Department of Biotechnology, Panjab University, Chandigarh, India; Department of Cellular & Molecular Medicine, Institute of Liver and Biliary Sciences, New Delhi, India.
Department of Biotechnology, Panjab University, Chandigarh, India.
Biochem Biophys Res Commun. 2020 May 14;525(4):941-947. doi: 10.1016/j.bbrc.2020.03.009. Epub 2020 Mar 12.
In hepatocellular carcinoma (HCC), the poor response to the chemotherapeutic agents is partially attributed to the chemoresistance property of cancer stem cells (CSCs). NOTCH signaling pathway plays a crucial role in the chemoresistance through the maintenance of the CSCs. We observed that the NOTCH pathway was activated in HCC CD133 cells treated with vincristine (VIN) and 5-fluorouracil (5-FU). Therefore, we examined whether inhibition of the NOTCH can improve sensitization of HCC CD133 cells to VIN and 5-FU. The Huh7 cell line was pre-incubated γ-secretase DAPT, as a NOTCH inhibitor, and then treated with IC dose of VIN or 5-FU. The CD133 cells were then isolated and analyzed for the cell viability, apoptosis, migration and spheroid formation capacities, and gene and protein expression. It was observed that pre-incubation with DAPT significantly downregulated the expression of NOTCH-related genes and led to a significant reduction in VIN- and 5-FU-CD133 population. In addition, DAPT pre-incubated VIN- and 5-FU-treated-CD133 cells formed fewer spheroids in 3D culture and had a lesser migration capacity in 2D culture. Importantly, DAPT enhanced the apoptosis rate of VIN- and 5-FU-treated CD133 cells for 3- and 2-fold, which was correlated with the enhanced expression of pro-apoptotic BBC3 (BCL-2-binding component 3) and decreased expression of HES1 that was reported to regulate BBC3 negatively. Collectively, it was observed that NOTCH inhibition sensitized the HCC CD133 cells to VIN and 5-FU through enhancing BBC3-mediated apoptosis. The results highlighted the role of NOTCH/HES1/BBC3 axis in resistance of CD133 cells to VIN and 5-FU. Understanding the molecular mechanisms underlying chemoresistance in HCC CD133 cells may help in designing the novel targeted therapies to chemosensitize them.
在肝细胞癌（HCC）中，化疗药物反应不良部分归因于癌症干细胞（CSC）的耐药性。NOTCH 信号通路通过维持 CSCs 在化疗耐药中起关键作用。我们观察到，在用长春新碱（VIN）和 5-氟尿嘧啶（5-FU）处理的 HCC CD133 细胞中，NOTCH 途径被激活。因此，我们研究了抑制 NOTCH 是否可以提高 HCC CD133 细胞对 VIN 和 5-FU 的敏感性。Huh7 细胞系先用 NOTCH 抑制剂γ-分泌酶 DAPT 孵育，然后用 IC 剂量的 VIN 或 5-FU 处理。然后分离和分析 CD133 细胞的活力、凋亡、迁移和球体形成能力以及基因和蛋白表达。结果观察到，DAPT 孵育可显著下调 NOTCH 相关基因的表达，导致 VIN 和 5-FU-CD133 群体显著减少。此外，DAPT 孵育的 VIN 和 5-FU 处理的-CD133 细胞在 3D 培养中形成的球体更少，在 2D 培养中的迁移能力更小。重要的是，DAPT 增强了 VIN 和 5-FU 处理的 CD133 细胞的凋亡率，分别为 3 倍和 2 倍，这与促凋亡 BBC3（BCL-2 结合成分 3）的表达增强和报告的负调控 BBC3 的 HES1 表达降低有关。总之，NOTCH 抑制通过增强 BBC3 介导的凋亡使 HCC CD133 细胞对 VIN 和 5-FU 敏感。结果突出了 NOTCH/HES1/BBC3 轴在 CD133 细胞对 VIN 和 5-FU 耐药中的作用。了解 HCC CD133 细胞化疗耐药的分子机制可能有助于设计新型靶向治疗方法使其化疗增敏。
