Involvement of extracellular signal-regulated kinase/mitogenactivated protein kinase pathway in multidrug resistance induced by HBx in hepatoma cell line

AIM:To investigate the molecular mechanism of the influenceof HBx protein on multidrug resistance associated genes:multidrug resistance 1(MDR-1),multidrug related protein(MRP-1),lung resistance related protein(LRP)in hepatomacells and the potential role of extracellular signal-regulatedkinase/mitogen-activated protein kinase(ERK/MAPK)pathwayin this process.METHODS:A cell model stably expressing the HBx proteinwas established by liposome-mediated transfection of HBxgene into HepG2 cell line.The expression of multidrugresistance associated genes and proteins was detected byRT-PCR and Westem blot.AnnexinV-FITC/PI assay was usedto confirm the multidrug resistance(MDR)phenotype oftransfected cells by fluorescence cytometry(FACS).TheERK/MAPK pathway activation was measured by Westernblot through comparing the ratio of phosphorylation ofERK/MAPK to total ERK/MAPK protein.After treated withthe ERK/MAPK pathway inhibitor U0126,the HBx-expressingcells were harvested.Then RT-PCR,Western blot and FACSwere used to analyze the alterations in the expression ofmultidrug resistance associated genes and the MDRphenotype after exposure.RESULTS:Compared with the control group,the transfectedcells showed a higher expression of MDR associated genesand proteins.Marked elevations in MDR-1(64.3%),MRP-1(87.5%)and LRP(90.8%)were observed in the transfectedcells(P<0.05).RT-PCR revealed that the over-expressionof MDR associated proteins was due to amplification ofsuch genes(MDR12.9 fold,MRP1 1.67 fold,LRP1.95 fold).Furthermore,we found that the ERK/MAPK activity wasremarkably high in the HBx-expressing cells.The activationof ERK/MAPK,as measured by the ratio of phosphorylatedERK bands normalized to the total ERK bands,was increasedby 2.3-fold in HBx-transfected cells compared with cellstransfected with the empty vector.After treated with theERK/MAPK pathway inhibitor,the level of MDR associatedgenes and proteins in the transfected cells decreased tosome extent.Compared with controls,a significant decreasein MDR-1 mRNA(53.3%),MRP-1 mRNA(59.7%)as well as LRP rnRNA(56.4%)was observed in the U0126 treatedtransfected cells after 12 h.Western blot also demonstratedthat the protein expression of these MDR associated genesslightly reduced after treated with U0126 for 12 h(MDR-140.1%,MRP-1 29.4%,LRP35.7%).This change wasaccompanied with the rise of cell apoptosis ratio confirmedby Annexin V-PI detection.The apoptosis index of U0126-treated cells increased by 1.28 fold,compared with that oftransfected cells.Obviously,the MDR phenotype of thesecells was obviously related with increased activities of theERK/MAPK pathway.CONCLUSION:HBx protein might be one of the causesfor the occurrence of MDR in HCC,and ERK/MAPK pathwaymight be involved in this change.