Decreased hepatocyte membrane potential differences and... : Hepatology (original) (raw)

Original Articles

Decreased hepatocyte membrane potential differences and GABAa-β3 expression in human hepatocellular carcinoma

Minuk, Gerald Y.1*; Zhang, Manna1; Gong, Yuewen1; Minuk, Leonard1; Dienes, Hans2; Pettigrew, Norman1; Kew, Michael3; Lipschitz, Jeremy4; Sun, Dongfeng1

1_Section of Hepatology, Department of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada_

2_Department of Pathology, University of Cologne, Cologne, Germany_

3_Department of Pathology, University of the Witwatersrand, Johannesburg, South Africa_

4_Department of Surgery, University of Manitoba, Winnipeg, Manitoba, Canada_

* Address reprint requests to: Head, Section of Hepatology, University of Manitoba, John Buhler Research Centre, 803-715 McDermot Avenue, Winnipeg, Manitoba R3E 3P4, Canada

Email:[email protected]

Received 22 December 2005; Accepted 15 November 2006

Published online in Wiley InterScience (www.interscience.wiley.com).

Supported by Canadian Institutes of Health Research.

Potential conflict of interest: Nothing to report.

fax: 204-789-3971

Abstract

To determine whether hepatocyte membrane potential differences (PDs) are depolarized in human HCC and whether depolarization is associated with changes in GABAA receptor expression, hepatocyte PDs and γ-aminobutyric acid (GABA)A receptor messenger RNA (mRNA) and protein expression were documented in HCC tissues via microelectrode impalement, real-time reverse-transcriptase polymerase chain reaction, and Western blot analysis, respectively. HCC tissues were significantly depolarized (−19.8 ± 1.3 versus −25.9 ± 3.2 mV, respectively [ P < 0.05]), and GABAA-β3 expression was down-regulated (GABAA-β3 mRNA and protein expression in HCC; 5,693 ± 1,385 and 0.29 ± 0.11 versus 11,046 ± 4,979 copies/100 mg RNA and 0.62 ± 0.16 optical density in adjacent tumor tissues, respectively [ P = 0.002 and P < 0.0001, respectively]) when compared with adjacent nontumor tissues. To determine the physiological relevance of the down-regulation, human malignant hepatocytes deficient in GABAA-β3 receptor expression (Huh-7 cells) were transfected with GABAA-β3 complementary DNA (cDNA) or vector alone and injected into nu/nu nude mice (n = 16-17 group). Tumors developed after a mean (± SD) of 51 ± 6 days (range: 41-60 days) in 7/16 (44%) mice injected with vector-transfected cells and 70 ± 12 days (range: 59-86 days) in 4/17 (24%) mice injected with GABAA-β3 cDNA-transfected cells ( P < 0.005). Conclusion: The results of this study indicate that (1) human HCC tissues are depolarized compared with adjacent nontumor tissues, (2) hepatic GABAA-β3 receptor expression is down-regulated in human HCC, and (3) restoration of GABAA-β3 receptor expression results in attenuated in vivo tumor growth in nude mice. (Hepatology 2007;45:735–745.)

Abbreviations: ATPase, adenosine triphosphatase; GABA, γ-aminobutyric acid; PD, potential differences; RT-PCR, reverse-transcription PCR.