Inhibition of cellular enzymes by equine catechol estrogens in human breast cancer cells: specificity for glutathione S-transferase P1-1.
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, 4,17beta-hydroxyequilenin (4,17beta-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 K(i) value (20.8 microM) compared to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 52.4 microM), P450 reductase (PR, 77.4 microM), pyruvate kinase (PK, 159 microM), glutathione reductase (GR, 230 microM), superoxide dismutase (SOD, 448 microM), catalase (562 microM), GST M1-1 (620 microM), thioredoxin reductase (TR, 694 microM), and glutathione peroxidase (GPX, 1410 microM). 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.
Department of Medicinal Chemistry and Pharmacognosy (M/C 781), College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612, USA.
Jiaqin
J
Yao
Minsun
M
Chang
Yan
Y
Li
Emily
E
Pisha
Xuemei
X
Liu
Dan
D
Yao
Ebrahim C
EC
Elguindi
Sylvie Y
SY
Blond
Judy L
JL
Bolton
D000818:Animals; D001943:Breast Neoplasms / Q000201:enzymology* / Q000473:pathology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004855:Equilenin / Q000031:analogs & derivatives* / Q000494:pharmacology*; D002393:Estrogens, Catechol / Q000494:pharmacology*; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors*; D006736:Horses; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors*; D007700:Kinetics; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D002393:Estrogens, Catechol; D007527:Isoenzymes; C065250:4-hydroxy-equilenin; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase; D004855:Equilenin