A Preliminary Study On Triptolide Inhibiting The Invasion And Migration Of Drug-resistant Ovarian Cancer Cells By Inducing Autophagy

Background:Ovarian cancer,as the disease with the highest mortality rate of female malignant tumors,is not easy to be diagnosed in the early stage.Once it is found that most of the cases are in the advanced stage,drug resistance to chemotherapy often appears in the process of clinical diagnosis and treatment,leading to drug resistance in most ovarian cancer patients after treatment,and the final prognosis of the patients is poor.Triptolide(TPL)is one of the effective components extracted from traditional Chinese medicine Triptolide Wilfordii,which has anti-inflammatory,immunomod ulatory,anti-tumor and other pharmacological activities.A large number of studies have shown that TPL has an inhibitory effect on a variety of tumor cells,and can inhibit the migration and invasion of drug-resistant ovarian cancer cells,which was also confirmed by our previous study.Autophagy is a method used by most mammalian cells(including tumor cells)to maintain their own survival.Continual excessive autophagy is detrimental to cell survival.A huge number of studies have shown that the migration and invasion of many malignant tumors are related to the changes in the level of autophagy of tumor cells.Is the inhibition of TPL on the invasion and migration of SKOV3/DDP cells also related to autophagy?Objective:To explore the relationship between TPL and autophagy and its effect on the invasion and migration behavior of SKOV3/DDP cells and its mechanism,provide experimental evidence for TPL to be expected to be an adjuvant therapy for drug-resistant ovarian cancer.Methods:1.Treat SKOV3/DDP cells with different concentrations of TPL(0,25,50,100 n M)for 24 hours,and determine the effect of TPL on the migration and invasion ability of SKOV3/DDP cells by Transwell experiment;Western blot was used to detect the expression of epithelial-mesenchymal transition(EMT)related proteins(E-cadherin,N-cadherin)and MMP9 of SKOV3/DDP cells by TPL.2.The effect of TPL on autophagy flow in SKOV3/DDP cells was detected by m RFP-GFP-LC3 dual fluorescence-labeled adenovirus transfection.The regulation of LC3 protein expression by TPL was detected by Western blot.3.Transfection of si-Beclin1 down-regulates the autophagy level of tumor cells.Tranwell test detects the changes in the migration and invasion of SKOV3/DDP cells after TPL,si-Beclin1 and their combination,and Western blot detects N-cadherin and E-cadherin,MMP9,Beclin1 and LC3 expression.4.The JAK2/STAT3 signaling pathway was activated by IL-6,and the effects of IL-6,TPL and TPL+IL-6 treatment groups on the migration and invasion of SKOV3/DDP cells were determined by Tranwell experiment.Western blot was used to detect N-cadherin,E-cadherin,MMP9 And the level of LC3.5.Using AG490 to inhibit JAK2/JAK3,the experiment was divided into control group,AG490 treatment group,TPL treatment group and TPL+AG490 treatment group;Western blot was used to detect the expression levels of N-cadherin,E-cadherin,MMP9,p-JAK2,JAK2,p-STAT3,STAT3,and LC3;Transwell experiment detects the changes of cell migration and invasion ability.Results:1.TPL can inhibit the migration and invasion of SKOV3/DDP cells,and is positively correlated with the concentration of TPL;TPL down-regulates the expression of N-cadherin and MMP9,and up-regulates the expression of E-cadherin.2.Under fluorescence microscope,we observed that the number of autophagosomes in SKOV3/DDP cells was positively correlated with TPL concentration,and LC3 level increased with the increase of TPL concentration.3.si-Beclin1 up-regulates the migration and invasion ability of SKOV3/DDP cells,up-regulates the expression of E-cadherin protein,inhibits the expression of N-cadherin and MMP9,and decreases Beclin1 and LC3 protein levels;the effect of TPL is antagonistic compared with si-Beclin1 effect.4.IL-6 up-regulates the migration and invasion levels of SKOV3/DDP cells,it can inhibit the levels of E-cadherin and LC3,and up-regulate the expression of N-cadherin and MMP9;in the combined treatment group of IL-6 and TPL,The promoting effect of IL-6 on the migration and invasion of SKOV3/DDP cells was inhibited by TPL,and it increased the levels of E-cadherin and LC3,and down-regulated the levels of N-cadherin and MMP9.5.Using AG490 alone not only down-regulated the expression of p-JAK2 protein and p-STAT3 protein,up-regulated the expression of LC3 protein,but also inhibited the migration and invasion ability of SKOV3/DDP cells,and inhibited the expression of N-cadherin and MMP9 protein,and promoted E-cadherin expression of cadherin protein.The combined application of TPL and AG490 has a synergistic effect.Conclusion:1.TPL has the ability to inhibit the migration and invasion of SKOV3/DDP cells in a dose-dependent manner.2.TPL induced autophagy in SKOV3/DDP cells,which was positively correlated with dose within a certain range.3.TPL may induce autophagy in SKOV3/DDP cells by inhibiting the JAK2/STAT3 signaling pathway,and then inhibit the migration and invasion of SKOV3/DDP cells.