GX15–070 (Obatoclax), a Bcl-2 family proteins inhibitor engenders apoptosis and pro-survival autophagy and increases Chemosensitivity in neuroblastoma

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.


Background
Neuroblastoma (NB) is a pediatric tumor of the sympathetic nervous system, with a median age of 17 months at diagnosis it is responsible for over 15% of pediatric cancer deaths [1]. In 50% of patients, it localises in in the adrenal medulla or paraspinal ganglia [1]. The NB is caracterised by it's clinical and biological heterogeneity [1]. Indeed, according to clinical, biological and pathological tumor datas, patients with tumors classified in the low-risk group present a good survival and can show a spontanous tumor regression while in patients with high-risk NB, the long term survival is poor despite aggressive chemotherapy and radiotherapy treatments, and stem cells transplants [2]. It is therefore essential to develop new therapeutic strategies to improve the survival of patients with high-risk NB.
The concept of cancers stem cells (CSCs) appears relevant to explain the high level of metastasis, chemoresistance and unfavorable prognosis commonly observed in NB. Many studies suggest that malignant tumors contain subpopulations of cells that share characteristics with normal stem cells, that is, they are capable of selfrenewal, differentiation, and initiation of tumors [3].
These CSCs are thought to be responsible for metastasis and are more resistant to chemotherapeutic agents, thereby permitting the recurrence of tumors after chemotherapy and radiotherapy [4]. CD133 has been identified as a marker of a subset of neural stem cells in the adult central nervous system as well as of glioblastoma stem-like cells [5,6]. In NB, CD133 expression is associated with poor prognosis, chemoresistance [7] and the cells expressing this protein present stemness property [8].
Apoptosis, also called programmed cell death, is essential for the normal regulation of cell survival by suppressing superfluous or damaged cells. A defect in the apoptosic pathway is now considered to be the foundation of most cancers [9]. Apoptosis occurs in response to the disturbance of one of the two interconnected pathways known to be the extrinsinc death receptors or the intrinsic mitochondrial pathway. The mitochondrial pathway of apoptosis is regulated by Bcl-2 family proteins which regulate cell survival and is comprised of Bcl-2, Bcl-XL, Bcl-W, Bcl-2-A1, and Mcl-1. Studies show that deregulation of the Bcl-2 as primary oncogenic event (initiator) or secondary (promoter) is crucial for the tumoral development, maintenance and chemoresistance [10]. In NB, in vitro studies have demonstrated that Bcl-2 is highly expressed, and its expression is inversely correlated to the number of apoptotic cells and the level of differentiation of the tumor [11]. Also, overexpression of Bcl-2 in NB cell lines is associated with chemoresistance [12]. The high level of expression of Bcl-2 is also correlated with unfavorable histology, MYCN amplification and poor prognosis [12]. The analysis of the anti-apoptotic protein Bcl-2 and Mcl-1 have demonstrated a strong expression in NB [13]. It was shown that the anti-apoptotic genes such as Bcl-2 and Bcl-xL were overexpressed in CSC while the expression of pro-apoptotic genes such as Bax were downregulated in CSC of glioblastoma [14]. Also, overexpression of Bcl-2 gene contribute to the resistance of medulloblastoma CSC to radiotherapy [15]. Therefore, Bcl-2 is an attractive target in the treatment of NB. Bcl-2 inhibitor could be used alone or in combination with others drugs to potentiate treatment efficiency [16].
GX 15-070, by activating the intrinsic pathway of apoptosis, is an inhibitor of all anti-apoptotic Bcl-2 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 tumors are primed for death with sequestration of Bim, a direct activator of apoptosis, by either Bcl-2 or Mcl-1, providing a survival dependency that predicts the activity of Bcl-2 antagonists [18]. Analogous to its predecessor ABT-737, ABT-263 possesses high affinity for Bcl-xL, Bcl-2, and Bcl-w, but not for Mcl-1 or A1 [19]. Also in NB, Mcl-1 seems to be the principal mediator of classical Bcl-2 antagonist resistance. GX 15-070 with its large spectrum of inhibition including Mcl-1 represents a potential interest in the treatment of NB. In other tumors, in vitro preclinical studies have shown efficacy of GX 15-070 as monotherapy or in combination with other anticancer agents like in refractory mantle cell lymphoma [20] or in antiestrogen-resistant breast cancer cells [21]. Moreover, GX 15-070 selectively eradicates quiescent human leukemia stem cells [22] and radiosensitizes glioblastoma stem-like cells [23].
Another mechanism implicated in tumor cell survival and drug resistance is autophagy whitch is activated by metabolic stress [24,25]. This lysosomal degrative pathway, caracterised by autophagosomes formation, seems to be involved in the unsuccessful therapeutic effectiveness because of tumor masses vessel, nutrient heterogenecities, and hypoxic tumor regions that undergo autophagy. Since several studies have shown that autophagy is crucial as a survival mechanism in different tumors with defects in the apoptotic pathway, the modulation of these pathways could be an interesting avenue for improvement of NB treatments [26]. Autophagy contributes to modulating the cytotoxicities of Bcl-2 homology domain-3 mimetics [27]. Beclin 1, a Bcl-2 homology 3 (BH3) domain only protein is an essential initiator of autophagy. In addition, Beclin 1 is a key determining factor as to whether cells undergo autophagy or apoptosis [28]. Beclin 1 has been shown to interact via its BH3 domain with Bcl-2 family members. The dual role of Bcl-2 and Bcl-xL in inhibiting both apoptosis and autophagic-associated cell death makes these proteins ideal chemotherapeutic targets. BH3 mimetics, GX 15-070 and ABT-737, disrupt the Bcl-2-Beclin1 interaction. GX15-070 induces pro-survival autophagy in head and neck squamous cell carcinoma cells [29] whereas the combination of GX15-070 with chloroquine, a specific autophagy inhibitor, results in synergistic cytotoxicity against pancreatic cancer cells [20].
The aim of our study was to evaluate the activity of Obatoclax against NB cells used in monotherapy or in combination with conventional drugs or with an autophagy inhibitor, hydroxychloroquine (HCQ). To test the importance of caspase-mediated apoptosis in cell death induced by GX 50-170, SK-N-DZ and IGR-NB8 cells were treated with increased concentration of GX 50-170 (0.05-5 μM) for 48 h, with or without Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone) 20 μM, a cell-permanent pancaspase inhibitor, that irreversibly binds to the catalytic site of caspase proteases. In the same way, to evaluate the effect of autophagy activation induced by GX 50-170, cell lines were treated with increased concentrations of GX 15-070 or combined with (30 μM) of hydroxychloroquine (HCQ), a specific inhibitor of the late phase of autophagy, prepared in PBS for 48 h.

Protein extraction and Western blotting
Cells previously treated were lysed with lysis buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.5% Nonidet P-40, 1 mM sodium orthovanadate and 1 mM sodium fluoride), 30 min at 4°C. Protein concentration was measured using the BCA assay kit (Pierce, Rockford, IL). Equal amounts of cell lysate protein were resolved by SDS-PAGE. After University Hospital Center. All mice were housed in ventilated cages under standard conditions of controlled temperature and humidity, and exposed to a 12-hourly light/dark cycle. NSG were established in 6-to 8-weekold mice by injection into the left adrenal gland of five NSG mice, respectively, under general anesthesia with 2% isofluran/O 2 . A clinical analysis (weight, vital signs, and behavior) was performed once a week. Mice injected orthotopically were treated intraperitonealy (i.p) with GX 15-070 alone (n = 5) or in combination with HCQ (n = 5), delivered at 3 mg/kg and 60 mg/kg or with PBS used as negative controls (n = 5) respectively for 5 days (from day 18 to 22 post NB cells injection). After 4 weeks, mice were sacrificed in a CO 2 chamber. A necropsy was done and the left adrenal gland (injection site) as well as liver, lungs, brain and femur (all potential sites of metastasis) were removed, fixed in 10% phosphatebuffered formalin and embedded in paraffin. Tumor size was determined and volumes were measured by caliper and calculated using the formula: Tumor volume = ( length x width 2 )/2.

Statistical analysis
All data are represented as the mean ± SEM. Cell survival statistical analyses were performed using Student's t-test with Graphpad Prism software (Version 5.00). Tumor volume statistics were analyzed by one-way analysis of variance (ANOVA) followed by bonferroni posttest. P < 0.05 was considered statistically significant. Synergism was also evaluated by median dose effect analysis using the Chou-Talaly method and a software program (CalcuSyn, Biosoft, Ferguson, MO) [30]. Cells were treated with Cis or Dox or HCQ and obatoclax at fixed concentration ratios for 48 h and cell death was monitored by MTT. Combination index (CI) values and isobolograms were computed using CalcuSyn software. CI values less than 1.0 denote synergism.

GX 15-070 used in monotherapy in NB cells
The basal levels of important pro and antiapoptotic BCL-2 family proteins were assessed by western blotting (Fig. 1a). IGR-N91 expressed low levels of MCL-1 protein as compared to the aothers cell lines. Notably, all the cell lines expressed relatively higher levels of at least two of the three predominant antiapoptotic BCL-2 family proteins. All NB cell lines exposed for 24 or 48 h to GX 15-070 exhibited dose-response curves characteristic (Fig. 1b) including those with MYCN amplification (NB-10, SK-N-DZ, IGR-N91 and IGR-NB8). They showed IC50 inferior to 1 μM and were considered sensitive to this drug used in monotherapy (Table 1). For two cell lines (IGR-NB8 and IGRN91), IC50 was less than 100 nM. The survival of SK-N-FI cell line (without MYCN amplification) is significantly decreased after a longer exposure (48 h) (Fig. 1b) while for the other cell lines, the sensitivity of the cells was time independent.

GX 15-070 induced neuroblastoma apoptosis
To verify if GX 15-070 induces cell death by caspase dependant apoptosis, western blots were performed on two cell lines (SK-N-DZ and IGR NB-8) treated with GX 15-070. In both cell lines, there was a correlation between the level of cleaved caspase 3 and the concentration of GX 15-070 used in monotherapy (Fig. 2a). The analysis by Annexin V/7-AAD staining confirmed that GX 15-070 induces increased apoptosis in a dose dependent manner (early and late apoptosis) (p < 0.0001) (Fig. 2b). The difference with control was already significant in both cell lines at a very low concentration of GX 15-070 (75 nM). Subsequent treatment of both cell lines with the pan-caspase inhibitor, Z-VAD-FMK, significantly inhibited the cell death indicating that apoptosis is mainly caspase-mediated (Fig. 2c). This activated apoptosis was not correlated with modification of Bcl-2 expression (Fig. 2a).

Autophagy is also activated by GX 15-070 treatment
The level of autophagy in cells treated with GX 15-070 was then evaluated by the study of LC3 protein cleavage by western blots (Fig. 3a). The LC3-II/LC3-I ratio increased in SK-N-DZ and IGR-NB8 cells treated with GX 15-070 compared to control (Fig. 3a). The specific inhibition of the late autophagy by HCQ significantly increased cells sensitivity to GX 15-070 (Fig. 3b).
The cancer stem cells were less sensitive to  SK-N-DZ cells were sorted into CD133 high and CD133 low populations and expressed two CSC markers of CSCs: CD133 and vimentin [31]. They were exposed for 48 h to varying concentration of GX 15-070.
CD133 high cells were found to be more resistant to treatment than their matched CD133 low counterparts (Fig. 4a). This data was in correlation with the higher expression of Bcl2 in CSCs (Fig. 4b).

GX 15-070 used in combined therapy in NB cells
Co-administration of GX 15-070 concentrations as low as 0.05 μM significantly increased cell death, when it is combined with Cis and Dox, chemotherapeutic agents classically used in NB treatments in both cell lines used in the present study ( Fig. 5a and b). Interestingly, the presence of 0.05 μM of GX 15-070 significantly decrease the IC50 for Dox and Cis in both cell lines (Table 2 and Table 3). The decrease in cell survival associated with the Cis and GX 15-070 combination is associated with caspase mediated apoptosis clearly demonstrated by the study of caspase 3 cleavage but without modifying the level of BCL-2 expression (Fig. 5d). More interestingly, this association clearly increased the level of autophagy detected by LC3II/LC3I ratio but did not modify ATG5 expression (Fig. 5e). Median Dose Effect analysis was used to evaluate synergism. Median Dose Effect analysis revealed Combination Index values < 1.0, indicating synergistic interactions. Synergistic effects of the combination of GX 15-070 with Cis or Dox or HCQ on cell death were observed in both cell lines. The combination indices (CI) of GX 15-070 with Cis were synergistic in SK-N-DZ and IGR-NB8 (CI 0.51 and CI 0.53, respectively) (Fig. 5c), those with Dox were synergistic in SK-N-DZ and IGR-NB8 (CI 0.45 and CI 0.32, respectively) (Fig. 5c), and those with HCQ were synergistic in SK-N-DZ and IGR-NB8 (CI 0.48 and CI 0.78, respectively) (Fig. 3c).

Effect of GX 15-070 used alone or in combination with HCQ on tumor volume
The in vivo study confirmed the sensitivity of the tumor cells to GX 15-070. Four weeks after orthotopic injections of SK-N-DZ cells in mice adrenal gland, the size of the tumor was significantly less in mice treated with GX 15-070 or with GX 15-070 combined with HCQ than control (p < 0.05 and p < 0.01 respectively) (Fig. 6a, b and Table 4). Moreover, the liver and lung metastases were less frequent in mice treated with GX 15-070 than in control and were absent in mice treated with the synergistic drugs (GX 15-070 and HCQ) ( Table 4).
(See figure on previous page.) Fig. 4 The effect of GX 15-070 used alone in NB cancer stem cells viability (a) and expression of Bcl-2 in NB CSCs (b). Cells viability of CD133 sorted SK-N-DZ cells treated for 48 h with GX 15-070 were measured using the standard MTT (The statistical difference between CD133 high and CD133 low was calculated with Fisher's exact test) (a). Immunoblotting analyses were performed on protein lysates from SK-N-DZ cells. Anti-CD133 and Bcl-2 antibodies were used (b)

Discussion
Increased expression of the anti-apoptotic members of the BCL-2 family proteins correlates with chemotherapy resistance in NB [13]. These major anti-apoptotic proteins are known to inhibit cell apoptosis, and therefore targeting them may restore the capacity of NB cells to undergo apoptosis.  [20]. This group showed that cell sensitivities to GX 15-070 treatment correlated with protein levels of different Bcl-2 protein members. In fact, cells that downexpressed Bcl-xL were the most sensitive, while those strongly expressed Mcl-1, Bcl-2 and Bcl-xL were more resistant to GX 15-070 treatment. A direct relationship was also demonstrated between the dose-response curves and the absence or near absence of Bak protein in the cell. Therefore, GX 15-070 used alone will be of greater efficiency in tumors that have reduced, absent or inactivated expression of Bcl-xL and Bak proteins [20]. In NB, a similar study was carried out with another inhibitor of Bcl-2, ABT-737, a mimetic molecule of proapoptotic BH3-only, which binds specifically Bcl-2, Bcl-xL and Bcl-w but has no affinity for Bcl-2-A1 and Mcl-1 [25]. Their results showed cellular sensitivity varied from 0.1 μM to 100 μM. This reduced sensitivity in NB cell lines was also found in a second study involving the ABT-737 monotherapy [32]. Due to its cellular heterogeneity, NB treatment should be more effective with GX 15-070 rather than ABT-737 because of the potential to inhibit all the antiapoptotic proteins, including Mcl-1 [33]. The dose-dependent cytotoxic effects of GX 15-070 and the heterogeneities underlying each tumor suggests that the most convincing therapeutic effects would be a combination of GX 15-070 with other anti-cancer agents [34,35]. We also observed a significant decrease cell survival when NB cell lines were treated with GX 15-070 in combination with Cis. This platinum-based agent that forms bridges between the DNA strands activates DNA repair mechanisms and, if the repair is too large, apoptosis is activated through the mitochondrial pathway [36]. Crawford and collegues showed a significant decrease in resistance when small lung cancer cells, already resistant to cisplatin, were treated with a combination of Cis and 0.5 μM of GX 15-070 [37]. In NB, a combination of ABT-737 with different chemotherapeutic agents like Cis, vincristine, etoposide or Dxr was done. All these agents activated the intrinsic apoptotic pathway and their combination with Bcl-2 inhibitor synergistically increased cell line toxicity [36,38]. Therfore, it seems that Bcl-2 inhibitors are able to sensitize tumor cells to conventional chemotherapeutic agents and reverse the resistance caused by the disregulation of the apoptotic machinery.
Our study also showed that GX 15-070 induced apoptosis in NB cell lines. Moreover, when this pathway is blocked, cell survival is restored and significantly increased even if cells are treated with GX 15-070. A previous study done in acute myeloid leukemia cells showed that GX 15-070 induced apoptosis by many mitochondrial pathways [39]. On isolated mitochondria, GX 15-070 treatments significantly increased the release of cytochrome C in the cytoplasm [39]. Intersingly, this study demonstrated that the absence of Bax and Bak doesn't eliminate completely the apoptosis induced by    [39,[41][42][43][44]. There is a spatial organization of autophagy and apoptosis control in which BH3-only proteins exert two independent functions. On the one hand, they can induce apoptosis, by (directly or indirectly) activating the mitochondrionpermeabilizing function of pro-apoptotic multidomain proteins from the Bcl-2 family. On the other hand, they can activate autophagy by liberating Beclin 1 from its inhibition by Bcl-2/Bcl-X(L) at the level of the endoplasmic reticulum [45]. Our study demonstrated that autophagy is increased when NB cells are treated with GX 15-070 alone or in combination with Cis. Also, we observed a significant decrease in the primary tumor volume when mice are treated with GX 15-070 alone or in combination with an autophagy inhibitor, HCQ, compared to controls. A study in prostate cancer showed a significant decrease in the tumor growth when mice are treated with dual inhibition of apoptosis and autophagy [26].

Conclusions
Our study supports the interest of using GX 15-070 alone or in combination with other conventional chemotherapeutic agents in the treatment of NB. Our data also illustrates that activating apoptosis and inhibiting autophagy pathways simultaneously have a synergistic antiproliferative potential and have to be considered in further studies surrounding NB treatment.