Study On The Inhibitory Effect Of Low Dose Tripterygium Wilfordii Polyglycosides On Human Epithelial Drug-resistant Ovarian Cancer A2780/DDP And Its Mechanism

Background and Objective:Background and Objective:As a common malignant tumor in gynecology,epithelial ovarian cancer has a high fatality rate,which poses a serious threat to the physical and mental health of women.Studies have shown that the overall remission rate in epithelial ovarian cancer is between 10%and35%,and the remission period is relatively short.Despite significant progress in the treatment of epithelial ovarian cancer,most patients with advanced disease still inevitably experience recurrence and eventually die as a result of chemotherapy resistance,with platinum resistance being the highest.Clinically,the treatment of human epithelial cisplatin-resistant ovarian cancer and advanced ovarian cancer has always been a difficult problem.In addition,cisplatin resistance of epithelial ovarian cancer often presents a multi-factorial mechanism,which is complex and has not been fully understood and recognized.Therefore,the main objective of the current treatment of platinum resistant/platinum refractory ovarian cancer is to maintain quality of life and alleviate symptoms through sequential monotherapy,while the current challenge still needs to be broken through clinical trials of innovative therapies.The research group led by my tutor has been exploring in the field of traditional Chinese medicine,trying to dig out effective traditional Chinese medicine or traditional Chinese medicine compound that can assist the treatment and improve the efficacy of these ovarian cancer patients from the treasure house of traditional Chinese medicine,hoping to be used in the treatment of these advanced or drug-resistant ovarian cancer.Therefore,it is of great significance to seek safe and effective Chinese medicine for the treatment of cisplatin-resistant ovarian cancer.In this study,a low dose of Tripterygium wilfordii polyglycoside（GTW）was applied to human epithelial cisplatin resistant ovarian cancer cell line A2780/DDP,and the inhibitory effects of low dose of GTW on the growth,invasion and migration of human epithelial cisplatin resistant ovarian cancer cell line A2780/DDP were observed through in vivo and in vitro experiments,and the mechanism of GTW in inhibiting epithelial drug resistant ovarian cancer was revealed.To provide experimental data and in vitro evaluation model for the clinical application of GTW in adjuvant treatment of advanced or epithelial drug resistant ovarian cancer.Part I Effect of GTW on Human Epithelial Cisplatin Resistant OvarianCancer CellsObjectiveTo evaluate the effect of GTW on the invasion and migration of tumor cells at the cellular level.Methods1.Culture,cryopreservation and counting of A2780/DDP cells:A2780/DDP cells were cultured in medium containing streptomycin,10%FBS and penicillin.Culture conditions:ambient temperature 37℃,5%CO₂,90%air humidity,liquid change period of 2-3d.Cell digestion and passage were performed when the adherent rate of cells reached about 90%.When the cells became round in shape,RPMI-1640 medium was added and digestion was stopped.Take out the supernatant and get the cell precipitate.According to the ratio of 9:1,FBS 900μl and DMSO100μl were added to make 1m L of cell freeze solution,and the cell concentration was adjusted.The adjusted cryopreservation solution and cell precipitate were evenly mixed and then cryopreservation was carried out.10%fetal bovine serum was added to the cell precipitate,the cell suspension was dispersed,and the cell suspension density was adjusted to 5×10⁴/m L for cell count.2.The survival rate of human epithelial cisplatin-resistant ovarian cancer A2780/DDP cell lines treated with different concentrations of GTW and DDP was detected by CCK8 method,and the IC₅₀ determination of A2780/DDP cells treated with GTW and DDP was calculated.3.Transwell migration and invasion assay was used to detect the changes of invasion and metastasis ability of tumor cells before and after the treatment of A2780/DDP cells with different concentrations of GTW.Group the same as before.The experiment was repeated three times,and the mean value of transmembrane cells was taken as the indicator of cell migration ability.4.One-way analysis of variance or two-way analysis of variance was used for statistical processing,and±SD was represented by mean value.P<0.05 indicated statistically significant difference.Results1.A2780/DDP cells were treated with different concentrations of GTW（50μg/m L,200μg/m L,800μg/m L,1600μg/m L,3200μg/m L）for 24h.The lowest effective inhibitory concentration of GTW was 200μg/m L.With the increase of GTW concentration,the cell survival rate of each experimental group showed a progressive decreasing trend（P<0.05）.The inhibitory effect of Tripterygium wilfordii polyglycosides on the growth of A2780/DDP cells showed a gradually increasing trend,and the IC₅₀ was 882.1ug/m L.These results indicated that GTW could inhibit the proliferation of A2780/DDP cells in a dose-dependent manner.2.A2780/DDP cells were treated with different concentrations of DDP（0.625g/m L,1.25 g/m L,2.5 g/m L,5 g/m L,10 g/m L,20 g/m L）for 24h,and the lowest effective inhibitory concentration of DDP was 1.25 g/m L（P<0.05）.The survival rate of cells decreased with the increase of DDP concentration（P<0.05）.The IC₅₀ of DDP on A2780/DDP cells was 1.891μg/m L.The results showed that DDP could inhibit the proliferation of A2780/DDP cells in a dose-dependent manner.3.The inhibitory effect of low dose GTW on the invasion and migration of A2780/DDP cells gradually increased with the increase of the concentration.When the concentration was 200/800/1600/3200 microgram/ml,the difference was significant,with statistical significance（P<0.05）.ConclusionGTW has a significant dose-dependent inhibition on the proliferation,migration,and invasion of A2780/DDP cells.Part II The inhibition of cisplatin resistance by GTW through ILK/Akt/GSK3β/Slug signaling pathway in human epithelial ovarian carcinoma was studied ObjectiveTo investigate whether GTW can inhibit epithelial mesenchymal transformation by inhibiting ILK/Akt/GSK3β/Slug signaling pathway,so as to enhance the sensitivity of DDP to inhibit tumor cell invasion and metastasis and cisplatin resistance.Methods1.A2780/DDP cells were divided into blank control group,GTW group,DDP group and GTW+DDP group.Si RNA-ILK,si RNA-Slig,Akt inhibitor and GSK3βinhibitor were treated with each basic group respectively,and the changes（invasion and migration）of cells were detected by Transwell experiment.2.The expressions of ILK/Akt/GSK3β/Slug signaling pathway related proteins E-cadherin,N-cadheriin,ILK,Akt,p-Akt,GSK3β,p-GSK3βand Slug in each group were determined by Western blot.3.One-way analysis of variance（ANOVA）or two-way analysis of variance（ANOVA）was used for statistical processing,and±SD was represented by mean value.P<0.05 indicated statistically significant difference.Results1.Compared with the normal control group,the DDP group,GTW group and GTW+DDP group could effectively inhibit the expression of Slug,and the GTW+DDP group had a significant inhibitory effect（P<0.005）.After si RNA-slug transfection,the expression of N-cadherin was further down-regulated,while the expression of E-cadherin was further up-regulated,especially in the GTW+DDP group,the difference was statistically significant（P<0.005）.These results indicated that si RNA-slug synergistically enhanced the inhibitory effect of GTW+DDP group on the invasion and migration ability of A2780/DDP cells（P<0.005）.2.After si RNA-ILK transfection,the migration and invasion ability of A2780/DDP cells were weakened.Compared with the normal control group,the migration and invasion of A2780/DDP cells in the DDP group,GTW group and GTW+DDP group were inhibited to different degrees,and the inhibition effect was obvious in the GTW+DDP group（P<0.005）.Compared with normal control group,the expressions of ILK,p-Akt,p-GSK3βand Slug were down-regulated in each group before transfection.Especially in the GTW+DDP group,the inhibition effect was the most significant,and the difference was statistically significant（P<0.005）.After ILK silencing by transfection si RNA-ILK,the levels of ILK,p-AKT,p-GSK3βand Slig were down-regulated more significantly,and the expression of N-cadherin was down-regulated further,while the expression of E-cadherin was up-regulated further,especially in the GTW+DDP group.The difference was statistically significant（P<0.005）.These results indicated that si RNA-ILK synergistically enhanced the inhibitory effect of GTW+DDP group.3.Akt inhibitor（MK2206）inhibited the growth of A2780/DDP cells in a dose-dependent manner.Akt inhibitors synergistically promoted the inhibitory effects of GTW,DDP and GTW+DDP on the migration and invasion ability of A2780/DDP cells,and the difference was statistically significant（P<0.005）.Akt inhibitors promoted the further down-regulation of p-Akt,p-GSK3βand ILK protein levels in the GTW group,DDP group and GTW+DDP group.It promoted the up-regulation of E-cadherin level and the further down-regulation of N-cadherin hydrolevel in A2780/DDP cells,and the differences were statistically significant（P<0.005）.4.GSK3βinhibitor inhibited the growth of A2780/DDP cells in a dose-dependent manner.GSK3βinhibitor synergistically promoted the inhibitory effect of GTW,DDP and GTW+DDP on the migration and invasion of A2780/DDP cells,and the inhibitory effect was the most significant in the GTW+DDP group,with statistical significance（P<0.005）.GSK3βinhibitor induced the upregulation of Slug and p-GSK3βin GTW group,DDP group and GTW+DDP group,but did not affect p-AKT and ILK.GSK3βinhibitor synergically promoted the up-regulation of E-cadherin level and down-regulation of N-cadherin level in each group,with statistically significant differences（P<0.05）.Conclusion1.GTW can cooperate with DDP to inhibit the proliferation,migration and invasion of human epithelial cisplatin resistant ovarian cancer A2780/DDP cells,and increase the sensitivity of A2780/DDP cells to cisplatin.2.GTW can inhibit the proliferation,migration and invasion of cisplatin-resistant ovarian cancer A2780/DDP cells by targeting the ILK/Akt/GSK3β/Slug signaling pathway.By targeting the ILK/Akt/GSK3β/Slug signaling pathway,the levels of EMT-related protein E-cadherin were up-regulated,while the levels of N-cadherin,ILK,p-Akt,p-GSK3βand Slug were down-regulated.GTW+DDP group was especially obvious,indicating that GTW could enhance the sensitivity to DDP by ILK/AKT/GSK3β/Slug targeting to inhibit the EMT of tumor cells,thus inhibiting the resistance of tumor cells to cisplatin.Part III Inhibitory effect of GTW on tumorigenesis of human epithelialcisplatin-resistant ovarian cancer in nude miceObjectiveIn vivo experiments were conducted to further confirm that GTW inhibited EMT by inhibiting ILK/Akt/GSK3β/Slug signaling pathway,thereby inhibiting EMT-associated metastasis and chemotherapy resistance（DDP-resistance）of tumor cells.Methods1.Forty-eight female nude mice aged 4 to 6 weeks with body weight of 15 to20g were randomly divided into four groups with 12 mice in each group and domesticated for one week.2.Human epithelial cisplatin-resistant ovarian cancer A2780/DDP was routinely cultured.When modeling in the abdominal cavity of nude mice,0.2ml cell suspension was taken for inoculation,and the density was controlled to 2×10⁷ cells per ml.96h after inoculation,0.2 m L of blood was drawn from the tail vein of the nude mice.After centrifugation,the serum was extracted and stored at-80℃for later use.After blood drawing,when the tumor volume reached 50mm3,the following treatments were received:（1）Control group:0.2 m L of normal saline was given to the nude mice by gavage every other day for 10 times.（2）GTW group:1mg/kg/d GTW normal saline was diluted to a final volume of 0.2 m L,and intraperitoneal injection was performed for 14 times.（3）DDP group:Cisplatin 4mg/kg/d was intraperitoneally injected,on the 1st and 8th day,for a total of 2 days.（4）GTW+DDP group:1mg/kg/d GTW normal saline was diluted into a final volume of 0.2 m L,and intraperitoneal injection was performed for 14 times.On day 1 and day 8,DDP was intraperitoneally injected at a dose of 4mg/kg/d for a total of 2 days.3.After inoculation,the activity,skin color and mental state of the tumor-bearing nude mice were observed every day,and the body weight and abdominal circumference of the tumor-bearing nude mice were observed every other day from the 7th day of inoculation.When the abdominal circumference of the tumor-bearing nude mice increased,abdominal metastasis was considered to have occurred.The experiment ended the day after the last dose.Tumor volume was measured twice a week and mice were weighed.On the 22nd day of the experiment,3 mice from each group were taken for euthanasia and dissected,abdominal tumor formation and tumor location were observed,the number of tumors in the mesentery was counted,and the mesentery and the metastatic tumors above the mesentery were removed and weighed.Meanwhile,the transplanted tumor specimens were kept aseptic and stored at-80℃.The remaining mice were used to evaluate survival curves.4.The changes of serum tumor markers CA125 and HE4 in tumor bearing nude mice treated with GTW,DDP and their combination were detected by ELISA.5.The protein expression levels of E-cadherin,Viin-cadheriin,ILK,Akt,p-Akt,GSK3β,p-GSK3βand Slug in intraperitoneal transplanted tumor tissues of human epithelial cisplatin resistant ovarian cancer in nude mice were detected by Western blot.6.One-way analysis of variance（ANOVA）or two-way analysis of variance（ANOVA）was used for statistical processing,and±SD was expressed as mean value.P<0.05 indicated statistically significant difference.Results1.After intraperitoneal tumor formation in nude mice,the volume and weight of tumor in each group changed in different degrees.The average volume of each group was 1916.463±52.491mm3 in the control group,1014.021±52.553mm3 in the DDP group,1196.322±51.681mm3 in the GTW group,and 680.321±33.652mm3 in the DDP+GTW group.The tumor volume in the control group was the largest,and the volume in the DDP group,GTW group and combined group was significantly smaller than that in the control group,with statistical significance（P<0.05）,and the reduction degree in the DDP+GTW group was the most obvious.The tumor volume of GTW group and DDP group was the next（P<0.05）.Tumor weight of each group:the average weight of the control group was 0.8210±0.1260g,the average weight of the DDP group was 0.4797±0.0054g,the average weight of the GTW group was 0.5413±0.049g,and the average weight of the DDP+GTW group was 0.2777±0.0046g.The tumor weight in the control group was the largest,and the tumor weight in the GTW group,DDP group and combined（DDP+GTW）group was significantly lower than that in the control group（P<0.05）,and the results were statistically significant.The tumor weight of DDP+GTW group decreased most obviously.The tumor severity in GTW and DDP groups was the second（P<0.05）.2.In terms of tumor survival time,the mean and median tumor survival time in the control group were 34.6 days and 37 days,respectively;the mean and median tumor survival time in the DDP group were 41.4 days and 44 days,respectively;and the mean and median tumor survival time in the GTW group were 39.8 days and 40days,respectively.The mean and median survival time of the combination（DDP+GTW）group were 46.6 days and 49 days,respectively.DDP+GTW group had the longest survival time,which could reach 50 days.3.ELISA was used to detect the levels of CA125 and HE4 in each group.Compared with the control group,the serum levels of tumor markers CA125 and HE4in GTW group,DDP and GTW+DDP group were significantly decreased,and the decrease was significant in the GTW+DDP group,with statistical significance（P<0.05）.4.The protein expression levels of E-cadherin,N-cadheriin,ILK,Akt,p-Akt,GSK3β,p-GSK3βand Slug in intraperitoneal transplanted tumor tissues of human epithelial cisplatin resistant ovarian cancer in nude mice were detected by Western Blot.The results showed that the levels of E-cadherin in transplanted tumor tissues were up-regulated in both GTW group and DDP group,while the levels of N-cadherin,ILK,p-AKT,p-GSK3βand Slug were down-regulated.GTW+DDP had more obvious regulation on the expression of the above related proteins in tumor tissues,and the difference was statistically significant（P<0.05）.ConclusionsGTW inhibited EMT by inhibiting ILK/Akt/GSK3β/Slug signaling pathway,thus inhibiting EMT-associated metastasis and chemotherapeutic resistance（DDP-resistance）of tumor cells.