Increasing cell membrane potential and GABAergic activity inhibits malignant hepatocyte growth

Abstract

Increasing hepatocyte membrane potentials by augmenting GABAergic activity inhibits nonmalignant hepatocyte proliferative activity. The objectives of this study were to document 1) potential differences (PDs) of four malignant hepatocyte cell lines, 2) GABAA receptor mRNA expression in the same cell lines, and 3) effects of restoring malignant hepatocyte PDs to levels approximating those of resting, nonmalignant hepatocytes. Hepatocyte PDs were documented in nonmalignant and malignant (Chang, HepG2, HuH-7, and PLC/PRF/5) hepatocytes with a fluorescent voltage-sensitive dye and GABAA receptor expression by RT-PCR and Western blot analyses. Compared with nonmalignant human hepatocytes, all four malignant cell lines were significantly depolarized (P < 0.0001, respectively). Only PLC/PRF/5 cells had detectable GABAA-beta3 receptor mRNA expression and all cell lines were negative for GABAA-beta3 receptor protein by Western blot analysis. Stable transfection of Chang cells with GABAA-beta3 receptor cDNA resulted in significant increases in PD and decreases in proliferative activity as manifest by decreased [3H]thymidine and bromodeoxyurieine incorporation rates, 4-[3-(4-lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate activity, a lower mitotic index, prolongation of cell-doubling times, and attenuated growth patterns compared with cells transfected with vector alone. Colony formation in soft agar and the number of abnormal mitoses were also significantly decreased in GABAA-beta3 receptor transfected cells. The results of this study indicate 1) relative to healthy hepatocytes, malignant hepatocytes are significantly depolarized, 2) GABAA-beta3 receptor expression is absent in malignant hepatocyte cell lines, and 3) increasing the PD of malignant hepatocytes is associated with less proliferative activity and a loss of malignant features.

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

“…Thus, our finding of a 24% decrease in HCC tissue PD is not only consistent with these reports but also reports describing depolarization in various rat and human malignant cell lines. 2,22 Although the mechanism whereby such depolarization might result in enhanced cell proliferation and tumor growth remains to be determined, electrogenic translocation of polyvalent cationic growth promoters such as polyamines from the relatively positive-charged environment of the cytoplasm to the negative-charged DNA of the nucleus has been implicated. 23 Perhaps the most striking finding of the present study was the consistent and significant decrease in GABA A -␤3 receptor expression in HCC compared with adjacent nontumor tissues.…”

“…This finding supports previous descriptions of down-regulated or absent expression of this receptor isoform in human malignant hepatocyte cell lines and regenerating livers following partial hepatectomy. 2,6 Although the correlation of decreased receptor expression with increased proliferative activity may represent an association rather than effect, previous in vitro studies have shown that restoration and/or activation of GABA A -␤3 receptors results in attenuation of proliferative activity in malignant hepatocytes. 2,24,25 Moreover, reports of chromosomal deletions in the region encoding the GABA A -␤3 receptor gene being associated with hepatic tumor development in humans also argues in favor of a causative role.…”

“…2,6 Although the correlation of decreased receptor expression with increased proliferative activity may represent an association rather than effect, previous in vitro studies have shown that restoration and/or activation of GABA A -␤3 receptors results in attenuation of proliferative activity in malignant hepatocytes. 2,24,25 Moreover, reports of chromosomal deletions in the region encoding the GABA A -␤3 receptor gene being associated with hepatic tumor development in humans also argues in favor of a causative role. 26 Finally, the results of the nude mice experiments described in this study in which restoration of GABA A -␤3 receptor expression in GABA A -␤3-deficient Huh-7 cells resulted in attenuated tumor growth compared with mice injected with Huh-7 cells transfected with vector alone also supports a causative role.…”

“…Plasmid construction and stable transfections were performed as described previously. 2 Briefly, the 1,640-bp cDNA containing the GABA A -␤3 receptor coding region was cut with XbaI from the plasmid pRK5-␤3 and cloned into the pcDNA3.1/ V5-His C vector producing a plasmid known as pcDNA-␤3. The cloned cDNA fragment is under the transcriptional control of the immediate early gene of the human cytomegalovirus promoter, and the vector contains polyadenylation signals and ampicillin-resistant and zencin-resistant genes.…”

“…In recent in vitro studies, we demonstrated that restoration of depolarized malignant hepatocyte cell membrane potential differences (PDs) toward those documented in nonmalignant hepatocytes results in a loss of malignant features, including decreased proliferative activity, absence of colony formation in soft agar, and normalization of phenotypic appearance. 2 Whether HCC tissues are depolarized relative to adjacent nontumor tissues and the mechanisms whereby such depolarization might exist have yet to be reported.…”