Clinical Cancer Research An Official Journal of the American Association for Cancer Research, 2001, 7(6):1782-9.

Differential sensitivity of normal and malignant breast epithelial cells to genistein is partly mediated by p21(WAF1)

Sunil Upadhyay, Samir Alhasan, et al

Department of Pathology, Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan

Genistein, a soy metabolite, is a potential chemopreventive agent against various types of cancer. There are several studies documenting molecular alterations leading to cell cycle arrest and induction of apoptosis in a variety of cancer cells; however, no studies, to date, have shown the effect of genistein in isogenic normal and malignant breast epithelial cells. In this study, we investigated whether genistein shows any differential sensitivity to normal (MCF10A and MCF12A) and malignant (MCF10CA1a and MDA-MB-231) breast epithelial cells. We found that genistein causes a greater degree of G(2)-M arrest and induces apoptosis in malignant cell lines compared with normal breast epithelial cells. After genistein treatment, flow cytometric analysis revealed a hyperdiploid population in malignant cells that was not observed in normal cells. Cell cycle regulator p21(WAF1), which is known to be up-regulated by genistein treatment, was greatly induced at RNA and protein levels in normal cells, whereas its level was only slightly induced in malignant MDA-MB-231 cells and not detectable in malignant MCF10CA1a cells. Therefore, we investigated the causal role of p21(WAF1) in the differential sensitivity of genistein among these cell lines. We examined the effects of genistein on p21(WAF1) -/- and p21(WAF1) +/+ HCT116 cells, which were used as controls prior to studies on breast cancer cells. We found that there was a greater degree of cell cycle arrest and apoptosis in p21(WAF1) -/- cells compared with p21(WAF1) +/+ HCT116 cells after genistein treatment. Flow cytometric analysis after genistein treatment showed a significant number of p21(WAF1) -/- cells in the hyperdiploid population, which are probably programmed to die through apoptotic processes. To further confirm the causal role of p21(WAF1) in genistein-mediated cell cycle arrest and apoptosis, we down-regulated p21(WAF1) by antisense p21(WAF1) cDNA transfection experiments. We found that both normal and malignant p21(WAF1) antisense (AS)-expressing clones became more sensitive to G(2)-M arrest after genistein treatment. Flow cytometric analysis showed an increase in the hyperdiploid population in the AS clones. Further evaluation showed an increase in apoptosis in malignant AS clones but not in normal breast epithelial AS clones. These results suggest that p21(WAF1) may play an important role in determining the sensitivity of normal and malignant breast epithelial cells to genistein.

美国《临床癌症研究美国癌症研究协会官方杂志》，2001年

正常和恶性乳腺上皮细胞对金雀异黄素的差异敏感性部分由p21（WAF1）介导

森尔 帕德亚，萨米尔 阿浩森，等

Karmanos癌症研究所病理学系，韦恩州立医科大学，密歇根州底特律

染料木黄酮，大豆代谢物，是一种潜在的抗各种癌症的化学预防剂。有几项研究记录了导致细胞周期阻滞和多种癌细胞凋亡诱导的分子改变;然而，到目前为止，还没有研究显示染料木素在同基因正常和恶性乳腺上皮细胞中的作用。在本研究中，我们调查了染料木素对正常（MCF10A和MCF12A）和恶性（MCF10CA1a和MDA-MB-231）乳腺上皮细胞的敏感性差异。与正常的乳腺上皮细胞相比，我们发现染料木素引起更大程度的G（2）-M阻滞并诱导恶性细胞系凋亡。在染料木素处理后，流式细胞术分析显示在正常细胞中未观察到的恶性细胞中的二倍体群体。已知通过染料木素处理上调的细胞周期调节因子p21（WAF1）在正常细胞中的RNA和蛋白质水平上被大大诱导，而其水平仅在恶性MDA-MB-231细胞中轻微诱导，并且不可检测在恶性MCF10CA1a细胞中。因此，我们调查了p21（WAF1）在这些细胞系中染料木素差异敏感性的因果作用。我们检查了染色质素对p21（WAF1） - / - 和p21（WAF1）+ / + HCT116细胞的影响，在乳腺癌细胞研究之前用作对照。我们发现与染料木素处理后的p21（WAF1）+ / + HCT116细胞相比，p21（WAF1） - / - 细胞的细胞周期停滞和凋亡程度更高。染料木素处理后的流式细胞分析显示，在二倍体体群体中p21（WAF1） - / - 细胞数量很多，这可能被编程为通过凋亡过程死亡。为了进一步证实p21（WAF1）在染料木黄酮介导的细胞周期阻滞和凋亡中的作用，我们通过反义p21（WAF1）cDNA转染实验下调p21（WAF1）。我们发现正常和恶性的p21（WAF1）反义（AS）表达克隆对染料木素处理后的G（2）-M阻滞变得更加敏感。流式细胞分析显示，AS克隆中的双倍体种群数量有所增加。进一步的评估显示恶性AS克隆中的细胞凋亡增加，但在正常乳腺上皮AS克隆中不增加。这些结果表明，p21（WAF1）可能在确定正常和恶性乳腺上皮细胞对染料木素的敏感性方面起重要作用。