Inhibition of NOTCH signaling pathway chemosensitizes HCC CD133+ cells to vincristine and 5-fluorouracil through upregulation of BBC3
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.
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.
References (31)
Epidemiology of hepatocellular carcinoma in India
J. Clin. Exp. Hepatol.
(2014)- et al.
Liver tumor-initiating cells as a therapeutic target for hepatocellular carcinoma
Canc. Lett.
(2013) - et al.
Blocking the NOTCH pathway can inhibit the growth of CD133-positive A549 cells and sensitize to chemotherapy
Biochem. Biophys. Res. Commun.
(2014) - et al.
Therapeutic targeting of HES1 transcriptional programs in T-ALL
Blood
(2015) - et al.
Recent advances in multidisciplinary management of hepatocellular carcinoma
World J. Hepatol.
(2015) - et al.
The incidence and epidemiology of hepatocellular carcinoma: a global and regional perspective
Oncol.
(2010) - et al.
International incidence and mortality trends of liver cancer: a global profile
Sci. Rep.
(2017) - et al.
Worldwide incidence of hepatocellular carcinoma cases attributable to major risk factors
Eur. J. Canc. Prev. Off. J. Eur. Canc. Prev. Organisat.
(2018) - et al.
CD133 positive hepatocellular carcinoma cells possess high capacity for tumorigenicity
Int. J. Canc.
(2007) - et al.
CD133+ HCC cancer stem cells confer chemoresistance by preferential expression of the Akt/PKB survival pathway
Oncogene
(2007)
Mechanisms of chemoresistance in cancer stem cells
Clin. Transl. Med.
The Notch signaling pathway as a mediator of tumor survival
Carcinogenesis
Inhibition of gamma-secretase affects proliferation of leukemia and hepatoma cell lines through Notch signaling
Anti Canc. Drugs
others, Notch signaling in hepatocellular carcinoma: molecular targeting in an advanced disease
Hepatoma Res.
Chemotherapy for hepatocellular carcinoma: current status and future perspectives
Jpn. J. Clin. Oncol.
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