ToxSci Advance Access originally published online on November 13, 2007
Toxicological Sciences 2008 102(1):120-128; doi:10.1093/toxsci/kfm281
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PPAR
and PPARβ Are Differentially Affected by Ethanol and the Ethanol Metabolite Acetaldehyde in the MCF-7 Breast Cancer Cell Line
School of Pharmacy, The University of Queensland, Brisbane, Queensland 4072, Australia
1 To whom correspondence should be addressed at School of Pharmacy, Steele Building (03), The University of Queensland, Brisbane, Queensland 4072, Australia. Fax: +61-7-3365-1688. E-mail: sarahrt{at}uq.edu.au
Received October 3, 2007; accepted October 30, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The activity and/or the level of the peroxisome proliferator–activated receptors (PPARs) in liver and oligodendrocytes are regulated by ethanol. Despite the association between ethanol consumption and breast cancer risk, and the increasing evidence for an involvement of PPARs in some cancers, there have been no studies on the effect of ethanol or its metabolite acetaldehyde on PPARs in breast cancer. Using the MCF-7 breast cancer cell line, we examined the relationship between ethanol and its metabolite acetaldehyde on PPAR
and PPARβ transactivation. Ethanol (20mM) reduced the potency of the PPARβ ligand GW0742, evident by a rightward shift in the GW0742 dose-response curve, whereas for PPAR activation by GW7647, ethanol mediated its effects primarily through reducing efficacy as evidenced by a reduction in maximal response. Using the enzyme inhibitors 4-methylpyrazole and cyanamide and the metabolite acetaldehyde, we showed that PPAR and PPARβ are differentially modulated by ethanol and acetaldehyde. While acetaldehyde is responsible for the inhibition of PPAR ligand inhibition with a concentration that inhibits 50% of activity (IC50) of 111 nM, acetaldehyde has no effect on PPARβ or its ligand activation. Instead, inhibition of PPARβ transactivation is mediated directly by ethanol. The differential effect of ethanol and acetaldehyde on PPAR and PPARβ further underscores the differences between these receptors and may indicate the relevance of PPARs in the effects of ethanol in the human breast. Key Words: ethanol; acetaldehyde; PPAR; proliferation; breast; cell lines; MCF-7.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Peroxisome proliferator–activated receptors (PPARs) are transcription factors that control the transcription of genes containing a peroxisome proliferator response element (PPRE) in their 5' region (Michalik et al., 2006
). PPARs are ligand activated and agonists include a range of endogenous and exogenous compounds including fatty acids, eicosanoids, fibrates, thiazolidinediones, and plasticizers (Michalik et al., 2006). Three different genes encode for the PPARs and give rise to PPAR, PPAR
, and PPARβ (Michalik et al., 2006). While there is some degree of overlap in ligand specificity and tissue expression between the isoforms, each transcription factor has preferential ligands and a distinct tissue distribution. PPAR is important in the metabolism/catabolism of free fatty acids and has an important clinical role in the treatment of hyperlipidemia (Michalik et al., 2006; Staels and Fruchart, 2005). PPAR agonists such as the thiazolidinidione drugs are used to control insulin sensitivity in the treatment of diabetes (Michalik et al., 2006; Staels and Fruchart, 2005). More recently, a role for PPAR has also been proposed in the modulation of insulin sensitivity indicating that it may have a dual clinical functionality (Michalik et al., 2006). PPARβ is ubiquitously expressed and is also a metabolic regulator important in energy homeostasis and with a role in inflammation (Barish et al., 2006; Michalik et al., 2006). PPARβ is also associated with the regulation of proliferation, apoptosis, and differentiation (Michalik et al., 2006). The PPAR isoforms share a common mechanism of transcriptional activation, involving the formation of a heterodimer with the retinoid X receptor (RXR) and the involvement of coregulators (Michalik et al., 2006). The final physiological response to PPAR activation will also be influenced by the degree of cross talk between PPARs and other transcription factors (Ogawa et al., 2005). PPARs are increasingly identified not only as drug targets but also as receptors that may mediate toxic responses and with an involvement in tumorigenesis (Peraza et al., 2006; Roberts-Thomson, 2000; Sertznig et al., 2007).
Along with a family history of breast cancer (Kelsey and Berkowitz, 1988), estrogen exposure (Kelsey et al., 1993), and dietary fat (Thiebaut et al., 2007), epidemiological evidence supports a positive correlation between ethanol consumption and breast cancer (Singletary and Gapstur, 2001). The suggestion is that risk increases with increasing ethanol consumption (Baan et al., 2007). Mechanistically, the action of ethanol in increasing breast cancer risk is largely unknown. However, a recent release on behalf of the World Health Organization (WHO) International Agency for Research on Cancer Monograph Working Group suggests that the ethanol metabolite acetaldehyde contributes to the etiology of malignant esophageal tumors (Baan et al., 2007). In vitro studies involving breast cancer cell lines and rat mammary gland studies also support a possible role for acetaldehyde in the mechanism of ethanol-induced mammary gland cancer (Barnes et al., 2000; Castro et al., 2001). Evidence also exists for a direct action of ethanol in breast cancer. One such example is ethanol-mediated increases in the transcriptional activity and expression of the estrogen receptor- (ER) and downregulation of the expression of the tumor suppressor BRCA1 (Fan et al., 2000). Ethanol also modulates breast cancer cell invasion via erbB2- and MMP2-modulated pathways (Aye et al., 2004; Luo and Miller, 2000; Ma et al., 2003). Thus, ethanol appears to act via several different pathways to enhance the risk of breast cancer in women who drink alcohol.
The interaction between ethanol and the change in activity and transcription of the ER shows that ethanol can modulate transcription factors and gene transcription associated with breast cancer (Fan et al., 2000). This interaction is specific, as other transcription factors such as E2F1 or Sp1 are not altered by ethanol under similar conditions (Fan et al., 2000). The PPARs are members of the same superfamily of nuclear hormone receptors as is the ER, and there is an increasing body of evidence suggesting that the mechanism of ethanol in liver diseases such as alcoholic fatty liver is via the PPARs, primarily through alterations in lipid metabolism (Crabb et al., 2004). Further evidence of a link between ethanol and PPARs is the modulation of PPARβ in oligodendrocyte cells by ethanol (Leisewitz et al., 2003). Given the ability of ethanol to modulate specific transcription factors linked to breast cancer and the emerging reports of an association between PPARs and ethanol, we sought in this study to explore the relationship between PPAR, PPARβ, and ethanol using the MCF-7 breast cancer cell line. Particular attention was paid to the potential isoform-specific regulation of PPAR and PPARβ by ethanol in the context of transcriptional changes, changes in activity, and sensitivity to the major ethanol metabolite acetaldehyde.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Chemicals.
Cell culture media Dulbecco's modified Eagle's medium (DMEM) with phenol red and fetal bovine serum was obtained from Sigma-Aldrich (Sydney, NSW, Australia). DMEM media without phenol red with 25mM HEPES buffer, cell culture media supplements and antibiotics L-glutamine, penicillin, streptomycin sulfate, and hygromycin were all purchased from Invitrogen (Mount Waverly, Vic, Australia); G418 was obtained from Promega (Annandale, NSW, Australia); and doxycycline hydrochloride (dox) was purchased from BD Biosciences (Clontech, Palo Alto, CA). Ethanol (absolute 200% proof, cell culture grade), acetaldehyde, dimethyl sulfoxide (DMSO), fatty acid–free bovine serum albumin, 4-methylpyrazole (4-MP), cyanamide, and PPARβ ligand GW0742 were all obtained from Sigma-Aldrich, and PPAR
ligand GW7647 was purchased from Calbiochem (Merck, Vic, Australia). RNA isolation kits were purchased from Qiagen (Clifton Hill, Vic, Australia), and real-time PCR reagents were purchased from Applied Biosystems (Scorseby, Vic, Australia). The CellTiter 96 AQueous One Solution Cell Proliferation Assay (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS), Glo lysis buffer, and the Bright-Glo luciferase assay system were all purchased from Promega.
Cell culture.
The MCF-7 breast cancer cell line was purchased from the American Type Culture Collection (Manassas, VA) and routinely cultured in growth media comprising high glucose DMEM supplemented with 10% fetal bovine serum, L-glutamine (4mM), penicillin G (100 U/ml), and streptomycin sulfate (100 µg/ml). MCF-7 Tet-off cells were purchased from BD Biosciences (Clontech) and maintained in growth media as above supplemented with G418 (100 µg/ml). For all experiments involving a closed chamber, environment cells were cultured in DMEM media without phenol red and with 25mM HEPES buffer, L-glutamine (4mM), penicillin G (100 U/ml), and streptomycin sulfate (100 µg/ml) and 10% fetal bovine serum where required. The MCF-7 cell line conditionally overexpressing PPAR under the control of a tetracycline-responsive element (MCF-7PPAR(C12)) has previously been described (Faddy et al., 2006); see section below for the description of the production of an MCF-7 cell line conditionally overexpressing PPARβ. MCF-7 Tet-off cells stably expressing PPAR or PPARβ were maintained in growth media supplemented with G418 (100 µg/ml), hygromycin (110 µg/ml), and dox (10 ng/ml or 1 µg/ml). When expression of the PPAR was required for experiments, dox was removed from the culture media upon plating for the experiment. All cell lines were maintained at 37°C in a humidified 5% CO2/95% air incubator.
Generation of a PPARβ MCF-7 Tet-off cell line.
MCF-7 Tet-off cells were plated at 5 x 106 cells per 10-cm dish and cotransfected with the plasmids pBI-GhPPARβ (Gopisetty Venkata et al., 2006) and pTK-Hyg (Clontech) using Lipofectamine 2000 (Invitrogen). Hygromycin B–resistant clones were selected and expanded. Colonies were screened for tetracycline-regulated β-galactosidase (β-gal) expression as a positive marker for the expression of PPARβ using the β-gal enzyme assay system (Promega) as previously described (Gopisetty Venkata et al., 2006). Colony MCF-7PPARβ(C7.4) was used for all experiments. For maintenance of the colony, see the section above regarding cell culture.
Ethanol incubations.
Due to the volatility of ethanol and concerns of the ability of the in vitro culture system to maintain consistency of ethanol concentrations over the experimental time (Adickes et al., 1988), we initially compared the concentration of ethanol in culture after 24 h in an open and closed environment system (Luo and Miller, 2000; Ma et al., 2003) using the NAD-ADH assay (Sigma-Aldrich). The results (data not shown) suggest that the closed environment system maintained ethanol concentrations; hence, for all reported studies involving ethanol, a closed system was used. The culture plates containing the cells and media were placed on a rack in a sealed plastic container (22 x 16 x 6 cm) containing a reservoir of 200-ml water with the same concentration of ethanol as in the culture media. A separate container was used for each ethanol concentration.
Real-time reverse transcription–PCR.
For quantitation of PPAR and PPARβ expression, MCF-7 cells were plated at 3 x 105 cells per well in six well plates and cultured in the presence of ethanol in a closed system (0–300mM) for 24 h. For cyclin D1, MCF-7PPARβ(C7.4) cells were plated at 2.5 x 105 cells per well in six well plates, dox was removed, and GW0742 (10nM) was added 24 h prior to RNA isolation. RNA was isolated using the RNeasy mini kit. PPAR and PPARβ mRNAs were amplified as previously described (Suchanek et al., 2002a,b), and cyclin D1 was amplified using the Applied Biosystems TaqMan Gene Expression Assay Hs00277039_m1. All amplifications were performed using an Applied Biosystems 7500 Real-Time PCR System. The amplification of the target mRNA was normalized to the endogenous control 18S rRNA level (
Ct) and relative to the appropriate control (Ct). The fold change was calculated using the formula 2–Ct (Suchanek et al., 2002b).
PPAR transactivation.
MCF-7 Tet-off cells stably expressing PPAR or PPARβ were plated at 4 x 104 cells per well into 96-well plates and allowed to adhere for 24 h. The cells were then transfected with the PPRE reporter construct (pTK3XPPRELuc; 2 µg per well) using Lipofectamine 2000 (0.8 µl per well) in phenol red–free, serum-free, and antibiotic-free media (Gopisetty Venkata et al., 2006). After 5 h, the transfection media was removed and the cells were incubated in a closed system with ethanol, acetaldehyde, GW7647, GW0742, 4-MP, or cyanamide as appropriate. GW7647 and GW0742 were dissolved in DMSO with the final concentration of DMSO no greater than 0.05% and standardized across all treatments and controls. 4-MP and cyanamide were made fresh in water with a final concentration no greater than 0.5%, which was also standardized across all treatments and controls. All treatments were added in phenol red–free, serum-free, and antibiotic-free DMEM media with 25mM HEPES buffer supplemented with 1.5% fatty acid–free bovine serum albumin. After 19 h, the cells were lysed with the Glo lysis buffer (100 µl) according to the manufacturer's protocol. The lysates were then transferred to a single 96-well plate, and the Bright-Glo luciferase assay reagent (100 µl) was added and the luciferase activity measured using a NOVOstar fluorescence microplate reader (BMG Lab Technologies, Germany). The luciferase data were corrected with the viable cell numbers determined using the MTS assay and data normalized to the control wells to obtain fold change. For the MTS assay, the CellTiter 96 AQueous One Solution Reagent (20 µl) was directly added to each well and the plates were incubated for 2 h at 37°C in a humidified 5% CO2/95% air incubator. After 2 h, the absorbance was read at 490 nm with a Bio-Rad Model 550 microplate reader.
Proliferation.
MCF-7PPARβ(C7.4) cells were plated into 96-well plates at a density of 500 cells per well in growth media supplemented with dox. The cells were allowed to adhere for 24 h and then moved to a closed system with media containing the presence or absence of dox and GW0742 (0, 2nM, 10nM, and 1µM). The media were replaced with fresh media every 48 h. On day 10, cell number was approximated using an MTS absorbance assay as previously described (Faddy et al., 2006).
Statistical analyses.
Data were analyzed for statistical significance at p < 0.05 using GraphPad Prism V4.03 software. Specific statistical tests are noted in the figure legends where relevant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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To examine the effects of ethanol on PPARs, in this study we analyzed the ability of ethanol to alter the transcription of PPAR
and PPARβ as well as the ability of ethanol and acetaldehyde to modulate the transactivation of PPAR and PPARβ using MCF-7 breast cancer cells.
Over a range of ethanol concentrations up to 300mM, ethanol was able to dose dependently and significantly (p < 0.05) increase the expression of PPAR mRNA in MCF-7 cells (Fig. 1A). Ethanol also modestly increased the mRNA for PPARβ with a significant (p < 0.05) increase seen at 30 and 300mM, although not at 100mM (Fig. 1B). The increased expression for PPARβ mRNA was only in the order of twofold in contrast to the approximately sevenfold increase seen for PPAR compared with the absence of ethanol (Fig. 1).
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Given that ethanol changed PPAR
and PPARβ transcription, we then examined the ability of ethanol at a high concentration (100mM) and a more physiologically relevant level of 20mM (Szabo et al., 1996) to modulate the function of the PPARs using a gene reporter assay in MCF-7 cells stably overexpressing either PPAR or PPARβ. MCF-7 Tet-off cells overexpressing PPAR have previously been described and characterized (Faddy et al., 2006). PPARβ overexpressing MCF-7 Tet-off cells were generated for this study, and characterization experiments are shown in Figure 2. The removal of dox induces PPARβ transcription with over a 50-fold increase in PPARβ mRNA (Fig. 2A). Transient transfection of the PPRE reporter construct showing PPARβ-mediated transactivation (Fig. 2B) demonstrates significant induction of PPARβ activity in the absence of dox and the presence of the PPARβ ligand GW0742.
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High concentrations of ethanol (100mM) inhibited the activation of both PPAR
and PPARβ by their specific ligands GW7647 and GW0742, respectively, with clear effects on the maximum response for both isoforms (Figs. 3A and B). However, in the presence of a lower concentration of ethanol (20mM), PPAR transactivation by isoform-specific ligands was differentially affected (Fig. 4). PPAR activation by GW7647 in the presence of 20mM ethanol had a lower maximal response (Fig. 4A), showing that 20mM ethanol reduces the efficacy of GW7647, whereas there was a rightward shift in the dose-response curve for PPARβ activation by GW0742 in the presence of 20mM ethanol (Fig. 4B). This reduction in the potency of GW0742 by 20mM ethanol lead to a shift in the concentration required to induce the half maximal effect (EC50) from 2.2 x 10–9M (95% confidence interval 1.6 x 10–9 to 2.9 x 10–9M) in the absence of ethanol to 10.7 x 10–9M (95% confidence interval 6.2 x 10–9 to 18.4 x 10–9M) in the presence of ethanol with the log EC50 values being significantly different between the two curves (p < 0.05).
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PPARβ activation is reported to increase the proliferation of some breast cancer cell lines and the transcription of cell cycle–associated genes, such as cyclin D1 (Stephen et al., 2004
). Using our model system, we examined the effects of PPARβ transactivation by GW0742 on MCF-7 cell proliferation and cyclin D1 transcription (Fig. 5). No effects on proliferation were seen in the absence (Fig. 5A) or presence (Fig. 5B) of PPARβ induction and activation or was there any effect seen on cyclin D1 expression after PPARβ overexpression and activation (Fig. 5C). The physiological consequence of PPARβ activation and modulation by ethanol requires further investigation as does the consequences of PPARβ activation on cancer cell lines generally as previously noted (Hollingshead et al., 2007). Given the recent literature, such studies should focus on the ability of ethanol to modulate PPARβ induction of differentiation (Kim et al., 2006).
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To determine whether the differential effects seen on PPAR transactivation by a physiological concentration of ethanol were due to ethanol or its major metabolite acetaldehyde, we used the enzyme inhibitors 4-MP and cyanamide. 4-MP inhibits alcohol dehydrogenase preventing the metabolic conversion of ethanol to acetaldehyde, whereas cyanamide inhibits acetaldehyde dehydrogenase, the enzyme involved in the conversion of acetaldehyde to acetic acid. For PPARβ, neither 4-MP (Fig. 6A) nor cyanamide (Fig. 6B) modulated the activation of PPARβ by GW0742 in the absence of ethanol, and in the presence of ethanol, neither compound altered the ethanol-induced inhibition of GW0742-mediated transactivation of PPARβ beyond that already seen in the dose-response studies, suggesting that ethanol itself, rather than a metabolite, mediates this inhibition.
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For PPAR
, 4-MP significantly (p < 0.05) decreased GW7647 transactivation (Fig. 7A), suggesting that it interferes with PPAR transactivation per se and precluding its use to determine the effects of ethanol on PPAR function. Our studies indicate that either 4-MP is an isoform-specific modulator of PPAR activity or that the enzymes with reported sensitivities to 4-MP, such as alcohol dehydrogenase and CYP2E1 (Clejan and Cederbaum, 1990; Ueng et al., 2004), produce substrates that are modulators of PPAR activity. Our studies show that these modulators are PPAR selective since 4-MP had no effect on the ability of GW0742 to activate PPARβ (Fig. 6A). Cyanamide, however, did not interfere with GW7647 activation of PPAR in the absence of ethanol and was therefore an appropriate tool. In the presence of ethanol, while GW7647, and GW7647 with cyanamide, were not significantly different, there was a modest inhibition of PPAR activity when cyanamide was present, suggesting the possible involvement of acetaldehyde (Fig. 7B). To explore this possible effect, the regulation of PPAR and PPARβ activation by acetaldehyde was assessed directly.
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We examined the ability of acetaldehyde to regulate the transactivation of PPAR
and PPARβ using the gene reporter assay (Fig. 8). Acetaldehyde alone was unable to activate PPAR (Fig. 8A) or PPARβ (Fig. 8B) and did not modulate the activation of PPARβ by its ligand GW0742 (Fig. 8B). However, acetaldehyde inhibited the activation of PPAR by GW7647 (Fig. 8A) with an IC50 of 111nM.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Our study explored the interaction between PPAR isoforms
and β and ethanol in the MCF-7 breast cancer cell line. Ethanol modulated the transcription of the PPARs and was also able to interfere with ligand-mediated PPAR transactivation. At lower (more physiologically relevant) concentrations of ethanol (20mM), pronounced isoform specificity in this modulation of PPAR transactivation was observed. This isoform selectivity was due to pronounced differences in sensitivity to the ethanol metabolite acetaldehyde, with PPAR transactivation potently inhibited by acetaldehyde while PPARβ transactivation was acetaldehyde resistant.
The metabolism of ethanol has been well characterized in the liver, where the enzyme alcohol dehydrogenase plays a major role with other minor pathways identified, including CYP2E1 and catalase (Nagy, 2004). The degree of acetaldehyde accumulation may vary among tissues, and of particular relevance to these studies is the dose-dependent accumulation of acetaldehyde in the rat mammary gland, where levels remain significantly higher than in plasma for several hours following ethanol administration (Castro et al., 2007). The relatively high levels are likely a combination of acetaldehyde production by the same enzyme systems present in the liver, as well as other contributors to ethanol metabolism and a lower level of enzymes capable of detoxifying acetaldehyde in this gland (Castro et al., 2001, 2007; Iscan et al., 2001). Aside from the known mutagenic and carcinogenic ability of acetaldehyde (Poschl and Seitz, 2004), we now show that it is capable of significantly inhibiting PPAR but not PPARβ in a human breast cancer cell line. Given the importance of PPAR in lipid homeostasis (Michalik et al., 2006), this finding is likely to have implications on the lipid profile of milk produced by the lactating woman as well as the association between breast cancer incidence and ethanol consumption.
Previous studies examining ethanol and PPAR isoforms are essentially confined to liver-specific effects. Rat liver H4IIEC3 hepatoma cells transfected with PPAR respond to ethanol (20mM) by decreasing both basal and agonist-stimulated transactivation (Galli et al., 2001). A similar effect is observed using primary rat hepatocytes, although when CV1 cells are used, which lack alcohol-metabolizing enzymes, ethanol has no effect on PPAR transactivation suggesting a role for acetaldehyde (Galli et al., 2001). Further studies implicate acetaldehyde as the mediator of the rat liver PPAR effects, possibly via inhibition of PPAR DNA-binding ability (Galli et al., 2001). Although there are differences between rodent and human PPAR in terms of the potential for ligand activation and carcinogenicity (Peters et al., 2005), our results suggest that similarly human PPAR basal and agonist-stimulated activity is modulated by acetaldehyde rather than ethanol. We also observed an increase in endogenous PPAR mRNA in MCF-7 cells with an increasing concentration of ethanol. Chronic ethanol feeding to mice results in fatty livers (Crabb et al., 2004), and depending on the study and concentration of ethanol used (Crabb et al., 2004), ethanol may either cause no change in the level of liver PPAR (Fischer et al., 2003) or cause a decrease in expression (Wan et al., 1995). Hence, the effect of ethanol in human breast cancer cells on PPAR expression appears different to that of the liver.
The effect of ethanol on PPARβ is less well studied and although we see an increase in endogenous PPARβ mRNA in MCF-7 cells in the presence of ethanol, this effect was modest and not seen at all concentrations. Opposite to our findings in MCF-7 cells, ethanol treatment of B12 oligodendrocyte-like cells derived from a rat brain tumor decreases PPARβ mRNA by increasing mRNA degradation (Leisewitz et al., 2003), suggesting tissue-specific effects of ethanol on PPARβ mRNA. In our MCF-7 cell studies, 20mM ethanol alters the response of ligand-activated PPARβ, evident by a rightward shift in the GW0742 dose-response curve. This effect was not mediated by acetaldehyde. Although in vitro experiments with murine PPARβ suggest that preincubation with 1mM acetaldehyde affects the ability of PPARβ to bind DNA (Galli et al., 2001), this concentration is well above the IC50 we observed for inhibition of ligand-mediated PPAR transactivation (111nM), and our studies did not detect any inhibition of PPARβ at 1mM in intact MCF-7 cells.
The studies presented in this paper reinforce the isoform-specific behavior of PPAR and PPARβ. Such differences between PPAR and PPARβ occur with neurotoxicity (Smith et al., 2001, 2004) and expression changes with sodium butyrate treatment of human colon cancer HT-29 cells and breast cancer MCF-7 cells (Aung et al., 2006). There are many other examples of functional differences between the isoforms ranging from downstream gene activation to differences in coregulators required for activation (Michalik et al., 2006). Similarly, pharmacological and endogenous agonists for the PPARs may exhibit specific activity for an individual isoform or may have pan agonism or dual agonism across isoforms (Michalik et al., 2006; Peraza et al., 2006). The differential effect of ethanol and acetaldehyde on PPAR and PPARβ further underscores the differences between these receptors, and our studies suggest that this may be of particular significance in the breast. Indeed, the PPAR polymorphism PPAR2Pro12Ala was recently determined to be important in a Danish population with regards to alcohol and breast cancer risk (Vogel et al., 2007).
The regulation of PPAR and PPARβ by ethanol and/or its metabolite acetaldehyde adds them to, among others ER (Fan et al., 2000), BRCA1 (Meng et al., 2000), erbB2 (Luo and Miller, 2000; Ma et al., 2003), and MMP2 (Aye et al., 2004) as proteins with possible roles in the promotion of breast cancer by ethanol (Singletary and Gapstur, 2001). Given that many of the pathways involving these proteins could act cooperatively, there is likely to be a complex interplay in the way ethanol and/or acetaldehyde acts via the PPARs and other proteins to influence tumorigenic relevant pathways such as proliferation, resistance to apoptosis, and invasiveness.
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FUNDING |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Australian Brewers Foundation Alcohol-Related Medical Research Grant Scheme and the award of a UQ Graduate School Scholarship to N.G.V.
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ACKNOWLEDGMENTS |
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We would like to thank Dr Ron Evans, Howard Hughes Medical Institute, The Salk Institute for Biological Studies, San Diego, for the kind gift of the pTK3XPPREluc plasmid.
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REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
Adickes ED, Mollner TJ, Lockwood SK. Closed chamber system for delivery of ethanol to cell cultures. Alcohol Alcohol (1988) 23:377–381.
Aung CS, Faddy HM, Lister EJ, Monteith GR, Roberts-Thomson SJ. Isoform specific changes in PPAR alpha and beta in colon and breast cancer with differentiation. Biochem. Biophys. Res. Commun. (2006) 340:656–660.[CrossRef][Web of Science][Medline]
Aye MM, Ma C, Lin H, Bower KA, Wiggins RC, Luo J. Ethanol-induced in vitro invasion of breast cancer cells: The contribution of MMP-2 by fibroblasts. Int. J. Cancer (2004) 112:738–746.[CrossRef][Web of Science][Medline]
Baan R, Straif K, Grosse Y, Secretan B, El Ghissassi F, Bouvard V, Altieri A, Cogliano V. Carcinogenicity of alcoholic beverages. Lancet Oncol. (2007) 8:292–293.[CrossRef][Web of Science][Medline]
Barish GD, Narkar VA, Evans RM. PPAR{delta}: A dagger in the heart of the metabolic syndrome. J. Clin. Invest. (2006) 116:590–597.[CrossRef][Web of Science][Medline]
Barnes SL, Singletary KW, Frey R. Ethanol and acetaldehyde enhance benzo[a]pyrene-DNA adduct formation in human mammary epithelial cells. Carcinogenesis (2000) 21:2123–2128.
Castro GD, Delgado de Layno AMA, Costantini MH, Castro JA. Cytosolic xanthine oxidoreductase mediated bioactivation of ethanol to acetaldehyde and free radicals in rat breast tissue. Its potential role in alcohol-promoted mammary cancer. Toxicology (2001) 160:11–18.[CrossRef][Web of Science][Medline]
Castro GD, Layno AM, Fanelli SL, Maciel ME, Gomez MI, Castro JA. Acetaldehyde accumulation in rat mammary tissue after an acute treatment with alcohol. J. Appl. Toxicol. (2007) (in press).
Clejan LA, Cederbaum AI. Oxidation of pyrazole by reconstituted systems containing cytochrome P-450 IIE1. Biochim. Biophys. Acta (1990) 1034:233–237.[Medline]
Crabb DW, Galli A, Fischer M, You M. Molecular mechanisms of alcoholic fatty liver: Role of peroxisome proliferator-activated receptor alpha. Alcohol (2004) 34:35–38.[CrossRef][Web of Science][Medline]
Faddy HM, Robinson JA, Lee WJ, Holman NA, Monteith GR, Roberts-Thomson SJ. Peroxisome proliferator-activated receptor alpha expression is regulated by estrogen receptor alpha and modulates the response of MCF-7 cells to sodium butyrate. Int. J. Biochem. Cell Biol. (2006) 38:255–266.[CrossRef][Web of Science][Medline]
Fan S, Meng Q, Gao B, Grossman J, Yadegari M, Goldberg ID, Rosen EM. Alcohol stimulates estrogen receptor signaling in human breast cancer cell lines. Cancer Res. (2000) 60:5635–5639.
Fischer M, You M, Matsumoto M, Crabb DW. Peroxisome proliferator-activated receptor alpha (PPAR alpha) agonist treatment reverses PPARalpha dysfunction and abnormalities in hepatic lipid metabolism in ethanol-fed mice. J. Biol. Chem. (2003) 278:27997–28004.
Galli A, Pinaire J, Fischer M, Dorris R, Crabb DW. The transcriptional and DNA binding activity of peroxisome proliferator-activated receptor alpha is inhibited by ethanol metabolism. A novel mechanism for the development of ethanol-induced fatty liver. J. Biol. Chem. (2001) 276:68–75.
Gopisetty Venkata N, Robinson JA, Cabot PJ, Davis B, Monteith GR, Roberts-Thomson SJ. Mono(2-ethylhexyl)phthalate and mono-n-butyl phthalate activation of peroxisome proliferator activated-receptors alpha and gamma in breast. Toxicol. Lett. (2006) 163:224–234.[CrossRef][Web of Science][Medline]
Hollingshead HE, Killins RL, Borland MG, Girroir EE, Billin AN, Willson TM, Sharma AK, Amin S, Gonzalez FJ, Peters JM. Peroxisome proliferator-activated receptor-{beta}/{delta} (PPAR{beta}/{delta}) ligands do not potentiate growth of human cancer cell lines. Carcinogenesis (2007) (in press).
Iscan M, Klaavuniemi T, Coban T, Kapucuoglu N, Pelkonen O, Raunio H. The expression of cytochrome P450 enzymes in human breast tumours and normal breast tissue. Breast Cancer Res. Treat. (2001) 70:47–54.[CrossRef][Web of Science][Medline]
Kelsey JL, Berkowitz GS. Breast cancer epidemiology. Cancer Res. (1988) 48:5615–5623.
Kelsey JL, Gammon MD, John EM. Reproductive factors and breast cancer. Epidemiol. Rev. (1993) 15:36–47.
Kim DJ, Bility MT, Billin AN, Willson TM, Gonzalez FJ, Peters JM. PPARbeta/delta selectively induces differentiation and inhibits cell proliferation. Cell Death Differ. (2006) 13:53–60.[CrossRef][Web of Science][Medline]
Leisewitz AV, Jung JE, Perez-Alzola P, Fuenzalida KM, Roth A, Inestrosa NC, Bronfman M. Ethanol specifically decreases peroxisome proliferator activated receptor beta in B12 oligodendrocyte-like cells. J. Neurochem. (2003) 85:135–141.[Web of Science][Medline]
Luo J, Miller MW. Ethanol enhances erbB-mediated migration of human breast cancer cells in culture. Breast Cancer Res. Treat. (2000) 63:61–69.[CrossRef][Web of Science][Medline]
Ma C, Lin H, Leonard SS, Shi X, Ye J, Luo J. Overexpression of ErbB2 enhances ethanol-stimulated intracellular signaling and invasion of human mammary epithelial and breast cancer cells in vitro. Oncogene (2003) 22:5281–5290.[CrossRef][Web of Science][Medline]
Meng Q, Gao B, Goldberg ID, Rosen EM, Fan S. Stimulation of cell invasion and migration by alcohol in breast cancer cells. Biochem. Biophys. Res. Commun. (2000) 273:448–453.[CrossRef][Web of Science][Medline]
Michalik L, Auwerx J, Berger JP, Chatterjee VK, Glass CK, Gonzalez FJ, Grimaldi PA, Kadowaki T, Lazar MA, O'Rahilly S, et al. International Union of Pharmacology. LXI. Peroxisome proliferator-activated receptors. Pharmacol. Rev. (2006) 58:726–741.
Nagy LE. Molecular aspects of alcohol metabolism: Transcription factors involved in early ethanol-induced liver injury. Annu. Rev. Nutr. (2004) 24:55–78.[CrossRef][Web of Science][Medline]
Ogawa S, Lozach J, Benner C, Pascual G, Tangirala RK, Westin S, Hoffmann A, Subramaniam S, David M, Rosenfeld MG, et al. Molecular determinants of crosstalk between nuclear receptors and toll-like receptors. Cell (2005) 122:707–721.[CrossRef][Web of Science][Medline]
Peraza MA, Burdick AD, Marin HE, Gonzalez FJ, Peters JM. The toxicology of ligands for peroxisome proliferator-activated receptors (PPAR). Toxicol. Sci. (2006) 90:269–295.
Peters J, Cheung C, Gonzalez F. Peroxisome proliferator-activated receptor- and liver cancer: Where do we stand? J. Mol. Med. (2005) 83:774–785.[CrossRef][Web of Science][Medline]
Poschl G, Seitz HK. Alcohol and cancer. Alcohol Alcohol (2004) 39:155–165.
Roberts-Thomson SJ. Peroxisome proliferator-activated receptors in tumorigenesis: Targets of tumour promotion and treatment. Immunol. Cell Biol. (2000) 78:436–441.[CrossRef][Medline]
Sertznig P, Seifert M, Tilgen W, Reichrath J. Present concepts and future outlook: Function of peroxisome proliferator-activated receptors (PPARs) for pathogenesis, progression, and therapy of cancer. J. Cell. Physiol. (2007) 212:1–12.[CrossRef][Web of Science][Medline]
Singletary KW, Gapstur SM. Alcohol and breast cancer: Review of epidemiologic and experimental evidence and potential mechanisms. JAMA (2001) 286:2143–2151.
Smith SA, May FJ, Monteith GR, Roberts-Thomson SJ. Activation of the peroxisome proliferator-activated receptor-alpha enhances cell death in cultured cerebellar granule cells. J. Neurosci. Res. (2001) 66:236–241.[CrossRef][Web of Science][Medline]
Smith SA, Monteith GR, Robinson JA, Venkata NG, May FJ, Roberts-Thomson SJ. Effect of the peroxisome proliferator-activated receptor beta activator GW0742 in rat cultured cerebellar granule neurons. J. Neurosci. Res. (2004) 77:240–249.[CrossRef][Web of Science][Medline]
Staels B, Fruchart JC. Therapeutic roles of peroxisome proliferator-activated receptor agonists. Diabetes (2005) 54:2460–2470.
Stephen RL, Gustafsson MC, Jarvis M, Tatoud R, Marshall BR, Knight D, Ehrenborg E, Harris AL, Wolf CR, Palmer CN. Activation of peroxisome proliferator-activated receptor delta stimulates the proliferation of human breast and prostate cancer cell lines. Cancer Res. (2004) 64:3162–3170.
Suchanek KM, May FJ, Lee WJ, Holman NA, Roberts-Thomson SJ. Peroxisome proliferator-activated receptor beta expression in human breast epithelial cell lines of tumorigenic and non-tumorigenic origin. Int. J. Biochem. Cell Biol. (2002a) 34:1051–1058.[CrossRef][Web of Science][Medline]
Suchanek KM, May FJ, Robinson JA, Lee WJ, Holman NA, Monteith GR, Roberts-Thomson SJ. Peroxisome proliferator-activated receptor alpha in the human breast cancer cell lines MCF-7 and MDA-MB-231. Mol. Carcinog. (2002b) 34:165–171.[CrossRef][Web of Science][Medline]
Szabo G, Mandrekar P, Girouard L, Catalano D. Regulation of human monocyte functions by acute ethanol treatment: Decreased tumor necrosis factor alpha, interleukin-lbeta; and elevated interleukin-10, and transforming growth factor-beta production. Alcohol Clin. Exp. Res. (1996) 20:900–907.[Web of Science][Medline]
Thiebaut ACM, Kipnis V, Chang S-C, Subar AF, Thompson FE, Rosenberg PS, Hollenbeck AR, Leitzmann M, Schatzkin A. Dietary fat and postmenopausal invasive breast cancer in the National Institutes of Health-AARP Diet and Health Study cohort. J. Natl. Cancer Inst. (2007) 99:451–462.
Ueng YF, Hsieh CH, Don MJ, Chi CW, Ho LK. Identification of the main human cytochrome P450 enzymes involved in safrole 1'-hydroxylation. Chem. Res. Toxicol. (2004) 17:1151–1156.[CrossRef][Web of Science][Medline]
Vogel U, Christensen J, Nexo BA, Wallin H, Friis S, Tjonneland A. Peroxisome profilerator-activated receptor{gamma}2 Pro12Ala, interaction with alcohol intake and NSAID use, in relation to risk of breast cancer in a prospective study of Danes. Carcinogenesis (2007) 28:427–434.
Wan YJ, Morimoto M, Thurman RG, Bojes HK, French SW. Expression of the peroxisome proliferator-activated receptor gene is decreased in experimental alcoholic liver disease. Life Sci. (1995) 56:307–317.[CrossRef][Web of Science][Medline]
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