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Long‐term ethanol exposure causes human liver cancer cells to become resistant to mitomycin C treatment through the inactivation of bad‐mediated apoptosis
Author(s) -
Huang ChingShui,
Lee YiRu,
Chen ChingShyang,
Tu ShihHsin,
Wang YingJan,
Lee ChiaHwa,
Chen LiChing,
Chang HuiWen,
Chang ChienHsi,
ChihMing Su,
Wu ChihHsiung,
Ho YuanSoon
Publication year - 2010
Publication title -
molecular carcinogenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.254
H-Index - 97
eISSN - 1098-2744
pISSN - 0899-1987
DOI - 10.1002/mc.20648
Subject(s) - biology , apoptosis , mitomycin c , flow cytometry , cell culture , cancer research , hepatocellular carcinoma , microbiology and biotechnology , cell cycle , protein kinase b , biochemistry , genetics
The aim of this study was to test whether long‐term ethanol consumption confers therapeutic resistance to human liver cancer patients infected with hepatitis B virus (HBV). Chronic ethanol‐treated cells were established by consecutively culturing a human hepatocellular carcinoma cell line, Hep 3B, which contains integrated HBV sequences, for 20–40 passages with or without 10 mM ethanol (designated as E20–E40 and C20–C40, respectively). Flow cytometry analysis demonstrated that a growth promoting effect of long‐term ethanol treatment was induced in the E40 cells through preferential acceleration of S‐phase in these cells. Lower protein expression levels of p16, p21/Cip1, and p27/Kip1 were detected in the ethanol‐treated E40 cells. We further demonstrated that long‐term ethanol‐treated E40 cells develop drug resistance in response to mitomycin C (MMC) treatment (>8 µM). Immunoblot analysis revealed that caspase‐8‐mediated mitochondrial apoptotic signals (such as Bad) were inactivated in the MMC‐resistant E40 cells. Immunoprecipitation experiments demonstrated that the sequestration of phosphorylated Bad (Ser‐112) through its binding with 14‐3‐3 was detected more profoundly in the MMC‐resistant E40 cells. Next, we examined the therapeutic efficacy of MMC (10 mg MMC/kg body weight, three times per week) in severe combined immunodeficient (SCID) mice bearing E40‐ and C40‐xenografted tumors. Significant reductions (>3‐fold) in tumor growth were detected in MMC‐treated C40‐xenografted mice. In vivo and in vitro studies demonstrated that AKT‐ and extracellular signal‐regulated kinase (ERK)‐mediated survival factors inhibited the Bad‐induced mitochondrial apoptotic signals that were involved in E40 tumor cells and that conferred resistance to MMC. © 2010 Wiley‐Liss, Inc.