Involvement of extracellular signal-regulated kinase/mitogen-activated protein kinase pathway in multidrug resistance induced by HBx in hepatoma cell line.
OBJECTIVE
To investigate the molecular mechanism of the influence of HBx protein on multidrug resistance associated genes: multidrug resistance 1 (MDR-1), multidrug related protein (MRP-1), lung resistance related protein (LRP) in hepatoma cells and the potential role of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway in this process.


METHODS
A cell model stably expressing the HBx protein was established by liposome-mediated transfection of HBx gene into HepG2 cell line. The expression of multidrug resistance associated genes and proteins was detected by RT-PCR and Western blot. AnnexinV-FITC/PI assay was used to confirm the multidrug resistance (MDR) phenotype of transfected cells by fluorescence cytometry (FACS). The ERK/MAPK pathway activation was measured by Western blot through comparing the ratio of phosphorylation of ERK/MAPK to total ERK/MAPK protein. After treated with the ERK/MAPK pathway inhibitor U0126, the HBx-expressing cells were harvested. Then RT-PCR, Western blot and FACS were used to analyze the alterations in the expression of multidrug resistance associated genes and the MDR phenotype after exposure.


RESULTS
Compared with the control group, the transfected cells showed a higher expression of MDR associated genes and proteins. Marked elevations in MDR-1 (64.3%), MRP-1 (87.5%) and LRP (90.8%) were observed in the transfected cells (P<0.05). RT-PCR revealed that the over-expression of MDR associated proteins was due to amplification of such genes (MDR1 2.9 fold, MRP1 1.67 fold, LRP1.95 fold). Furthermore, we found that the ERK/MAPK activity was remarkably high in the HBx-expressing cells. The activation of ERK/MAPK, as measured by the ratio of phosphorylated ERK bands normalized to the total ERK bands, was increased by 2.3-fold in HBx-transfected cells compared with cells transfected with the empty vector. After treated with the ERK/MAPK pathway inhibitor, the level of MDR associated genes and proteins in the transfected cells decreased to some extent. Compared with controls, a significant decrease in MDR-1 mRNA (53.3%), MRP-1 mRNA (59.7%) as well as LRP mRNA (56.4%) was observed in the U0126 treated transfected cells after 12 h. Western blot also demonstrated that the protein expression of these MDR associated genes slightly reduced after treated with U0126 for 12 h (MDR-1 40.1%, MRP-1 29.4%, LRP35.7%). This change was accompanied with the rise of cell apoptosis ratio confirmed by Annexin V-PI detection. The apoptosis index of U0126-treated cells increased by 1.28 fold, compared with that of transfected cells. Obviously, the MDR phenotype of these cells was obviously related with increased activities of the ERK/MAPK pathway.


CONCLUSIONS
HBx protein might be one of the causes for the occurrence of MDR in HCC, and ERK/MAPK pathway might be involved in this change.
Guan
Jian
J
Hepatic Surgery Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China.
Chen
Xiao-Ping
XP
Zhu
Hong
H
Luo
Shun-Feng
SF
Cao
Bin
B
Ding
Lei
L
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology; D045744:Cell Line, Tumor; D018432:Drug Resistance, Multiple / Q000235:genetics*; D019008:Drug Resistance, Neoplasm / Q000502:physiology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology; D020935:MAP Kinase Signaling System / Q000502:physiology*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics; D009363:Neoplasm Proteins / Q000235:genetics; D010641:Phenotype; D014162:Transfection; D020394:Vault Ribonucleoprotein Particles / Q000235:genetics; D000099187:Major Vault Protein
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D048049:Extracellular Signal-Regulated MAP Kinases; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins; D020394:Vault Ribonucleoprotein Particles; D000099187:Major Vault Protein; C421109:multidrug resistance-associated protein 1