* Department of Biochemistry and Molecular Pharmacology and
Mary Babb Randolph Cancer Center, West Virginia University Health Sciences Center, P.O. Box 9142, Morgantown, West Virginia 265069142; and
National Institute of Occupational Safety and Health, Health Effects Laboratory Division, Morgantown, West Virginia 26505
Received September 3, 2002; accepted November 27, 2002
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ABSTRACT |
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Key Words: acetaminophen; Ishikawa cells; estrogen receptors; alkaline phosphatase; endocrine disruption.
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INTRODUCTION |
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Other studies suggest acetaminophen may alter some hormone-regulated processes. In rats, high doses of acetaminophen reduce testis DNA synthesis resulting in decreased testicular weight, decreased number of spermatocytes, and deterioration of sperm chromatin structures (Wiger et al., 1995). In humans, acetaminophen use is associated with a decreased risk of ovarian cancer (Cramer et al., 1998a
), as well as reduced gonadotropin and estradiol (E2) levels (Cramer et al., 1998b
). Although acetaminophen does not exert a positive or negative effect in rodent E2-dependent uterotrophic (wet weight) assays (Harnagea-Theophilus et al., 1999a
; Isenhower et al., 1986
; Patel and Rosengren, 2001
), some studies indicate therapeutic levels of this drug can specifically alter E2-regulated processes. In cultured trout liver cells acetaminophen decreases E2-dependent vitellogenin mRNA and protein production in a concentration-dependent manner (Miller et al., 1999
). Similarly, in immature mice moderate doses of acetaminophen reduce E2-induced uterine peroxidase activity and nuclear progesterone receptor protein levels (Patel and Rosengren, 2001
). Furthermore, therapeutic concentrations of acetaminophen stimulate proliferation of estrogen receptor (ER)-positive breast cancer cells (MCF-7, T47D, ZR-75-1), but not ER-negative (MDA-MB-231, HS578T) breast cancer cells, via a mechanism inhibited by antiestrogens (Harnagea-Theophilus and Miller, 1998
; Harnagea-Theophilus et al., 1999a
,b
). It is not yet clear why acetaminophen exerts antiestrogenic activity on some processes in some cells, while it mimics estrogen in stimulating proliferation of breast cancer cells.
To further explore the influence of acetaminophen on E2-regulated processes in human cells, the hypothesis that acetaminophen would alter expression of alkaline phosphatase activity in Ishikawa cells was determined. Ishikawa cells are endometrial adenocarcinoma cells that express functional ER and ERß (Bhat and Pezzuto, 2001
; Enmark et al., 1997
; Kassan et al., 1989
). Estradiol has a variety of effects upon Ishikawa cells including upregulation of progesterone receptors (Holinka et al., 1986b
) and c-fos mRNA (Gadd et al., 2002
); increased c-erbB2/NEU proto-oncogene protein expression (Markogiannakis et al., 1997
); increased intracellular endorphin release (Makrigiannakis et al., 1992
); modest stimulation of cell proliferation (Holinka et al., 1986a
; Holinka et al., 1989
; Nishada et al., 1985
); and increased alkaline phosphatase enzyme activity and mRNA (Albert et al., 1990
; Holinka et al., 1986b
; Littlefield et al., 1990
). The sensitivity of Ishikawa cell alkaline phosphatase activity to induction by E2 provided a well-characterized system in which to assess the E2-agonistic and E2-antagonistic activity of acetaminophen. In addition, this system was used to determine the extent to which acetaminophen impacted the effect of an antiestrogen, 4-hydroxy-tamoxifen, on expression of an E2-regulated gene. Because many of the cells/tissues in which acetaminophen alters E2-regulated processes express different levels of ER
and ERß, the ability of acetaminophen to directly bind the ligand-binding site of purified ER
and ERß is reported for the first time.
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MATERIALS AND METHODS |
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Alkaline phosphatase activity and protein determination.
Ishikawa cells in estrogen-free medium were trypsinized and plated in CoStar 96-well plates at 7 x 103 cells/well. Twenty-four h later the indicated compounds were added in fresh medium, and cells were exposed to these compounds for four days, with one medium change two days after the first addition of compounds. At the end of the exposure period wells were assayed for cellular alkaline phosphatase activity as previously described (Littlefield et al., 1990) using a Spectramax 340PC plate reader. Alkaline phosphatase activity was determined for n = 5 wells per treatment in each experiment and is presented as mean pmol p-nitrophenol (pNP) formed/min/well ± SE. Total cellular protein in cultures was determined using fluorescamine reagent by measuring fluorescence (360 nm ex/460 nm em) in a Cytofluor 400, as previously described (Udenfriend et al., 1972
). Total cellular protein determinations were performed in triplicate and the amount of protein was extrapolated from an albumin standard curve. Results are presented as mean µg protein/well ± SE. The effects of compounds on alkaline phosphatase activity and on total cellular protein are reported as independent parameters, to illustrate how the compounds differentially altered each of these parameters.
To assess the effect of acetaminophen directly on alkaline phosphatase activity, an extract from E2-treated cells was prepared. Alkaline phosphatase activity in the extract was measured in the absence and presence of various concentrations of acetaminophen.
Lactate dehydrogenase cytotoxicity assay.
Ishikawa cells were plated into CoStar 6-well plates at 1.3 x 106 cells/well and allowed to adhere for 24 h. Cells were then treated with various concentrations of acetaminophen for indicated periods of time. Medium was carefully removed from cells, and lactate dehydrogenase (LDH) released from cells was measured in culture medium. Cellular LDH activity was determined by disrupting cells with 1 ml 0.1% Triton X-100 and measuring LDH in cell lysates. LDH activity was measured as described in Reeves and Fimognari (1966), and protein in cell lysates was determined by Bio-Rad Protein Assay (Bio-Rad, CA). LDH activity in both cell supernatant and cell lysate was normalized to protein concentration in cell lysates.
ER binding assays.
In vitro competition binding assays utilized purified human recombinant ER and ERß (PanVera). Briefly, reactions containing 1 pmol ER, 1 pmol [3H]-E2 (Amersham), and indicated concentrations of competing, unlabeled E2 or acetaminophen in 10 mM Tris, pH 7.5, 10% glycerol, 2 mM dithiothreitol (DTT), 1 mg/ml bovine serum albumin were incubated in a 37°C water bath for 30 min. ER with bound [3H]-E2 was separated from free [3H]-E2 by the addition of hydroxylapatite and repeated centrifugation and washing. Liquid scintillation counting determined the amount of [3H]-E2 bound to ER.
Western blots.
Western blots were used to assess the relative abundance of ER in MCF-7 cells and in Ishikawa cells, using antibodies specific for ER
(Santa Cruz). Ishikawa and MCF-7 (human breast cancer) cells were plated into 100-mm dishes and maintained in E2-free medium for four days with one medium change on day 2. On day 4, cells were rinsed with ice-cold phosphate buffered saline and lysed in 1 ml of SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% sodium dodecylsulfate (SDS), 10% glycerol, 50 mM DTT, 0.1% bromphenol blue). Protein concentration was determined by 50% trichloroacetic acid precipitation of an aliquot of cell lysate, followed by the Coomassie Plus Protein Assay (Pierce). Proteins were separated via electrophoresis in denaturing 7.5% polyacrylamide gels and electroblotted to PVDF membrane. Blots were blocked with 5% powdered milk for 1 h at room temperature and then incubated overnight at 4°C with anti-ER
antibody (Santa Cruz) diluted in 5% albumin. Visualization was achieved through incubation with horseradish peroxidase-linked donkey antirabbit IgG (Pierce) in 5% powdered milk for 1 h at room temperature, followed by chemiluminescence detection (SuperSignal West Pico Chemiluminescent Substrate, Pierce). Blots were stripped and then reprobed for ß-catenin (Santa Cruz). Optimas 6.2 software was used to quantitate the optical density of each band of interest.
Statistical analysis.
Statistical analysis of alkaline phosphatase activity, protein concentration, and LDH assays were conducted by one-way ANOVA followed by Tukeys Test; significant differences (p < 0.05) between group means are indicated. For ER-binding assays, one-way ANOVA was performed, followed by the Dunnetts test; significant differences (p < 0.05) are indicated.
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RESULTS |
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Hydroxy-tamoxifen was used in further studies because it reduced alkaline phosphatase activity without significantly altering cellular protein. Figure 5A shows that 0.01 µM hydroxy-tamoxifen, a nontoxic concentration (Fig. 4
), reduced the E2-induced alkaline phosphatase activity
60%, and acetaminophen further reduced alkaline phosphatase activity in a concentration-dependent manner between 0.1 and 1.0 mM. Although acetaminophen also reduced the amount of protein in cell cultures (Fig. 5B
), the reduced cellular protein only partially accounted for the decreased alkaline phosphatase activity mediated by 0.11.0 mM acetaminophen.
Acetaminophen Does Not Compete with E2 Binding ER or ERß
Ishikawa cells express both high- and low-affinity E2-binding sites (Kassan et al., 1989), and recent studies demonstrated that both ER
and ERß are expressed in these cells (Bhat and Pezzuto, 2001
). To determine if the acetaminophen-mediated reduction of E2-regulated alkaline phosphatase in Ishikawa cells may be attributed to nonproductive binding of acetaminophen to ER
or ERß, competition binding studies were performed using purified human ER
and ERß. Even the highest concentrations of acetaminophen tested (106-fold molar excess) did not compete significantly with [3H]-E2 for binding ER
or ERß (Fig. 6
). In addition, the acetaminophen-mediated inhibition of E2-induced alkaline phosphatase activity (Fig. 3
) was not significantly altered by increasing the concentration of E2 100-fold (not shown), further indicating that acetaminophen and E2 are not directly competing for binding ER. The relative level of ER
expression in Ishikawa and MCF-7 cells was also investigated. Figure 7
demonstrates that ER
is expressed at a lower level in Ishikawa cells than in MCF-7 cells. In two independent studies, the optical density of immunoreactive ER
was
3-fold higher in MCF-7 cells than in Ishikawa cells with equal amounts of total cellular protein analyzed; in contrast, the level of ß-catenin was
1.5-fold higher in Ishikawa cells than in MCF-7 cells.
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DISCUSSION |
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The mechanism by which acetaminophen inhibits E2-induced gene expression without binding ER or ERß is not established, but may be mediated by PPAR (peroxisome proliferator-activated receptor) and RXR (retinoid X receptor). PPAR and RXR have been shown to be expressed in ER-positive tissues (Nunez et al., 1997
), and PPAR*RXR heterodimers can modulate transcription of genes containing PPREs (PPAR response elements) or EREs (estrogen response elements; Keller et al., 1995
; Nunez et al., 1997
). Binding of PPAR*RXR heterodimers to natural ERE-containing promoters inhibits transactivation by E2*ER complex through competition for ERE binding (Keller et al., 1995
). The addition of acetaminophen, a known peroxisome proliferator (Lee et al., 1997
), to Ishikawa cells could initiate PPAR*RXR heterodimer formation and binding to ERE(s) in the alkaline phosphatase gene promoter, resulting in inhibition of E2-induced alkaline phosphatase. On the other hand, acetaminophen may inhibit E2-induction of alkaline phosphatase by altering an arachidonic acid metabolism pathway. Acetaminophen is a weak inhibitor of cyclooxygenase 1 (COX-1) and COX-2 and a more potent inhibitor of COX-3 (Chandrasekharan et al., 2002
), reducing prostaglandin production in most tissues (Prescott, 1996
). However, acetaminophen elevates COX-2 and prostaglandin production in liver (Reilly et al., 2001
). Alteration of arachidonic acid metabolism via COX can generate potent mediators (prostaglandins, leukotrienes, hydroxyeicosapentaenoic acids) that could alter other signal transduction pathways (Serhan et al., 2000
), possibly culminating in inhibition of E2-induced alkaline phosphatase.
In addition, ER and ERß are differentially expressed in different tissues (Enmark et al., 1997
; Kuiper et al., 1997
); and gene expression can be differentially regulated by a ligand binding to ER
or ERß (Paech et al., 1997
). Therefore, the distribution of ER
and ERß in different cells/tissues may alter the biological response to an ER-ligand. Thus, the observations that acetaminophen stimulates human breast cancer cell proliferation, mimicking E2 (Harnagea-Theophilus and Miller, 1998
; Harnagea-Theophilus et al., 1999a
,b
), but exerts E2-antagonistic activity in human endometrial adenocarcinoma cells (Figs. 1 and 3
), as well as in trout liver (Miller et al., 1999
) and mouse uterus (Patel and Rosengren, 2001
), could in part be due to differential expression of ER
and ERß in those cells/tissues. Ishikawa cells express both ER
and ERß (Bhat and Pezzuto, 2001
), whereas MCF-7 cells predominantly express ER
; and ER
is expressed
3-fold higher in MCF-7 cells than in Ishikawa cells (Fig. 7
). The density of ERß is estimated to be
10-fold higher than ER
in Ishikawa cells (Kassan et al., 1989
); therefore we estimate that total ER (
+ ß) may be
34-fold higher in Ishikawa cells than in MCF-7 cells. Differences in the proportion of ER
and ERß or the total level of ER in Ishikawa and MCF-7 cells may impact the response to acetaminophen. However, acetaminophen does not directly compete significantly with E2 binding human ER
or ERß (Fig. 6
), indicating acetaminophen does not differentially interact with the ligand-binding domain of these receptors. This result does not eliminate the possibility that acetaminophen (or a metabolite) may differentially interact directly or indirectly with an ER-accessory protein/factor in the different cells. Alternatively, because the membrane-associated forms of ER
or ERß may exhibit ligand specificity that differs from that of the soluble receptor forms (Wade et al., 2001
), acetaminophen may differentially affect signaling via the membrane-associated forms of ER
or ERß in different cells.
The ability of acetaminophen to exert mitogenic activity on select cells may depend on both the internal cell environment and the presence of ER. Acetaminophen and E2 both stimulate ER-positive breast cancer cells (MCF-7, T47D, ZR-75-1) to proliferate via an ER-mediated mechanism (Harnagea-Theophilus and Miller, 1998; Harnagea-Theophilus et al., 1999a
,b
) and induce c-myc expression (Gadd et al., 2002
). In contrast, in Ishikawa cells E2 marginally stimulates c-myc expression
20% (Gadd et al., 2002
) and cell proliferation (Holinka et al., 1986a
), while acetaminophen slightly reduces c-myc expression (Gadd et al., 2002
) and does not stimulate proliferation, reflected in cellular protein (Fig. 1
), at any concentration tested. In addition, ER-positive breast cancer cells are relatively resistant to acetaminophen toxicity (Harnagea-Theophilus and Miller, 1998
; Harnagea-Theophilus et al., 1999a
,b
), while Ishikawa cells are more sensitive to acetaminophen toxicity (Table 1
), likely reflecting differences in metabolism of acetaminophen to the toxic NAPQI. Furthermore, a recent report indicates acetaminophen-stimulated cell proliferation is not restricted to ER-positive breast cancer cells. Acetaminophen was reported to stimulate the proliferation of human endometrioid ovarian cancer (MDAH 2774) cells (Bilir et al., 2002
), which are reported to lack ER (Thompson et al., 1991
). Therefore the presence of ER may not be sufficient or necessary for acetaminophen to induce cell proliferation. MDAH cells carry mutations in p-53 and MSH-2 genes (Orth et al., 1994
; Santoso et al., 1995
), prompting the idea that alteration of the oxidative state of these cells by acetaminophen may promote a mitogenic signal (Bilir et al., 2002
). In breast cancer cells, acetaminophen may activate a proliferation signal transduction pathway that requires ER to culminate in a mitotic signal.
Tamoxifen is a chemotherapeutic agent commonly used in the treatment of E2-responsive breast cancer; tamoxifen and metabolites, including 4-hydroxy-tamoxifen exert their beneficial effects as ER-antagonists. Because women receiving tamoxifen often take acetaminophen to manage pain, it was of interest to determine the effect of the combination of these agents on an ER-regulated process. Therapeutic and toxic concentrations of acetaminophen (0.10.3 and 1 mM, respectively) augmented the hydroxy-tamoxifen inhibition of Ishikawa cell alkaline phosphatase activity (Fig. 5). Additional studies are required to determine if the combination of tamoxifen (or other antiestrogens) chemotherapy and the use of acetaminophen can affect the drug efficacy or patient health.
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NOTES |
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