Fu Haifeng, Xing Feiyue, Lv Yi, Zeng Boning, You Pengtao, Liu Jing
Institute of Tissue Transplantation and Immunology, Department of Immunobiology, Jinan University, Guangzhou 510632, China.
Institute of Tissue Transplantation and Immunology, Department of Immunobiology, Jinan University, Guangzhou 510632, China.
Tissue Cell. 2018 Oct;54:65-71. doi: 10.1016/j.tice.2018.08.004. Epub 2018 Aug 18.
The Notch signaling pathway plays a key role in cell proliferation and development that is closely related to an inverted CCAAT box binding protein (ICBP90), but little is known about whether there is a correlation between Notch signaling and ICBP90. The aim of the current study was to elucidate this. MTT assay and flow cytometry were used to determine the proliferation, cell cycle and apoptosis of HepG2 or Hepa1-6 cells treated by N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), a specific inhibitor of the Notch pathway. RT-PCR, Western Blot and in situ immunofluorescence staining were employed to examine expression of ICBP90 in the cells. DAPT caused inhibition of the activation of the Notch signaling pathway, followed by preventing the cells at the G0/G1 phases to enter S and G2/M phases. ICBP90 and Hes-1 proteins were highly expressed in the untreated cells. The reduced levels of Notch intracellular domain (NICD) protein were observed in the DAPT-treated cells, thereby bringing about the down-regulation of ICBP90 with the increment of the DAPT dose. Consistent with this, knockdown of the Hes-1 gene, which encodes a critical transcriptional factor in the Notch pathway, also led to the attenuation of ICBP90. On the contrary, Jagged-1, a Notch ligand, facilitated ICBP90 production. Adriamycin could result in the reduction of ICBP90, which was not accompanied with the alteration of Hes-1. ICBP90 was almost fully distributed within the nuclei, but Hes-1 was visible within both the cytoplasm and nuclei. Our novel findings strongly indicate that inactivation of the Notch signaling pathway impedes hepatocellular carcinoma progress via reduction of ICBP90.
Notch信号通路在细胞增殖和发育中起关键作用，这与一种反向CCAAT盒结合蛋白（ICBP90）密切相关，但关于Notch信号与ICBP90之间是否存在关联却知之甚少。本研究的目的是阐明这一问题。采用MTT法和流式细胞术来测定经Notch通路特异性抑制剂N-[N-(3,5-二氟苯乙酰基)-L-丙氨酰基]-S-苯甘氨酸叔丁酯（DAPT）处理的HepG2或Hepa1-6细胞的增殖、细胞周期和凋亡情况。运用RT-PCR、蛋白质免疫印迹法和原位免疫荧光染色来检测细胞中ICBP90的表达。DAPT导致Notch信号通路的激活受到抑制，随后阻止细胞从G0/G1期进入S期和G2/M期。ICBP90和Hes-1蛋白在未处理的细胞中高表达。在经DAPT处理的细胞中观察到Notch细胞内结构域（NICD）蛋白水平降低，从而随着DAPT剂量的增加导致ICBP90下调。与此一致的是，编码Notch通路中关键转录因子的Hes-1基因的敲低也导致ICBP90减弱。相反，Notch配体Jagged-1促进ICBP90的产生。阿霉素可导致ICBP90减少，而这并未伴随Hes-1改变。ICBP90几乎完全分布于细胞核内，但Hes-1在细胞质和细胞核中均可见。我们的新发现有力地表明，Notch信号通路的失活通过降低ICBP90来阻碍肝癌进展。
