Fate of 3,3′-diindolylmethane in cultured MCF-7 human breast cancer cells
Fate of 3,3′-diindolylmethane in cultured MCF-7 human breast cancer cells
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DOI：
10.1021/tx050325z
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发表时间：
2006-03-01
影响因子：
4.1
通讯作者：
Bjeldanes, LF
中科院分区：
文献类型：
--
作者：
Staub, RE;Onisko, B;Bjeldanes, LF
3，3 '-二吲哚基甲烷（DIM）是在芸苔属蔬菜中发现的癌症预防剂吲哚-3-甲醇的主要体内产物。在这里，我们报告了放射性标记的DIM在MCF-7细胞中的代谢命运。DIM缓慢代谢为主要在培养基中检测到的氧化DIM产物的几种硫酸盐结合物。在直至72 h处理的所有时间间隔，细胞中检测到的放射性主要为未修饰的DIM（81-93%）。用槲皮素共处理MCF-7细胞减慢了氧化DIM产物在培养基中积累的速率，而吲哚[3，2-B]咔唑（ICZ）共处理加速了它们的产生。ICZ是P450 1A 2的诱导剂，而槲皮素是该亚型的特异性抑制剂，表明P450 1A 2主要负责DIM的氧化，可能通过类似于3-甲基吲哚的2，3-环氧化。氧化的DIM代谢物的硫酸盐缀合物通过硫酸酯酶消化裂解，并通过LC/MS鉴定为3-（1H-吲哚-3-基甲基）-2-羟吲哚（2-ox-DIM），双（1H-吲哚-3-基）甲醇（3-亚甲基羟基-DIM），3-[羟基-（1H-吲哚-3-基）-甲基]-1，3-二氢-2-羟吲哚（3-亚甲基羟基-2-ox-DIM）和3-羟基-3-（1H-吲哚-3-基甲基）-2-羟吲哚（3-羟基-2-ox-DIM）。2-ox-DIM衍生物在硫酸酯酶消化培养基中的放射性大于30%。虽然羟吲哚的形成是MCF-7细胞的主要代谢途径，合成的2-ox-DIM在4-ERE-荧光素酶报告基因分析中无活性，因此，可能不是以前观察到的DIM的雌激素活性的原因。未修饰的DIM迅速积累在核膜中，在0.5-2 h处理后约占放射性的35-40%。放射性标记的DIM的摄取似乎是一种被动分配到核膜，并不依赖于细胞胞质溶胶。DIM的核摄取是不饱和的，不能被未标记的DIM（100 μ M）的预处理阻断。此外，在无血清培养基中的处理增加了MCF-7细胞对放射性标记的DIM的摄取。这些发现表明，膜对DIM的摄取显著增加了其局部浓度，这可能有助于其生物活性。
3,3'-Diindolylmethane (DIM) is a major in vivo product of the cancer preventative agent indole-3-carbinol that is found in vegetables of the genus Brassica. Here, we report on the metabolic fate of radiolabeled DIM in MCF-7 cells. DIM was slowly metabolized to several sulfate conjugates of oxidized DIM products that were primarily detected in the medium. The radioactivity detected in cells was predominantly unmodified DIM (81-93%) at all time intervals up to 72 h treatment. Co-treatment of MCF-7 cells with quercetin slowed the rate that oxidized DIM products accumulated in the medium, while indole[3,2-b]carbazole (ICZ) co-treatment accelerated their production. ICZ is an inducer of P450 1A2, while quercetin is a specific inhibitor of this isoform, suggesting that P450 1A2 is primarily responsible for the oxidation of DIM, probably through 2,3-epoxidation similar to 3-methylindole. Sulfate conjugates of oxidized DIM metabolites were cleaved by sulfatase digestion and identified by LC/MS as 3-(1H-indole-3-ylmethyl)-2-oxindole (2-ox-DIM), bis(1H-indol-3-yl)methanol (3-methylenehydroxy-DIM), 3-[hydroxy-(1H-indol-3-yl)-methyl]-1,3-dihydro-2-oxindole (3-methylenehydroxy-2-ox-DIM), and 3-hydroxy-3-(1H-indole-3-ylmethyl)-2-oxindole (3-hydroxy-2-ox-DIM). Derivatives of 2-ox-DIM represented greater than 30% of the radioactivity in the sulfatase-digested medium. Although oxindole formation was the primary metabolic pathway in MCF-7 cells, synthetic 2-ox-DIM was inactive in a 4-ERE-luciferase reporter assay and, therefore, probably not responsible for the estrogenic activity previously observed for DIM. Unmodified DIM rapidly accumulated in the nuclear membranes representing approximately 35-40% of the radioactivity after 0.5-2 h treatment. Uptake of radiolabeled DIM appeared to be a passive partitioning into the nuclear membranes and was not dependent upon the cell cytosol. The nuclear uptake of DIM was not saturable and could not be blocked by pretreatment with unlabeled DIM (100 mu M). Further, treatments in serum-free medium increased the uptake of radiolabeled DIM by the MCF-7 cells. These findings show that the uptake of DIM by membranes significantly increases its localized concentration, which may contribute to its biological activities.