Study On The Anti-ovarian Cancer Effect Of Xiaoaiping Injection Combined With Paclitaxel Via Regulating PXR

Paclitaxel is first-line chemotherapeutic for ovarian cancer,though there are drug resistance,recurrence and tumor metastasis in the clinical applying process.Xiaoaiping injection has been exclusively used on curing malignant tumor and as supplementary drug for chemo-drug,such as paclitaxel.However,it’s unclear whether Xiaoaiping injection and paclitaxel have synergistic interaction and its molecular mechanism.Objective:To explore the regularoty effect of Xiaoaiping injection on nuclear receptor PXR and its downstream drug metabolic enzymes CYP2C8,CYP3A4/5 and transporter P-gp,clarifying the synergistic effect and molecular mechanism of Xiaoaiping injection with paclitaxel on anti-ovarian cancer by regulating nuclear receptor PXR,so as to provide new idea for the prevention and treatment of ovarian cancer.Methods:The proliferation rate of treating with Xiaoaiping injection and paclitaxel alone or in combination on ovarian cancer cells A2780 and SKOV3 was determined by thiazole blue assay and 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.Fluorescence inverted microscope was used to observe the cell morphology and flow cytometry was used to detect the apoptosis and cell cycle distribution.qRT-PCR and Western blot were used to detect the expression levels of nuclear receptor PXR,CAR and downstream metabolic enzymes CYP2C8,CYP3A4,CYP3A5,transporter P-gp and the apoptosis-related proteins Bcl-2 family.Flow cytometry was used to detect the accumulation of rhodamine 123 in the treated cells and P-gp activity could be inferred.Transfected PXR plasmids,the expression of PXR in ovarian cancer cells A2780 and SKOV3 were overexpressed,which verified the regulatory effect of Xiaoaiping injection on PXR and its downstream related molecules.Ovarian cancer SKOV3 cells were injected into nude mice to establish a xenograft tumor model.And the effect of the combination of two drugs on tumor growth was evaluated.TUNEL staining was used to determine the proportion of apoptotic cells in tumor tissues.qRT-PCR and Western blot were used to detect the mRNA and protein expression levels of nuclear receptor PXR and downstream metabolic enzymes CYP2C8,CYP3A4,CYP3A5,transporter P-gp and apoptosis-related proteins of tumor tissues.Results:1.Xiaoaiping injection combined with paclitaxel has synergistic effect on ovarian cancer cells.Compared with paclitaxel alone,Xiaoaiping injection combined with paclitaxel significantly enhanced the proliferation inhibition on A2780 and SKOV3 cells.When Xiaoaiping concentration is lower than 160 mg/mL and paclitaxel concentration is lower than 160 nmol/L within treating 24h and Xiaoaiping concentration is lower than40 mg/mL and paclitaxel concentration is lower than 10 nmol/L within treating 48h,the combination index is less than 1,indicating that the two drugs show synergistic effect.After treated with paclitaxel in combination with Xiaoaiping injection,cells were significantly less than paclitaxel alone group,arranged sparsely in irregular shape,accompanied by more floating dead cells and cell debris.Nuclear dye results showed plenty of dense nucleus of debris and part of the dyed the nucleus shape without rules,indicating that a large number of cell nucleus pycnosis and cells had entered the stage of apoptosis.After the combination of Xiaoaiping injection and paclitaxel,the apoptosis rate of A2780 cells significantly increased from 8.66%of paclitaxel alone to 15.49%and 88.0%.And the apoptosis rate of SKOV3 cells increased from 7.85%of paclitaxel alone to 10.52%and 19.36%.The cell distribution of SKOV3 cells have been significantly inhibited in the G₂+M phase and increased in the G₀+G₁ and S phase.2.Xiaoaiping injection combined with paclitaxel enhances the anti-ovarian cancer effect via PXR.qRT-PCR results showed that,compared to control group,paclitaxel significantly up-regulated the mRNA expression of PXR,CAR,CYP2C8 and CYP3A4 in A2780and SKOV3 cells and the mRNA expression levels of these molecules were inhibited directly when treated with Xiaoaiping injection combination.Western blot results showed that paclitaxel can activate the expression of PXR,CAR,CYP2C8,P-gp and anti-apoptotic proteins Bcl-2 and Bcl-xl in A2780 and SKOV3 cells and the expression of these molecules was significantly inhibited after the combination of Xiaoaiping injection.In ovarian cancer cells A2780 and SKOV3 transfected with PXR plasmid,the expressions of PXR,CYP2C8,P-gp,Bcl-2 and Bcl-xl were increased correspondingly.The addition of Xiaoaiping injection could inhibit the protein and mRNA levels and the effect of Xiaoaiping injection at high dose was stronger.Flow cytometry results showed that paclitaxel reduced the accumulation of rhodamine 123 in cells,while Xiaoaiping injection could reverse this effect with the fluorescence intensity increasing after combined use,verifying P-gp activity was inhibited.3.Xiaoaiping injection combined with paclitaxel enhances anti-tumor effect on ovarian cancer in vivo.In 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.qRT-PCR and Western blot results showed that paclitaxel can increase the expression of PXR,CAR,CYP2C8,CYP3A4,P-gp and apoptosis related proteins the Bcl-2,Bcl-xl,Bad,Bax in nude mice in vivo,after combined with Xiaoaiping injection,the activated PXR,CAR,CYP2C8,CYP3A4,P-gp and the suppressing apoptosis protein Bcl-2,Bcl-xl were restrained with the mRNA and protein levels drop while the expression of activating apoptosis protein Bax and Bad increased.Conclusion:Xiaoaiping injection could increase the anti-tumor effect of paclitaxel,and its molecular mechanism may be related to Xiaoaiping injection inhibit the expression of nuclear receptor PXR,thus affecting the expression and activity of metabolic enzyme CYP2C8,transporter P-gp and the suppression of apoptosis protein Bcl-2,Bcl-xl.This study will provide a theoretical basis and help to enrich the theory of the combined treatment of traditional Chinese medicine and chemotherapy drugs for malignant tumors.