Kim Su-Hyeong, Singh Shivendra V
Department of Pharmacology & Chemical Biology, and University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, 2.32A Hillman Cancer Center Research Pavilion, 5117 Centre Avenue, Pittsburgh, PA, 15213, USA.
Breast Cancer Res Treat. 2015 Feb;149(3):681-92. doi: 10.1007/s10549-015-3279-5. Epub 2015 Feb 8.
We showed previously that garden cress constituent benzyl isothiocyanate (BITC) inhibits self-renewal of breast cancer stem cells (bCSC) in vitro and in vivo. The present study offers novel insights into the mechanism by which BITC inhibits bCSC. Flow cytometry and mammosphere assay were performed to quantify bCSC fraction. Protein expression was determined by western blotting. Apoptosis was assessed by flow cytometry using Annexin V-propidium iodide method. Cell migration was determined by Boyden chamber assay. BITC treatment resulted in a marked decrease in protein level of polycomb group protein B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi-1) in cultured human breast cancer cells (MCF-7, SUM159, MDA-MB-231, and MDA-MB-361) and MDA-MB-231 xenografts in vivo. Overexpression (MCF-7) or knockdown (SUM159, and MDA-MB-231) of Bmi-1 protein had no meaningful impact on the BITC's ability to inhibit cell viability and cell migration and/or induce apoptosis. On the other hand, inhibition of bCSC markers (aldehyde dehydrogenase 1 activity and mammosphere frequency) resulting from BITC exposure was significantly altered by Bmi-1 overexpression and knockdown. BITC was previously shown to cause activation of Notch1, Notch2, and Notch4 in association with induction of γ-secretase complex component Nicastrin, which are also implicated in maintenance of cancer stemness. BITC-mediated inhibition of bCSC was augmented by knockdown of Notch4 and Nicastrin, but not by RNA interference of Notch1 or Notch2. The present study highlights important roles for Bmi-1 and Notch4 in BITC-mediated suppression of bCSC.
我们之前的研究表明，水田芥成分苄基异硫氰酸酯（BITC）在体外和体内均可抑制乳腺癌干细胞（bCSC）的自我更新。本研究为BITC抑制bCSC的机制提供了新的见解。通过流式细胞术和乳腺球形成实验对bCSC比例进行定量。采用蛋白质印迹法测定蛋白质表达。使用膜联蛋白V-碘化丙啶法通过流式细胞术评估细胞凋亡。通过Boyden小室实验测定细胞迁移。BITC处理导致培养的人乳腺癌细胞（MCF-7、SUM159、MDA-MB-231和MDA-MB-361）以及体内MDA-MB-231异种移植瘤中多梳蛋白组蛋白B淋巴细胞莫洛尼鼠白血病病毒插入区1（Bmi-1）的蛋白质水平显著降低。Bmi-1蛋白的过表达（MCF-7）或敲低（SUM159和MDA-MB-231）对BITC抑制细胞活力和细胞迁移及/或诱导凋亡的能力没有显著影响。另一方面，Bmi-1的过表达和敲低显著改变了BITC暴露导致的bCSC标志物（醛脱氢酶1活性和乳腺球形成频率）的抑制作用。先前研究表明，BITC可导致Notch1、Notch2和Notch4激活，并伴有γ-分泌酶复合物成分Nicastrin的诱导，这些也与癌症干性的维持有关。Notch4和Nicastrin的敲低增强了BITC介导的对bCSC的抑制作用，但Notch1或Notch2的RNA干扰则没有这种作用。本研究突出了Bmi-1和Notch4在BITC介导的bCSC抑制中的重要作用。
