title: Ethanol-TGFalpha-MEK signaling promotes growth of human hepatocellular carcinoma.
abstract: BACKGROUND
  Chronic ethanol intake is a significant risk factor for the development of cirrhosis and hepatocellular carcinoma (HCC). The effects of ethanol on extracellular signal-regulated kinase (ERK) activation, transforming growth factor alpha (TGF-alpha), and HCC growth were examined in this study.
  
  
  METHODS
  HepG2, SKHep, Hep3B human HCC cells, or normal human hepatocytes were treated with ethanol (0-100 mM), exogenous TGF-alpha, TGF-alpha neutralization antibody or the MEK inhibitor U0126. TGF-alpha levels were quantified by ELISA. Growth was determined by trypan blue-excluded cell counts. Cell cycle phase distribution was determined by flow cytometry. Protein expression was determined by Western blot.
  
  
  RESULTS
  Ethanol treatment (10-40 mM) increased ERK activation in HepG2 and SKHep HCC cells but not in Hep3B or human hepatocyte cells. Growth increased in HepG2 (174 +/- 29%, P < 0.05) and SKHep (149 +/- 12%, P < 0.05) cells in response to ethanol treatment. Correspondingly, ethanol increased S phase distribution in these cells. U0126 suppressed ethanol-induced growth increases. Ethanol treatment for 24 h also raised TGF-alpha levels in HepG2 cells (118%-198%) and SKHep cells (112%-177%). Exogenous administration of recombinant TGF-alpha mimicked the ethanol-induced growth in HepG2 and SKHep cells; TGF-alpha neutralization antibody effectively abrogated this effect. The TGF-a neutralization antibody also prevented ERK activation by ethanol in HepG2 cells.
  
  
  CONCLUSIONS
  These data demonstrate that clinically relevant doses of ethanol stimulate ERK-dependent proliferation of HCC cells. Ethanol up-regulates TGF-alpha levels in HCC cells and enhances growth through cell cycles changes, which appear to be mediated through TGF-alpha-MEK-ERK signaling. Ethanol-MEK signaling in normal hepatocytes is absent, suggesting that ethanol promotion of HCC growth may in part depend upon the acquisition of cancer-specific signaling by hepatocytes.
authors.affiliation: Department of Surgery, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors.forename: Matthew
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authors.lastname: Hennig
authors.forename: Michele T
authors.initials: MT
authors.lastname: Yip-Schneider
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authors.lastname: Klein
authors.forename: Sabrina
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authors.lastname: Wentz
authors.forename: Jesus M
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authors.lastname: Matos
authors.forename: Courtney
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authors.lastname: Doyle
authors.forename: Jennifer
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authors.forename: Huangbing
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authors.lastname: Wu
authors.forename: Amanda
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authors.lastname: O'Mara
authors.forename: Alex
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authors.lastname: Menze
authors.forename: Stephen
authors.initials: S
authors.lastname: Noble
authors.forename: Iain H
authors.initials: IH
authors.lastname: McKillop
authors.forename: C Max
authors.initials: CM
authors.lastname: Schmidt