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Effect of ethanol on proliferation and estrogen receptor-α expression in human breast cancer cells
Cancer Letters 165 (2001) 131±137
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Effect of ethanol on proliferation and estrogen receptor-a expression in human breast cancer cells Keith W. Singletary*, Randall S. Frey, Wen Yan Department of Food Science and Human Nutrition, University of Illinois, 467 Bevier Hall, 905 South Goodwin Avenue, Urbana-Champaign, IL 61801, USA Received 13 November 2000; received in revised form 17 January 2001; accepted 18 January 2001
Abstract There is substantial epidemiological evidence suggesting that alcohol consumption is associated with increased risk for breast cancer. However, possible biological mechanisms have not been clearly established. In the present studies, a direct effect of ethanol on the proliferation and intracellular content of cyclic AMP (cAMP) in two estrogen receptor-positive (ER1) and two estrogen receptor-negative (ER2) human breast cancer cell lines was examined. Treatment of ER1 human breast cancer cells (MCF-7 and ZR75.1) with ethanol at concentrations between 10 and 100 mM was associated with increased cell numbers compared to controls. The ERa content and the amount of intracellular cAMP also increased in ER1 cells exposed to ethanol, compared to controls. On the other hand, ethanol treatment did not increase cell proliferation or cAMP levels in the ER2 (BT20 and MDA-MB-231) human breast cancer cells. Therefore, ethanol added at physiologically relevant concentrations to ER1 human breast cancer cell cultures can enhance cell proliferation and increase the content of ERa. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ethanol; Breast cancer; Cyclic AMP; Estrogen receptor
1. Introduction Breast cancer is a leading cause of cancer-related death among American women. Although the topic is controversial, a majority of epidemiological studies report that alcohol consumption is positively associated with breast cancer risk even at moderate levels of intake [1,2]. Several mechanisms of action have been put forth to explain this effect of alcohol on breast cancer, including the association of alcohol consumption with enhanced circulating levels of estradiol and other steroid hormones [3±7], impaired * Corresponding author. Tel.: 11-217-333-5549; fax: 11-217244-2455. E-mail address: [email protected] (K.W. Singletary).
immune defenses, increased mammary gland proliferation, and altered carcinogen metabolism [8]. Of recent interest is evidence that an interaction between alcohol intake and other breast cancer risk factors may be occurring [9]. In particular, subgroups of alcoholconsuming women may differ in their risk for breast cancer depending on their type of breast tumor as determined by hormone receptor status. For example, alcohol intake has been linked to increased risk for breast tumors possessing estrogen receptors (ER1) or estrogen and progesterone receptors (ER1/PR1) [10±12]. In contrast, others have reported, a positive association of alcohol consumption with ER2/PR2 tumors, an inverse relationship between alcohol intake and prevalence of ER2 rich tumors, or no association at all [10,13±15]. Only one report has indi-
0304-3835/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(01)00419-0
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cated that exposure to ethanol in vitro increases proliferation of ER1 human breast cancer cells [16]. The ER and other steroid hormone receptors are members of the nuclear receptor superfamily capable of eliciting a variety of cell responses and in¯uencing signal transduction pathways both in ligand-dependent and ligand-independent manners [17±19]. Thus, the interaction of alcohol with speci®c subtypes of human breast cancer cells (based on hormone receptor status) deserves further attention. The present studies, therefore, were conducted to examine a direct effect of ethanol exposure on the proliferation of two ER1 human mammary cancer cell lines (MCF-7 and ZR75-1) and two ER2 human mammary cell lines (BT20 and MDA-MB-231), and to determine whether an effect of ethanol on ER1 breast cancer cells is associated with changes in ERa levels. 2. Material and methods The human breast cancer cell lines MCF-7, ZR-751, BT-20 and MDA-MB-231 were obtained from the American Type Culture Collection (Rockville, MD). MCF-7, ZR-75-1 and BT-20 cells were grown (378C, 5% CO2) in Eagle's minimum essential medium containing 5% fetal bovine serum, 10 mM HEPES buffer, 1% MEM non-essential amino acids, 2 mM glutamine penicillin (100 units/ml), streptomycin (100 mg/ml), hydrocortisone (3.75 ng/ml) and insulin (6 ng/ml). MDA-MB-231 cells were grown in Leibovitz's medium L-15 supplemented as described by Berthois et al. [20]. For cell proliferation experiments, cells (2±5 £ 104 ) were plated in 60-mm tissue culture plates containing 3 ml of medium lacking ethanol. After 24 h of cell attachment, the medium was replaced and supplemented with ethanol at concentrations ranging from 0 to 100 mM. Concentrations in this range correspond to blood levels of ethanol in humans, which could result from low to high intakes of alcohol. Cells in triplicate dishes per treatment group were exposed to ethanol for periods up to 10 days. Fresh ethanol-containing medium was added to cells daily. Cells were then collected and counted using a hemocytometer, after ensuring that cells were well dispersed. For measurement of ERa, MCF-7 and ZR 75.1 cells were exposed to ethanol for the speci®ed number
of days and then collected in lysis buffer (1% Na deoxycholate, 1% Triton X-100, 0.01% SDS, 150 mM NaCl, 50 mM Tris (pH 7.5), 0.5 mM NaVO4, 1 mM PMSF and 20 mg/ml aprotonin, leupeptin, and pepstatin), sonicated and centrifuged. The pellet was discarded and protein content of the lysate determined. Equal amounts of protein were loaded on a 10% acrylamide gel and SDS±PAGE performed. After electrophoresis, the gel was stained with zinc stain (BioRad) and photographed to con®rm equal protein loading. The gel was then destained and membrane transfer was accomplished. After transfer to nitrocellulose membrane, ERa was detected by use of a mouse anti-human monoclonal ERa antibody (AB-10, Neomarkers, Union City, CA) and quantitated by ECL. Cyclic AMP was quantitated by radioimmunoassay as described by Aronica et al. [21]. Brie¯y, cells were scraped from plates into TNE buffer (40 mM Tris± HCl (pH 7.5); 140 mM NaCl; 1.5 mM EDTA) on ice and pelleted. Cell pellets were resuspended in 200 ml ice-cold cAMP extraction buffer (50 mM Tris±HCl (pH 7.5); 4 mM EDTA) and sonicated. Extracts remained on ice for 15 min, with regular mixing. An aliquot of extract was analyzed for protein content by the BCA assay (Pierce) and the remaining portion was boiled for 10 min. Cell debris was pelleted and supernatant removed for cAMP quantitation using a cAMP radioimmunoassay kit (Amersham). Statistically signi®cant differences between treatments were determined by analysis of variance and the application of Fisher's least signi®cant difference test for post hoc comparisons. Mean differences at P , 0:05 were considered statistically signi®cant. 3. Results Exposure of ER1 cell lines to increasing concentrations of ethanol was associated with an increase in cell proliferation (Table 1). For example, ethanol added to cultures of cells at concentrations of 20±50 mM signi®cantly stimulated proliferation of MCF-7 and ZR75.1 cells by 53±91% following 7 and 10 days of treatment, compared to controls. For both cell lines, cell proliferation was signi®cantly enhanced at both days 7 and 10 of ethanol treatment, with no consistent increase in proliferation being observed following 2
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Table 1 Effect of ethanol concentration on proliferation of ER1 human breast cancer cell lines (MCF-7 and ZR-75-1) a Ethanol concentration (mM)
Cell number/plate ( £ 10 4) MCF-7
0 5 10 20 50 100
ZR-75-1
Day 7
Day 10
Day 7
Day 10
50.0 ^ 1.1 a 51.7 ^ 0.9 a 68.0 ^ 2.3 b 77.0 ^ 1.5 c 80.3 ^ 0.8 c 93.3 ^ 2.9 d
94.3 ^ 0.9 a 118.3 ^ 4.1 b 127.7 ^ 3.4 b 144.3 ^ 3.8 c 172.7 ^ 6.6 d 201.3 ^ 4.3 e
11.0 ^ 1.1 a 14.7 ^ 0.7 b 15.7 ^ 1.2 c 17.7 ^ 0.9 c,d 21.0 ^ 1.5 d 25.7 ^ 1.2 e
38.6 ^ 1.7 a 40.3 ^ 0.9 a 49.3 ^ 1.2 b 59.3 ^ 1.8 c 69.7 ^ 1.4 d 86.3 ^ 0.9 e
a Values represent mean ^ SE of three determinations, and are representative of at least two separate experiments. Means among treatment groups sharing unlike superscripts are signi®cantly different.
mM increased ERa levels by approximately 2.1±5fold, compared to controls, although only the 50 mM values were statistically different. No effect of ethanol on ERa levels in either cell line was observed after only 1 day treatment with ethanol. The amount of cAMP in all cells also was measured, because of reports that cAMP is elevated in liver cells that exhibit increased proliferation in response to ethanol exposure [22±24]. The cellular content of cAMP in the MCF-7 and ZR75.1 cell lines increased in response to ethanol supplementation (Table 3), although the timing of the increase differed between the two cell lines. For MCF-7 cells, incubation with 25 or 100 mM ethanol for 2, 4 and 8 days signi®cantly increased intracellular cAMP content 1.3±1.8-fold, compared with controls. Exposure of
days of treatment with ethanol. No changes in viability, size or morphology of cells in these two lines were discernible following treatment with ethanol, compared with controls. In contrast to that for the ER1 cell lines, neither of the ER2 cell lines (BT20 and MDA-MB-231) responded to ethanol exposure for 6 and 10 days with an increase in cell number (Table 2). To determine whether treatment with ethanol affected ER levels in the ER1 human breast cancer cell lines, ZR 75.1 and MCF-7 cells were treated with several concentrations of ethanol for either 1 or 7 days. After 7 days of treatment with ethanol, an increase in the immunoreactive content of ERa was observed for both cell lines (Fig. 1). Exposure of ZR 75.1 and MCF-7 cells to ethanol doses of 25 and 50
Table 2 Effect of ethanol concentration on proliferation of ER2 human breast cancer cell lines (MDA-MB-231 and BT-20) a Ethanol concentration (mM)
Cell number/plate ( £ 10 4) MDA-MB-231 Day 6
0 5 10 20 50 100
BT-20 Day 10
a
25.0 ^ 1.5 25.0 ^ 2.5 a 25.0 ^ 1.1 a 26.3 ^ 1.7 a 25.0 ^ 1.1 a 26.7 ^ 1.2 a
Day 6 a
55.3 ^ 2.9 52.0 ^ 2.3 a 52.0 ^ 2.1 a 52.3 ^ 2.9 a 59.3 ^ 1.8 a 57.3 ^ 1.2 a
Day 10 a
40.7 ^ 1.2 42.3 ^ 0.9 a 43.0 ^ 2.5 a 43.7 ^ 1.8 a 42.3 ^ 0.9 a 41.0 ^ 0.6 a
111.7 ^ 3.4 a 104.7 ^ 0.9 a 104.0 ^ 1.5 a 106.7 ^ 2.0 a 107.3 ^ 2.2 a 113.7 ^ 1.4 a
a Values represent mean ^ SE of three determinations and are representative of at least two separate experiments. Means among treatment groups sharing unlike superscripts are signi®cantly different.
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ZR75.1 cells to ethanol at 25 and 100 mM concentrations was associated with signi®cant increases in cellular cAMP after 1- and 2-day incubation periods
(Table 3), but not at day 8. In contrast to the ER1 cells, the content of cAMP in the ER2 cell lines did not increase after exposure to ethanol at any of the concentrations examined from 5 to 100 mM for 2±7 days (data not shown). 4. Discussion
Fig. 1. In¯uence of ethanol treatment (7 days) on ERa expression in cultures of ZR75.1 (a,b) and MCF-7 (c,d) cells. Values and densitometric scans are representative of two separate experiments. *Signi®cant at P , 0:05.
The present studies indicate that treatment with ethanol is associated with increased proliferation of two ER1 human breast cancer cell lines. The concentrations of ethanol at which signi®cant increases in proliferation were observed correspond to blood levels in humans that are attainable after moderate to high intakes of ethanol. For instance, 54 kg women consuming up to three drinks per day have been reported to have blood ethanol concentrations from 5 to 24 mM, and those women drinking daily 0.75 g/kg body weight peak blood ethanol levels of 20±24 mM [25,26]. In contrast to that for ER1 cells, no increase in cell proliferation was observed for two ER2 cell lines supplemented with the same ethanol concentrations. Our ®ndings are similar to those reported by Przylipiak et al. [16], who observed that ethanol added to cultures of MCF-7 cells at concentrations of 0.001±10% signi®cantly enhanced DNA synthesis. We are not aware of other reports in which a stimulating effect of ethanol on human breast cancer cell multiplication was observed, although an enhancement of cell division in rat mammary, liver and rectal cells in response to ethanol and/or acetaldehyde exposure has been reported [22,27±29]. The selective stimulation of ER1 cells in our studies is noteworthy, since there is recent evidence that ethanol intake by women may enhance the risk for ER1 or ER1/PR1 breast tumors [11,12]. However, our reported association of ethanol with increased proliferation of ER1 human cancer requires additional study before ®rm comparisons with human ®ndings can be drawn. First, it is not clear whether the presence or lack of ER or PR represent different diseases or different stages in the progression of the same disease [30], making it dif®cult to interpret how exposure to ethanol will impact the relative balance of ER2 and ER1 cells in the tumors of women. Also, some of the data on ER status of tumors in postmenopausal women is dependent on whether hormone
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Table 3 In¯uence of ethanol concentration on intracellular cAMP content of MCF-7 and ZR-75-1 cells a Ethanol concentration (mM)
cAMP content (pmol cAMP/mg protein) MCF-7
0 5 25 100
ZR-75-1
Day 2
Day 4
Day 8
Day 1
Day 2
Day 8
19.4 ^ 1.5 a 23.2 ^ 1.8 a,b 26.0 ^ 0.5 b 25.7 ^ 0.5 b
21.9 ^ 1.3 a 22.5 ^ 1.4 a 32.4 ^ 7.7 a,b 37.8 ^ 9.2 b
36.7 ^ 3.1 a 49.6 ^ 9.0 a,b 55.5 ^ 6.0 b 67.2 ^ 2.5 b
6.5 ^ 0.7 a 7.4 ^ 0.2 a,b 8.3 ^ 0.3 b 8.6 ^ 0.6 b
6.0 ^ 0.8 a 8.2 ^ 1.1 a 11.4 ^ 0.8 b 13.1 ^ 0.6 b
3.7 ^ 0.8 a 3.9 ^ 0.4 a 3.6 ^ 0.3 a 4.1 ^ 0.4 a
a Values represent mean ^ SE of three determinations and are representative of data from two separate experiments. Means among treatment groups sharing unlike superscripts are signi®cantly different.
(estrogen) replacement therapy is being used [10]. Thus, the effect of both ethanol dose and hormone exposure on the breast cancer cells needs to be more carefully quanti®ed. The mechanism whereby ethanol stimulates cell proliferation in ER1 human breast cancer cells in this study nor why the effect is time-dependent is not known. Although ethanol treatment of ZR 75.1 and MCF-7 cells was associated with an increase in cell cAMP content, the temporal change in intracellular cAMP content was not consistent between cell lines and did not necessarily coincide with the increases in cell number. For ZR 75.1 cells, cell proliferation was not signi®cantly elevated at 1 or 2 days of exposure to ethanol, yet cAMP content increased signi®cantly at days 1 and 2 of treatment but not by 8 days of ethanol exposure. On the other hand for MCF-7 cells, ethanol treatment at concentrations greater than 25 mM was associated with an increase in cAMP throughout the 8-day period. This suggests that continuous elevation of cellular cAMP may not be necessary for the increased cell proliferation in response to ethanol, or that other ethanol-associated intracellular events are more important in increasing cell proliferation. The relationship of ethanol to intracellular cAMP is tissue-speci®c, with ethanol either stimulating or lowering cAMP production depending on the tissue examined [23,24,35]. Our results are similar to others [22±24] in which ethanol treatment of hepatocytes is associated with increased proliferation and cAMP production. We also observed that in these ER1 human breast cancer cells, ethanol's impact on cell numbers and on ERa levels both occur after several days of exposure to ethanol,
suggesting that an ethanol-associated increase in ERa may play a role in the enhancement of ER1 breast cancer cell proliferation. However, the increased content of ERa cannot completely explain the increase in cell proliferation, since increased cell numbers were observed in response to ethanol at doses which did not produce signi®cant increases in ERa content. This could indicate that ethanol-induced changes in ER activity may be important in addition to total amounts of ERa. In this regard, Fan et al. [31] recently reported that treatment of human breast cancer cells with ethanol increased both ERa levels and activity. There is evidence that alcohol may stimulate the content of nuclear estrogen receptors in the hypothalamus of the male rat [32], and may enhance estrogen-induced rat hepatocarcinogenesis by enhancing cell proliferation via alterations in estrogen and progesterone receptor kinetics [33,34]. It is known that the ER activity of human breast cancer cells is affected by multiple cell-signaling pathways, including those mediated by cAMP and IGF-1 [36]. For example, Aronica et al. [21] observed that estrogen treatment increases cellular cAMP content in ER1 human breast cancer cells, resulting in a stimulation of cAMP response element-mediated gene transcription. It was suggested that, since cAMP is known to regulate the activity of ER and PR in human breast cancer cells, increased cAMP content may activate protein kinase pathways leading to enhanced ER phosphorylation and ER2 mediated transactivation [18]. Thus, the capacity of ethanol to in¯uence signal transduction pathways regulating the proliferation, ER status, and ER activity of human breast cancer cells warrants further characterization.
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In summary, we report that supplementation of cultures of ER1 human breast cancer cell lines MCF-7 and ZR75.1 with ethanol is associated with increased cell proliferation and cellular cAMP content. In addition, in ZR75.1 and MCF-7 cells, ethanol treatment was associated with substantial increases in ERa content. In contrast, no increase in cell division or cAMP content was observed when cultures of the ER2 human breast cancer cell lines BT-20 and MDA-MB-231 were supplemented with the same concentrations of ethanol. The capacity of ethanol to stimulate proliferation of speci®c types of human breast cancer cells, rather than all breast cancer cells, might partly explain the generally modest increases in breast cancer risk reported in epidemiological studies, since an increased development of breast tumors may only be apparent for a subset of tumors with speci®c hormone receptor characteristics. The mechanism for the selective effect of ethanol on human breast cancer cell proliferation, cAMP content, and ER levels warrants further study.
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www.elsevier.com/locate/canlet
Effect of ethanol on proliferation and estrogen receptor-a expression in human breast cancer cells Keith W. Singletary*, Randall S. Frey, Wen Yan Department of Food Science and Human Nutrition, University of Illinois, 467 Bevier Hall, 905 South Goodwin Avenue, Urbana-Champaign, IL 61801, USA Received 13 November 2000; received in revised form 17 January 2001; accepted 18 January 2001
Abstract There is substantial epidemiological evidence suggesting that alcohol consumption is associated with increased risk for breast cancer. However, possible biological mechanisms have not been clearly established. In the present studies, a direct effect of ethanol on the proliferation and intracellular content of cyclic AMP (cAMP) in two estrogen receptor-positive (ER1) and two estrogen receptor-negative (ER2) human breast cancer cell lines was examined. Treatment of ER1 human breast cancer cells (MCF-7 and ZR75.1) with ethanol at concentrations between 10 and 100 mM was associated with increased cell numbers compared to controls. The ERa content and the amount of intracellular cAMP also increased in ER1 cells exposed to ethanol, compared to controls. On the other hand, ethanol treatment did not increase cell proliferation or cAMP levels in the ER2 (BT20 and MDA-MB-231) human breast cancer cells. Therefore, ethanol added at physiologically relevant concentrations to ER1 human breast cancer cell cultures can enhance cell proliferation and increase the content of ERa. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ethanol; Breast cancer; Cyclic AMP; Estrogen receptor
1. Introduction Breast cancer is a leading cause of cancer-related death among American women. Although the topic is controversial, a majority of epidemiological studies report that alcohol consumption is positively associated with breast cancer risk even at moderate levels of intake [1,2]. Several mechanisms of action have been put forth to explain this effect of alcohol on breast cancer, including the association of alcohol consumption with enhanced circulating levels of estradiol and other steroid hormones [3±7], impaired * Corresponding author. Tel.: 11-217-333-5549; fax: 11-217244-2455. E-mail address: [email protected] (K.W. Singletary).
immune defenses, increased mammary gland proliferation, and altered carcinogen metabolism [8]. Of recent interest is evidence that an interaction between alcohol intake and other breast cancer risk factors may be occurring [9]. In particular, subgroups of alcoholconsuming women may differ in their risk for breast cancer depending on their type of breast tumor as determined by hormone receptor status. For example, alcohol intake has been linked to increased risk for breast tumors possessing estrogen receptors (ER1) or estrogen and progesterone receptors (ER1/PR1) [10±12]. In contrast, others have reported, a positive association of alcohol consumption with ER2/PR2 tumors, an inverse relationship between alcohol intake and prevalence of ER2 rich tumors, or no association at all [10,13±15]. Only one report has indi-
0304-3835/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(01)00419-0
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cated that exposure to ethanol in vitro increases proliferation of ER1 human breast cancer cells [16]. The ER and other steroid hormone receptors are members of the nuclear receptor superfamily capable of eliciting a variety of cell responses and in¯uencing signal transduction pathways both in ligand-dependent and ligand-independent manners [17±19]. Thus, the interaction of alcohol with speci®c subtypes of human breast cancer cells (based on hormone receptor status) deserves further attention. The present studies, therefore, were conducted to examine a direct effect of ethanol exposure on the proliferation of two ER1 human mammary cancer cell lines (MCF-7 and ZR75-1) and two ER2 human mammary cell lines (BT20 and MDA-MB-231), and to determine whether an effect of ethanol on ER1 breast cancer cells is associated with changes in ERa levels. 2. Material and methods The human breast cancer cell lines MCF-7, ZR-751, BT-20 and MDA-MB-231 were obtained from the American Type Culture Collection (Rockville, MD). MCF-7, ZR-75-1 and BT-20 cells were grown (378C, 5% CO2) in Eagle's minimum essential medium containing 5% fetal bovine serum, 10 mM HEPES buffer, 1% MEM non-essential amino acids, 2 mM glutamine penicillin (100 units/ml), streptomycin (100 mg/ml), hydrocortisone (3.75 ng/ml) and insulin (6 ng/ml). MDA-MB-231 cells were grown in Leibovitz's medium L-15 supplemented as described by Berthois et al. [20]. For cell proliferation experiments, cells (2±5 £ 104 ) were plated in 60-mm tissue culture plates containing 3 ml of medium lacking ethanol. After 24 h of cell attachment, the medium was replaced and supplemented with ethanol at concentrations ranging from 0 to 100 mM. Concentrations in this range correspond to blood levels of ethanol in humans, which could result from low to high intakes of alcohol. Cells in triplicate dishes per treatment group were exposed to ethanol for periods up to 10 days. Fresh ethanol-containing medium was added to cells daily. Cells were then collected and counted using a hemocytometer, after ensuring that cells were well dispersed. For measurement of ERa, MCF-7 and ZR 75.1 cells were exposed to ethanol for the speci®ed number
of days and then collected in lysis buffer (1% Na deoxycholate, 1% Triton X-100, 0.01% SDS, 150 mM NaCl, 50 mM Tris (pH 7.5), 0.5 mM NaVO4, 1 mM PMSF and 20 mg/ml aprotonin, leupeptin, and pepstatin), sonicated and centrifuged. The pellet was discarded and protein content of the lysate determined. Equal amounts of protein were loaded on a 10% acrylamide gel and SDS±PAGE performed. After electrophoresis, the gel was stained with zinc stain (BioRad) and photographed to con®rm equal protein loading. The gel was then destained and membrane transfer was accomplished. After transfer to nitrocellulose membrane, ERa was detected by use of a mouse anti-human monoclonal ERa antibody (AB-10, Neomarkers, Union City, CA) and quantitated by ECL. Cyclic AMP was quantitated by radioimmunoassay as described by Aronica et al. [21]. Brie¯y, cells were scraped from plates into TNE buffer (40 mM Tris± HCl (pH 7.5); 140 mM NaCl; 1.5 mM EDTA) on ice and pelleted. Cell pellets were resuspended in 200 ml ice-cold cAMP extraction buffer (50 mM Tris±HCl (pH 7.5); 4 mM EDTA) and sonicated. Extracts remained on ice for 15 min, with regular mixing. An aliquot of extract was analyzed for protein content by the BCA assay (Pierce) and the remaining portion was boiled for 10 min. Cell debris was pelleted and supernatant removed for cAMP quantitation using a cAMP radioimmunoassay kit (Amersham). Statistically signi®cant differences between treatments were determined by analysis of variance and the application of Fisher's least signi®cant difference test for post hoc comparisons. Mean differences at P , 0:05 were considered statistically signi®cant. 3. Results Exposure of ER1 cell lines to increasing concentrations of ethanol was associated with an increase in cell proliferation (Table 1). For example, ethanol added to cultures of cells at concentrations of 20±50 mM signi®cantly stimulated proliferation of MCF-7 and ZR75.1 cells by 53±91% following 7 and 10 days of treatment, compared to controls. For both cell lines, cell proliferation was signi®cantly enhanced at both days 7 and 10 of ethanol treatment, with no consistent increase in proliferation being observed following 2
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Table 1 Effect of ethanol concentration on proliferation of ER1 human breast cancer cell lines (MCF-7 and ZR-75-1) a Ethanol concentration (mM)
Cell number/plate ( £ 10 4) MCF-7
0 5 10 20 50 100
ZR-75-1
Day 7
Day 10
Day 7
Day 10
50.0 ^ 1.1 a 51.7 ^ 0.9 a 68.0 ^ 2.3 b 77.0 ^ 1.5 c 80.3 ^ 0.8 c 93.3 ^ 2.9 d
94.3 ^ 0.9 a 118.3 ^ 4.1 b 127.7 ^ 3.4 b 144.3 ^ 3.8 c 172.7 ^ 6.6 d 201.3 ^ 4.3 e
11.0 ^ 1.1 a 14.7 ^ 0.7 b 15.7 ^ 1.2 c 17.7 ^ 0.9 c,d 21.0 ^ 1.5 d 25.7 ^ 1.2 e
38.6 ^ 1.7 a 40.3 ^ 0.9 a 49.3 ^ 1.2 b 59.3 ^ 1.8 c 69.7 ^ 1.4 d 86.3 ^ 0.9 e
a Values represent mean ^ SE of three determinations, and are representative of at least two separate experiments. Means among treatment groups sharing unlike superscripts are signi®cantly different.
mM increased ERa levels by approximately 2.1±5fold, compared to controls, although only the 50 mM values were statistically different. No effect of ethanol on ERa levels in either cell line was observed after only 1 day treatment with ethanol. The amount of cAMP in all cells also was measured, because of reports that cAMP is elevated in liver cells that exhibit increased proliferation in response to ethanol exposure [22±24]. The cellular content of cAMP in the MCF-7 and ZR75.1 cell lines increased in response to ethanol supplementation (Table 3), although the timing of the increase differed between the two cell lines. For MCF-7 cells, incubation with 25 or 100 mM ethanol for 2, 4 and 8 days signi®cantly increased intracellular cAMP content 1.3±1.8-fold, compared with controls. Exposure of
days of treatment with ethanol. No changes in viability, size or morphology of cells in these two lines were discernible following treatment with ethanol, compared with controls. In contrast to that for the ER1 cell lines, neither of the ER2 cell lines (BT20 and MDA-MB-231) responded to ethanol exposure for 6 and 10 days with an increase in cell number (Table 2). To determine whether treatment with ethanol affected ER levels in the ER1 human breast cancer cell lines, ZR 75.1 and MCF-7 cells were treated with several concentrations of ethanol for either 1 or 7 days. After 7 days of treatment with ethanol, an increase in the immunoreactive content of ERa was observed for both cell lines (Fig. 1). Exposure of ZR 75.1 and MCF-7 cells to ethanol doses of 25 and 50
Table 2 Effect of ethanol concentration on proliferation of ER2 human breast cancer cell lines (MDA-MB-231 and BT-20) a Ethanol concentration (mM)
Cell number/plate ( £ 10 4) MDA-MB-231 Day 6
0 5 10 20 50 100
BT-20 Day 10
a
25.0 ^ 1.5 25.0 ^ 2.5 a 25.0 ^ 1.1 a 26.3 ^ 1.7 a 25.0 ^ 1.1 a 26.7 ^ 1.2 a
Day 6 a
55.3 ^ 2.9 52.0 ^ 2.3 a 52.0 ^ 2.1 a 52.3 ^ 2.9 a 59.3 ^ 1.8 a 57.3 ^ 1.2 a
Day 10 a
40.7 ^ 1.2 42.3 ^ 0.9 a 43.0 ^ 2.5 a 43.7 ^ 1.8 a 42.3 ^ 0.9 a 41.0 ^ 0.6 a
111.7 ^ 3.4 a 104.7 ^ 0.9 a 104.0 ^ 1.5 a 106.7 ^ 2.0 a 107.3 ^ 2.2 a 113.7 ^ 1.4 a
a Values represent mean ^ SE of three determinations and are representative of at least two separate experiments. Means among treatment groups sharing unlike superscripts are signi®cantly different.
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ZR75.1 cells to ethanol at 25 and 100 mM concentrations was associated with signi®cant increases in cellular cAMP after 1- and 2-day incubation periods
(Table 3), but not at day 8. In contrast to the ER1 cells, the content of cAMP in the ER2 cell lines did not increase after exposure to ethanol at any of the concentrations examined from 5 to 100 mM for 2±7 days (data not shown). 4. Discussion
Fig. 1. In¯uence of ethanol treatment (7 days) on ERa expression in cultures of ZR75.1 (a,b) and MCF-7 (c,d) cells. Values and densitometric scans are representative of two separate experiments. *Signi®cant at P , 0:05.
The present studies indicate that treatment with ethanol is associated with increased proliferation of two ER1 human breast cancer cell lines. The concentrations of ethanol at which signi®cant increases in proliferation were observed correspond to blood levels in humans that are attainable after moderate to high intakes of ethanol. For instance, 54 kg women consuming up to three drinks per day have been reported to have blood ethanol concentrations from 5 to 24 mM, and those women drinking daily 0.75 g/kg body weight peak blood ethanol levels of 20±24 mM [25,26]. In contrast to that for ER1 cells, no increase in cell proliferation was observed for two ER2 cell lines supplemented with the same ethanol concentrations. Our ®ndings are similar to those reported by Przylipiak et al. [16], who observed that ethanol added to cultures of MCF-7 cells at concentrations of 0.001±10% signi®cantly enhanced DNA synthesis. We are not aware of other reports in which a stimulating effect of ethanol on human breast cancer cell multiplication was observed, although an enhancement of cell division in rat mammary, liver and rectal cells in response to ethanol and/or acetaldehyde exposure has been reported [22,27±29]. The selective stimulation of ER1 cells in our studies is noteworthy, since there is recent evidence that ethanol intake by women may enhance the risk for ER1 or ER1/PR1 breast tumors [11,12]. However, our reported association of ethanol with increased proliferation of ER1 human cancer requires additional study before ®rm comparisons with human ®ndings can be drawn. First, it is not clear whether the presence or lack of ER or PR represent different diseases or different stages in the progression of the same disease [30], making it dif®cult to interpret how exposure to ethanol will impact the relative balance of ER2 and ER1 cells in the tumors of women. Also, some of the data on ER status of tumors in postmenopausal women is dependent on whether hormone
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Table 3 In¯uence of ethanol concentration on intracellular cAMP content of MCF-7 and ZR-75-1 cells a Ethanol concentration (mM)
cAMP content (pmol cAMP/mg protein) MCF-7
0 5 25 100
ZR-75-1
Day 2
Day 4
Day 8
Day 1
Day 2
Day 8
19.4 ^ 1.5 a 23.2 ^ 1.8 a,b 26.0 ^ 0.5 b 25.7 ^ 0.5 b
21.9 ^ 1.3 a 22.5 ^ 1.4 a 32.4 ^ 7.7 a,b 37.8 ^ 9.2 b
36.7 ^ 3.1 a 49.6 ^ 9.0 a,b 55.5 ^ 6.0 b 67.2 ^ 2.5 b
6.5 ^ 0.7 a 7.4 ^ 0.2 a,b 8.3 ^ 0.3 b 8.6 ^ 0.6 b
6.0 ^ 0.8 a 8.2 ^ 1.1 a 11.4 ^ 0.8 b 13.1 ^ 0.6 b
3.7 ^ 0.8 a 3.9 ^ 0.4 a 3.6 ^ 0.3 a 4.1 ^ 0.4 a
a Values represent mean ^ SE of three determinations and are representative of data from two separate experiments. Means among treatment groups sharing unlike superscripts are signi®cantly different.
(estrogen) replacement therapy is being used [10]. Thus, the effect of both ethanol dose and hormone exposure on the breast cancer cells needs to be more carefully quanti®ed. The mechanism whereby ethanol stimulates cell proliferation in ER1 human breast cancer cells in this study nor why the effect is time-dependent is not known. Although ethanol treatment of ZR 75.1 and MCF-7 cells was associated with an increase in cell cAMP content, the temporal change in intracellular cAMP content was not consistent between cell lines and did not necessarily coincide with the increases in cell number. For ZR 75.1 cells, cell proliferation was not signi®cantly elevated at 1 or 2 days of exposure to ethanol, yet cAMP content increased signi®cantly at days 1 and 2 of treatment but not by 8 days of ethanol exposure. On the other hand for MCF-7 cells, ethanol treatment at concentrations greater than 25 mM was associated with an increase in cAMP throughout the 8-day period. This suggests that continuous elevation of cellular cAMP may not be necessary for the increased cell proliferation in response to ethanol, or that other ethanol-associated intracellular events are more important in increasing cell proliferation. The relationship of ethanol to intracellular cAMP is tissue-speci®c, with ethanol either stimulating or lowering cAMP production depending on the tissue examined [23,24,35]. Our results are similar to others [22±24] in which ethanol treatment of hepatocytes is associated with increased proliferation and cAMP production. We also observed that in these ER1 human breast cancer cells, ethanol's impact on cell numbers and on ERa levels both occur after several days of exposure to ethanol,
suggesting that an ethanol-associated increase in ERa may play a role in the enhancement of ER1 breast cancer cell proliferation. However, the increased content of ERa cannot completely explain the increase in cell proliferation, since increased cell numbers were observed in response to ethanol at doses which did not produce signi®cant increases in ERa content. This could indicate that ethanol-induced changes in ER activity may be important in addition to total amounts of ERa. In this regard, Fan et al. [31] recently reported that treatment of human breast cancer cells with ethanol increased both ERa levels and activity. There is evidence that alcohol may stimulate the content of nuclear estrogen receptors in the hypothalamus of the male rat [32], and may enhance estrogen-induced rat hepatocarcinogenesis by enhancing cell proliferation via alterations in estrogen and progesterone receptor kinetics [33,34]. It is known that the ER activity of human breast cancer cells is affected by multiple cell-signaling pathways, including those mediated by cAMP and IGF-1 [36]. For example, Aronica et al. [21] observed that estrogen treatment increases cellular cAMP content in ER1 human breast cancer cells, resulting in a stimulation of cAMP response element-mediated gene transcription. It was suggested that, since cAMP is known to regulate the activity of ER and PR in human breast cancer cells, increased cAMP content may activate protein kinase pathways leading to enhanced ER phosphorylation and ER2 mediated transactivation [18]. Thus, the capacity of ethanol to in¯uence signal transduction pathways regulating the proliferation, ER status, and ER activity of human breast cancer cells warrants further characterization.
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In summary, we report that supplementation of cultures of ER1 human breast cancer cell lines MCF-7 and ZR75.1 with ethanol is associated with increased cell proliferation and cellular cAMP content. In addition, in ZR75.1 and MCF-7 cells, ethanol treatment was associated with substantial increases in ERa content. In contrast, no increase in cell division or cAMP content was observed when cultures of the ER2 human breast cancer cell lines BT-20 and MDA-MB-231 were supplemented with the same concentrations of ethanol. The capacity of ethanol to stimulate proliferation of speci®c types of human breast cancer cells, rather than all breast cancer cells, might partly explain the generally modest increases in breast cancer risk reported in epidemiological studies, since an increased development of breast tumors may only be apparent for a subset of tumors with speci®c hormone receptor characteristics. The mechanism for the selective effect of ethanol on human breast cancer cell proliferation, cAMP content, and ER levels warrants further study.
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