Inhibition of Cellular Enzymes by Equine Catechol Estrogens in

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Chem. Res. Toxicol. 2002, 15, 935-942

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Inhibition of Cellular Enzymes by Equine Catechol Estrogens in Human Breast Cancer Cells: Specificity for Glutathione S-Transferase P1-1 Jiaqin Yao, Minsun Chang, Yan Li, Emily Pisha, Xuemei Liu, Dan Yao,† Ebrahim C. Elguindi, Sylvie Y. Blond, and Judy L. Bolton* Department of Medicinal Chemistry and Pharmacognosy (M/C 781), College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612 Received February 22, 2002

Glutathione S-transferases (GSTs) are a family of detoxification isozymes that protect cells by conjugating GSH to a variety of toxic compounds, and they may also play a role in the regulation of both cellular proliferation and apoptosis. We have previously shown that human GST P1-1, which is the most widely distributed extrahepatic isozyme, could be inactivated by the catechol estrogen metabolite 4-hydroxyequilenin (4-OHEN) in vitro [Chang, M., Shin, Y. G., van Breemen, R. B., Blond, S. Y., and Bolton, J. L. (2001) Biochemistry 40, 4811-4820]. In the present study, we found that 4-OHEN and another catechol estrogen, 4,17β-hydroxyequilenin (4,17β-OHEN), significantly decreased GSH levels and the activity of GST within minutes in both estrogen receptor (ER) negative (MDA-MB-231) and ER positive (S30) human breast cancer cells. In addition, 4-OHEN caused significant decreases in GST activity in nontransformed human breast epithelial cells (MCF-10A) but not in the human hepatoma HepG2 cells, which lack GST P1-1. We also showed that GSH partially protected the inactivation of GST P1-1 by 4-OHEN in vitro, and depletion of cellular GSH enhanced the 4-OHEN-induced inhibition of GST activity. In addition, 4-OHEN GSH conjugates contributed about 27% of the inactivation of GST P1-1 by 4-OEHN in vitro. Our in vitro kinetic inhibition experiments with 4-OHEN showed that GST P1-1 had a lower Ki value (20.8 µM) compared to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 52.4 µM), P450 reductase (PR, 77.4 µM), pyruvate kinase (PK, 159 µM), glutathione reductase (GR, 230 µM), superoxide dismutase (SOD, 448 µM), catalase (562 µM), GST M1-1 (620 µM), thioredoxin reductase (TR, 694 µM), and glutathione peroxidase (GPX, 1410 µM). In contrast to the significant inhibition of total GST activity in these human breast cancer cells, 4-OHEN only slightly inhibited the cellular GAPDH activity, and other cellular enzymes including PR, PK, GR, SOD, catalase, TR, and GPX were resistant to 4-OHEN-induced inhibition. These data suggest that GST P1-1 may be a preferred protein target for equine catechol estrogens in vivo.

Introduction Estrogen replacement therapy has been associated with an increased risk of developing cancers in several tissues, particularly the breast and endometrium (1-3); however, the molecular mechanism(s) involved in the carcinogenic action of estrogens is (are) still unclear. Metabolic activation of estrogens to either redox and/or electrophilic metabolites may play an etiologic role in tumor formation. 4-Hydroxyequilenin (4-OHEN)1 is the major phase I catechol metabolite of the equine estrogens equilenin and equilin which can make up ∼50% of the most widely prescribed estrogen replacement formulation, Premarin (Wyeth-Ayerst) (4, 5). We have previously showed that 4-OHEN rapidly autoxidized to 4-OHEN-oquinone (Figure 1), which in turn formed a redox couple with the semiquinone radical generating reactive oxygen * Address correspondence to this author at the Department of Medicinal Chemistry and Pharmacognosy (M/C 781), College of Pharmacy, University of Illinois at Chicago, 833 S. Wood St., Chicago, IL 60612. Tel: (312) 996-5280, Fax: (312) 996-7107, E-mail: [email protected]. † Present address: Merck & Co. Inc., 126 E. Lincoln Ave., Building RY80R, Room 142, Rahway, NJ 07065.

species (ROS) (4, 6). The quinoids and ROS could cause depletion of reducing equivalents, alkylation/oxidation of detoxification enzymes, and DNA damage (7). 4-OHEN has been shown to induce a variety of DNA lesions in vitro including formation of bulky stable adducts, apurinic sites, and oxidation of the phosphate sugar backbone and purine/pyrimidine bases (4, 8-10). In human breast cancer cells, 4-OHEN induced DNA damage and apoptosis, all of which were enhanced by redox cycling (11). Similar DNA damage was also observed in vivo in the mammary tissues of rats treated with 4-OHEN (12). Another catechol estrogen, 4,17β-hydroxyequilenin (4,17β1 Abbreviations: ARE, antioxidant response element; BSA, bovine serum albumin; BSO, L-buthionine-(S,R)-sulfoximine; CDNB, 1-chloro2,4-dinitrobenzene; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); DTT, dithiothreitol; ECL, enhanced chemiluminescence; ER, estrogen receptor; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPX, glutathione peroxidase; GR, glutathione reductase; GSTs, glutathione S-transferases; 4-OHE, 4-hydroxyestrone; 4-OHEN, 4-hydroxyequilenin; 4,17β-OHEN, 4,17β-hydroxyequilenin; JNKs, c-Jun N-terminal kinases; NBT, nitro blue tetrazolium; PBS, phosphate-buffered saline; PK, pyruvate kinase; PR, P450 reductase; PSF, penicillin-streptomycin-fungizome; ROS, reactive oxygen species; SOD, superoxide dismutase; TR, thioredoxin reductase; XRE, xenobiotic response element.

10.1021/tx020018i CCC: $22.00 © 2002 American Chemical Society Published on Web 06/11/2002

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of interest to test whether 4-OHEN might also inactivate these enzymes. The kinetic data with purified enzymes and experiments in breast cancer cells suggest that GST P1-1 may be a preferred target for equine catechol estrogens in vivo.

Materials and Methods

Figure 1. Possible mechanisms for the toxic effects induced by 4-OHEN.

OHEN), has also been shown to induce more extensive oxidative DNA damage as compared to 4-OHEN (13). In addition, recent data suggest that 4-OHEN has the potential to be a much more effective tumor promoter and complete carcinogen in vitro in comparison with the endogenous catechol estrogen, 4-hydroxyestrone (4-OHE) (14). Glutathione S-transferases (GSTs) catalyze the conjugation of GSH with both physiological and xenobiotic electrophilic compounds, generally performing a detoxificative function (15, 16). However, overexpression of GSTs and increased GST activity have been found to confer cellular resistance to antineoplastic agents (17). Human GSTs are a family of isozymes which includes at least seven distinct classes: alpha (A), mu (M), pi (P), sigma (S), theta (T), kappa (K), and zeta (Z) (18). GST P1-1 is the most widely distributed extrahepatic isozyme (19), and it has also been shown to be an endogenous inhibitor of c-Jun N-terminal kinases (JNKs), a multimember family of stress kinases (20). Since changes in JNK’s activity could influence key cellular functions, including growth, apoptosis, and transformation (20, 21), GSTs may play a role in regulation of both cellular proliferation and apoptosis. Previously, we have demonstrated in vitro that 4-OHEN autoxidized to an o-quinone that reacted with GSH to form conjugates and inhibited the activity of recombinant human GST M1-1 (22). 4-OHEN also inactivated human GST P1-1 in vitro by covalent modification of cysteine residues and by oxidative damage, resulting in disulfide bond formation (23). It is the purpose of the present study to examine whether 4-OHEN inhibits GST activity in human breast cancer cells and to further understand the possible mechanisms of 4-OHEN-induced inactivation of GST P1-1. Besides the GSH/GST system, mammalian cells have evolved other detoxification enzymes, such as superoxidase dismutase (SOD), catalase, glutathione peroxidase (GPX), glutathione reductase (GR), and P450 reductase (PR). Human thioredoxin reductase (TR) is a powerful NADPH-dependent disulfide oxidoreductase closely related to glutathione reductase (24). Glyceraldehyde-3phosphate dehydrogenase (GAPDH) is an abundant S-thiolated protein during conditions of oxidative stress, and oxidation of the essential sulfhydryl group in the active site of GAPDH results in loss of enzyme activity (25). Pyruvate kinase (PK), which catalyzes the transfer of phosphate groups from phosphoenolpyruvate to ADP to form ATP and plays a critical role in glycolysis regulation, contains a cysteine residue that is the most likely target for irreversible modification (26). Since the status of cysteine residues is involved in both the structure and function of these and other enzymes, it was

Materials. Caution: The catechol estrogens were handled in accordance with NIH guidelines for the Laboratory Use of Chemical Carcinogens (27). All chemicals were purchased from Sigma (St. Louis, MO), Aldrich (Milwaukee, WI), or Fisher Scientific (Itasca, IL) unless stated otherwise. 4-OHEN was synthesized by treating equilin with Fremy’s salt as described previously (28, 29) with minor modifications (6). 4,17β-OHEN was prepared by the reduction of 4-OHEN with lithium tri-tertbutoxyaluminohydride (30). The 4-OHEN GSH conjugates were prepared by incubating 4-OHEN (0.5 mM) with 5.0 mM GSH in 1 mL of 50 mM potassium phosphate buffer (pH 7.4) at 25 °C for 5 min (22). The conjugates were isolated from the aqueous phase on a PreSep-C18 extraction column (Fisher) and eluted with methanol. After drying with N2, the conjugates were dissolved in 500 µL of DMSO to give a final concentration of 1 mM. Fetal bovine serum (FBS) was purchased from Atlanta Biologicals (Atlanta, GA). Purified human SOD, catalase, GPX, GAPDH, bovine GR, rabbit PK, and E. coli TR were purchased from Sigma, and human recombinant PR was obtained from Panvera (Madison, WI). Cell Culture Conditions. The MDA-MB-231 cells were obtained from American Type Culture Collection (Rockville, MD) and maintained in MEME medium supplemented with 1% penicillin-streptomycin-fungizome (PSF, GibcoBRL, Grand Island, NY), 6 µg/L insulin, 1% glutaMAX (GibcoBRL), 5% FBS, and 5% CO2 at 37 °C. The S30 cell line, which was a generous gift from V. C. Jordan’s laboratory (Northwestern University, Chicago, IL), was grown in MEME medium without phenol red supplemented with 1% PSF, 6 µg/L insulin, 1% glutaMAX, 500 mg/L G418, 5% charcoal-dextran-treated FBS, and 5% CO2 at 37 °C. Human hepatoma HepG2 cells (American Type Culture Collection) were grown in MEME medium supplemented with 1% PSF, 1 µM sodium pyruvate, 1× nonessential amino acid, 10% FBS, and 5% CO2 at 37 °C. Human nontransformed breast epithelial MCF-10A cells (American Type Culture Collection) were grown in DMEM-H/F12 medium with 1% PSF, 10 µg/mL insulin, 100 ng/mL cholera toxin, 20 ng/mL epidermal growth factor, 500 ng/mL hydrocortisone, 5% FBS, and 5% CO2 at 37 °C. The medium was routinely changed every 3 or 4 days. For all experiments, the cells were grown for 24-48 h after splitting to maintain logarithmic growth, and then treated with DMSO or various concentrations of catechol estrogens freshly prepared in DMSO. Measurement of GSH Levels in Cells. The cellular GSH levels were measured by an enzymatic recycling procedure (31), with modifications suitable for the use of 96-well plates. An NADPH generating system (NADP+, glucose-6-phosphate, and glucose-6-phosphate dehydrogenase), instead of NADPH, was used to avoid substrate depletion to increase the linear range. Briefly, the cells on 96-well plates were incubated with 4-OHEN or DMSO. In some cases, the cells were pretreated for 24 h with 50 µM L-buthionine-(S,R)-sulfoximine (BSO) prior to addition of 4-OHEN. After treatment, the medium was removed, and the cells were lysed by two repetitive freeze-thaw cycles (-80 °C for at least 3 h, 37 °C for 2 min; -80 °C for 10 min, 37 °C for 2 min). In each cell-containing well, 40 µL of buffer (125 mM NaH2PO4 and 6.3 mM EDTA, pH 7.5) was added, and various concentrations of GSH in 40 µL of buffer were added to blank wells to prepare a standard curve for calculation. All wells were mixed with 170 µL of a freshly prepared reaction mixture consisting of 20 µL of 6 mM 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), 10 µL of 5 units/mL GR, and 140 µL of NADPH generating system (25 mM Tris-HCl, pH 7.4, 0.01% Tween 20,

Inhibition of GST P1-1 by Equine Catechol Estrogens 1 mM glucose-6-phosphate, 30 µM NADP+, and 2 units/mL glucose-6-phosphate dehydrogenase). The absorbance at 405 nm was measured at 25 °C using a microplate scanning spectrophotometer (Bio-Tck Instruments, Winooski, VT) 5 min after the addition of the reaction mixture. The GSH levels were calculated from the standard curve. Measurement of Enzyme Activity in Cells. All assays were conducted at 25 °C. Total cellular GST activity was measured spectrophotometrically by the method of Habig (32) with minor modifications (33). Briefly, the cells on 96-well plates were incubated with 4-OHEN or DMSO. In some cases, the cells were pretreated for 30 min with 10 µM S-hexylglutathione, or for 24 h with 50 µM BSO prior to addition of 4-OHEN. After treatment, the medium was removed, and cells were lysed by two repetitive freeze-thaw cycles as described above. In each well, 25 µL of 100 mM potassium phosphate buffer (pH 6.5) was added. Following reaction for 45 or 60 min with 100 µL of freshly prepared reaction mixture containing 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) and 2.5 mM GSH, the absorbance at 340 nm was measured. To determine the ability of 4-OHEN to inhibit the activity of other cellular enzymes, cell lysates were prepared as follows after treatment with 10 µM 4-OHEN for 0, 5, 10, or 30 min. Cells were washed with phosphate-buffered saline (PBS) twice and then collected in cold PBS by scraping. The cell pellet (5 × 107 cells) was resuspended in 1 mL of 50 mM potassium phosphate buffer, pH 7.0. After sonication, the lysates were centrifuged at 2000g for 15 min at 4 °C. Aliquots of the supernatants were stored at -80 °C for enzyme activity assays. The protein concentration of lysates was determined by using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). The activity of total SOD and Mn SOD was measured by the nitro blue tetrazolium (NBT) method (34) with minor modifications. This method is based on the inhibition of the reduction of NBT by SOD. The reaction mixture (200 µL) in a 96-well plate contained 20 µL of serially diluted cell lysates, 1 mM diethylenetriaminepentaacetic acid, 26 µg of bovine serum albumin (BSA), 1 unit of catalase, 56 µM NBT, 100 µM xanthine, 50 µM bathocuproinedisulfonic acid, and 6 units/L xanthine oxidase, in 50 mM potassium phosphate buffer, pH 7.8. To measure Mn SOD, 5 mM NaCN, which specifically inhibits the Cu/Zn SOD activity, was included in the reaction mixture, and the solution was incubated at 25 °C for 45 min before the addition of xanthine oxidase. The change in absorbance at 560 nm was measured from 30 to 210 s at 30 s intervals, and the amount of protein which gave half-maximum inhibition was calculated. The activity of catalase was measured as described previously (35) with minor modifications. Diluted cell lysates (20 µL) were reacted with 200 µL of 10 mM H2O2 in 50 mM potassium phosphate buffer, pH 7.0, in a 96-well plate. The change in absorbance at 240 nm at 30 and 210 s was monitored. The activity of cellular GPX was measured by the method of Gunzler and Flohe (36) with minor modifications. Briefly, the incubation mixture (200 µL) in a 96-well plate contained 20 µL of cell lysates, 0.15 mM NADPH, 0.15 mM H2O2, 0.5 mM EDTA, 0.5 mM NaN3, 0.24 unit/mL GR, and 1 mM GSH in 100 mM potassium phosphate buffer, pH 7.0. The nonenzymatic oxidation of GSH was determined under the same conditions with the substitution of reaction buffer for the lysates. The rate of GSH oxidation was measured at 340 nm after starting the reaction by the addition of H2O2. The GPX activity was obtained by subtracting the rate of nonenzymatic GSH oxidation from the overall rate of GSH oxidation. The activity of GR was measured as described previously (37) with minor modifications. Cell lysates (20 µL) were mixed with 20 µL of 6 mM EDTA, 20 µL of 22 mM GSSG, and 120 µL of 180 mM sodium phosphate buffer, pH 7.2. After 5 min incubation at 25 °C, the reaction was started by the addition of 20 µL of 4.5 mM NADPH, and the rate of disappearance of NADPH was measured at 340 nm for 3 min. The activity of PR was determined spectrophotometrically by measuring the rate of NADPH-cytochrome c reduction as

Chem. Res. Toxicol., Vol. 15, No. 7, 2002 937 described previously (38) with minor modifications. The incubation mixture (200 µL) in a 96-well plate contained 20 µL of cell lysates, 40 µM cytochrome c, and 100 µM NADPH in 50 mM potassium phosphate buffer, pH 7.4. The reaction was started by the addition of NADPH, and the change in absorbance at 550 nm was measured for 5 min. The activity of TR was measured by the DTNB reduction assay as described previously (24). The reaction mixture (1 mL) contained 100 µL of cell lysates, 100 mM potassium phosphate, 2 mM EDTA, pH 7.4, 5 mM DTNB, and 200 µM NADPH. The reaction was initiated with NADPH, and the change in absorbance at 412 nm was monitored using a Hewlett-Packard (Palo Alto, CA) model 8452 diode array UV/vis spectrophotometer. The activity of GAPDH was measured in a 1 mL reaction mixture containing 100 mM potassium phosphate (pH 7.4), 1 mM NAD+, 10 mM EDTA, 0.1 mM DTT, 4 mM glyceraldehyde3-phosphate, and 50 µL of cell lysates (39). NADH formation was monitored at 340 nm. The activity of PK was assayed by recording NADH oxidation at 340 nm as described previously (40). The reaction mixture (1 mL) contained 50 mM Tris-HCl, pH 7.4, 100 mM KCl, 5 mM MgCl2, 1 mM ADP, 2.5 international units of L-lactate dehydrogenase, 0.17 mM NADH, 0.5 mM phosphoenolpyruvate, and 100 µL of cell lysates. Western Blotting. After treatment with 10 µM 4-OHEN for the indicated times, the cells were washed with cold PBS and harvested in a lysis buffer (10 mM Tris-HCl, pH 7.4, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 100 µM Na3VO4, 2 mM iodoacetic acid, 5 mM ZnCl2, 1 mM phenylmethylsulfonyl fluoride, and 0.5% Triton-X 100). Cell lysates were prepared as described previously (41). To detect cellular GST P1-1 protein, cell lysates (15 µg) were separated by 10% mini SDS-PAGE and transferred to a PVDF membrane (Millipore, Bedford, MA). After blocking with 5% nonfat dry milk, the membrane was probed with anti-human polyclonal antibody (MBL 312, Panvera, Madison, WI) overnight at 4 °C and then incubated with a horseradish peroxidase-linked anti-mouse IgG antibody (Amersham, Arlington Heights, IL) at 25 °C for 1 h. The protein was visualized using enhanced chemiluminescence (ECL) reagents (Amersham). Inhibition of Purified Human GST P1-1 in Vitro. Recombinant human GST P1-1 was prepared and reduced with dithiothreitol (DTT) as described previously (23). GST P1-1 (200 µg/mL) was preincubated at 25 °C in 100 mM potassium phosphate buffer containing 1 mM EDTA, pH 6.5, with 10 µM 4-OHEN GSH conjugates, 10 µM S-hexylglutathione, 10 µM 4-OHEN, 10 µM 4-OHEN plus 10 µM S-hexylglutathione, or 10 µM 4-OHEN plus 100 µM GSH. Aliquots (10 µL) were removed at various times, and the GST activity was measured at 340 nm in a total volume of 1 mL containing 1 mM CDNB, 2.5 mM GSH, 1 mM EDTA, and 100 mM potassium phosphate, pH 6.5 (23). The percent inhibition was calculated from the difference in rates compared to the DMSO-treated enzyme. Irreversible Inhibition Kinetics of Purified Enzymes. Inhibition kinetic studies were performed according to Kitz and Wilson (42). Purified human GST P1-1, SOD, catalase, GPX, PR, GAPDH, bovine GR, rabbit PK, or TR from E. coli was preincubated at 25 °C for various times with different concentrations of 4-OHEN (5 µM to 1 mM, depending on the sensitivity of the enzyme). Aliquots (10 µL) were removed and diluted 100fold to a total volume of 1 mL. The initial rate of each sample was determined spectrophotometrically as described below. For GST P1-1, the reaction mixture contained 2 µg/mL GST P1-1, 1 mM CDNB, 2.5 mM GSH, 1 mM EDTA, and 100 mM potassium phosphate, pH 6.5, and the change in absorbance was measured at 340 nm. For PR, the reaction mixture contained 1 µg/mL PR, 40 µM cytochrome c, and 100 µM NADPH in 50 mM potassium phosphate buffer, pH 7.4, and the change in absorbance was measured at 550 nm. For catalase, the reaction mixture contained 10 units/mL catalase and 10 mM H2O2 in 50 mM potassium phosphate buffer, pH 7.0, and the change in absorbance was monitored at 240 nm. For GPX, the reaction mixture

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contained 25 milliunits/mL GPX, 0.15 mM NADPH, 0.15 mM H2O2, 0.5 mM EDTA, 0.5 mM NaN3, 0.24 unit/mL GR, and 1 mM GSH in 0.1 M potassium phosphate buffer, pH 7.0, and the rate of GSH oxidation was measured at 340 nm. For GR, the reaction mixture contained 25 nM GR, 0.6 mM EDTA, 2.2 mM GSSG, and 0.45 mM NADPH in 100 mM sodium phosphate buffer, pH 7.2, and the rate of disappearance of NADPH was measured at 340 nm. For GAPDH, the reaction mixture contained 2 µg/mL GAPDH, 1 mM NAD+, 10 mM EDTA, 0.1 mM DTT, and 4 mM glyceraldehyde-3-phosphate in 100 mM potassium phosphate buffer, pH 7.4, and the rate of NADH formation was monitored at 340 nm. For PK, the reaction mixture contained 2 µg/mL PK, 100 mM KCl, 5 mM MgCl2, 1 mM ADP, 2.5 international units of l-lactate dehydrogenase, 0.17 mM NADH, and 0.5 mM phosphoenolpyruvate in 50 mM Tris-HCl buffer, pH 7.4, and the rate of NADH oxidation was measured at 340 nm. For TR, the reaction mixture contained 10 µg/mL TR, 5 mM DTNB, and 200 µM NADPH in 100 mM potassium phosphate and 2 mM EDTA buffer, pH 7.4, and the change in absorbance was monitored at 412 nm. For SOD, the initial rate of samples was assayed by the pyrogallol autoxidation method according to Marklund (43) with minor modifications. The reaction mixture contained 0.4 µg/mL SOD, 200 µM pyrogallol, 0.1 µM catalase, and 1 mM diethylenetriaminepentaacetic acid in air-equilibrated 50 mM Tris-HCl buffer, pH 8.2, and the change in absorbance at 420 nm was measured. The percent inhibition of each enzyme, except SOD, was further calculated from the difference in rates compared to DMSO-treated enzymes. The percent inhibition of SOD by 4-OHEN was calculated as: [(a - b)/(a - c)] × 100, where a is the control slope without SOD, b is the slope with SOD and 4-OHEN, and c is the slope with SOD. The dissociation constant for the reversible enzyme-inhibitor complexes (Ki) and the rate constant for the conversion of the reversible enzyme-inhibitor complexes to the irreversibly inhibited enzymes (k2) of each enzyme were obtained as described previously (22, 42).

Yao et al.

Figure 2. Decrease in cellular GSH levels by 4-OHEN in human breast cancer cells. (A) 10 µM 4-OHEN (squares) or 4-OHE (circles) treatment in MDA-MB-231 cells (closed) and S30 cells (open). (B) 10 min treatment with various concentrations of 4-OHEN (squares) or 4-OHE (circles) in MDA-MB-231 cells (closed) and S30 cells (open). GSH levels after each treatment were measured as described under Materials and Methods. Data represent the mean ( SD from two independent experiments with triplicate measurements.

Results Effect of Catechol Estrogens on GSH Levels in Human Breast Cancer Cells. Previously, we have shown that 4-OHEN reacted with GSH in vitro to form conjugates (22). Since GSH represents the major intracellular nonprotein thiol whose role is to protect cells from oxidative injury, we examined the effect of 4-OHEN and another catechol estrogen, 4,17β-OHEN, on GSH content in human breast cancer cells. We found 4-OHEN (10 µM) significantly decreased GSH levels within minutes in both estrogen receptor (ER) negative cells (MDA-MB-231) and ER positive cells (S30, MDA-MB-231 cells stably transfected with ERR) (Figure 2A). 4-OHEN also caused a dose-dependent decrease in GSH levels in both cell lines (Figure 2B). In contrast, the endogenous catechol estrogen 4-OHE, which does not autoxidize to an o-quinone, did not cause any change in GSH levels in both cell lines (Figure 2). Like 4-OHEN, 4,17β-OHEN had a similar effect on GSH levels in both cell lines (data not shown). Effect of Catechol Estrogens on GST Activity in Human Breast Cancer Cells. 4-OHEN has also been demonstrated to inhibit the activity of purified human GST M1-1 and P1-1 in vitro (22, 23). In the present study, we further examined the effect of 4-OHEN and 4,17βOHEN on GST activity in human breast cancer cells. Similar to the effect on GSH levels, both catechol estrogens at the 10 µM dose significantly inhibited the total GST activity within minutes in S30 cells (Figure 3A). They also caused a dose-dependent decrease in GST activity in S30 cells (Figure 3B). In contrast, 4-OHE did not cause any time- or dose-dependent change in GST

Figure 3. Inhibition of GST activity by 4-OHEN in S30 cells. (A) 10 µM 4-OHEN (squares), 4,17β-OHEN (triangles), or 4-OHE (circles). (B) 10 min treatment with various concentrations of 4-OHEN (squares), 4,17β-OHEN (triangles), or 4-OHE (circles). GST activity after each treatment was measured as described under Materials and Methods. Data represent the mean ( SD from two independent experiments with triplicate measurements.

activity (Figure 3). Similar effects on GST activity were also observed in MDA-MB-231 cells (data not shown), suggesting that the inhibition of GST activity in human breast cancer cells was independent of the presence of ERR.

Inhibition of GST P1-1 by Equine Catechol Estrogens

Chem. Res. Toxicol., Vol. 15, No. 7, 2002 939 Table 1. Kinetic Parameters of Irreversible Inhibition of Enzymes by 4-OHENa enzyme

Ki (µM)

k2 × 103 (s-1)

k2/Ki (M-1 s-1)

GST P1-1

20.8 ( 1.2 18.3 ( 0.3b 52.4 ( 4.6 77.4 ( 3.8 159 ( 6 230 ( 30 448 ( 31 562 ( 53 620 ( 80 694 ( 12 1410 ( 210

1.4 ( 0.1 1.72 ( 0.02b 3.3 ( 0.3 8.2 ( 0.4 9.2 ( 0.4 33 ( 4 4.1 ( 0.3 6.1 ( 0.6 7.3 ( 0.9 6.4 ( 0.1 2.4 ( 0.4

67 94 63 106 58 143 9.2 11 12 9.2 1.7

GAPDH PR PK GR SOD catalase GST M1-1c TR GPX

a Purified enzymes were preincubated with different concentrations of 4-OHEN for various times. Aliquots were removed and diluted 100-fold into assay buffer. The initial rate was determined spectrophotometrically as described under Materials and Methods. The dissociation constants for the reversible enzyme-inhibitor complexes (Ki) and the rate constants for the conversion of the reversible enzyme-inhibitor complexes to the irreversibly inhibited enzymes (k2) were calculated as described (22, 42). b Data from reference (23). c Data from reference (22).

Figure 4. Inhibition of human GST P1-1 by 4-OHEN. (A) Recombinant GST P1-1 was incubated with 10 µM S-hexylglutathione (diamonds), 10 µM 4-OHEN in the presence of 100 µM GSH (circles), 10 µM 4-OHEN GSH conjugates (closed triangles), 10 µM 4-OHEN in the presence of 10 µM Shexylglutathione (squares), or with 10 µM 4-OHEN (open triangles). Aliquots were removed at various times, and the GST activity was measured as described under Materials and Methods. (B) GST activity in MDA-MB-231 cells after treatment of 10 µM 4-OHEN (triangles), 10 µM 4-OHEN following 24 h treatment of 50 µM BSO (circles), or 10 µM 4-OHEN in the presence of 10 µM S-hexylglutathione (squares). Experimental details were described under Materials and Methods. Data represent the mean ( SD from two independent experiments with triplicate measurements.

In MDA-MB-231 cells, GST P1-1 is the dominant form of GSTs (44). However, we did not observe any timedependent (10-240 min) decrease in GST P1-1 protein as determined by Western blotting after 10 µM 4-OHEN treatment in MDA-MB-231 cells or S30 cells (Supporting Information, Figure A). Interestingly, 4-OHEN did not cause a significant change in GST activity in HepG2 cells, which lack GST P1-1 (45, 46); a time- and dose-dependent inhibition of GST activity by 4-OHEN was observed in MCF-10A cells, which contain GST P1-1 (47) (Supporting Information, Figure B), suggesting that the inhibition of total GST activity in MDA-MB-231 and S30 cells by 4-OHEN is primarily due to inhibition of GST P1-1. Modulation of 4-OHEN-Mediated Inhibition of Human GST P1-1. As expected, the inactivation of GST P1-1 in vitro by 10 µM 4-OHEN was significantly decreased in the presence of 10-fold higher concentration of GSH (100 µM) (Figure 4A). In addition, depletion of GSH by BSO, a GSH synthesis inhibitor, slightly enhanced the inhibition of GST activity by 4-OHEN in MDA-MB-231 cells (Figure 4B). Our results also showed that 4-OHEN GSH conjugates contributed about 27% to the inactivation of GST P1-1 by 10 µM 4-OHEN in vitro (Figure 4A). S-Hexylglutathione, a known competitive inhibitor of GSTs, at a noninhibitory concentration (10 µM), decreased the inhibition of human GST P1-1 by 10 µM 4-OHEN about one-third in vitro (Figure 4A). In MDA-MB-231 cells, treatment with S-hexylglutathione

alone (10 µM) only slightly inhibited GST activity; GST activity remained greater than 90% at 30 and 60 min after the treatment (data not shown). In contrast to the in vitro assay, the inhibition of GST activity by 10 µM 4-OHEN was slightly enhanced in the presence of Shexylglutathione (10 µM) in cells (Figure 4B). These data suggest that the major mechanism for the inactivation of GST P1-1 is modification of sulfhydryl groups primarily by 4-OHEN although the 4-OHEN GSH conjugates could also contribute to the inhibition mechanism. Kinetics of Irreversible Inhibition. We performed kinetic experiments by incubating 4-OHEN with purified human GST P1-1, SOD, catalase, GPX, PR, GAPDH, bovine GR, rabbit PK, or TR from E. coli to examine whether 4-OHEN inactivated these enzymes in vitro (Table 1). All enzyme inhibition time-courses followed first-order kinetics (data not shown). The data showed that human GST P1-1 had the lowest dissociation constant Ki value (20.8 µM) among all the enzymes tested (Table 1), indicating that GST P1-1 is the most sensitive to 4-OHEN-induced irreversible inactivation. GAPDH was the second most affected enzyme with a 2.5-fold higher Ki value than GST P1-1. The Ki values of catalase, GST M1-1, TR, and GPX were over 500 µM. Effect of 4-OHEN on Other Cellular Enzymes in Human Breast Cancer Cells. To further examine whether 4-OHEN inhibits these enzymes in breast cancer cells, we measured the activity of cellular GAPDH, PR, PK, GR, total SOD, Mn SOD, catalase, TR, and GPX after 10 µM 4-OHEN treatment for 5, 10, or 30 min in MDAMB-231 and S30 cells. Figure 5 showed that the total GST activity was significantly inhibited by 10 µM 4-OHEN treatment in MDA-MB-231 cells, which was consistent with the low Ki value obtained with the purified enzyme (Table 1). GAPDH, which was the second most sensitive enzyme, was slightly inhibited by about 20% by 30 min in 10 µM 4-OHEN-treated MDA-MB-231 cells. Other cellular enzymes tested were resistant to 4-OHEN-induced inhibition compared to GST in MDAMB-231 cells (Figure 5). Similar results were also observed in S30 cells after 10 µM 4-OHEN treatment (data not shown).

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Figure 5. Activity of GST, GAPDH, PR, PK, GR, total SOD, Mn SOD, catalase, TR, and GPX after 10 µM 4-OHEN treatment in MDA-MB-231 cells. The activity of cellular enzymes at 5 min (open bar), 10 min (bar with horizontal stripes), or 30 min (solid bar) after 4-OHEN treatment was measured as described under Materials and Methods. Data represent the mean ( SD of triplicate measurements (GAPDH, PR, PK, GR, catalase, and GPX) or from 2 to 4 independent experiments (GST, SOD, Mn SOD, and TR).

Discussion Previously, we demonstrated that 4-OHEN rapidly autoxidized in vitro to 4-OHEN-o-quinone, which could react with GSH to form conjugates, and inhibited the activity of human GST M1-1 (22), as well as human GST P1-1, by covalent modification of cysteine residues and disulfide bond formation (23). In the present study, we further showed that both 4-OHEN and 4,17β-OHEN decreased the cellular GSH levels and inhibited the GST activity in human breast cancer cells and that the GSTs in these cells were more sensitive to the 4-OHEN-induced inhibition compared to other cellular enzymes including GAPDH, PR, PK, GR, total SOD, Mn SOD, catalase, TR, and GPX. 4-OHEN also inhibited the GST activity in human nontransformed breast epithelial MCF-10A cells; however, no change in GST activity was observed in human hepatoma HepG2 cells, which do not contain GST P1-1 (45, 46). These data suggest that rapid and significant inhibition of GST activity in human breast cancer cells by 4-OHEN was due to the presence of GST P1-1, the dominant form of GSTs in these cells. However, since these cells contain other GST isoforms, such as GST M11, which are not sensitive to the inhibition of 4-OHEN, we found only about a 60% inhibition of GST activity after 10 µM 4-OHEN treatment for 10 min in both S30 and MDA-MD-231 and 73% inhibition at a dose of 50 µM 4-OHEN (Figures 3 and 4). The regulation of GST genes has been widely investigated. Pearson et al. have demonstrated a tissue-specific induction of murine GST mRNAs by butylated hydroxyanisole (48). Induction of GST gene expression by phenobarbital, 3-methylcholanthrene, and dithiolethiones in cultured human hepatocytes has also been observed (49). GST expression was transcriptionally regulated in HBVtransfected HepG2 cells (50), and hypermethylation of the GST P1-1 promoter down-regulated GST P1-1 expression in human prostate cancer cells (51). Activation of GST gene expression by antioxidants and other inducers could be mediated through the antioxidant response element (ARE) and/or the xenobiotic response element (XRE) (52). ROS-generating agents activated GST P1-1 gene transcription via the JNK/Jun cascade in cells (53, 54). Unlike these examples, in the present study we found that the rapid and significant inhibition of GST activity by 4-OHEN did not result from a decrease in GST P1-1 protein concentration. A change in GST gene transcrip-

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tion was unlikely due to the inhibition occurring within minutes after treatment in cells. Protein modification, which impairs GST activity, was the most likely scenario. In this study, we determined the 4-OHEN-induced GST inactivation was dependent on GSH concentration. The inactivation of GST P1-1 by 4-OHEN in vitro was lessened significantly in the presence of GSH (Figure 4A), and depletion of GSH by BSO slightly enhanced GST inhibition by 4-OHEN in MDA-MB-231 cells (Figure 4B). We further showed that 4-OHEN GSH conjugates only partially contributed to the 4-OHEN-induced inactivation of human GST P1-1 in vitro (Figure 4A). In addition, S-hexylglutathione provided protection against GST P1-1 inactivation by 4-OHEN in vitro (Figure 4A), suggesting that the irreversible inhibition of human GST P1-1 by 4-OHEN could occur at or near the region of the GSH binding site rather than random modification at other sites (55). Previously, we showed in vitro that 4-OHEN modified Cys47, which is located near the GSH binding site and is important in maintaining the proper intact protein structure for catalysis (23). These data indicate that protein modification of GST P1-1 plays an important role, which is supported by our previous studies demonstrating that 4-OHEN inactivated human GST P1-1 in vitro by covalent modification of cysteine residues and disulfide bond formation (23). Our in vitro kinetic inhibition experiments showed that human GST P1-1 had the lowest Ki value among all the enzymes tested (Table 1), suggesting that GST P1-1 has the highest affinity for 4-OHEN. The Ki and k2 values of GST P1-1 with 4-OHEN in this study were consistent with those reported previously [Table 1, (23)]. The k2/Ki values obtained for GST P1-1, GAPDH, and PK were similar while PR and GR had even higher values; however, inhibition of GST activity by 10 µM 4-OHEN was much greater than inhibition of the other cellular enzymes in human breast cancer cells (Figure 5). An explanation for the selectivity of 4-OHEN for GST P1-1 in vivo is likely a combination of the abundance of this enzyme relative to the others in breast cancer cells and its very low Ki value. Similarly, others have reported that haloenol lactone is a selective and irreversible inhibitor of mouse GST P1-1 with a t1/2 of about 2 min in vitro (55), and is an irreversible inhibitor of GST P1-1 in human renal carcinoma cells (56). Taken together, our data suggest that GST P1-1 may be a preferred protein target for equine catechol estrogens in vivo and modification of sulfhydryl groups could represent the major mechanism for the inactivation of GST P1-1 by 4-OHEN. 4-OHEN induced DNA damage and apoptosis in MDAMB-231 and S30 cells, and agents which reduce GSH or catalyze redox cycling enhance both effects (11, 13). The inhibition of GST activity in cells reported in the present study may be associated with early events that trigger apoptosis. Not only is GST a detoxification enzyme providing protection against products of oxidative stress, it has also been shown to be involved in cellular regulation through the JNKs signaling pathway. Adler et al. have demonstrated that human GST P1-1 is an endogenous inhibitor of JNKs, which belong to the multimember family of stress kinases and play a role in cell growth, apoptosis, and transformation (20). It has been reported that GST inhibitors (S-hexylglutathione and ethacrynic acid) caused significant male germ cell apoptosis and that apoptosis, induced by H2O2, could be enhanced by the presence of GST inhibitors (57). Baez

Inhibition of GST P1-1 by Equine Catechol Estrogens

et al. have also shown that GST can protect cells against apoptosis induced by o-quinones derived from catecholamines (58). Consistently, the endogenous catechol estrogen 4-OHE, which did not decrease the intercellular GSH levels nor inhibit GST activity in cells (Figures 2 and 3), has been shown to be ineffective at inducing apoptosis in human breast cancer cells (11). However, we did not observe any significant change in the amount of phosphorylated JNKs protein within 4 h after 10 µM 4-OHEN treatment in both MDA-MB-231 and S30 cells (data not shown), although the cellular GST activity was significantly inhibited within minutes. It will be the subject of future work to study the signal transduction pathways activated after the decrease in cellular GSH levels and the inhibition of GST activity by catechol estrogens in cells. In summary, we found that both 4-OHEN and 4,17βOHEN significantly decreased GSH levels and GST activity in human breast cancer cells within minutes. GSH partially protected the inactivation of GST P1-1 by 4-OHEN in vitro, and depletion of GSH in cells enhanced the inhibition of GST activity. In addition, 4-OHEN GSH conjugates partially contributed to 4-OHEN-induced inactivation of GST P1-1. In vitro kinetic inhibition experiments with 4-OHEN demonstrated that GST P1-1 had a much lower Ki value compared to GAPDH, PR, PK, GR, SOD, catalase, GST M1-1, TR, and GPX. In contrast to the significant inhibition of GST activity in human breast cancer cells, 4-OHEN only slightly inhibited the GAPDH activity, and other enzymes including PR, PK, GR, total SOD, Mn SOD, catalase, TR, and GPX were resistant to 4-OHEN-induced inhibition. Our data indicated that GST P1-1 is a preferred target for inhibition by equine catechol estrogens in vivo.

Acknowledgment. This research was supported by NIH Grant CA73638. We are grateful to Dr. V. C. Jordan (Northwestern University) for the gift of the S30 cell line. In addition, we greatly appreciate access to the Bioassay Research Facility under the direction of Dr. John. M. Pezzuto and helpful discussions with Dr. Andrew Mesecar (University of Illinois at Chicago).

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Supporting Information Available: Two figures showing Western blotting analysis of GST P1-1 after 10 µM 4-OHEN treatment and inhibition of GST activity after 10 µM 4-OHEN treatment. This material is available free of charge via the Internet at http://pubs.acs.org.

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