3,3′-Diindolylmethane (DIM) induces a G1 cell cycle arrest in human breast cancer cells that is accompanied by Sp1-mediated activation of p21WAF1/CIP1 expression
3,3′-Diindolylmethane (DIM) induces a G1 cell cycle arrest in human breast cancer cells that is accompanied by Sp1-mediated activation of p21WAF1/CIP1 expression
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DOI：
10.1093/carcin/23.8.1297
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发表时间：
2002-08-01
期刊：
影响因子：
4.7
通讯作者：
Bjeldanes, LF
中科院分区：
文献类型：
--
作者：
Hong, CB;Kim, HA;Bjeldanes, LF
3，3 '-二吲哚基甲烷（DIM）是一种来源于芸苔属食用植物的有前途的癌症化学预防剂。为了确定这种天然吲哚是否对人乳腺癌细胞具有直接的生长抑制作用，我们研究了DIM在雌激素依赖性（MCF-7）和雌激素非依赖性（MDA-MB-231）人乳腺癌细胞系中的细胞周期调节作用。流式细胞术研究的结果表明，DIM处理产生了显着增加（从51%至79%）的细胞周期的G（1）期的细胞比例，无论雌激素受体的状态。G（1）-作用细胞周期组分的分析表明，细胞周期蛋白依赖性激酶（CDK）2的酶活性也强烈降低。Western印迹分析显示，与DIM诱导的细胞周期停滞同时，DIM刺激CDK抑制剂p21（WAF 1/CIP 1）（p21）水平的快速和显著增加。北方印迹分析表明，DIM增加p21 mRNA的表达与最大的6-7倍的诱导，和暴露于放线菌酮没有阻断的反应。在MCF-7和MDA-MB-231人乳腺癌细胞中观察到p21蛋白和mRNA表达的类似增加，表明DIM诱导p21表达不依赖于雌激素受体信号传导和p53。瞬时转染p21启动子的5 ′-缺失构建体证明，含有6个启动子特异性转录因子1（Sp1）元件的近端启动子的第一个291 bp片段保持DIM响应性。与Sp1在此响应中的作用一致，由三个共识Sp1结合位点驱动的报告构建体对DIM有响应。此外，电泳迁移率变动分析表明DIM诱导了Sp1和Sp3与共识Sp1响应元件的结合。因此，我们的观察揭示了DIM的抗增殖途径，其涉及Sp1/Sp3诱导的p21表达作为人乳腺癌细胞中细胞周期控制的靶点。
3,3'-Diindolylmethane (DIM) is a promising cancer chemopreventive agent derived from Brassica food plants. To determine whether this natural indole has a direct growth inhibitory effect on human breast cancer cells, we examined the cell cycle regulatory effects of DIM in estrogendependent (MCF-7) and estrogen-independent (MDA-MB-231) human breast cancer cell lines. Results of flow cytometry studies showed that DIM treatment produced a marked increase (from 51 to 79%) in the proportion of cells in the G(1) phase of the cell cycle, regardless of estrogen-receptor status. Analyses of G(1)-acting cell cycle components indicated that the enzymatic activity of cyclin-dependent kinase (CDK) 2 was also strongly reduced. Western blot analyses showed that, concurrent with the DIM-induced cell cycle arrest, DIM stimulated a rapid and pronounced increase in levels of the CDK inhibitor, p21(WAF1/CIP1) (p21). Northern blot analysis demonstrated that DIM increased p21 mRNA expression with a maximal 6-7-fold induction, and exposure to cycloheximide did not block the response. Similar increases in expression of p21 protein and mRNA were observed in both MCF-7 and MDA-MB-231 human breast cancer cells, suggesting that DIM induction of p21 expression is independent of estrogen-receptor signaling and p53. Transient transfection of 5'-deletion constructs of the p21 promoter demonstrated that the first 291 bp segment of the proximal promoter, which contains six promoter specific transcription factor 1 (Sp1) elements, maintained DIM responsiveness. Consistent with a role for Sp1 in this response, a reporter construct driven by three consensus Sp1 binding sites was responsive to DIM. In addition, electrophoretic mobility shift assays showed that DIM induced the binding of Sp1 and Sp3 to the consensus Sp1 responsive element. Thus, our observations have uncovered an antiproliferative pathway for DIM that implicates Sp1/Sp3-induced expression of p21 as a target for cell cycle control in human breast cancer cells.