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Regulation of HMG-CoA reductase in MCF-7 cells by genistein, EPA, and DHA, alone and in combination with mevastatin
Cancer Letters 224 (2005) 221–228 www.elsevier.com/locate/canlet
Regulation of HMG-CoA reductase in MCF-7 cells by genistein, EPA, and DHA, alone and in combination with mevastatin Robin E. Duncana, Ahmed El-Sohemya, Michael C. Archera,b,* a
Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, FitzGerald Building, 150 College Street, Toronto, Ont., Canada M5S 3E2 b Department of Medical Biophysics, Faculty of Medicine, University of Toronto, FitzGerald Building, 150 College Street, Toronto, Ont., Canada M5S 3E2 Received 2 September 2004; received in revised form 11 October 2004; accepted 1 November 2004
Abstract We investigated the regulation of HMG-CoA reductase in MCF-7 human breast cancer cells by genistein, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). All three compounds down-regulated reductase activity, primarily through posttranscriptional effects. In mevastatin-treated cells, only genistein and DHA abrogated the induction of reductase activity caused by this competitive inhibitor. Diets rich in soy isoflavones and fish oils, therefore, may exert anti-cancer effects through the inhibition of mevalonate synthesis in the breast. Genistein and DHA, in particular, may augment the efficacy of statins, increasing the potential for use of these drugs in adjuvant therapy for breast cancer. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: HMG-CoA reductase; Breast cancer; Genistein; Docosahexaenoic acid; Eicosapentaenoic acids; Mevastatin
1. Introduction Breast cancer rates in North America exceed those in Japan by several-fold [1]. Studies in migrant populations demonstrate a rise in breast cancer risk within 10 years of migration from a region of lower risk to a region of higher risk, suggesting that these differences are due to environment and lifestyle rather * Corresponding author. Address: Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, FitzGerald Building, 150 College Street, Toronto, Ont., Canada M5S 3E2. Tel.: C1 416 978 8195; fax: C1 416 971 2366. E-mail address: [email protected] (M.C. Archer).
than genetic factors [1]. The traditional diet of women living in Japan provides a significantly greater intake of soy isoflavones [2], of which genistein is the most abundant [3], and fish oils [4] that are a rich source of the long-chain n-3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Soy isoflavones have been shown to inhibit the growth of breast cancer cells in culture [5–9] and mammary tumors in animals [10,11], and are associated with a reduced risk of breast cancer in human studies [12–14]. EPA and DHA also inhibit the growth of breast cancer cells in culture [15] and mammary tumors in nude mice [16,17], and are associated with breast cancer
0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.11.031
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protective effects in humans [18–20]. Mechanisms underlying the anti-cancer effects of genistein, EPA, and DHA, however, are not fully understood. The purpose of this study was to investigate the regulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase by genistein, EPA, and DHA. HMG-CoA reductase catalyzes the formation of mevalonate that is required for the synthesis of sterols, such as cholesterol, and the non-sterol isoprenoids. These compounds function in diverse cellular processes including membrane formation, hormone biosynthesis, and the activation of growth regulatory proteins and oncoproteins [21,22]. Depletion of cellular mevalonate by competitive inhibitors of HMG-CoA reductase (e.g. statins) arrests the growth of breast cancer cells in culture [23], inhibits the growth of mammary tumors in rodents [24,25], and is associated with a reduced risk of breast cancer in some studies [26–28], although not all studies have found this [29–31]. Conversely, increased mevalonate has been shown to promote the growth of breast cancer cells in vitro and in vivo [32]. We have previously reported that a diet rich in long-chain n-3 PUFAs down-regulates HMG-CoA reductase activity in normal rat mammary glands [33]. Others have shown that genistein inhibits cholesterol synthesis in HepG2 hepatocarcinoma cells [34], and competitively inhibits the activity of recombinant HMG-CoA reductase in vitro [35]. The regulation of HMG-CoA reductase in malignant breast cells by genistein, DHA, or EPA, however, has not been reported. Here we describe the effects of these compounds on reductase activity and mRNA expression in MCF-7 human breast adenocarcinoma cells. In response to mevalonate depletion by statins, cells undergo a compensatory induction of HMGCoA reductase protein [21]. This induced enzyme is inactive in cells because it is inhibited by the statin, but it can be detected in reductase assays of cellular extracts after the statin has been removed by washing and dilution [21]. Statins inhibit the growth of human breast cancer cells in culture [36] and have shown promising anti-cancer effects in rodent mammary tumorigenesis experiments [24,25], but cannot be used as chemotherapeutic agents due to dose-limiting myotoxicity [37]. Compounds that can attenuate the induction of reductase by statins may augment the effect of these competitive inhibitors, resulting in
increased efficacy at lower doses. Therefore, we also examined whether EPA, DHA, or genistein could temper the induction of reductase in cells treated with mevastatin.
2. Materials and methods 2.1. Materials Unless otherwise indicated, all chemicals and reagents were purchased from Sigma Chemical Company (Mississauga, Ont., Canada). Fetal bovine serum (FBS) was from Gibco BRL (Grand Island, NY); [14C]HMG-CoA was from Perkin Elmer Life Sciences Inc (Mississauga, Ont., Canada); the cell proliferation kit (enzyme-linked immunosorbent assay (ELISA)) was from Roche Molecular Biochemicals (Indianapolis, IN); silica gel G plates for chromatography were from Analtech Inc. (Newark, DE). Genistein from Sigma was dissolved in dimethylsulfoxide (DMSO) and stored at K20 8C. EPA and DHA were supplied by Sigma as free fatty acids, dissolved in 100% ethanol, and stored at K20 8C under nitrogen in light-proof containers. 2.2. Cell culture MCF-7 human breast cancer cells were purchased from the American Type Cell Culture collection and routinely cultured in 150 dL flasks at 37 8C and 5% CO2 in 1:1 DME/F12 with 1% penicillin/streptomycin, and 10% FBS. Experiments with EPA and DHA were carried out in 1:1 DME/F12 with 2% FBS and 2 g/L fatty acid-free bovine serum albumin that was added as a carrier for the fatty acids. Final ethanol concentration in test and control cells in fish oil experiments was 0.1%. Experiments with genistein were done in DME:F12 (1:1) with 5% FBS. DMSO was adjusted to the same concentration in test and control cells in each experiment to a maximum of 0.05%. Cells were given fresh treatments every 1 to 2 days. 2.3. Cell proliferation MCF-7 cells were seeded in 96 well plates in complete medium at an average density of 2!103
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cells per well. On the following day, medium was replaced with treatment medium. Fresh treatment medium was provided every 1 to 2 days. Proliferation rates on day 7 were measured according to manufacturer’s instructions by quantifying bromodeoxyuridine incorporation into DNA of actively proliferating cells using the ELISA kit. 2.4. HMG-CoA reductase activity After 7 days of growth in treatment medium, cells were harvested by trypsinization, washed repeatedly with PBS, and then disrupted with a glass minihomogenizer in 20 mM Tris-HCl (pH 7.2) containing 0.25 M sucrose, 70 mM KCl, 5 mM EDTA, 5 mM EGTA, 50 mM leupeptin, and 1 mM DTT. HMG-CoA reductase activity in samples of supernatant prepared by centrifugation of homogenates at 5000!g for 1 min was determined as described previously [33]. Briefly, 100 mg samples of total protein were preincubated at 37 8C for 5 min in 100 mM phosphate (pH 7.4) containing 70 mM KCl, 10 mM dithiothreitol, 5 mM EDTA, 5 mM EGTA, and 50 mM leupeptin. After a further 5 min incubation with an NADPH regenerating system (1 U glucose-6-phosphate dehydrogenase, 20 mM glucose-6-phosphate, 2 mM NADP), HMG-CoA containing 8.12 nCi/nmol [14C]HMG-CoA was added to yield a final concentration of 55.4 mM and assay mixtures (75 mL final volume) were incubated at 37 8C for 30 min. The assay was terminated by addition of 5 mL of 10 N HCL containing 16 mM mevalonolactone as a carrier for thin-layer chromatography. Samples were incubated for 1 h to allow complete lactonization of product, and then centrifuged for 1 min at 3000!g to remove denatured protein. Forty microliters of supernatant was applied to a silica gel G plate and chromatographed in toluene/acetone (1:1). Plates were then autoradiographed for 24–48 h using an Instant Imagerw (Canberra Packard Canada, Mississauga, Ont., Canada), after which bands corresponding to mevalonolactone (Rf 0.7) were visualized and quantified in counts per minute. Enzyme activity in samples treated with test compounds was normalized to the mean activity of the control samples in each individual experimental set. Relative enzyme activity was then expressed as a percentage of control values.
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2.5. Real time polymerase chain reaction Total RNA was harvested using TRI reagent (Sigma, Mississauga, Ont., Canada) according to the manufacturer’s protocol from cells grown for 7 days in the presence of 50 mM EPA or DHA, or 40 mM genistein, with or without 5 or 10 mM mevastatin, respectively. cDNA was synthesized from 5 mg of total RNA by random hexamer priming using the Superscript First Strand reverse transcriptase kit from Gibco. The expression of the HMG-CoA reductase and b-actin genes was then determined by real time polymerase chain reaction (PCR) using an Applied Biosystems Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). PCR amplification of 1 mL of cDNA in TaqMan Universal PCR Master Mix (total volume 25 mL) was performed using the universal temperature cycles: 10 min at 94 8C, followed by 40 two-temperature cycles (15 s at 94 8C and 1 min at 60 8C). Product amplification was measured using sequence-specific probes labeled with the fluorescent reporter molecule FAM at their 5 0 ends and with non-fluorescent quencher molecules at their 3 0 ends, that were designed to anneal to a sequence located between the forward and reverse primers. Probes and primers were obtained from Applied Biosystems for both HMG-CoA reductase and b-actin (Assay-by-Demand product #Hs00168352_m1 and #Hs99999903_m1, respectively). Expression of HMG-CoA reductase and b-actin were quantified by measuring the threshold cycle (CT), defined as the fractional cycle number at which the fluorescence exceeds a fixed threshold [38]. The expression of HMG-CoA reductase was then normalized to expression of b-actin using the method outlined in the ABI PRISM 7700 Sequence Detection System: User Bulletin #2 [39].
3. Results and discussion We have previously reported that HMG-CoA reductase activity is elevated in rat mammary tumors compared to normal mammary glands, and is resistant to feedback regulation by sterols [40]. Increased activity of HMG-CoA reductase in malignant cells has been shown to result not only from increased expression of reductase protein, but also increases in
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Fig. 1. The dietary compounds (A) genistein (B) EPA and (C) DHA inhibit MCF-7 cell proliferation and down-regulate HMG-CoA reductase activity after 1 week of treatment, relative to controls. (†P!0.05, *P!0.01 when compared to control by ANOVA with Dunnett’s post-hoc test).
the proportion of the active form and the catalytic efficiency of the enzyme [41,42]. The occurrence of changes that elevate the specific activity of the enzyme suggests that increased synthesis of mevalonate may contribute to the development of the malignant phenotype, rather than merely reflect an up-regulation of enzyme activity in response to accelerated growth [41]. Compounds that can inhibit
HMG-CoA reductase in tumor cells, therefore, may be effective as adjuvant chemotherapeutic agents. Here we have shown that treatment with the isoflavone genistein or the long chain n-3 PUFAs DHA or EPA significantly inhibits the proliferation of MCF-7 human breast cancer cells in a concentrationdependent manner (Fig. 1). Under the same treatment conditions, genistein, EPA and DHA also downregulated MCF-7 cell HMG-CoA reductase as measured by the enzyme activity of cell-free supernatants (Fig. 1). A dose-dependent response was seen for genistein on HMG-CoA reductase activity (P!0.001 for linear trend). Growth inhibition by this compound precluded testing the effects of concentrations higher than 50 mM. A significant dosedependent effect of DHA and EPA on HMG-CoA reductase was also evident (P!0.001 for linear trend for both n-3 PUFAs). Inhibition of cell growth and HMG-CoA reductase activity was seen at concentrations of n-3 PUFAs that were an order of magnitude lower than serum concentrations reported in a Japanese cohort [43]. Dietary sources, therefore, may provide EPA and DHA at levels sufficient to alter breast cancer cell growth and reductase regulation in vivo. Levels of genistein utilized in this study are in the range of maximum plasma concentrations reported in women following ingestion of a pharmacological dose of isoflavones [44]. This dose provided O20 times the amount of genistein consumed daily in the average Japanese diet [44]. Supplemental genistein may, therefore, be necessary to inhibit growth and HMG-CoA reductase activity in breast cancer cells in vivo. We utilized real time RT-PCR analysis of mRNA normalized to b-actin to investigate whether effects of the test compounds on HMG-CoA reductase may have been mediated transcriptionally. MCF-7 cells were treated with compounds at a concentration that had been shown to cause significant inhibition of cell proliferation and down-regulation of reductase activity. There was no significant change in reductase gene expression in cells treated with either 50 mM EPA or DHA or 40 mM genistein (Fig. 2). While this does not preclude the possibility that gene expression may be altered at higher concentrations, it does strongly suggest that down-regulation of HMG-CoA reductase by genistein, EPA, and DHA is mediated primarily at a post-transcriptional level. Our findings
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activity that follows mevastatin treatment. Treatment of MCF-7 cells with mevastatin alone resulted in a 10to 15-fold induction of HMG-CoA reductase activity (Fig. 3) in association with a 2.5- to 3.5-fold induction of HMG-CoA reductase mRNA expression (Fig. 4). Genistein significantly abrogated the induction of reductase, the activity of which was w40% lower in cells treated concomitantly with genistein and mevastatin compared to cells treated with mevastatin alone (Fig. 3). The magnitude of this effect was similar to that observed in cells grown in the absence of a competitive inhibitor, where 40 mM genistein was shown to down-regulate HMG-CoA reductase activity by w35% compared to untreated control cells (Fig. 1). Reductase mRNA, measured by real-time PCR, was unchanged by genistein in mevastatintreated cells. This indicates that down-regulation of reductase activity by genistein in these cells was
Fig. 2. HMG-CoA reductase mRNA expression, measured by realtime PCR, relative to b-actin in MCF-7 cells treated for 1 week with (A) 40 mM genistein or (B) 50 mM DHA or EPA, expressed relative to untreated control cells.
are similar to those reported for dietary tocotrienols that have been shown in HepG2 cells to regulate reductase activity post-transcriptionally [45]. However, it is clear that the effect of genistein in MCF-7 cells is different from that in HepG2 cells, in which this isoflavone has been shown to increase expression of HMG-CoA reductase mRNA [34]. Statin-induced mevalonate depletion in Chinese hamster ovary cells causes an adaptive induction of HMG-CoA reductase protein by up to 200-fold [21]. This induction is mediated by multiplicative effects, including an increase in the rate of reductase gene transcription (by up to 8-fold), an increase in the efficiency of mRNA translation (by up to 5-fold), and a decrease in the rate of reductase degradation (also by up to 5-fold) [21]. Since genistein, DHA and EPA down-regulated HMG-CoA reductase in MCF-7 cells post-transcriptionally through effects on translation and/or degradation, we examined whether these compounds may attenuate the induction of reductase
Fig. 3. HMG-CoA reductase activity in MCF-7 cells treated for 1 week with mevastatin alone or in combination with (A) genistein or (B) EPA or DHA at the concentrations indicated (*P!0.02, **P! 0.01 by Student’s t-test versus cells treated only with mevastatin).
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In summary, our results indicate that genistein, DHA, and EPA are able to down-regulate HMG-CoA reductase activity, primarily through post-transcriptional mechanisms. Diets rich in genistein and long chain n-3 PUFAs, therefore, may act to inhibit the growth of breast cancer, at least in part, through down-regulation of HMG-CoA reductase. Furthermore, DHA and genistein, but not EPA, attenuated the induction of reductase activity in mevastatin-treated cells. This suggests that dietary genistein or DHA may lower the dose of statin required to achieve the same level of reductase inhibition, thereby increasing the potential for use of these drugs in adjuvant therapy for breast cancer.
Acknowledgements
Fig. 4. HMG-CoA reductase mRNA expression, measured by realtime PCR, relative to b-actin in MCF-7 cells treated concomitantly with mevastatin and either (A) genistein or (B) DHA for 1 wk, relative to controls.
mediated entirely through post-transcriptional events (Fig. 4). DHA also significantly attenuated the induction of HMG-CoA reductase activity by mevastatin in MCF-7 cells. Reductase activity was w15% lower in cells treated concomitantly with DHA and mevastatin, compared to cells treated with mevastatin alone (Fig. 3). The magnitude of this down-regulation, however, was smaller than that observed in cells grown in the absence of mevastatin, in which DHA produced an w30% reduction in HMG-CoA reductase activity (Fig. 1). DHA caused no change in reductase mRNA in mevastatin-treated cells, indicating that regulation of reductase activity by DHA was mediated entirely at a post-transcriptional level. Unlike DHA or genistein, EPA produced no change in HMG-CoA reductase in mevastatin-treated cells (Fig. 3).
This research was supported by the US Army Medical Research Acquisition Activity, 820 Chandler Street, Fort Detrick, MD 21702-5014, Grant No. DAMD17-99-1-9409. The content of the information does not necessarily reflect the position or the policy of the US Government, and no official endorsement should be inferred. MCA is the recipient of a Natural Sciences and Engineering Research Council of Canada Industrial Research Chair, and acknowledges support from the member companies of the program in Food Safety, Nutrition and Regulatory Affairs of the University of Toronto. The authors thank HyeonJoo Lee for assistance.
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Regulation of HMG-CoA reductase in MCF-7 cells by genistein, EPA, and DHA, alone and in combination with mevastatin Robin E. Duncana, Ahmed El-Sohemya, Michael C. Archera,b,* a
Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, FitzGerald Building, 150 College Street, Toronto, Ont., Canada M5S 3E2 b Department of Medical Biophysics, Faculty of Medicine, University of Toronto, FitzGerald Building, 150 College Street, Toronto, Ont., Canada M5S 3E2 Received 2 September 2004; received in revised form 11 October 2004; accepted 1 November 2004
Abstract We investigated the regulation of HMG-CoA reductase in MCF-7 human breast cancer cells by genistein, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). All three compounds down-regulated reductase activity, primarily through posttranscriptional effects. In mevastatin-treated cells, only genistein and DHA abrogated the induction of reductase activity caused by this competitive inhibitor. Diets rich in soy isoflavones and fish oils, therefore, may exert anti-cancer effects through the inhibition of mevalonate synthesis in the breast. Genistein and DHA, in particular, may augment the efficacy of statins, increasing the potential for use of these drugs in adjuvant therapy for breast cancer. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: HMG-CoA reductase; Breast cancer; Genistein; Docosahexaenoic acid; Eicosapentaenoic acids; Mevastatin
1. Introduction Breast cancer rates in North America exceed those in Japan by several-fold [1]. Studies in migrant populations demonstrate a rise in breast cancer risk within 10 years of migration from a region of lower risk to a region of higher risk, suggesting that these differences are due to environment and lifestyle rather * Corresponding author. Address: Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, FitzGerald Building, 150 College Street, Toronto, Ont., Canada M5S 3E2. Tel.: C1 416 978 8195; fax: C1 416 971 2366. E-mail address: [email protected] (M.C. Archer).
than genetic factors [1]. The traditional diet of women living in Japan provides a significantly greater intake of soy isoflavones [2], of which genistein is the most abundant [3], and fish oils [4] that are a rich source of the long-chain n-3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Soy isoflavones have been shown to inhibit the growth of breast cancer cells in culture [5–9] and mammary tumors in animals [10,11], and are associated with a reduced risk of breast cancer in human studies [12–14]. EPA and DHA also inhibit the growth of breast cancer cells in culture [15] and mammary tumors in nude mice [16,17], and are associated with breast cancer
0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.11.031
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protective effects in humans [18–20]. Mechanisms underlying the anti-cancer effects of genistein, EPA, and DHA, however, are not fully understood. The purpose of this study was to investigate the regulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase by genistein, EPA, and DHA. HMG-CoA reductase catalyzes the formation of mevalonate that is required for the synthesis of sterols, such as cholesterol, and the non-sterol isoprenoids. These compounds function in diverse cellular processes including membrane formation, hormone biosynthesis, and the activation of growth regulatory proteins and oncoproteins [21,22]. Depletion of cellular mevalonate by competitive inhibitors of HMG-CoA reductase (e.g. statins) arrests the growth of breast cancer cells in culture [23], inhibits the growth of mammary tumors in rodents [24,25], and is associated with a reduced risk of breast cancer in some studies [26–28], although not all studies have found this [29–31]. Conversely, increased mevalonate has been shown to promote the growth of breast cancer cells in vitro and in vivo [32]. We have previously reported that a diet rich in long-chain n-3 PUFAs down-regulates HMG-CoA reductase activity in normal rat mammary glands [33]. Others have shown that genistein inhibits cholesterol synthesis in HepG2 hepatocarcinoma cells [34], and competitively inhibits the activity of recombinant HMG-CoA reductase in vitro [35]. The regulation of HMG-CoA reductase in malignant breast cells by genistein, DHA, or EPA, however, has not been reported. Here we describe the effects of these compounds on reductase activity and mRNA expression in MCF-7 human breast adenocarcinoma cells. In response to mevalonate depletion by statins, cells undergo a compensatory induction of HMGCoA reductase protein [21]. This induced enzyme is inactive in cells because it is inhibited by the statin, but it can be detected in reductase assays of cellular extracts after the statin has been removed by washing and dilution [21]. Statins inhibit the growth of human breast cancer cells in culture [36] and have shown promising anti-cancer effects in rodent mammary tumorigenesis experiments [24,25], but cannot be used as chemotherapeutic agents due to dose-limiting myotoxicity [37]. Compounds that can attenuate the induction of reductase by statins may augment the effect of these competitive inhibitors, resulting in
increased efficacy at lower doses. Therefore, we also examined whether EPA, DHA, or genistein could temper the induction of reductase in cells treated with mevastatin.
2. Materials and methods 2.1. Materials Unless otherwise indicated, all chemicals and reagents were purchased from Sigma Chemical Company (Mississauga, Ont., Canada). Fetal bovine serum (FBS) was from Gibco BRL (Grand Island, NY); [14C]HMG-CoA was from Perkin Elmer Life Sciences Inc (Mississauga, Ont., Canada); the cell proliferation kit (enzyme-linked immunosorbent assay (ELISA)) was from Roche Molecular Biochemicals (Indianapolis, IN); silica gel G plates for chromatography were from Analtech Inc. (Newark, DE). Genistein from Sigma was dissolved in dimethylsulfoxide (DMSO) and stored at K20 8C. EPA and DHA were supplied by Sigma as free fatty acids, dissolved in 100% ethanol, and stored at K20 8C under nitrogen in light-proof containers. 2.2. Cell culture MCF-7 human breast cancer cells were purchased from the American Type Cell Culture collection and routinely cultured in 150 dL flasks at 37 8C and 5% CO2 in 1:1 DME/F12 with 1% penicillin/streptomycin, and 10% FBS. Experiments with EPA and DHA were carried out in 1:1 DME/F12 with 2% FBS and 2 g/L fatty acid-free bovine serum albumin that was added as a carrier for the fatty acids. Final ethanol concentration in test and control cells in fish oil experiments was 0.1%. Experiments with genistein were done in DME:F12 (1:1) with 5% FBS. DMSO was adjusted to the same concentration in test and control cells in each experiment to a maximum of 0.05%. Cells were given fresh treatments every 1 to 2 days. 2.3. Cell proliferation MCF-7 cells were seeded in 96 well plates in complete medium at an average density of 2!103
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cells per well. On the following day, medium was replaced with treatment medium. Fresh treatment medium was provided every 1 to 2 days. Proliferation rates on day 7 were measured according to manufacturer’s instructions by quantifying bromodeoxyuridine incorporation into DNA of actively proliferating cells using the ELISA kit. 2.4. HMG-CoA reductase activity After 7 days of growth in treatment medium, cells were harvested by trypsinization, washed repeatedly with PBS, and then disrupted with a glass minihomogenizer in 20 mM Tris-HCl (pH 7.2) containing 0.25 M sucrose, 70 mM KCl, 5 mM EDTA, 5 mM EGTA, 50 mM leupeptin, and 1 mM DTT. HMG-CoA reductase activity in samples of supernatant prepared by centrifugation of homogenates at 5000!g for 1 min was determined as described previously [33]. Briefly, 100 mg samples of total protein were preincubated at 37 8C for 5 min in 100 mM phosphate (pH 7.4) containing 70 mM KCl, 10 mM dithiothreitol, 5 mM EDTA, 5 mM EGTA, and 50 mM leupeptin. After a further 5 min incubation with an NADPH regenerating system (1 U glucose-6-phosphate dehydrogenase, 20 mM glucose-6-phosphate, 2 mM NADP), HMG-CoA containing 8.12 nCi/nmol [14C]HMG-CoA was added to yield a final concentration of 55.4 mM and assay mixtures (75 mL final volume) were incubated at 37 8C for 30 min. The assay was terminated by addition of 5 mL of 10 N HCL containing 16 mM mevalonolactone as a carrier for thin-layer chromatography. Samples were incubated for 1 h to allow complete lactonization of product, and then centrifuged for 1 min at 3000!g to remove denatured protein. Forty microliters of supernatant was applied to a silica gel G plate and chromatographed in toluene/acetone (1:1). Plates were then autoradiographed for 24–48 h using an Instant Imagerw (Canberra Packard Canada, Mississauga, Ont., Canada), after which bands corresponding to mevalonolactone (Rf 0.7) were visualized and quantified in counts per minute. Enzyme activity in samples treated with test compounds was normalized to the mean activity of the control samples in each individual experimental set. Relative enzyme activity was then expressed as a percentage of control values.
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2.5. Real time polymerase chain reaction Total RNA was harvested using TRI reagent (Sigma, Mississauga, Ont., Canada) according to the manufacturer’s protocol from cells grown for 7 days in the presence of 50 mM EPA or DHA, or 40 mM genistein, with or without 5 or 10 mM mevastatin, respectively. cDNA was synthesized from 5 mg of total RNA by random hexamer priming using the Superscript First Strand reverse transcriptase kit from Gibco. The expression of the HMG-CoA reductase and b-actin genes was then determined by real time polymerase chain reaction (PCR) using an Applied Biosystems Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). PCR amplification of 1 mL of cDNA in TaqMan Universal PCR Master Mix (total volume 25 mL) was performed using the universal temperature cycles: 10 min at 94 8C, followed by 40 two-temperature cycles (15 s at 94 8C and 1 min at 60 8C). Product amplification was measured using sequence-specific probes labeled with the fluorescent reporter molecule FAM at their 5 0 ends and with non-fluorescent quencher molecules at their 3 0 ends, that were designed to anneal to a sequence located between the forward and reverse primers. Probes and primers were obtained from Applied Biosystems for both HMG-CoA reductase and b-actin (Assay-by-Demand product #Hs00168352_m1 and #Hs99999903_m1, respectively). Expression of HMG-CoA reductase and b-actin were quantified by measuring the threshold cycle (CT), defined as the fractional cycle number at which the fluorescence exceeds a fixed threshold [38]. The expression of HMG-CoA reductase was then normalized to expression of b-actin using the method outlined in the ABI PRISM 7700 Sequence Detection System: User Bulletin #2 [39].
3. Results and discussion We have previously reported that HMG-CoA reductase activity is elevated in rat mammary tumors compared to normal mammary glands, and is resistant to feedback regulation by sterols [40]. Increased activity of HMG-CoA reductase in malignant cells has been shown to result not only from increased expression of reductase protein, but also increases in
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Fig. 1. The dietary compounds (A) genistein (B) EPA and (C) DHA inhibit MCF-7 cell proliferation and down-regulate HMG-CoA reductase activity after 1 week of treatment, relative to controls. (†P!0.05, *P!0.01 when compared to control by ANOVA with Dunnett’s post-hoc test).
the proportion of the active form and the catalytic efficiency of the enzyme [41,42]. The occurrence of changes that elevate the specific activity of the enzyme suggests that increased synthesis of mevalonate may contribute to the development of the malignant phenotype, rather than merely reflect an up-regulation of enzyme activity in response to accelerated growth [41]. Compounds that can inhibit
HMG-CoA reductase in tumor cells, therefore, may be effective as adjuvant chemotherapeutic agents. Here we have shown that treatment with the isoflavone genistein or the long chain n-3 PUFAs DHA or EPA significantly inhibits the proliferation of MCF-7 human breast cancer cells in a concentrationdependent manner (Fig. 1). Under the same treatment conditions, genistein, EPA and DHA also downregulated MCF-7 cell HMG-CoA reductase as measured by the enzyme activity of cell-free supernatants (Fig. 1). A dose-dependent response was seen for genistein on HMG-CoA reductase activity (P!0.001 for linear trend). Growth inhibition by this compound precluded testing the effects of concentrations higher than 50 mM. A significant dosedependent effect of DHA and EPA on HMG-CoA reductase was also evident (P!0.001 for linear trend for both n-3 PUFAs). Inhibition of cell growth and HMG-CoA reductase activity was seen at concentrations of n-3 PUFAs that were an order of magnitude lower than serum concentrations reported in a Japanese cohort [43]. Dietary sources, therefore, may provide EPA and DHA at levels sufficient to alter breast cancer cell growth and reductase regulation in vivo. Levels of genistein utilized in this study are in the range of maximum plasma concentrations reported in women following ingestion of a pharmacological dose of isoflavones [44]. This dose provided O20 times the amount of genistein consumed daily in the average Japanese diet [44]. Supplemental genistein may, therefore, be necessary to inhibit growth and HMG-CoA reductase activity in breast cancer cells in vivo. We utilized real time RT-PCR analysis of mRNA normalized to b-actin to investigate whether effects of the test compounds on HMG-CoA reductase may have been mediated transcriptionally. MCF-7 cells were treated with compounds at a concentration that had been shown to cause significant inhibition of cell proliferation and down-regulation of reductase activity. There was no significant change in reductase gene expression in cells treated with either 50 mM EPA or DHA or 40 mM genistein (Fig. 2). While this does not preclude the possibility that gene expression may be altered at higher concentrations, it does strongly suggest that down-regulation of HMG-CoA reductase by genistein, EPA, and DHA is mediated primarily at a post-transcriptional level. Our findings
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activity that follows mevastatin treatment. Treatment of MCF-7 cells with mevastatin alone resulted in a 10to 15-fold induction of HMG-CoA reductase activity (Fig. 3) in association with a 2.5- to 3.5-fold induction of HMG-CoA reductase mRNA expression (Fig. 4). Genistein significantly abrogated the induction of reductase, the activity of which was w40% lower in cells treated concomitantly with genistein and mevastatin compared to cells treated with mevastatin alone (Fig. 3). The magnitude of this effect was similar to that observed in cells grown in the absence of a competitive inhibitor, where 40 mM genistein was shown to down-regulate HMG-CoA reductase activity by w35% compared to untreated control cells (Fig. 1). Reductase mRNA, measured by real-time PCR, was unchanged by genistein in mevastatintreated cells. This indicates that down-regulation of reductase activity by genistein in these cells was
Fig. 2. HMG-CoA reductase mRNA expression, measured by realtime PCR, relative to b-actin in MCF-7 cells treated for 1 week with (A) 40 mM genistein or (B) 50 mM DHA or EPA, expressed relative to untreated control cells.
are similar to those reported for dietary tocotrienols that have been shown in HepG2 cells to regulate reductase activity post-transcriptionally [45]. However, it is clear that the effect of genistein in MCF-7 cells is different from that in HepG2 cells, in which this isoflavone has been shown to increase expression of HMG-CoA reductase mRNA [34]. Statin-induced mevalonate depletion in Chinese hamster ovary cells causes an adaptive induction of HMG-CoA reductase protein by up to 200-fold [21]. This induction is mediated by multiplicative effects, including an increase in the rate of reductase gene transcription (by up to 8-fold), an increase in the efficiency of mRNA translation (by up to 5-fold), and a decrease in the rate of reductase degradation (also by up to 5-fold) [21]. Since genistein, DHA and EPA down-regulated HMG-CoA reductase in MCF-7 cells post-transcriptionally through effects on translation and/or degradation, we examined whether these compounds may attenuate the induction of reductase
Fig. 3. HMG-CoA reductase activity in MCF-7 cells treated for 1 week with mevastatin alone or in combination with (A) genistein or (B) EPA or DHA at the concentrations indicated (*P!0.02, **P! 0.01 by Student’s t-test versus cells treated only with mevastatin).
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In summary, our results indicate that genistein, DHA, and EPA are able to down-regulate HMG-CoA reductase activity, primarily through post-transcriptional mechanisms. Diets rich in genistein and long chain n-3 PUFAs, therefore, may act to inhibit the growth of breast cancer, at least in part, through down-regulation of HMG-CoA reductase. Furthermore, DHA and genistein, but not EPA, attenuated the induction of reductase activity in mevastatin-treated cells. This suggests that dietary genistein or DHA may lower the dose of statin required to achieve the same level of reductase inhibition, thereby increasing the potential for use of these drugs in adjuvant therapy for breast cancer.
Acknowledgements
Fig. 4. HMG-CoA reductase mRNA expression, measured by realtime PCR, relative to b-actin in MCF-7 cells treated concomitantly with mevastatin and either (A) genistein or (B) DHA for 1 wk, relative to controls.
mediated entirely through post-transcriptional events (Fig. 4). DHA also significantly attenuated the induction of HMG-CoA reductase activity by mevastatin in MCF-7 cells. Reductase activity was w15% lower in cells treated concomitantly with DHA and mevastatin, compared to cells treated with mevastatin alone (Fig. 3). The magnitude of this down-regulation, however, was smaller than that observed in cells grown in the absence of mevastatin, in which DHA produced an w30% reduction in HMG-CoA reductase activity (Fig. 1). DHA caused no change in reductase mRNA in mevastatin-treated cells, indicating that regulation of reductase activity by DHA was mediated entirely at a post-transcriptional level. Unlike DHA or genistein, EPA produced no change in HMG-CoA reductase in mevastatin-treated cells (Fig. 3).
This research was supported by the US Army Medical Research Acquisition Activity, 820 Chandler Street, Fort Detrick, MD 21702-5014, Grant No. DAMD17-99-1-9409. The content of the information does not necessarily reflect the position or the policy of the US Government, and no official endorsement should be inferred. MCA is the recipient of a Natural Sciences and Engineering Research Council of Canada Industrial Research Chair, and acknowledges support from the member companies of the program in Food Safety, Nutrition and Regulatory Affairs of the University of Toronto. The authors thank HyeonJoo Lee for assistance.
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