Research article
Docosahexaenoic acid induces apoptosis in CYP2E1-containing HepG2 cells by activating the c-Jun N-terminal protein kinase related mitochondrial damage

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Abstract

Docosahexaenoic acid (DHA) causes apoptosis of various cancer cells, but the mechanism of DHA-induced cell death is still unclear. We hypothesized that the early signaling of apoptosis may be important in causing cell death as well as the production of free radical metabolites. DHA caused time- and dose-dependent cell death in human HepG2 hepatoma cells transduced with CYP2E1 (E47) but not in C34 (without CYP2E1), suggesting an important role of CYP2E1 in the DHA-mediated damage. DHA increased the c-Jun N-terminal protein kinase (JNK) activity until 8 h without activating other mitogen-activated protein kinases. The contents of proapoptotic Bad and FasL at 4 h and cytochrome c and caspase 3 activity at 8 h were increased and accompanied by the JNK activation in a successive manner. In contrast, Bax and Bcl-2 were not changed. Levels of lipid peroxides (LPOs) were elevated three- and fivefold at 8 and 24 h, respectively, in DHA-induced E47 cells. However, pretreatment with chlormethiazole (CMZ), a specific inhibitor of CYP2E1, significantly reduced the levels of LPO, CYP2E1, JNK activity and the rate of cell death. In addition, pretreatment with quercetin (one as a JNK inhibitor and one as an antioxidant) significantly reduced the cell death rate and JNK and SEK-1 activities. Our results indicated that DHA-mediated apoptosis in E47 cells was induced through the activation of the JNK-related cell death pathway, which may be involved in the production of LPO or reactive oxygen species during the CYP2E1 catalytic cycle, followed by mitochondrial injury and apoptosis.

Introduction

Docosahexaenoic acid (DHA), a w-3 fatty acid, is present in high amounts as a major component of brain gray matter and of the retina in most mammalian species [1] and is considered essential for normal neurological and cellular developments. Dietary DHA has been demonstrated to reduce the incidences of atherosclerosis, cardiac arrhythmia and coronary heart diseases [2]. Furthermore, DHA is known to lower the incidence of cancer when it is used alone or combined with other components of dietary fish oils such as eicosapentaenoic acid (EPA). DHA and EPA have been demonstrated to decrease tumor cell proliferation in both in vitro and in vivo models [3], [4], [5].

There are several mechanisms by which DHA might induce apoptosis in cancer cells. DHA can cause translocation of phosphatidylserine, which is an early event in apoptosis followed by protein-phosphatase-mediated processes and the systematic destruction of cellular proteins by caspase 3 in apoptotic events [6]. Alternatively, it is possible that the alteration of mitogen-activated protein (MAP) kinases plays an important role in DHA-mediated apoptosis, as demonstrated by the DHA-mediated apoptosis of vascular smooth muscle cells through stimulation of p38 MAP kinase [7]. The activation of extracellular signal-regulated kinase (ERK) is usually associated with cell growth and survival pathways, whereas c-Jun N-terminal protein kinase (JNK)/stress-activated protein kinase (SAPK) and p38 MAP kinase are related to cell death. Because of this generally accepted theory [8], [9], we hypothesized that MAP kinases may play a key role in the DHA-induced apoptosis of HepG2 cells, although the role of MAP kinases in DHA-mediated damage in hepatoma cells has not been studied.

Ethanol-inducible P450 2E1 (CYP2E1, a loosely bound P450 enzyme), present in the liver and other extrahepatic tissues, is known to metabolize various small molecules of potentially toxic substrates: ethanol, acetaldehyde, dimethyl nitrosamine, acetaminophen, CCl4, benzene, unsaturated fatty acids and so forth [10]. CYP2E1-mediated metabolism of these substrates usually leads to the production of more toxic or carcinogenic metabolites such as reactive or free radical metabolites and lipid peroxides (LPOs) [10], [11], [12]. Cederbaum et al. established the stable human E47 HepG2 hepatoma cell line (transduced CYP2E1) to study the role of CYP2E1 in cell damage caused by its substrates, including fatty acids [13], [14], [15], [16]. Arachidonic acid (AA; w-6 fatty acid), as CYP2E1 substrates, was shown to induce much more damage to E47 cells than C34 control hepatoma cells, demonstrating the role of CYP2E1 in the metabolic activation and the subsequent cell damage caused by CYP2E1 substrates [15].

Despite extensive studies on apoptosis of cancer cells caused by DHA [3], [17], [18], [19], [20], [21], the early signaling mechanism important in DHA-mediated cell death pathway has not been studied systematically. The goal of this study was to investigate the cytotoxic effects of DHA on HepG2 cells expressing CYP2E1 (E47 cells) and the roles of CYP2E1 in the apoptotic mechanisms of the DHA-mediated E47 cells, compared with C34 control cells. In addition, the importance of the levels of LPO in the DHA-mediated apoptosis is compared with that of the early signaling pathway to establish the linkages between CYP2E1, DHA, LPO and cytotoxicity.

Section snippets

Materials

Minimal essential media (MEM) and other materials for cell culture including gentamicin, antibiotics, l-glutamine and fetal bovine serum (FBS) were purchased from Invitrogen (San Diego, CA, USA). C34 and E47 HepG2 cells were kindly provided by Dr. A. Cederbaum (Mount Sinai Medical School, New York, NY). MTT cell proliferation kit was purchased from Roche Diagnostics GmbH (Indianapolis, USA). LPO kit and SP100625 were purchased from Calbiochem Co. (San Diego, USA). DHA, quercetin, indomethacin,

DHA concentration- and time-dependent apoptosis

To study the effect of DHA on growth rates of hepatoma cells with (E47) or without CYP2E1 (C34), we determined the dose- and time-dependent changes in cell morphology and cell viability. Generally, more cells died after longer exposure and higher concentrations of DHA than shorter exposure and lower DHA concentrations. For instance, less than 5% of E47 cells died after exposure to 25 μmol/L DHA for 12 h, 25% died after exposure to 100 μmol/L DHA for 12 h or 50 μmol/L DHA for 24 h and more than

Discussion

Previous studies indicate that dietary PUFA such as DHA can trigger apoptosis of various cancer cells, although the cell death depends on the cell type, concentration and the duration of treatment. Our results revealed that the DHA-mediated cell death of E47 cells took place mainly via apoptosis, although necrosis could occur at higher levels of DHA. Chen et al. reported that AA efficiently caused cell death of E9 HepG2 cells, which continuously express CYP2E1, possibly through the production

Acknowledgment

This work was supported by the Korea Research Foundation Grant KRF-2001-D00077. We thank to Drs. Byung-Jun Song and Norman Salem, Jr., for supporting this study in NIH. We are also grateful to Dr. Cederbaum for providing E47 and C34 cells and to Dr. Joong-Ick Yang for providing the chlormethiazole used in this study.

References (37)

  • R.M. Laethem et al.

    Formation of 19(S)-, 19(R)-, and 18(R)-hydroxyeicosatetraenoic acids by alcohol-inducible cytochrome P450 2E1

    J Biol Chem

    (1993)
  • E. Yang et al.

    Bad, a heterodimeric partner for Bcl-XL and Bcl-2. Displaces Bax and promotes cell death

    Cell

    (1995)
  • A. Mansouri et al.

    Sustained activation of JNK/p38MAPK pathways in response to cisplatin leads to Fas ligand induction and cell death in ovarian carcinoma cells

    J Biol Chem

    (2003)
  • A.A. Caro et al.

    Role of calcium and calcium-activated proteases in CYP2E1-dependent toxicity in HepG2 cells

    J Biol Chem

    (2002)
  • P.A. Sandstrom et al.

    Lipid hydroperoxides induce apoptosis in T cells displaying a HIV-associated glutathione peroxidase deficiency

    J Biol Chem

    (1994)
  • JX. Kangl et al.

    Regulation of sodium channel gene expression by class I antiarrhythmic drugs and n-3 polyunsaturated fatty acids in cultured neonatal rat cardiac myocytes

    Proc Natl Acad Sci U S A

    (1997)
  • G. Calviello et al.

    Dietary supplementation with eicosapentaenoic and docosahexaenoic acid inhibits growth of Morris hepatocarcinoma 3924A in rats: effects on proliferation and apoptosis

    Int J Cancer

    (1998)
  • A. Colguhoum et al.

    Gamma-linoleic acid and eicosapetanoic acid induce modification in mitochondrial metabolism, reactive oxygen species generation, lipid peroxidation and apoptosis in Walker 256 rat carcinoma cells

    Biochim Biophys Acta

    (2001)
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