BMC Medical Genomics, 2008, 1(1):1-13.

The molecular basis of genistein-induced mitotic arrest and exit of self-renewal in embryonal carcinoma and primary cancer cell lines

Christian RA Regenbrecht, James Adjaye, et al

Max Planck Institute for Molecular Genetics, Department for Vertebrate Genomics, Ihnestr. 73, D-14195 Berlin, Germany

BACKGROUND:

Genistein is an isoflavonoid present in soybeans that exhibits anti-carcinogenic properties. The issue of genistein as a potential anti-cancer drug has been addressed in some papers, but comprehensive genomic analysis to elucidate the molecular mechanisms underlying the effect elicited by genistein on cancer cells have not been performed on primary cancer cells, but rather on transformed cell lines. In the present study, we treated primary glioblastoma, rhabdomyosarcoma, hepatocellular carcinoma and human embryonic carcinoma cells (NCCIT) with mu-molar concentrations of genistein and assessed mitotic index, cell morphology, global gene expression, and specific cell-cycle regulating genes. We compared the expression profiles of NCCIT cells with that of the cancer cell lines in order to identify common genistein-dependent transcriptional changes and accompanying signaling cascades.

METHODS:

We treated primary cancer cells and NCCIT cells with 50 muM genistein for 48 h. Thereafter, we compared the mitotic index of treated versus untreated cells and investigated the protein expression of key regulatory self renewal factors as OCT4, SOX2 and NANOG. We then used gene expression arrays (Illumina) for genome-wide expression analysis and validated the results for genes of interest by means of Real-Time PCR. Functional annotations were then performed using the DAVID and KEGG online tools.
RESULTS:

We found that cancer cells treated with genistein undergo cell-cycle arrest at different checkpoints. This arrest was associated with a decrease in the mRNA levels of core regulatory genes, PBK, BUB1, and CDC20 as determined by microarray-analysis and verified by Real-Time PCR. In contrast, human NCCIT cells showed over-expression of GADD45 A and G (growth arrest- and DNA-damage-inducible proteins 45A and G), as well as down-regulation of OCT4, and NANOG protein. Furthermore, genistein induced the expression of apoptotic and anti-migratory proteins p53 and p38 in all cell lines. Genistein also up-regulated steady-state levels of both CYCLIN A and B.

CONCLUSION:

The results of the present study, together with the results of earlier studies show that genistein targets genes involved in the progression of the M-phase of the cell cycle. In this respect it is of particular interest that this conclusion cannot be drawn from comparison of the individual genes found differentially regulated in the datasets, but by the rather global view of the pathways influenced by genistein treatment.

英国《BMC医学基因组学》，2008年

金雀异黄素诱导胚胎癌和原代癌细胞系停止有丝分裂和退出自我更新的分子原理

克里斯提娜 RA 瑞格本迟，詹姆斯 阿迪耶，等

马克斯·普朗克分子遗传学研究所，德国脊椎动物基因组系

背景：

染料木黄酮是存在于大豆中的异黄酮，具有抗致癌性质。染料木素作为潜在的抗癌药物的问题药物已在一些论文中得到解决，但综合基因组分析阐明了影响的分子机制由染料木黄酮引起的癌细胞没有在原代癌细胞上进行，而是在转化的细胞系上进行。在目前的研究中，我们用mu-mole治疗原发性胶质母细胞瘤，横纹肌肉瘤，肝细胞癌和人胚胎癌细胞（NCCIT）染料木素的浓度和有丝分裂指数，细胞形态，全球基因表达和特异性细胞周期调控基因。我们将NCCIT细胞的表达谱与癌细胞系的表达谱进行比较，以鉴定常见的染料木黄酮依赖性转录变化和伴随信号级联。

方法：

我们用50μM染料木素处理原代癌细胞和NCCIT细胞48小时。此后，我们比较了治疗的有丝分裂指数未处理细胞，并研究了OCT4，SOX2和NANOG等关键调节自我更新因子的蛋白表达。然后我们使用基因表达阵列（Illumina）进行全基因组表达分析，并通过实时PCR验证感兴趣基因的结果。然后使用DAVID和KEGG在线工具执行功能注释。

结果：

我们发现用染料木素处理的癌细胞在不同的检查点进行细胞周期停滞。这次逮捕与减少有关在通过微阵列分析测定并通过Real-Time PCR验证的核心调节基因，PBK，BUB1和CDC20的mRNA水平。在对比，人类NCCIT细胞显示GADD45A和G（生长停滞和DNA损伤诱导蛋白45A和G）的过度表达，如以及OCT4的下调和NANOG蛋白。此外，染料木黄酮诱导凋亡和抗迁移蛋白的表达p53和p38在所有细胞系中。染料木黄酮也上调了CYCLIN A和B的稳态水平。

结论：

本研究结果与早期研究结果一致，表明染料木黄酮靶向参与进化的基因细胞周期的M期。在这方面，特别感兴趣的是，不能从个人的比较中得出这一结论在数据集中发现差异调控的基因，但受到染料木素治疗影响的途径的全球观点。