Study On The Mechanism Of Xiaoaiping Injection To Reverse Paclitaxel Resistance Of Ovarian Cancer Via Regulating TAB1/TAK1/p38MAPK

Paclitaxel is the first-line chemotherapeutic agent for the treatment of ovarian cancer,although it is easy to cause drug resistance during clinical application and lead to treatment failure.Xiaoaiping injection is often combined with chemotherapy drugs such as paclitaxel to treat multiples tumors.Our previous study found that Xiaoaiping injection could reverse the resistance of ovarian cancer cells to paclitaxel.However,the effect and molecular mechanism of Xiaoaiping injection combined with paclitaxel in reversing paclitaxel resistance in ovarian cancer remains to be elucidated.Objective:To study the effect of Xiaoaiping injection on the reversal of paclitaxel resistance in ovarian cancer,and further investigate the regulation of TAB1 protein and related TAK1 protein as well as the downstream p38MAPK.Elucidating the synergistic effect and molecular mechanism of Xiaoaiping injection combined with paclitaxel associated with TAB1/TAK1/p38MAPK pathway will provide a new direction for the prevention and treatment of ovarian cancer chemotherapy resistance.Methods:The proliferation rate of treating with Xiaoaiping injection and paclitaxel alone or in combination on ovarian cancer cells A2780 and paclitaxel-resistant cells A2780/T was determined by MTT assay.Label-Free quantitative proteomics was used to identify and screen differentially expressed proteins in the two groups of cells,and bioinformatics analysis was used to determine the potential target for paclitaxel resistance in ovarian cancer.qRT-PCR and Western blot were used to validate the expression levels of the target and downstream m RNA and protein.Ovarian cancer A2780 cells and A2780/T cells were injected into nude mice to establish a xenograft tumor model.The effect of the combination of Xiaoaiping injection and paclitaxel on tumor growth was evaluated.HE staining was used to observe the cell pathology in tumor tissue.Four C₂₁steroids in Xiaoaiping injection?Tenacissoside I,Tenacissoside G,Tenacissoside H,17?-Tenacigenin B?were chose to combine with paclitaxel to intervene A2780/T cells.The proliferation rate was determined by MTT assay.The effect of combination of two drugs on the proliferation of ovarian cancer cells was evaluated by using the medium effect principle to calculate the CI of two drugs.Results:1.The combination of Xiaoaiping injection and paclitaxel reversing the resistance of paclitaxel to ovarian cancer is related to TAB1Compared with paclitaxel alone,Xiaoaiping injection combined with paclitaxel significantly inhibited the proliferation of ovarian cancer A2780 cells and paclitaxel-resistant A2780/T cells.The expression of TAB1 in A2780/T cells was lower than that in A2780 cells.After combined treatment with Xiaoaiping injection and paclitaxel in A2780/T cells,the intracellular TAB1 expression increased significantly compare with A2780 cells.According to GO enrichment function analysis and KEGG pathway analysis,TAK1 and p38MAPK were closely related to TAB1.The protein expression site assay showed TAB1 was located in ovary.Thus,TAB1 may be the key molecule for reversing paclitaxel resistance of ovarian cancer with Xiaoaiping injection.2.Xiaoaiping injection regulates the expression of TAB1,TAK1 and downstream p38in paclitaxel-resistant ovarian cancer cellsThe results of qRT-PCR showed that the m RNA expression of TAB1,TAK1 and p38were significantly lower in ovarian cancer A2780/T cells compared with A2780 cells.When A2780/T cells were treated with Xiaoaiping injection with concentrations gradient of 40,80,160 mg/m L alone,or combined with 1?mol/L paclitaxel and 4?mol/L paclitaxel,respectively,with Xiaoaiping injection with concentrations gradient of 40,80 mg/m L,the expression of m RNA levels of TAB1 and TAK1 in cells did not change.After treating A2780/T cells with 1?mol/L paclitaxel or 4?mol/L paclitaxel and 160 mg/m L Xiaoaiping injection,the m RNA expression of TAB1 and TAK1increased significantly,while the expression of p38 decreased with the increased concentration of paclitaxel and Xiaoaiping injection.Western blot results showed that compared with A2780 cell,the expression of TAB1 protein in A2780/T cells was significantly lower,and the expression of TAK1 and p38remained unchanged.After treating A2780/T cells with Xiaoaiping injection with a concentration gradient of 40,80,160 mg/m L alone,or with 1?mol/L paclitaxel combined with Xiaoaiping injection with concentrations gradient of 40,80,160 mg/m L,there was no change in the expression of TAB1,TAK1 and p38 protein levels.After treating A2780/T cells with 4?mol/L paclitaxel combined with Xiaoaiping injection with concentrations gradient of 40,80 mg/m L,there was no change in the expression of TAB1,TAK1 and p38 protein levels.After treating A2780/T cells with 4?mol/L paclitaxel combined with 160 mg/m L Xiaoaiping injection,the expression of TAB1 and TAK1 was increased significantly while the expression of p38 was decreased significantly.3.Xiaoaiping injection enhances the anti-ovarian cancer effect of paclitaxel in nude miceIn vivo experiments of tumor xenograft model in nude mice,it was found that the combination of Xiaoaiping injection and paclitaxel could significantly inhibit the tumor growth and the effect was better than that of paclitaxel alone and Xiaoaiping injection alone.At the same time,the combination of Xiaoaiping high-dose group and paclitaxel showed the greatest effect on the pathological morphology of A2780 cells and A2780/T cells in the tumor tissue.The combination of Xiaoaiping high-dose group and paclitaxel caused a large number of tumor cell necrosis,nuclear shrinkage and changes in cell morphology.4.The combination of C₂₁steroids and paclitaxel in Xiaoaiping injection has the synergistic effect of reversing paclitaxel resistance in ovarian cancer.After treating A2780/T cells with four C₂₁steroids and paclitaxel alone or in combination with different concentration for 24 h and 48 h,cell proliferation decreased with increasing drug concentrations.Compared with the two drugs used alone,Tenacissoside I and 17?-Tenacigenin B combined with PTX respectively did not significantly increase the inhibitory effect on A2780/T,while the inhibition effect of Tenacissoside G and Tenacissoside H on A2780/T was significantly increased when treated with PTX for 48h,and the inhibition rate reached about 50%.When A2780/T cells were treated with Tenacissoside G or Tenacissoside H combined with PTX for 48 h,the Fa ranges from 0.2 to 0.95?Tsd-G concentration is 1.2?222404.1?M,PTX concentration is 1.0?177922.3?M?or 0.1 to 0.95?Tsd-H concentration is 0.1?337006.7?M,PTX concentration is 0.1?177922.3?M?,respectively.The CI is less than 1,which indicate the two drugs have a synergistic effect.Conclusion:Xiaoaiping injection enhanced the anti-ovarian cancer effect of paclitaxel and reversed the resistance of ovarian cancer to paclitaxel.The molecular mechanism may be related to the upregulation of TAB1 and TAK1 expression by Xiaoaiping injection.C₂₁steroids Tenacissoside G and Tenacissoside H may be associated with Xiaoaiping injection to reverse the drug resistance.The research will provide a theoretical basis for the combination of traditional Chinese medicine and chemotherapeutic drugs in the treatment of malignant tumors,and help to enrich the theory of integrated traditional Chinese and western medicine in the treatment of tumors.