title: Short exposure to millimolar concentrations of ethanol induces apoptotic cell death in multicellular HepG2 spheroids.
abstract: OBJECTIVE
  We have shown previously that 1 mM ethanol reduces cell proliferation and increases apoptosis in monolayers of human hepatocellular carcinoma (HepG2) cells. However, in vivo liver tumors are usually three-dimensional and multicellular. The purpose of this study was therefore to determine the effect of ethanol in multicellular tumor spheroids (MCTS) as a model system in vitro.
  
  
  METHODS
  After the application of 1 mM ethanol for 24 h and 48 h, viable, apoptotic and necrotic cells within MCTS were stained with specific fluorescent dyes, and their amount and distribution within the MCTS were assessed by confocal laser scanning microscopy. To evaluate the effect on HepG2 cell migration and cell proliferation, the outgrowth potential after 1 week in culture was evaluated.
  
  
  RESULTS
  As assessed by YO-PRO-1 staining, ethanol increased the number of apoptotic cells from 21.5 units (U) in control spheroids to 364 U and 482.2 U after 24 h and 48 h in ethanol-treated spheroids, respectively (P < 0.001). Merocyanine staining fluorescence increased from 10.7 U in the control to 122 U after 24 h and 293.2 U after 48 h (P < 0.001). Cell viability, as determined by staining with the acetoxymethyl ester of calcein, decreased from 578.5 U in the control to 236 U and 73.4 U after 24 h and 48 h of ethanol exposure respectively (P < 0.001). Necrosis showed an increase from 2 U in control to 24.9 after 24 h and 54 U after 48 h. MCTS treated with ethanol showed almost complete inhibition of outgrowth potential after 1 week in culture, compared to controls (P < 0.005).
  
  
  CONCLUSIONS
  Small concentrations of ethanol (1 mM) induced apoptosis in HepG2 MCTS with a concomitant inhibition on outgrowth potential, accompanied with a low degree of necrosis. These findings suggest that low concentrations of ethanol may already be sufficient for the treatment of hepatocellular carcinoma.
authors.affiliation: Max-Planck-Institut für molekulare Physiologie, Abteilung Epithelphysiology, Dortmund, Germany. castaned@mpi-dortmund.mpg.de
authors.forename: F
authors.initials: F
authors.lastname: Castañeda
authors.forename: R K
authors.initials: RK
authors.lastname: Kinne
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000473:pathology; D000431:Ethanol / Q000494:pharmacology*; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000473:pathology; D018613:Microscopy, Confocal; D009336:Necrosis; D009994:Osmolar Concentration; D018874:Spheroids, Cellular / Q000187:drug effects; D013997:Time Factors; D014407:Tumor Cells, Cultured / Q000187:drug effects
chemical_list: D000970:Antineoplastic Agents; D000431:Ethanol