory effect.(2)In PC9 cells,YFSJP and Gefitinib significantly inhibited their migration ability,and the effect of combined administration group was better.2.3 Transwell cell invasion test(1)Gefitinib group significantly inhibited the invasion of A549,but there was no significant inhibitory effect in YFSJP group and combined administration group.(2)In PC9,YFSJP group and Gefitinib group significantly inhibited the migration ability,also combined administration group was better than the former two groups.(3)These results suggest that YFSJP has a more significant effective on the migration and invasion of sensitive strain PC9 than A549,which may be related to the inhibition of EMT functioned by YFSJP.2.4 WB test(1)In A549,YFSJP reduced the phosphorylation level of Akt and Vimentin expression level,and inhibited the process of EMT to some extent,but did not further enhance the effect of Gefitinib.(2)In PC9,the combined application of YFSJP and Gefitinib significantly inhibited the phosphorylation level of p-p38,it’s one of the downstream targets of EGFR.Moreover,the combined administration had further increased the expression level of E-cadherin and decreased the expression level of Vimentin,thus inhibited the process of EMT.Conclusion1.Because raw rabbit serum has protective components on NSCLC cells A549 and PC9,although YFSJP has a certain inhibitory effect on their proliferation,it is not obvious.2.Compared with NSCLC non sensitive strain A549,sensitive strain PC9 in EMT related migration and invasion experiment is more obvious than the former,which shows that YFSJP and Gefitinib significantly inhibit its migration ability,and the effect of combined administration group is better.3.YFSJP and combined administration can enhance the EGFR sensitivity of NSCLC cells A549 and PC9 through p38 signal pathway in vitro.4.YFSJP and combined administration can inhibit the increase of transplanted tumor volume of NSCLC cell PC9 in nude mice through p38 signal pathway in vivo.