Cournoyer et al. BMC Cancer (2019) 19:1018 https://doi.org/10.1186/s12885-019-6195-y RESEARCH ARTICLE Open Access GX15–070 (Obatoclax), a Bcl-2 family proteins inhibitor engenders apoptosis and pro-survival autophagy and increases Chemosensitivity in neuroblastoma Sonia Cournoyer1, Anissa Addioui1,2, Assila Belounis1,2, Mona Beaunoyer1,3, Carine Nyalendo1, Roxane Le Gall1, Pierre Teira1,4, Elie Haddad1, Gilles Vassal5 and Hervé Sartelet1,2,6* Abstract Background: Neuroblastoma (NB) is a frequent pediatric tumor associated with poor prognosis. The disregulation of Bcl-2, an anti-apoptotic protein, is crucial for the tumoral development and chemoresistance. Autophagy is also implicated in tumor cell survival and chemoresistance. The aim of our study was to demonstrate therapeutic efficiency of GX 15–070, a pan-Bcl-2 family inhibitor, used alone and in combination with conventional drugs or with hydroxychloroquine (HCQ), an autophagy inhibitor. Methods: Five neuroblastoma cell lines were tested for the cytotoxic activity of GX 15–070 alone or in combination with cisplatin, doxorubicin, HCQ or Z-VAD-FMK a broad-spectrum caspase inhibitor. Apoptosis and autophagy levels were studied by western-blot and FACS. Orthotopic injections were performed on NOD/LtSz-scid/IL-2Rgamma null mice that were treated with either GX 15–070 alone or in combination with HCQ. Results: Synergistic cytotoxicity was observed for the drug combination in all of the 5 neuroblastoma cell lines tested, including MYCN amplified lines and in cancer stem cells. GX 15–070 significantly increased apoptosis and autophagy in neuroblastoma cells as evidenced by increased levels of the autophagy marker, LC3-II. Inhibition of autophagy by HCQ, further increased the cytotoxicity of this combinatorial treatment, suggesting that autophagy induced by these agent plays a cytoprotective role. In vivo, GX 15–070 combined with HCQ significantly decreased the growth of the tumor and the number of distant metastases. Conclusions: Based on the synergistic effect of HCQ and GX 15–070 observed in this study, the combination of these two drugs may be utilized as a new therapeutic approach for neuroblastoma. Keywords: Bcl-2, Neuroblastoma, Apoptosis, Autophagy, GX15–070 Background [1]. Indeed, according to clinical, biological and patho- Neuroblastoma (NB) is a pediatric tumor of the sympa- logical tumor datas, patients with tumors classified in thetic nervous system, with a median age of 17 months the low-risk group present a good survival and can show at diagnosis it is responsible for over 15% of pediatric a spontanous tumor regression while in patients with cancer deaths [1]. In 50% of patients, it localises in in high-risk NB, the long term survival is poor despite ag- the adrenal medulla or paraspinal ganglia [1]. The NB is gressive chemotherapy and radiotherapy treatments, and caracterised by it’s clinical and biological heterogeneity stem cells transplants [2]. It is therefore essential to de- velop new therapeutic strategies to improve the survival * Correspondence: [email protected] of patients with high-risk NB. 1Research Center, Sainte Justine University Hospital Center, Montreal, QC, Canada The concept of cancers stem cells (CSCs) appears rele- 2Department of Pathology and Cellular Biology, Université de Montréal, vant to explain the high level of metastasis, chemoresis- Montreal, QC, Canada tance and unfavorable prognosis commonly observed in Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Cournoyer et al. BMC Cancer (2019) 19:1018 Page 2 of 14 NB. Many studies suggest that malignant tumors contain These CSCs are thought to be responsible for metastasis subpopulations of cells that share characteristics with and are more resistant to chemotherapeutic agents, normal stem cells, that is, they are capable of self- thereby permitting the recurrence of tumors after renewal, differentiation, and initiation of tumors [3]. chemotherapy and radiotherapy [4]. CD133 has been Fig. 1 a Basal level expression of important pro and antiapoptotic BCL-2 family proteins in human neuroblastoma. β-actin served as loading control. b. The effect of GX 15–070 used in monotherapy in NB cell viability in vitro. IGR-NB8, IGR-91, NB-10, SK-N-DZ and SK-N-FI cells treated 24 and 48 h with increasing concentrations of GX 15–070 (0.078 μM, 0.15 μM, 0.3 μM, 0.6 μM, and 5 μM).were added to the medium for 24 and 48 h and cell viability determined by the standard methyl-thiazoldiphenyl tetrazolium (MTT) assay. Each value represents the mean ± SEM (n =3) Cournoyer et al. BMC Cancer (2019) 19:1018 Page 3 of 14 identified as a marker of a subset of neural stem cells in resistance. GX 15–070 with its large spectrum of in- the adult central nervous system as well as of glioblast- hibition including Mcl-1 represents a potential inter- oma stem-like cells [5, 6]. In NB, CD133 expression is est in the treatment of NB. In other tumors, in vitro associated with poor prognosis, chemoresistance [7] and preclinical studies have shown efficacy of GX 15– the cells expressing this protein present stemness prop- 070 as monotherapy or in combination with other erty [8]. anticancer agents like in refractory mantle cell Apoptosis, also called programmed cell death, is es- lymphoma [20] or in antiestrogen-resistant breast sential for the normal regulation of cell survival by cancer cells [21]. Moreover, GX 15–070 selectively suppressing superfluous or damaged cells. A defect in eradicates quiescent human leukemia stem cells [22] the apoptosic pathway is now considered to be the and radiosensitizes glioblastoma stem-like cells [23]. foundation of most cancers [9]. Apoptosis occurs in Another mechanism implicated in tumor cell response to the disturbance of one of the two inter- survival and drug resistance is autophagy whitch is connected pathways known to be the extrinsinc death activated by metabolic stress [24, 25]. This lysosomal receptors or the intrinsic mitochondrial pathway. The degrative pathway, caracterised by autophagosomes mitochondrial pathway of apoptosis is regulated by formation, seems to be involved in the unsuccessful Bcl-2 family proteins which regulate cell survival and therapeutic effectiveness because of tumor masses is comprised of Bcl-2, Bcl-XL, Bcl-W, Bcl-2-A1, and vessel, nutrient heterogenecities, and hypoxic tumor Mcl-1. Studies show that deregulation of the Bcl-2 as regions that undergo autophagy. Since several studies primary oncogenic event (initiator) or secondary (pro- have shown that autophagy is crucial as a survival moter) is crucial for the tumoral development, main- mechanism in different tumors with defects in the tenance and chemoresistance [10]. In NB, in vitro apoptotic pathway, the modulation of these pathways studies have demonstrated that Bcl-2 is highly could be an interesting avenue for improvement of expressed, and its expression is inversely correlated to NB treatments [26]. Autophagy contributes to modu- the number of apoptotic cells and the level of differ- lating the cytotoxicities of Bcl-2 homology domain-3 entiation of the tumor [11]. Also, overexpression of mimetics [27]. Beclin 1, a Bcl-2 homology 3 (BH3) Bcl-2 in NB cell lines is associated with chemoresis- domain only protein is an essential initiator of au- tance [12]. The high level of expression of Bcl-2 is tophagy. In addition, Beclin 1 is a key determining also correlated with unfavorable histology, MYCN factor as to whether cells undergo autophagy or apop- amplification and poor prognosis [12]. The analysis of tosis [28]. Beclin 1 has been shown to interact via its the anti-apoptotic protein Bcl-2 and Mcl-1 have dem- BH3 domain with Bcl-2 family members. The dual onstrated a strong expression in NB [13]. It was role of Bcl-2 and Bcl-xL in inhibiting both apoptosis shown that the anti-apoptotic genes such as Bcl-2 and autophagic-associated cell death makes these pro- and Bcl-xL were overexpressed in CSC while the ex- teins ideal chemotherapeutic targets. BH3 mimetics, pression of pro-apoptotic genes such as Bax were GX 15–070 and ABT-737, disrupt the Bcl-2-Beclin1 downregulated in CSC of glioblastoma [14]. Also, interaction. GX15–070 induces pro-survival autophagy overexpression of Bcl-2 gene contribute to the resist- in head and neck squamous cell carcinoma cells [29] ance of medulloblastoma CSC to radiotherapy [15]. whereas the combination of GX15–070 with chloro- Therefore, Bcl-2 is an attractive target in the treat- quine, a specific autophagy inhibitor, results in syner- ment of NB. Bcl-2 inhibitor could be used alone or in gistic cytotoxicity against pancreatic cancer cells [20]. combination with others drugs to potentiate treat- The aim of our study was to evaluate the activity ment efficiency [16]. of Obatoclax against NB cells used in monotherapy GX 15–070, by activating the intrinsic pathway of or in combination with conventional drugs or with apoptosis, is an inhibitor of all anti-apoptotic Bcl-2 an autophagy inhibitor, hydroxychloroquine (HCQ). proteins. GX 15–070 inhibits specifically Bcl-XL, Bcl-2, MCL-1, Bcl-w, A1 and Bcl-B [17]. Recent – study desmonstrated that NB cell lines and primary Table 1 IC50 of NB cell lines treated with GX 15 070 μ μ tumors are primed for death with sequestration of NB cell IC50 ( M) IC50 ( M) lines Bim, a direct activator of apoptosis, by either Bcl-2 24 h 48 h or Mcl-1, providing a survival dependency that pre- SK-N-DZ 0,213 0,164 dicts the activity of Bcl-2 antagonists [18].