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Chronic Ethanol Exposure Promotes MEK-ERK Dependent Growth in Human Hepatocellular Carcinoma (HCC)
Matthew Hennig, Patrick J. Klein, Alex Menze, Jesus Matos, Indiana University, Indianapolis, IN; Jean Campbell, Nelson Fausto, University of Washington, Seattle, WA; Iain McKillop, University of North Carolina - Charlotte, Charlotte, NC; Jim Sitzmann, C M. Schmidt, Indiana University, Indianapolis, IN

Background:
Ethanol use results in cirrhosis, a major risk factor for HCC. Whether ethanol directly promotes HCC, however, is unclear. Previous work has implicated MEK-ERK signaling in hepatocarcinogenesis. The aims of this study were to determine the effects of physiologic chronic ethanol exposure on MEK-ERK activity/expression and growth in HCC lines compared to control hepatocytes.

Methods:
Three HCC cell lines of varying differentiation: SKHep (poor), Hep3B (moderate, HepB+), HepG2 (well), and two murine hepatocyte lines, NMH and TAMH, were used. The NMH line is a non-tumorigenic, immortalized hepatocyte line (CD1 background). TAMH hepatocytes from a MT42-TGF (CD1) transgenic mouse are tumorigenic in athymic nude mice. All five cell lines underwent chronic ethanol (10-100mM; 24 hours) treatment in the presence/absence of MEK inhibitor, U0126 (5M). Total ERK, phosphorylated ERK (MEK activity), and actin (control) were measured by Western blot. Ethanol concentrations were confirmed by ELISA. Growth was assessed by trypan blue excluded cell counts.

Results:
Ethanol treatment at physiologic doses (10-40 mM = 0.04-0.18 BAL) increased phospho-ERK level (MEK activity) in all three human HCC lines. Correspondingly, ethanol induced a dose-dependent increase in cell growth (10-50%) as determined by cell counts. Higher concentrations of ethanol (60-100mM) showed no change in phospho-ERK. In contrast, hepatocyte lines demonstrated no increase in phospho-ERK at either physiologic or higher dose ranges of ethanol. Total ERK expression was unaffected at any of these concentrations of ethanol in all cell lines tested. U0126 effectively suppressed the ethanol-induced increase in phospho-ERK in all three HCC lines. Notably, despite an increase in phospho-ERK with EtOH alone, the combination of EtOH and U0126 decreased the phospho-ERK level to or below the level of U0126 alone.

Conclusions:
Physiologic doses of ethanol appear to promote growth of well to poorly differentiated HCC possibly through MEK-ERK dependent signaling. U0126 effectively suppresses EtOH-induced phospho-ERK signaling and this effect is enhanced in the presence of EtOH. In contrast, phospho-ERK in nonmalignant hepatocytes is unaffected with ethanol treatment, and the presence of the TGF transgene alone is insufficient to provoke the ethanol-MEK signaling seen in well differentiated HCC. Ethanol-MEK signaling may be a mechanism whereby ethanol directly promotes hepatocarcinogenesis.


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