Inhibition of NOTCH signaling pathway chemosensitizes HCC CD133+ cells to vincristine and 5-fluorouracil through upregulation of BBC3

Abstract

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)1 and 5-fluorouracil (5-FU)2. 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 IC50 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.