MacIndoe J H
Department of Internal Medicine, University of Iowa College of Medicine, Iowa City.
Endocrinology. 1988 Sep;123(3):1281-7. doi: 10.1210/endo-123-3-1281.
Reports of estrone (E1) and dehydroepiandrosterone (DHEA) sulfatase (sulfohydrolase) activities within many human breast cancers have prompted us to undertake the identification and partial characterization of these enzyme activities within MCF-7 human breast cancer cells. Enzyme assays were performed within subcellular preparations and intact cultures by quantifying the total nonpolar 3H-labeled metabolites formed from [3H]E1 sulfate (E1S) and [3H]DHEA sulfate (DHEAS). The results have shown that the hydrolysis of each steroid sulfate is mediated by different particulate enzymes, which demonstrate optimal activity between pH 6.0-7.0. The analysis of enzyme kinetic data showed the Km values of E1S and DHEAS for their enzymes to be approximately 6.3 and 3.6 microM/L, respectively. Neither enzyme was subject to product inhibition. Androsterone sulfate and pregnenolone sulfate produced significant inhibition of E1, but not DHEA, sulfatase activity. E1S inhibited DHEA sulfatase competitively, with an approximate Ki of 11 microM, whereas DHEAS inhibited E2 sulfatase in a noncompetitive fashion, demonstrating an approximate Ki of 0.6 microM. Studies carried out with intact MCF-7 cultures using physiological concentrations of 3H-labeled E1S (2 nM) or DHEAS (1 microM) showed the accumulation of nonpolar metabolites during a 20-h incubation period. When cultures were incubated with similar concentrations of both steroid sulfates the apparent intracellular activity of E1 sulfatase was reduced by approximately 70%, whereas DHEA sulfatase activity remained unchanged. The results of these studies confirm the ability of MCF-7 cells to hydrolyze extracellular E1S and DHEAS, indicate that these reactions are mediated by different enzymes, and demonstrate that DHEAS is a potent inhibitor of MCF-7 E1 sulfatase. Circulating DHEAS, therefore, may substantially limit the ability of most postmenopausal breast cancers to use E1S as a substrate for intracellular estrogen biosynthesis.
许多人类乳腺癌中存在雌酮（E1）和脱氢表雄酮（DHEA）硫酸酯酶（硫酸水解酶）活性的报道促使我们对MCF-7人乳腺癌细胞中的这些酶活性进行鉴定和部分特性分析。通过定量由[3H]E1硫酸盐（E1S）和[3H]DHEA硫酸盐（DHEAS）形成的总非极性3H标记代谢物，在亚细胞制剂和完整培养物中进行酶活性测定。结果表明，每种甾体硫酸盐的水解由不同的颗粒酶介导，这些酶在pH 6.0 - 7.0之间表现出最佳活性。酶动力学数据分析表明，E1S和DHEAS对其酶的Km值分别约为6.3和3.6 microM/L。两种酶均不受产物抑制。硫酸雄酮和硫酸孕烯醇酮对E1硫酸酯酶活性有显著抑制作用，但对DHEA硫酸酯酶活性无抑制作用。E1S竞争性抑制DHEA硫酸酯酶，近似Ki为11 microM，而DHEAS以非竞争性方式抑制E2硫酸酯酶，近似Ki为0.6 microM。使用生理浓度的3H标记E1S（2 nM）或DHEAS（1 microM）对完整的MCF-7培养物进行的研究表明，在20小时的孵育期内非极性代谢物会积累。当培养物与相似浓度的两种甾体硫酸盐一起孵育时，E1硫酸酯酶的表观细胞内活性降低了约70%，而DHEA硫酸酯酶活性保持不变。这些研究结果证实了MCF-7细胞水解细胞外E1S和DHEAS的能力，表明这些反应由不同的酶介导，并证明DHEAS是MCF-7 E1硫酸酯酶的有效抑制剂。因此，循环中的DHEAS可能会极大地限制大多数绝经后乳腺癌利用E1S作为细胞内雌激素生物合成底物的能力。
