Effects Of Bifenthrin On Pregnane X Receptor Pathway In HCC Cells And Its Mechanism In Sorafenib Metabolism And Clearance

Objective:To determine the effects of bifenthrin on the activity of pregnane X receptor transcription factor and its downstream genes（CYP3A4 and ABCB-1）and corresponding proteins.The effect of bifenthrin on the killing of hepatocellular carcinoma cell lines was elucidated by molecular targeted drugs and to explore the molecular mechanism of metabolism and clearance of sorafenib induced by bifenthrin in hepatocellular carcinoma cell lines.Methods:1.In vivo experiment:hepatocellular carcinoma cell lines（HepG2 and MHCC97-H cells）were used（1）Luciferase reporter gene experiments was used to demonstrate whether bifenthrin can induce PXR transcription factor activity（DR6-Luc,ER3-Luc,XREM-Luc and PXRE-Luc）.Then the median effective concentration(EC₅₀)of bifenthrin-induced activity was calculated when compared it with the effects of the PXR agonist rifampicin and the PXR antagonist ketoconazole.（2）Quantitative polymerase chain reaction was used to demonstrate whether bifenthrin can induce the expression of PXR downstream genes CYP3A4 and ABCB-1in a dose-dependent manner in hepatocellular carcinoma cell lines.Then the EC₅₀ of bifenthrin-induced activity was calculated when compared it with the effect of PXR agonist rifampicin and PXR antagonist ketoconazole.（3）Western blotting was used to detect whether bifenthrin could affect the expression of proteins（CYP3A4 and P-GP）encoded by PXR in hepatocellular carcinoma cell lines.The specificity of the effects of bifenthrin was confirmed when compared with the solvent control group（0.1%DMSO）.（4）Chromatin immunoprecipitation was used to detect whether bifenthrin could induce the recruitment of PXR in its downstream gene promoter region（XREM,-7836/-7208）and enhancer region（PXRE,-362/+52）in hepatocellular carcinoma cell lines.（5）Liquid chromatography-mass spectrometry was used to detect whether bifenthrin could accelerate the metabolism and clearance of sorafenib in hepatocellular carcinoma cell lines and subcutaneous tumors of nude mice.In addition,the half-life of sorafenib under the influence of bifenthrin was calculated.The effect was compared with control group.（6）MTT assay was used to detect whether the presence of bifenthrin could affect the killing of hepatocellular carcinoma cell lines by molecular targeted drug with different concentration gradients,then the half inhibitory concentration(IC₅₀)of molecular targeted drug killing hepatocellular carcinoma cell lines was calculated.The effect was compared with control group.2.In vivo experiment:Subcutaneous and liver in situ tumor models of Bal B/C^NULLnude mice were constructedThe nude mouse subcutaneous tumor model and liver tumor in situ model were used to confirm whether the presence of bifenthrin could affect the effect of sorafenib in killing subcutaneous and liver tumor in nude mice.Results:1.In vivo experiment:hepatocellular carcinoma cell lines（HepG2 and MHCC97-H cells）were used（1）Bifenthrin could induce the activity of PXR transcription factor in hepatocellular carcinoma cell lines.Luciferase reporter genes directly reflect that bifenthrin could induce the activity of PXR on its downstream gene binding elements or the luciferase reporter genes DR6-Luc,ER3-Luc,XREM-Luc and PXRE-Luc in the promoter region and enhancer region of CYP3A4,the most typical downstream genes.EC50 of HepG2 cells were 6.08μmol/L,5.61μmol/L,5.84μmol/L and 7.90μmol/L,respectively.The EC₅₀in MHCC97-H cells were 6.27μmol/L、6.16μmol/L、7.14μmol/L and 6.09μmol/L,respectively.Rifampicin,a PXR agonist as a positive control,was more active than bifenthrin in inducing luciferase reporter gene（P<0.05）.When the PXR antagonist ketoconazole was added,bifenthrin failed to induce luciferase reporter activity in a dose-dependent manner.（2）Bifenthrin could induce the expression of PXR downstream genes（CYP3A4and ABCB-1）in hepatocellular carcinoma cell lines.In HepG2 cells,bifenthrin could induce the expression of CYP3A4 and ABCB-1 in a dose-dependent manner.EC₅₀ was6.13μmol/L and 5.40μmol/L,and in MHCC97-H cells,its EC₅₀was 7.22μmol/L and7.04μmol/L.Rifampicin,a PXR agonist as a positive control,was more active in inducing PXR downstream genes than bifenthrin（P<0.05）.When the PXR antagonist ketoconazole was added,bifenthrin failed to induce the expression of PXR downstream genes in a dose-dependent manner.（3）Bifenthrin could induce the expression of proteins corresponding to downstream genes of PXR（CYP3A4 and P-GP）in hepatocellular carcinoma cell lines.In MHCC97-H cells,the expression of CYP3A4 protein in 3μmol/L and 10μmol/L bifenthrin groups was significantly up-regulated compared with the control group.The difference was statistically significant（P<0.05）,and there was no significant difference in protein expression between other bifenthrin treatment groups and control group（P>0.05）.（4）Chromatin immunoprecipitation confirmed that bifenthrin could induce the recruitment of PXR in its downstream gene promoter region（XREM,-7836/-7208）and enhancer region（PXRE,-362/+52）in HepG2 cells.This indicated that bifenthrin could up-regulate the transcription factor activity of PXR by inducing the recruitment of PXR in its downstream gene promoter and enhancer regions.（5）Bifenthrin could accelerate the metabolism and clearance rate of molecular targeted drug sorafenib in vivo and in vitro.HepG2 or MHCC97-H cells treated with10μmol/L bifenthrin could significantly accelerate the metabolism and clearance of sorafenib in this study.The half-life of metabolism and clearance in the two hepatocellular carcinoma cell lines were 10.12h and 9.70h,respectively,while the half-life of the control group was 17.37h and 18.63h,and the difference was statistically significant（P<0.05）.At the same time,the metabolism and clearance of sorafenib in the subcutaneous tumor tissue of nude mice also showed similar results.The half-life of sorafenib in the subcutaneous tumor tissue of HepG2 or MHCC97-H cells treated with bifenthrin were 23.00h and 22.70h,respectively,while the half-life of sorafenib in the control group was 34.35h and 30.99h,and the difference was statistically significant（P<0.05）.（6）Bifenthrin can affect the killing effect of molecular targeted drugs in hepatocellular carcinoma cell lines.After treatment with 10μmol/L bifenthrin,the IC₅₀of the four molecular targeted drugs in HepG2 cells were 5.94μmol/L,6.77μmol/L,5.95μmol/L and 5.63μmol/L,and the IC₅₀ in MHCC97-H cells were 5.55μmol/L,5.73μmol/L,5.56μmol/L and 4.50μmol/L,respectively.The IC₅₀ of each group was significantly higher than that of the control group,and the difference was statistically significant（P<0.05）.2.In vivo experiment:Subcutaneous and liver in situ tumor models of Bal B/C^NULLnude mice were constructedIn the subcutaneous and liver in situ tumor experimental groups of nude mice,the effect of oral sorafenib antitumor therapy in nude mice intragastric with 5mg/kg bifenthrin was significantly worse than that in nude mice not intragastric with bifenthrin.The tumor weight and volume of subcutaneous tumors and the relative tumor size of liver in situ tumors were significantly different（P<0.05）.Conclusion:1.Bifenthrin could induce the activity of PXR transcription factor in hepatocellular carcinoma cell lines and there is an obvious dose-response relationship.2.Bifenthrin could induce the expression of PXR downstream genes ABCB-1 and CYP3A4 and the expression of corresponding proteins（P-GP and CYP3A4）in a dose-dependent manner.3.Bifenthrin could induce the recruitment of PXR in its downstream gene promoter and enhancer regions.4.Bifenthrin could accelerate the metabolism and clearance rate of molecular targeted drug sorafenib in hepatocellular carcinoma cell lines and affect the antitumor effect of molecular targeting drug in vitro and vivo.