Study On Mechanism Of Action Of Gambogenic Acid In Enhanceing Sensitivity To Lenvatinib In Hepatocellular Carcinoma Via Regulating PXR

Hepatocellular carcinoma is the most common type of primary liver cancer and is the fourth leading cause of cancer-related death worldwide.The main treatments for hepatocellular carcinoma currently include resection of tumour lesions,chemotherapy,radiotherapy and liver transplantation.Lenvatinib is a novel first-line treatment for hepatocellular carcinoma,but current studies have shown that the overall effectiveness of Lenvatinib in HCC is only 24%,and cases of Lenvatinib resistance have been reported.The search for drug candidates that might sensitize Lenvatinib in the treatment of hepatocellular carcinoma is of clinical importance to improve the sensitivity of the targeted drug.Gambogenic acid（GNA）is an active ingredient isolated from the Chinese herbal medicine Gamboge,which can inhibit the proliferation of tumour cells.In this study,GNA was combined with lenvatinib to investigate whether it could enhance the inhibitory effect of lenvatinib on hepatocellular carcinoma and promote apoptosis in hepatocellular carcinoma,and to conduct a preliminary mechanistic study.Objective:To investigate whether Gambogenic acid can enhance the inhibition of hepatocellular carcinoma by lenvatinib,promote apoptosis in hepatocellular carcinoma and further explore the mechanism involved.Methods:Hepa RG cell viability was measured by CCK8,apoptosis was detected by DAPI staining and Annexin V-FITC/PI double-staining assay.The m RNA and protein levels of nuclear receptor PXR,downstream metabolite CYP3A4,transporter P-gp,BCRP and related apoptotic factors were measured by RT-q PCR and Western Blot assay respectively in Hepa RG cells to investigate the mechanisms involved.The effect of GNA and lenvatinib on tumor growth was assessed in a transplantation model using H₂₂hepatoma cells,and the effect of the combination of GNA and lenvatinib on major organs was investigated by HE staining.The protein expression of nuclear receptor PXR,metabolic enzyme CYP3A4 and transporters P-gp and BCRP in tumor tissues was detected by immunohistochemistry.Results:（1）The CCK8 results showed a dose-dependent decrease in cell survival with increasing drug concentrations.Cell survival was greater than 90%at non-toxic concentrations in the range of 0-2μmol/L,and subsequent experiments were conducted using 2μmol/L non-toxic concentrations.After DAPI staining,the cells showed apoptotic features,and the number of apoptotic cells was significantly higher after the combination of GNA（2μmol/L）and lenvatinib（10μmol/L）than in the single drug group.Fluorescence microscopy showed that GNA and lenvatinib alone had little effect on intracellular ROS,while the combination significantly increased the accumulation of ROS.In clonogenesis assays,2μmol/L GNA alone significantly inhibited clonogenic capacity,which was almost lost after the combination.（2）RT-q PCR results showed that GNA may inhibit the expression levels of PXR,P-gp,BCRP and CYP3A4 m RNA.Western Blot results showed that the expression of PXR and CYP3A4 proteins in Hepa RG cells was significantly reduced after 24 h of action at a GNA concentration of 2μmol/L compared to the control group.The expression of PXR,P-gp,BCRP and CYP3A4was further down-regulated in the combination group compared to the lenvatinib group alone;the expression levels of Bax and Cleaved-caspase-3 were significantly up-regulated and the expression of Bcl-2 protein was significantly inhibited in the combination group.To further confirm the effect of GNA on PXR,Hepa RG cells were treated with PXR agonist rifampicin（RFP）and inhibitor ketoconazole（KTZ）.30μmol/L of RFP significantly up-regulated the expression of PXR,P-gp,BCRP,CYP3A4 and Bcl-2 proteins in Hepa RG cells compared to the control group,and the combination with GNA（2μmol/L）significantly down-regulated PXR,P-gp,CYP3A4 and Bcl-2 proteins induced by RFP.Compared with the control group,KTZ significantly inhibited the expression of PXR,P-gp,CYP3A4 and Bcl-2,and the protein expression of PXR,P-gp and Bcl-2 inhibited by KTZ was further down-regulated after combination with GNA.（3）In vivo experiments in a mouse xenograft tumor model showed that the combination of GNA and lenvatinib significantly inhibited tumor growth compared to the model group and was superior to the GNA and lenvatinib groups;HE staining results showed that no significant pathological changes were observed in the major organs（heart,liver,spleen,lung and kidney）of the mice after administration of the drug.Western Blot results showed that the expression of PXR,P-gp,BCRP,CYP3A4 and Bcl-2 in the tumor tissues was significantly reduced after the combination of GNA and lenvatinib,and the expression of P53,Bax,Cleaved-caspase-2 and Bcl-2 was significantly reduced after the combination of GNA and lenvatinib.Cleaved-caspase-3-related apoptotic proteins were increased.Conclusions:GNA enhanced the sensitivity of hepatocellular carcinoma to lenvatinib,and the molecular mechanism may be related to the inhibition of nuclear receptor PXR expression by GNA,thus affecting the activity of metabolic enzyme CYP3A4,transporter P-gp,BCRP and related apoptotic proteins Bcl-2,Bax and Cleaved-caspase-3,and enhancing the proliferation inhibition and apoptosis induction of hepatocellular carcinoma by the targeted drug lenvatinib.apoptosis induction.It provides a basis for the combination of the two drugs in clinical practice and enriches the basic research on the combination of traditional Chinese medicine and targeted drugs in the treatment of hepatocellular carcinoma.