SULT1A1 catalyzes 2-methoxyestradiol sulfonation in MCF-7 breast cancer cells.
SULT1A1 catalyzes 2-methoxyestradiol sulfonation in MCF-7 breast cancer cells.
复制标题
DOI：
10.1093/carcin/21.11.1947
复制
发表时间：
2000-11
期刊：
影响因子：
4.7
通讯作者：
B. C. Spink;Barbara H. Katz;M. Hussain;Shaokun Pang;Shaokun Pang;Steven Connor;Kenneth M. Aldous;Kenneth M. Aldous;J. Gierthy;J. Gierthy;D. Spink;D. Spink
中科院分区：
文献类型：
--
作者：
B. C. Spink;Barbara H. Katz;M. Hussain;Shaokun Pang;Shaokun Pang;Steven Connor;Kenneth M. Aldous;Kenneth M. Aldous;J. Gierthy;J. Gierthy;D. Spink;D. Spink
在先前对9个人类乳腺细胞系的研究中，当培养物暴露于芳香烃受体激动剂2，3，7，8-四氯二苯并对二恶英时，17 β-雌二醇（E（2））的代谢率大大提高。观察到C-2、-4、-6 α和-15 α位置的E（2）羟基化速率升高，同时伴有细胞色素P450 1A 1和1B 1的诱导。在每种细胞系中，2-和4-羟基乙醇（2-和4-OHE（2））通过儿茶酚O：-甲基转移酶的作用转化为2-和4-甲氧基乙醇（2-和4-MeOE（2））。在这项研究中，这些雌激素代谢产物的共轭进行了研究。对使用和未使用粗品β-葡萄糖醛酸苷酶/硫酸酯酶制剂预先处理培养基测定的代谢物水平进行比较，结果表明，存在的大部分2-MeOE（2）为结合形式，而4-MeOE（2）、6 α-OHE（2）和15 α-OHE（2）的结合程度极低。抑制剂研究表明，是该制剂的硫酸酯酶活性水解了MCF-7细胞培养基中的2-MeOE（2）结合物;通过电喷雾离子阱质谱法证实了MCF-7培养基中2-MeOE（2）-3-硫酸盐的存在。为了鉴定催化这种结合的酶，通过使用逆转录-聚合酶链反应在9种细胞系中评价编码5种磺基转移酶（SULT 1A 1、SULT 1A 2、SULT 1A 3、SULT 1 E1和SULT 2A 1）的mRNA的表达。在这些细胞系中，只有SULT 1A 1 mRNA的表达与观察到的纳摩尔水平的2-MeOE结合相关（2）。对MCF-7细胞SULT 1A 1 cDNA的克隆和测序显示，编码两种先前鉴定的等位基因变体SULT 1A 1 *1（（213）Arg）和SULT 1A 1 *2（（213）His）的mRNA在这些细胞中表达。在通常不表达SULT 1A 1的MDA-MB-231细胞中，异源cDNA指导的任一变体的表达赋予2-MeOE（2）磺化活性。SULT 1A 1等位基因变体也在SF：9昆虫细胞中表达，使用微粒体后上清液测定SULT 1A 1 *1和SULT 1A 1 *2的K：（m）值，分别为0.90 +/- 0.12和0.81 +/- 0.06 μ M，以2-MeOE（2）为底物。这些结果表明，SULT 1A 1是2-MeOE（2）磺化的有效和选择性催化剂，因此，在调节最近描述的2-MeOE（2）的抗癌作用中可能是重要的。
In a previous study of nine human breast-derived cell lines, rates of metabolism of 17beta-estradiol (E(2)) were greatly enhanced when cultures were exposed to the aromatic hydrocarbon receptor agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin. Elevated rates of E(2) hydroxylation at the C-2, -4, -6alpha and -15alpha positions were observed concomitant with the induction of cytochromes P450 1A1 and 1B1. In each cell line, 2- and 4-hydroxyestradiol (2- and 4-OHE(2)) were converted to 2- and 4-methoxyestradiol (2- and 4-MeOE(2)) by the action of catechol O:-methyltransferase. In this study, conjugation of these estrogen metabolites was investigated. A comparison of the levels of metabolites determined with and without prior treatment of the media with a crude beta-glucuronidase/sulfatase preparation showed that most of the 2-MeOE(2) present was in conjugated form, whereas 4-MeOE(2), 6alpha-OHE(2) and 15alpha-OHE(2) were minimally conjugated. Inhibitor studies suggested that it was the sulfatase activity of the preparation that hydrolyzed the 2-MeOE(2) conjugates in MCF-7 cell media; the presence of 2-MeOE(2)-3-sulfate in MCF-7 culture media was confirmed by electrospray ion-trap mass spectrometry. To identify the enzyme catalyzing this conjugation, the expression of mRNAs encoding five sulfotransferases (SULT1A1, SULT1A2, SULT1A3, SULT1E1 and SULT2A1) was evaluated in the nine cell lines by use of the reverse transcription-polymerase chain reaction. Only expression of SULT1A1 mRNA correlated with the observed conjugation of nanomolar levels of 2-MeOE(2) in these cell lines. Cloning and sequencing of SULT1A1 cDNA from MCF-7 cells revealed that mRNAs encoding two previously identified allelic variants, SULT1A1*1 ((213)Arg) and SULT1A1*2 ((213)His), were expressed in these cells. Heterologous cDNA-directed expression of either variant in MDA-MB-231 cells, which do not normally express SULT1A1, conferred 2-MeOE(2) sulfonation activity. The SULT1A1 allelic variants were also expressed in SF:9 insect cells, from which post-microsomal supernatants were used to determine K:(m) values of 0.90 +/- 0.12 and 0.81 +/- 0.06 microM for SULT1A1*1 and SULT1A1*2, respectively, with 2-MeOE(2) as substrate. These results show that SULT1A1 is an efficient and selective catalyst of 2-MeOE(2) sulfonation and, as such, may be important in modulating the anticarcinogenic effects of 2-MeOE(2) that have been described recently.