Role of hypoxia-inducible factor-α in hepatitis-B-virus X protein-mediated MDR1 activation

doi:10.1016/j.bbrc.2007.04.012

Biochemical and Biophysical Research Communications

Volume 357, Issue 2, 1 June 2007, Pages 567-573

https://doi.org/10.1016/j.bbrc.2007.04.012 Get rights and content

Abstract

The transition from chemotherapy-responsive cancer cells to chemotherapy-resistant cancer cells is mainly accompanied by the increased expression of multi-drug resistance 1 (MDR1). We found that hepatitis-B-virus X protein (HBx) increases the transcriptional activity and protein level of MDR1 in a hepatoma cell line, H4IIE. In addition, HBx overexpression made H4IIE cells more resistant to verapamil-uptake. HBx stabilized hypoxia-inducible factor-1α (HIF-1α) and induced the nuclear translocation of C/EBPβ. Reporter gene analyses showed that HBx increased the reporter activity in the cells transfected with the reporter containing MDR1 gene promoter. Moreover, the luciferase reporter gene activity was significantly inhibited by HIF-1α siRNA but not by overexpression of C/EBP dominant negative mutant. These results imply that HBx increases the MDR1 transporter activity through the transcriptional activation of the MDR1 gene with HIF-1α activation, and suggest HIF-1α for the therapeutic target of HBV-mediated chemoresistance.

Section snippets

Materials and methods

Materials. The anti-MDR1 antibody was supplied from Calbiochem (Darmstadt, Germany). The horseradish peroxidase-conjugated donkey anti-rabbit IgG, anti-goat IgG, and alkaline phosphatase-conjugated donkey anti-mouse IgG were purchased from Jackson Immunoresearch Laboratories (WestGrove, PA). Most of the reagents used for molecular studies were purchased from Sigma (St. Louis, MO). siRNA targeting rat HIF-1α was purchased from Ambion (Austin, TX).

Cell culture. The H4IIE rat hepatoma cells were

HBx overexpression increases the activity and expression level of MDR1

After transfecting the H4IIE cells with the pCEP4-HBx (1.0 μg) or pCMV5 plasmid (1.0 μg), RT-PCR showed that HBx was expressed in the HBx-transfected H4IIE cells (Fig. 1A). There was no detectable band in the Mock-transfection group.

The expression level of MDR1 in the HBx-overexpressed cells was determined by Western blot analyses. HBx overexpression (0.05–5 μg) caused the induction of MDR1 in a concentration-dependent manner (Fig. 1B). However, the cells transfected with 5 μg of the pCEP4-HBx

Discussion

The development of HCC is a frequent event in patients with chronic liver diseases such as cirrhosis and hepatitis. Multi-drug resistance related to MDR1 expression restricts the effectiveness of the various chemotherapeutical treatments. Indeed, the increased expression of MDR1 is frequently observed in hepatic tissues from chronic liver disease patients including HCC [25]. Here, it is suggested that HBx is an important factor for inducing MDR1 during HCC development. MDR1 is involved in the

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We found that NOX1 induced HIF1α specifically increased MDR1, a member of ABC transporter family proteins. Consistent with our studies, HIF1α has been reported to be a direct regulator of MDR1 through binding its promoter and then mediates chemoresistance in liver cancer [26]. Moreover, either ROS scavenger treatment or HIF1α inhibition could faithfully reduce MDR1 expression.
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¹: These authors contributed equally to this work.

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Role of hypoxia-inducible factor-α in hepatitis-B-virus X protein-mediated MDR1 activation

Abstract

Section snippets

Materials and methods

HBx overexpression increases the activity and expression level of MDR1

Discussion

J. Lab. Clin. Med.

J. Biol. Chem.

J. Hepatol.

J. Pharm. Sci.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Exp. Mol. Med.

Multidrug resistance in cancer: role of ATP-dependent transporters

Nat. Rev.

Analysis of expression of cMOAT (MRP2), MRP3, MRP4, and MRP5, homologues of the multidrug resistance-associated protein gene (MRP1), in human cancer cell lines

Cancer Res.

In vitro and in vivo reversal of P-glycoprotein-mediated multidrug resistance by a novel potent modulator, XR9576

Cancer Res.

HBx gene of hepatitis B virus induces liver cancer in transgenic mice

Nature

Induction of cell cycle progression by hepatitis B virus HBx gene expression in quiescent mouse fibroblasts

J. Clin. Invest.

Overexpression of the MDR1 gene and P-glycoprotein in human hepatocellular carcinoma

J. Natl. Cancer Inst.