Study On The Mechanism Of Osthole Inhibiting The Growth Of Ovarian Cancer Cell

Objective:Epithelial ovarian cancer is one of the most common malignant tumors.Because there is no specific screening index and method for ovarian cancer,the vast majority of patients are in the advanced stage of ovarian cancer at the time of discovery.Because the tumor is often in advanced stage and the therapeutic drugs and means are limited,the therapeutic effect of most ovarian cancer is very poor.Pharmacological studies of Osthole have shown that it can inhibit the growth and metastasis of a variety of malignant tumors,but there are few reports on the treatment of ovarian cancer,and there is no in-depth mechanism research.We intend to apply different concentrations of Osthole to two common human epithelial ovarian cancer cell lines A2780 and OVCAR3 respectively,to explore whether Osthole can inhibit the proliferation,invasion and metastasis of ovarian cancer cells,to clarify the mechanism of Osthole induced apoptosis,apoptosis and scorch death of tumor cells,and to find effective targets,Through sequencing analysis and verification,we hope to find out the molecular mechanism and related signal pathways of Osthole in the treatment of ovarian cancer.Method:Part Ⅰ:Study on the inhibition of Osthole on proliferation and apoptosis of ovarian cancer cells1.Two ovarian cancer cell lines(A2780 and OVCAR3)and normal ovarian epithelial cells IOSE80 were treated with osthole at different concentrations for 24 hours.Cell proliferation activity test and Live/Head cell staining test were used.The nude mice were randomly divided into osthole group and control group.The mice were injected intraperitoneally with osthole or the same amount of solvent.The mice were killed and their main organs were taken for H&E staining analysis after 10 days.2.Two ovarian cancer cells(A2780 and OVCAR3)were treated with Osthole at different concentrations for 24 hours.The treated cells were collected and stained with annexin V-FITC/PI and Hoechst.At the same time,the expression of PARP protein before and after treatment was detected by Western blot.3.Two ovarian cancer cells(A2780 and OVCAR3)were treated with Osthole at different concentrations for 12 hours.The treated cells were collected and the reactive oxygen species of ovarian cancer cells before and after treatment were detected by fluorescent probe DCFH-DA.Mitochondrial membrane potential detection kit(JC-1)was used to detect the changes of mitochondrial membrane potential.The fluorescence intensity was observed by fluorescence microscope.Part Ⅱ:Study on Osthole Induced Autophagy and Coke Death of Ovarian Cancer Cells1.Two ovarian cancer cells(A2780 and OVCAR3)were treated with Osthole at different concentrations for 24 hours.The treated cells were collected and observed by transmission electron microscope.The autophagy bodies of treated ovarian cancer cells were stained and labeled with MDC staining.The expression of autophagy related light chain protein LC3-Ⅱ in treated ovarian cancer cells was detected by Western blot.2.Two ovarian cancer cells(A2780 and OVCAR3)were treated with Osthole at different concentrations for 24 hours.The treated cells were collected and the morphological changes of dead cells were observed by optical microscope.The expression of gsdme was detected by Western blot.Part Ⅲ:Study on the prediction of the regulatory pathway of Osthole inhibiting the progression of ovarian cancer1.After 24 hours of osthole intervention on ovarian cancer cell A2780,transcriptome sequencing was performed.Compared with the blank control group,principal component analysis and cluster thermogram analysis were performed on the transcriptome differential genes of ovarian cancer cells treated with osthole.The purpose of these things was to identify the differential genes significantly affected by osthole.2.KEGG pathway enrichment analysis,GO enrichment analysis and GSEA analysis were performed on transcriptome differential genes of A2780 ovarian cancer cells treated with osthole.The purpose of these things is to clarify that osthole affects significant signaling pathways.3.The differential genes of transcriptome of A2780 ovarian cancer cells treated with osthole were analyzed through STRING database.The purpose of these things is to clarify the interaction between proteins expressed by different genes.4.The differential genes of transcriptome sequencing analysis were verified by routine QPCR.The purpose of these things is to determine whether the differential genes of transcriptome sequencing analysis can be verified again.Part Ⅳ:Study on Osthole Regulating autophagy through the PI3K/AKT/mTOR pathway via MET1.We obtained the original data of transcriptome sequencing of ovarian cancer tissue and normal ovarian tissue from TCGA database,GTEX database and pan cancer database.The TPM values of these data were normalized,and then analyzed by bioinformatics.We mainly analyzed the difference of MET gene between ovarian cancer and normal ovarian tissue,and analyzed the clinical significance of MET gene in ovarian cancer.2.80uM osthole and the same amount of solvent were used to treat ovarian cancer cells A2780 and OVCAR3 for 24 hours.Western blot was used to detect the expression of MET protein,PI3K protein,p-PI3K protein,AKT protein and p-AKT protein in ovarian cancer cells.3.80 μM osthole,10 nM JNJ-38877605 or 20 μM CQ was used to treat ovarian cancer cells A2780 and OVCAR3 for 24 hours.Western blot was used to detect LC3 and other proteins in ovarian cancer cells.A2780 and OVCAR3 cells were intervened by 80 μM osthole for 24 hours and detected by protein immunoprecipitation method using LC3 antibody,MET antibody or IgG antibody.4.Ovarian cancer cells A2780 and OVCAR3 were 80 μM osthole and 5 nM rapamycin were intervened for 24 hours.MET,mTOR and EIF4EBP1 protein were detected by Western blot.When stimulated with HGF(40 ng/mL),cells were exposed to 80 μM osthole intervened for 24 hours and was treated with HGF for 2 hours,and then MET,mTOR and EIF4EBP1 protein were detected by Western blot.5.Ovarian cancer cells A2780 and OVCAR were divided into six groups:control group,autophagy inhibitor(CQ,20 μM)Group,Osthole(80 μM)Group,MET inhibitor group(Met In,50 nM),osthol+autophagy inhibitor group and MET inhibitor+autophagy inhibitor group.Add the above six groups of media containing different drugs to incubate with cells for 24 hours.The expression of LC3 in ovarian cancer cells was detected by immunofluorescence assay.The proliferative activity of ovarian cancer cells was detected by CCK8 method.The cloning ability of ovarian cancer cells was detected by cell plate cloning method.6.Ovarian cancer cells OVCAR3 were used to generate tumors on the back of nude mice and randomly divided into six groups:control group(normal saline),CQ(10 mg/kg)group,Osthole(40 mg/kg)group,Met-In(20 mg/kg)group,Osthole+CQ group and Met In+CQ group.Then,200 μL of the above drug of corresponding concentration is injected intraperitoneally every day,The tumor volume was measured and counted every three days.When the difference was significant on the 15th day,the tumor was taken out and weighed.7.Ovarian cancer cells A2780 were used to generate tumors on the back of nude mice and randomly divided into four groups:control group(normal saline),3-MA(10 mg/kg)group,Osthole(40 mg/kg)group and Osthole+3-MA group.Then,200mL of the above drug of corresponding concentration is injected intraperitoneally every day,and the volume of tumor is measured and counted every three days.When the difference is significant on the 17th day,the tumor is taken out and weighed.Result:Part Ⅰ:Osthole inhibits the progression of ovarian cancer by inhibiting cell proliferation and inducing apoptosis1.CCK8 results showed that after ovarian cancer cell lines A2780 and OVCAR3 were treated with different concentrations of osthole for 24h,the survival rate of ovarian cancer cells gradually decreased in a concentration dependent manner,and their IC50 were 73.58 μM(A2780)and 75.24 μ M(OVCAR3)(P<0.05).CCK8 results show that osthole is at the concentration of 80 μM,it has no obvious effect on the proliferation of normal ovarian epithelial cells.The HE results of animal experiments in vivo showed that the high dose of osthole had little effect on the main organs.2.The results of annexin V-FITC and PI staining showed that the apoptotic cells of ovarian cancer cell lines A2780 and OVCAR3 increased significantly with the increase of concentration after Osthole treatment.When the drug concentration is 80 μM,the apoptosis rate of A2780 reached more than 20%,while 50%of OVCAR3 cells underwent apoptosis(P<0.05).Hoechst staining showed that the DNA of two ovarian cancer cells was broken and pyknosis after treatment with 80μMOsthole for 24 hours.The results of Western blot showed that PARP protein was sheared in a concentration dependent manner after treatment with Osthole at different concentrations(P<0.05).3.Flow cytometry results show that the ROS levels of the two ovarian cancer cells increased significantly after treatment with Osthole 80 μM for 12 hours(P<0.05).JC-1 test results showed that the green fluorescence of ovarian cancer cells treated with Osthole at different concentrations was enhanced.When A2780 cells were treated with 80μMOsthole,the green fluorescence was significantly enhanced and 80μM osthole could increase the green fluorescence intensity by more than 50%.The results of fluorescence microscope showed that the red fluorescence decreased and the green fluorescence increased after Osthole treatment.Part Ⅱ:Osthole inhibits ovarian cancer progression and induces autophagy and pyroptosis.1.Transmission electron microscopy showed that autophagy bodies with different sizes could be seen in ovarian cancer cells treated with 80μMOsthole.MDC staining showed that compared with untreated cells,a large number of green fluorescent bodies appeared in A2780 and OVCAR3 cells treated with 80μMOsthole,and the green fluorescence in OVCAR3 was more obvious.The results of Western blot showed that Osthole had no effect on two ovarian cancer cells.With the increase of Osthole concentration,the expression of LC3-II in two ovarian cancer cells increased gradually.Similar results were observed by immunofluorescence staining.2.Light microscopic observation showed that many cell membranes swelled and ruptured in Osthole treated ovarian cancer cells,accompanied by the escape of vesicular structures from the cell membrane.This phenomenon is particularly significant when the concentration of Osthole reaches 80μM.Western blot showed that gsdme was cut into c-gsdme in two ovarian cancer cells treated with Osthole.The concentration of c-gsdme increased gradually with the increase of Osthole concentration.Part Ⅲ:Osthole may inhibit the progression of ovarian cancer through MET and other pathways1.Principal component analysis and cluster thermogram analysis showed that the gene expression of osthole treated group was significantly different from that of the blank control group.A total of 3005 differential genes were screened,including 1412 up-regulated genes and 1593 down regulated genes.2.The enrichment analysis of KEGG pathway showed that the differential pathway was enriched in proteoglycans,cell junctions,lysosomes,energy metabolism and cell cycle.GO enrichment analysis showed that the up-regulated genes were enriched in autophagy,endoplasmic reticulum stress,etc.The down regulated genes are enriched in cell division,cell proliferation and cell cycle.GSEA analysis showed that cell cycle,WNT pathway,DNA replication decreased and apoptosis pathway increased.3.Analysis of protein interaction network showed that some key tumor regulatory genes were found in the protein interaction network,such as MET,ENO1,SLC2A3,TRL4,TOP2A and CASP4.4.The differential genes of transcriptome sequencing analysis were verified by routine QPCR method.The results showed that the expression of important oncogenes TOP2A and MET decreased,and the expression of metabolism related genes SLC2A3 and ENOl decreased(P<0.05).The expression of RNA methylation related genes YTHDC2 and FTO decreased(P<0.05).The expression of apoptosis related gene CASP4 and immune response gene TLR4 increased(P<0.05).The results of QPCR were similar to those of sequencing.Part Ⅳ:Osthole down regulates MET expression to affect PI3K/AKT/mTOR pathway to regulate cell autophagy and ovarian cancer progression1.Transcriptome sequencing analysis of TCGA and other databases showed that MET gene was significantly overexpressed in ovarian cancer tissues,which was significantly higher than that in normal ovarian tissues(P<0.05).Further survival analysis showed that the survival rate of patients with high MET expression group was significantly lower than that of patients with low MET expression group(P<0.05).2.We treated A2780 ovarian cancer cells with 80uM osthole for 24h,and compared with the blank control group,the expression of MET protein,p-PI3K protein and p-AKT protein in the osthole treated group were down regulated.The results of OVCAR3 group were similar.3.After the ovarian cancer cells A2780 and OVCAR3 were treated with JNJ-38877605 and osthol respectively,the protein expression level of MET decreased significantly,while the expression of autophagy related protein p62 and LC3-Ⅱ increased.When autophagy was blocked by an autophagy inhibitor(CQ),the expression of LC3 in ovarian cancer cells treated with osthole was significant.The results of protein immunoprecipitation experiment showed that MET indeed interacted with LC3 protein.4.Western blot showed that the expression of LC3-Ⅱ in ovarian cancer cells was increased and autophagy was enhanced after rapamycin treatment alone,but it was lower than that induced by osthole.When osthole and rapamycin were used together,autophagy was reduced.Western blot analysis showed that in ovarian cancer cells treated with osthole,the expression of MET,p-mTOR and p-EIF4EBP1 was decreased.In ovarian cancer cells A2780 and OVCAR3 using HGF to stimulate the MET pathway,osthole could reduce the expression of MET,p-mTOR and p-EIF4EBP1 in ovarian cancer cells.5.CCK8 test results showed that the proliferation of ovarian cancer cells A2780 and OVCAR3 was further significantly inhibited by osthole and autophagy inhibitors(P<0.05).Similar results were obtained in the plate cloning experiment.After the combination of osthole and autophagy inhibitor,the proliferation of ovarian cancer cells A2780 and OVCAR3 was further significantly inhibited(P<0.05).The results of immunofluorescence showed that osthole combined with autophagy inhibitor could accumulate LC3-Ⅱ in cells to promote cell death.6.The results of tumor formation experiment of OVCAR3 cells in nude mice showed that when MET inhibitors or osthol combined with autophagy inhibitors were used in nude mice,the tumor volume and tumor weight were significantly inhibited compared with the control group and drug intervention alone(P<0.05).7.The results of tumor formation experiment of A2780 cell in nude mice showed that when the nude mice were treated with osthole combined with autophagy inhibitor 3-MA,the tumor volume and tumor weight were significantly inhibited compared with the control group and the drug intervention alone(P<0.05).This is similar to the tumor formation experiment of OVCAR3 in nude mice.Conclusion:1.Osthole can significantly inhibit the proliferation of ovarian cancer cells,induce apoptosis of ovarian cells and has a significant killing effect on ovarian cancer cells.2.Osthole inhibits the growth of ovarian cancer cells,while autophagy occurs in ovarian cancer cells.It is suggested that autophagy plays an important role in killing ovarian cancer cells.Some ovarian cancer cells die through autophagy,while others may have drug resistance protection through autophagy.3.Osthole can inhibit the expression of TOP2A,MET,SlC2A3,ENO1,YTHDC2 and FTO in ovarian cancer cells,and upregulate the expression of CASP4 and TLR4 in ovarian cancer cells.4.Osthole can induce autophagy of ovarian cancer cells induced by MET inhibition.Osthole can induce the reduction of MET in ovarian cancer cells,and affect the occurrence and level of autophagy by regulating the activation of PI3K/AKT/mTOR and other pathways.Osthole combined with autophagy inhibitors can synergistically inhibit the progression of ovarian cancer.