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

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Highlights

  • HCC CD133+ cancer stem cells poorly responded to vincristine and 5-florouracil.

  • NOTCH signaling pathway was upregulated in vincristine- and 5-fluorouracil-treated CD133+ cells.

  • NOTCH inhibition enhanced apoptosis in vincristine- and 5-fluorouracil-treated CD133+ cells.

  • The expression level of BBC3 enhanced after inhibition of the NOTCH signaling pathway.

  • NOTCH/HES1/BBC3 axis may regulate apoptosis in drug-resistant CD133+ cells.

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.

Graphical abstract

NOTCH inhibition-mediated apoptosis via HES1/BBC3. DAPT-mediated NOTCH inhibition downregulates HES1 which negatively regulate BBC3. Upregulation of BBC3 releases the inhibition of anti-apoptotic Bcl-2 family proteins which leads to activate BAX/BAK. Activation of BAX/BAK triggers mitochondria dysfunction and release cytochrome C leading to activate caspases and promote apoptosis.

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Introduction

Hepatocellular carcinoma (HCC) is the most common form of liver cancer [1] and the third leading cause of cancer-related mortality [2]. HCC accounts for 70%–90% of primary liver cancer cases in most countries [2,3]. India is among the top five countries with the largest number of HCC cases, with an incidence rate of 1.6% per year [4,5]. Chemotherapy-based treatment of HCC is not curative; therefore, the recurrence phenomenon prevails. This is also due to the presence of cancer stem cells and (CSCs)3 and their chemoresistance property. Several CSC markers including EpCAM, CD133, CD90, CD44, CD13, and CD24 have been identified in HCC cells [6]. The cells expressing surface glycoprotein CD133 (AC133 antigen) possess tumorigenicity, are resistant to conventional anti-cancer therapies [7,8] and exhibit activated NOTCH signaling pathway [9]. The NOTCH pathway plays a key role in the maintenance of the CSCs [10,11], and contributes to chemoresistance [10]. Gamma (γ)-secretase inhibitors such as DAPT (N-[N-(3,5-difluorophenacetyl)- L-alanyl]-S-phenylglycine t-butyl ester)4 which prevent proteolytic cleavage of the NOTCH intracellular domain (NICD), suppress the NOTCH activity [12,13].

Sorafenib is the only standard molecular-targeted therapy for advanced HCC; the other chemotherapeutic agents, such as doxorubicin, cisplatin, and 5-fluorouracil (5-FU) are being used in the form of mono- or polytherapy regimens [14]. 5-FU is a uracil analogue. Its active metabolites are misincorporated into DNA and RNA or form a complex with thymidylate synthase, which prevents the production of deoxythymidine monophosphate (dTMP) [15]. The reduction in the level of dTMP imbalances the ATP/dTTP ratio and leads to damage in DNA [16]. Vincristine (VIN) that belongs to Vinca alkaloid microtubule-targeted drugs depolymerizes microtubules or stabilizes dynamics of them, results in blocking or slowing down the mitosis process at the metaphase-anaphase transition, and subsequently induction of apoptotic cell death [17]. It was shown that CD133+ cells were more resistant to 5-FU and VIN compared to CD133- population [8,18,19]; however, the mechanisms underlying resistance to them in HCC CD133+ cells are still not well explored.

We previously reported that NOTCH is activated in HCC CSCs [20]. In this study, we aimed to explore the effect of DAPT-mediated NOTCH inhibition on the sensitization of HCC CD133+ cells to VIN and 5-FU. Our findings highlighted the role of NOTCH/HES1/BBC3 axis in maintaining chemoresistant CD133+ cells. Our results may help in understanding the molecular mechanisms underlying the role of NOTCH in chemoresistance of HCC CSCs.

Section snippets

Cell culture and drug treatment

Huh7 cell line was cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin, at 37 °C in a humidified incubator with 5% carbon dioxide. The cells were pre-incubated with 2 μM DAPT (Sigma Aldrich) for 24 h and then treated with IC50 dose of VIN (Sigma Aldrich) or 5-FU (Sigma Aldrich) for 48 h. The treated cells were washed, trypsinized, and used for sorting CD133+ cells.

Drug cytotoxicity and growth inhibition assay

The

VIN and 5-FU treatment induced NOTCH signaling in CD133+ cells leading to enhanced stemness properties

The MTT assay was performed to determine IC50 doses of VIN and 5-FU. It was found that 48 h IC50 doses of VIN and 5-FU were 44 μM and 316 μM (Supplementary Fig. 1). The Huh7 cells were then treated with IC50 doses of VIN and 5-FU, and their CD133+ population was isolated. After VIN treatment, CD133+ Huh7 cells showed a significant increase in the gene expression of NOTCH receptors NOTCH1 (6.2 ± 0.8 fold), NOTCH2 (3.2 ± 0.3 fold), NOTCH3 (5.1 ± 1 fold), and NOTCH4 (7.0 ± 0.5 fold) and ligand

Discussion

Chemotherapy is a palliative treatment to enhance the survival rate of HCC patients. The CSC-associated chemoresistance is the main reason for treatment failure and recurrence of HCC [23]. CD133 population in HCC has been shown to possess CSC properties [7]. The NOTCH signaling pathway plays a crucial role in the maintenance and tumorigenicity of CSCs [20]. Various studies have reported that NOTCH is activated in HCC and also in response to chemotherapy [20,[24], [25], [26]]. It has been shown

Declaration of competing interest

The authors declare that they have no competing interests.

Acknowledgment

We would like to thank the Department of Biotechnology, Ministry of Science and Technology, New Delhi, India, for funding support (Grant No. BT/PR5343/MED/31/157/2012). We gratefully acknowledge Dr. Gayatri Ramakrishna for her critical discussions.

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