Neuroblastoma is a common solid tumor of childhood and advanced disease carries a poor prognosis despite intensive multimodality therapy. Hypoxia is a common feature of solid tumors because of poorly organized tumor-induced neovasculature. Hypoxia is associated with advanced stage and poor outcome in a range of tumor types, and leads to resistance to clinically relevant cytotoxic agents in neuroblastoma and other pediatric tumors in vitro. Resistance to apoptosis is a common feature of tumor cells and leads to pleiotropic drug resistance, mediated by Bcl-2 family proteins. ABT-737 is a novel small-molecule inhibitor of Bcl-2 and Bcl-xL that is able to induce apoptosis in a range of tumor types. Neuroblastoma cell lines are relatively resistant to ABT-737–induced apoptosis in normoxia, but in contrast to the situation with conventional cytotoxic agents are more sensitive in hypoxia. This sensitization is because of an increase in ABT-737–induced apoptosis and is variably dependent upon the presence of functional hypoxia-inducible factor 1 (HIF-1) α. In contrast to the situation in colon carcinoma and non–small cell lung cancer cells, hypoxia does not result in downregulation of the known ABT-737 resistance factor, Mcl-1, nor any other Bcl-2 family proteins. ABT-737 sensitizes neuroblastoma cells to clinically relevant cytotoxic agents under normal levels of oxygen, and importantly, this sensitization is maintained under hypoxia when neuroblastoma cells are resistant to these agents. Thus rational combinations of ABT-737 and conventional cytotoxics offer a novel approach to overcoming hypoxia-induced drug resistance in neuroblastoma. Mol Cancer Ther; 10(12); 2373–83. ©2011 AACR.

Neuroblastoma is the most common extracranial solid tumor of childhood. Significant numbers of patients have widely disseminated disease and have poor survival despite intensive multiagent induction chemotherapy, myeloablative therapy, radiotherapy, surgery, and 13-cis retinoic acid (1), and for patients with an inadequate response to induction chemotherapy, the outcome is very poor (2).

Hypoxia, a reduction in the level of tissue oxygen, is a common feature of solid tumors because of reduced blood flow in the tumor-induced neovasculature (3). Hypoxia is associated with decreased survival and advanced stage in several types of adult tumor, including squamous cell carcinomas of the head and neck, and carcinoma of the cervix (4). Hypoxia has long been known to decrease the effectiveness of radiotherapy, and more recent studies have shown that cytotoxic agents are also less effective under conditions of low oxygen (5, 6). In neuroblastoma cells, hypoxia reduces etoposide and vincristine-induced apoptosis (7) and leads to resistance to cisplatin (8). Similar effects of hypoxia are seen in the common pediatric tumors rhabdomyosarcoma and Ewing sarcoma (9). The principle modulator of tumor cell response to hypoxia is the transcription factor hypoxia-inducible factor 1 (HIF-1; ref. 10). HIF-1 is a heterodimer of HIF-1α and HIF-1β; HIF-1β is constitutively expressed, but HIF-1α levels are kept low through proteasomal degradation in normoxia (11, 12). Under hypoxic conditions, HIF-1α is no longer targeted for degradation and is able to dimerize with HIF-1β and transactivate target genes (13). HIF-1α is stabilized in neuroblastoma cell lines in hypoxia, and HIF-1 target genes including VEGF and tyrosine hydroxylase are upregulated, both in vitro and as xenografts (14). Multiple angiogenic factors, including VEGF, have been shown to be expressed in neuroblastomas in vivo, and higher level expression correlates with advanced disease and poor outcome (15).

Failure of apoptosis is considered a hallmark of cancer (16). Commitment to apoptosis via the mitochondrial pathway is controlled by interactions between anti- and proapoptotic Bcl-2 family members that share homology via their BH-3 domains (17). The multidomain proapoptotic Bcl-2 family proteins Bax and Bak are essential for mitochondrial apoptosis and their activity is controlled by the BH-3–only proapoptotic Bcl-2 family proteins. Two models have been suggested for this activation of Bax and Bak. In the “direct model,” BH-3–only proteins directly activate Bax and Bak, whereas in the “indirect model,” BH-3–only proteins bind to antiapoptotic family members and prevent them from binding to and inhibiting Bak and Bax (18). Activation of Bax and Bak results in release of apoptogens from the mitochondrial inter membrane space and activation of an amplifying cascade of caspase-mediated proteolysis (19).

ABT-737 is a novel small molecule that mimics the BH-3 domain of Bad and binds with nanomolar affinity to the hydrophobic pocket of Bcl-2, Bcl-xL, and Bcl-w, disrupting their interactions with death-promoting Bcl-2 family members, and releasing these to activate Bax and Bak and engage apoptosis (ref. 20; see Supplementary Fig. S1 for the chemical structure of ABT-737 and other drugs used in this study). ABT-737 sensitizes a number of adult cancer cell types to cytotoxic agents (21), and has activity against acute myeloid leukemia, and small cell lung cancer (SCLC), as a single agent in preclinical models (20, 22, 23). ABT-737 binds poorly to Mcl-1 and thus tumor cell expression of Mcl-1 is associated with resistance to ABT-737 (24, 25). ABT-263, an orally bio-available counterpart with similar biologic activity, has been evaluated against the pediatric testing panel and has single agent activity against acute lymphoblastic leukemia (26). ABT-263 is now in clinical trial against adult tumors (27). In this study, we have evaluated the efficacy of ABT-737 against neuroblastoma cell lines in hypoxia, both as a single agent and in combination with clinically relevant cytotoxic agents.

Cell culture

Both MYCN-amplified and MYCN-nonamplified neuroblastoma cell lines were used. In addition, phenotypically distinct S and N-type subclones of SK-N-SH and LA-N-1 were studied. SH-EP1, SH-SY5Y, LA1-55n, and LA1-5s were the kind gift of Dr. Robert Ross (Fordham University, Bronx, NY), NGP cells were a kind gift from Dr. Deborah Tweddle (University of Newcastle-upon-Tyne), IMR-32 cells were purchased from American Type Culture Collection and LGC. All cell lines were authenticated by Cancer Research UK in July 2010 using STR profiling. All cell lines were maintained in Dulbecco's Modified Eagle's Medium/F12 with 10% FCS (Gibco, Invitrogen). SH-EP1 clones stably expressing mouse Bcl-2 or short hairpin RNA (shRNA) against Mcl-1 were a kind gift from Professor Simone Fulda (Institut für Experimentelle Tumorforschung in der Pädiatrie, Goethe-Universität, Frankfurt am Main, Germany). For hypoxia experiments, cells were cultured and treated in a sealed modular incubator (InVivo2 Hypoxia Workstation 400) flushed with 1% O2, 5% CO2, and 94% N2 (hereafter referred to as hypoxia).

Drug treatment

A stock solution of 10 mmol/L of ABT-737 (Abbott Laboratories) was dissolved in dimethyl sulfoxide (DMSO) and initial dose–response curves for all cell lines were generated using DMSO and a less potent enantiomer (20) as control. The sulforhodamine B (SRB) assay was used to determine total protein reflecting cell number after treatment. Cells were plated in exponential growth phase in 96-well plates and treated for 24 to 72 hours with varying concentrations of ABT-737 or conventional cytotoxics. The cells were fixed and stained according to standard SRB protocol (28), and absorbance was measured using a microplate reader (Labsystems Multiskan EX, Thermo Scientific) at 540 nm.

Short interfering RNA treatment

HIF-1α knockdown was achieved using siGENOME SMART pool from Dharmacon Thermo Scientific, and control oligos were nontarget pool from the same supplier. Cells were treated in 96- or in 6-well plates as per manufacturer instructions.

Western blotting

Cells were washed with PBS and lysed into either cell lysis buffer (Cell Signaling) supplemented with Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail I and II (Sigma), or directly into 2× Laemmli sample buffer. Loading was normalized by cell number or by protein concentration, which was determined by bicinchoninic acid assay (Thermo Scientific) following the manufacturer's instructions, both methods gave similar results. Samples were run on appropriate percentage polyacrylamide gels, and transferred to polyvinylidene difluoride membranes (Immobilon, Millipore). Membranes were blocked with 5% (w/v) non–fat-dried milk/0.05% (v/v) TBS-Tween 20 and probed with primary antibody in 0.05% (v/v) TBS-Tween 20 at 4°C overnight and then with secondary antibody conjugated to horseradish peroxidase in 5% (w/v) milk/0.05% (v/v) TBS-Tween 20 for 1 hour at room temperature. Blots were visualized with the enhanced chemiluminescence system (Amersham) and analyzed on a Fuji LAS-1000 Plus imaging system with AIDA software (Fuji). Primary antibodies used were HIF-1α (BD Pharmingen), carbonic anhydrase IX (CA IX; gift from Boehringer), glucose transporter 1 (GLUT-1; RD Systems), mouse Bcl-2 (Abcam), human Bcl-2 (Dako), Mcl-1 (BD Pharmingen), Bcl-xL (BD Transduction), Bcl-w (Axxora), Noxa (Imgenex), Bid (RD Systems), actin (AC-40, Sigma), cleaved caspase-3 (Cell Signalling), and PARP (Cell Signalling). Secondary antibodies were either goat antimouse horseradish peroxidase or goat antirabbit horseradish peroxidase (both from DAKO).

Fluorescence-activated cell-sorting analysis of apoptotic cells

Exponentially growing cells were treated for 18 to 48 hours with various concentrations of ABT-737 or etoposide as control. Cells were harvested by trypsinization and stained in 96-well plate with APC Annexin V and 7-aminoactinomycin D (7-AAD) to identify apoptotic cells. Data were collected on BD FACS Array and analyzed by FlowJo software.

Drug combination assays

Data from the SRB assay, carried out in triplicate, were used to calculate combination index (CI) with CalcuSyn software (Biosoft; ref. 29). This method is based on the multiple drug–effect equation of Chou–Talalay derived from enzyme kinetic models (30). On the basis of this approach, CI values less than 0.9 are considered synergistic, more than 1.1 are antagonistic, and values 0.9 to 1.1 are nearly additive. The ratios of ABT-737 and cytotoxic drugs were fixed using IC50 values obtained by the SRB assay. Cells were cotreated for 24 to 72 hours using ABT-737 and the cytotoxic drugs doxorubicin, cisplatin, etoposide, and vincristine. Six drug concentrations were used covering the concentration–effect. Linear correlation coefficients (r) were generated for each concentration response curve to determine the applicability of the data to the method of analysis. In all experiments, r was more than 0.9.

Statistical analysis

Two-way ANOVA followed by the Bonferroni test was used to establish whether significant differences existed between normoxic and hypoxic conditions over a range of doses. The Student t test calculations were carried out on single dose–response data and IC50 calculations.

ABT-737 is more effective in hypoxia in neuroblastoma cell lines

All 6 neuroblastoma cell lines were relatively resistant to ABT-737 in normoxia, with IC50 values in SRB assay ranging from 0.58 to 15.3 μmol/L (Fig. 1; Table 1). There was no correlation between this 26-fold variation in sensitivity to ABT-737 in normoxia and biology; 2 MYCN-amplified cell lines had an IC50 of more than 10 μmol/L and 2 MYCN-amplified lines had IC50 values below 1 μmol/L. However, in all 6 neuroblastoma cell lines ABT-737 was more effective against cells grown in 1% oxygen (hypoxia) than in 21% oxygen in SRB assay (Fig. 1; Table 1). In 5 of 6 cell lines, this difference reached statistical significance, whereas in the remaining cell line, LA1-5S, the trend was not significant, and this remained the case whether the difference in the dose–response curve between normoxia and hypoxia was analyzed (Fig. 1), or whether the IC50 values for ABT-737 in hypoxia or normoxia were compared by Student t test. Despite the wide variation in sensitivity of the neuroblastoma cell lines to ABT-737 in normoxia the degree of sensitization in hypoxia was relatively consistent, ranging from 1.4 to 3.2 fold. This sensitization in hypoxia was seen consistently despite the major differences in the biology of the cell lines studied, such that the MYCN no-amplified, non–1P-deleted pair, SH-EP1 and SH-SY5Y showed similar sensitization to the MYCN amplified, 1p-deleted lines LA1-55n and NGP.

Figure 1.

ABT-737 is more effective against neuroblastoma cell lines in hypoxia than in normoxia. SRB assays of cell number against ABT-737 concentration in normoxia (21% O2, open squares) compared with hypoxia (1% O2, closed diamonds). P < 0.05 for the comparison between normoxia and hypoxia by 2-way ANOVA for all cell lines except LA1-5S. Treatment durations were 24 hours for LA1-55n; 48 hours for SH-EP1, NGP, and IMR-32; and 72 hours for SH-SY5Y and LA1-5S.

Figure 1.

ABT-737 is more effective against neuroblastoma cell lines in hypoxia than in normoxia. SRB assays of cell number against ABT-737 concentration in normoxia (21% O2, open squares) compared with hypoxia (1% O2, closed diamonds). P < 0.05 for the comparison between normoxia and hypoxia by 2-way ANOVA for all cell lines except LA1-5S. Treatment durations were 24 hours for LA1-55n; 48 hours for SH-EP1, NGP, and IMR-32; and 72 hours for SH-SY5Y and LA1-5S.

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Table 1.

Comparison of SRB IC50 value for ABT-737 between normoxia and hypoxia in neuroblastoma cell lines

Cell lineMolecular changesABT-737 IC50 normoxia, μmol/LABT-737 IC50 hypoxia, μmol/LSensitization in hypoxia, fold change
SH-EP1 Single copy MYCN 11 3.1 3.2 
SH-SY5Y Single copy MYCN 4.2 1.5 2.8 
LA1-5S MYCN amp, p53 null 15.3 11.8 1.45 
LA1-55n MYCN amp, p53 null 0.58 0.28 2.1 
NGP MYCN amp, 1p deleted 10.6 7.4 1.43 
IMR-32 MYCN amp 0.75 0.41 1.82 
Cell lineMolecular changesABT-737 IC50 normoxia, μmol/LABT-737 IC50 hypoxia, μmol/LSensitization in hypoxia, fold change
SH-EP1 Single copy MYCN 11 3.1 3.2 
SH-SY5Y Single copy MYCN 4.2 1.5 2.8 
LA1-5S MYCN amp, p53 null 15.3 11.8 1.45 
LA1-55n MYCN amp, p53 null 0.58 0.28 2.1 
NGP MYCN amp, 1p deleted 10.6 7.4 1.43 
IMR-32 MYCN amp 0.75 0.41 1.82 

NOTE: P < 0.01 for all cell lines by Student t test.

Sensitization to ABT-737 in hypoxia is because of increased apoptosis

The SRB assay is a measure of protein, and as such will give information only about cell number. To address the mode of hypoxic sensitization to ABT-737, apoptosis was assayed. One-hour exposure to 5 μmol/L ABT-737 in MYCN-amplified NGP cells lead to an increase in the Annexin V–positive cell population in hypoxia within 8 hours of exposure to ABT-737 (15.5% of cells Annexin V positive in hypoxia compared with 12.2% of cells Annexin V positive in hypoxia) and this difference was maintained to 24 hours after ABT-737 exposure (35.9% of cells Annexin V positive in hypoxia compared with 17.3% of cells in normoxia; data not shown). Similar results were seen in the MYCN single copy SH-EP1 cell line where at 8 hours after exposure to 10 μmol/L ABT-737 there were 15.8% Annexin V–positive cells in hypoxia compared with 12.7% in normoxia, and again this difference remained at 24 hours after drug exposure (30.9% Annexin V positive in hypoxia compared with 21.3% positive in normoxia), and persisted at 48 hours after drug exposure (56.5% Annexin V positive in hypoxia compared with 38.1% positive in normoxia). This increase in ABT-737–induced apoptosis in hypoxia was seen in all 6 neuroblastoma cell lines at 18 to 48 hours after ABT-737 exposure (Fig. 2A). Immunoblotting for cleaved caspase-3 and cleaved PARP at 18 to 48 hours after exposure to ABT-737 revealed the same pattern of increased ABT-737–induced apoptosis in hypoxia (Fig. 2B). Furthermore, inhibition of ABT-737–induced apoptosis with the pan-caspase inhibitor Q-VD-Oph (QVD) ablates the sensitization of neuroblastoma cells to ABT-737 in hypoxia (Supplementary Fig. S2). Thus, the sensitization of neuroblastoma cell lines to ABT-737 in hypoxia is because of an increase in the amount of ABT-737–induced apoptosis in hypoxia.

Figure 2.

ABT-737 is more effective at inducing apoptosis in neuroblastoma cell lines in hypoxia than in normoxia. A, histograms showing percentage of Annexin V–positive cells at 18 to 48 hours after ABT-737 exposure in all 6 neuroblastoma cell lines in hypoxia and normoxia. B, representative Western blot analysis for HIF-1α, PARP, and cleaved caspase-3 in response to 24 to 72 hours of exposure to ABT-737 in varying concentration in normoxia and hypoxia in 4 neuroblastoma cell lines. Actin is shown as a loading control. Blots are representative of 3 independent experiments. NT, nontreated.

Figure 2.

ABT-737 is more effective at inducing apoptosis in neuroblastoma cell lines in hypoxia than in normoxia. A, histograms showing percentage of Annexin V–positive cells at 18 to 48 hours after ABT-737 exposure in all 6 neuroblastoma cell lines in hypoxia and normoxia. B, representative Western blot analysis for HIF-1α, PARP, and cleaved caspase-3 in response to 24 to 72 hours of exposure to ABT-737 in varying concentration in normoxia and hypoxia in 4 neuroblastoma cell lines. Actin is shown as a loading control. Blots are representative of 3 independent experiments. NT, nontreated.

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Differential expression of Bcl-2 family proteins does not account for differences in sensitivity to ABT-737 in neuroblastoma cell lines

Because of the wide variation in sensitivity to ABT-737 across the 6 neuroblastoma cell lines expression levels of Bcl-2 and Bcl-xL, known targets for ABT-737, and Mcl-1, a known resistance marker for ABT-737, were studied. As can be seen in Fig. 3A, there was considerable variation in expression of these proteins across the cell line panel. To some extent protein levels of the ABT-737 target Bcl-2 did seem to correlate with sensitivity to ABT-737, such that the 2 neuroblastoma cell lines with the lowest expression of Bcl-2, LA1-5S and SH-EP1, were the 2 most resistant to ABT-737. However NGP cells have a very similar IC50 to ABT-737 to SH-EP1 cells in normoxia (10.6 μmol/L compared with 11 μmol/L) but very different levels of expression of Bcl-2 protein. Less correlation was seen with levels of Bcl-xL protein, although the highest expressers of Bcl-xL were the most sensitive cell line (LA1-55n), the most resistant cell line (LA1-5S) also expressed far more Bcl-xL than the remaining cell lines. With regard to Mcl-1 protein levels the pattern was similar, thus the most sensitive cell line (LA1-55n) had the lowest level of Mcl-1, but the cell line with the highest expression of Mcl-1 was not the most resistant to ABT-737 (SH-SY5Y).

Figure 3.

Differential expression of Bcl-2 family proteins does not account for differences in sensitivity to ABT-737 in neuroblastoma cell lines. A, Western blot analysis showing variation in expression of the ABT-737 targets Bcl-2 and Bcl-xL and the recognized resistance marker Mcl-1 across the 6 neuroblastoma cell lines. Note the lack of change in protein level between normoxia (N) and after 48 hours in hypoxia (H). Actin is shown as a loading control. Blots are representative of 3 independent experiments. B, expression of mouse Bcl-2 protein in stably transfected SH-EP1mBcl-2 cells in comparison with vector-only controls (SHEP1-pMSCV). No difference in human Bcl-2 levels are seen with an antibody that recognizes only human Bcl-2 (hBcl-2), but higher expression of Bcl-2 is seen in SH-EP1mBcl2 cells with an antibody that recognizes both human and mouse Bcl-2 (h + mBcl-2). No changes in levels of either human or mouse Bcl-2 were seen over a 64-hour exposure to hypoxia. Actin is shown as a loading control. Blots are representative of 3 independent experiments. C, increased expression of Bcl-2 does not sensitize SH-EP1 cells to ABT-737. The response of wild-type SH-EP1 cells, vector-only controls (SHEP1-pMSCV), and SH-EP1 cells stably overexpressing mouse Bcl-2 (SH-EP1mBcl-2) to ABT-737 in SRB assay in normoxia is identical. D, increased expression of Bcl-2 does not sensitize SH-EP1 cells to ABT-737–induced apoptosis. No difference in percentage of Annexin V (AnV)/7-AAD–positive cells was seen after 24-hour exposure to ABT-737 at varying concentrations between vector-only controls (SHEP1-pMSCV) and SH-EP1 cells stably overexpressing mouse Bcl-2 (SH-EP1mBcl-2), in either normoxia or hypoxia. NT, nontreated.

Figure 3.

Differential expression of Bcl-2 family proteins does not account for differences in sensitivity to ABT-737 in neuroblastoma cell lines. A, Western blot analysis showing variation in expression of the ABT-737 targets Bcl-2 and Bcl-xL and the recognized resistance marker Mcl-1 across the 6 neuroblastoma cell lines. Note the lack of change in protein level between normoxia (N) and after 48 hours in hypoxia (H). Actin is shown as a loading control. Blots are representative of 3 independent experiments. B, expression of mouse Bcl-2 protein in stably transfected SH-EP1mBcl-2 cells in comparison with vector-only controls (SHEP1-pMSCV). No difference in human Bcl-2 levels are seen with an antibody that recognizes only human Bcl-2 (hBcl-2), but higher expression of Bcl-2 is seen in SH-EP1mBcl2 cells with an antibody that recognizes both human and mouse Bcl-2 (h + mBcl-2). No changes in levels of either human or mouse Bcl-2 were seen over a 64-hour exposure to hypoxia. Actin is shown as a loading control. Blots are representative of 3 independent experiments. C, increased expression of Bcl-2 does not sensitize SH-EP1 cells to ABT-737. The response of wild-type SH-EP1 cells, vector-only controls (SHEP1-pMSCV), and SH-EP1 cells stably overexpressing mouse Bcl-2 (SH-EP1mBcl-2) to ABT-737 in SRB assay in normoxia is identical. D, increased expression of Bcl-2 does not sensitize SH-EP1 cells to ABT-737–induced apoptosis. No difference in percentage of Annexin V (AnV)/7-AAD–positive cells was seen after 24-hour exposure to ABT-737 at varying concentrations between vector-only controls (SHEP1-pMSCV) and SH-EP1 cells stably overexpressing mouse Bcl-2 (SH-EP1mBcl-2), in either normoxia or hypoxia. NT, nontreated.

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To test whether differences in expression of the ABT-737 target Bcl-2 could account for differences in sensitivity to ABT-737, the responses of SH-EP1 cells stably overexpressing mouse Bcl-2 were investigated. ABT-737 is able to bind to and inhibit mouse Bcl-2 with a similar level of efficacy to human Bcl-2 (Saul Rosenberg, Abbott Laboratories, personal communication). SHEP-Bcl2 cells express detectably higher levels of Bcl-2 than vector transfected controls (SHEP-pMSCV), and this is not because of any difference in their levels of human Bcl-2 (Fig. 3B). However, this clear increase in level of Bcl-2 protein has no effect upon the response of SH-EP1 cells to ABT-737 in normoxia, in which no difference can be seen between wild-type SH-EP1 cells, SH-EP1 cells expressing mouse Bcl-2, and SH-EP1 cells containing vector alone in SRB assay (Fig. 3C). In addition, overexpression of Bcl-2 had no effect upon the induction of apoptosis by ABT-737 in SH-EP1 cells in normoxia as measured by flow cytometry for Annexin V, or on the sensitization of SH-EP1 cells to ABT-737–induced apoptosis in hypoxia (Fig. 3D). Thus levels of Bcl-2 do not modulate sensitivity to ABT-737 in normoxia in these neuroblastoma cell lines.

Sensitization of neuroblastoma cells to ABT-737 in hypoxia is variably dependent on HIF-1α

HIF-1 is the central regulator of cellular response to hypoxia, and we and others have previously shown that resistance to cytotoxic agents in hypoxia in neuroblastoma cell lines is dependent upon HIF-1α (7, 8). Our previous studies with ABT-737 in the colon carcinoma cell line HCT-116 had shown that sensitization to ABT-737 in hypoxia did not depend upon the presence of functional HIF-1 (31). To evaluate the importance of HIF-1 in the sensitization of neuroblastoma cells to ABT-737 in hypoxia SH-EP1 cells with HIF-1α transiently downregulated by short interfering RNA (siRNA) were generated. These cells showed no detectable protein expression of HIF-1α over the time course of either SRB or Annexin V assay, and no detectable protein expression of the known HIF-1 transcriptional targets CA IX and GLUT-1, indicating lack of functional HIF-1 (Fig. 4A). Loss of functional HIF-1 had no effect upon the response of SH-EP1 cells to ABT-737 in normoxia but, unlike the situation in the colon carcinoma cell line HCT-116, in SH-EP1 cells loss of functional HIF-1 lead to loss of sensitization to ABT-737 in hypoxia in SRB assay (Fig. 4B). In addition, while the loss of functional HIF-1 had no effect on ABT-737–induced apoptosis in normoxia, it significantly reduced ABT-737–induced apoptosis in hypoxia and lead to the loss of the previously highly significant difference between ABT-737–induced apoptosis in normoxia and hypoxia (Fig. 4C). However, in 3 other neuroblastoma cell lines, SH-SY5Y, IMR-32, and LA1-55n, knockdown of HIF1α with siRNA, while leading to functional inhibition of the HIF-1 pathway as measured by GLUT-1 expression, did not prevent sensitization to ABT-737 in hypoxia (Supplementary Fig. S3). Thus in some neuroblastoma cell lines sensitization to ABT-737 in hypoxia seems to be dependent upon functional HIF-1α, while in others it is not.

Figure 4.

Sensitization of neuroblastoma cells to ABT-737 in hypoxia is variably dependent on HIF-1α. A, SH-EP1 cells transiently transfected with siRNA to HIF-1α show no protein expression of HIF-1α over a 64-hour time course in hypoxia, and no hypoxia-induced upregulation of the known HIF-1 transcriptional targets CA IX and GLUT-1. Actin is shown as a loading control. Blots are representative of 3 independent experiments. B, Sensitization to ABT-737 by 48-hour exposure to hypoxia is lost in SH-EP1 cells transiently transfected with siRNA to HIF-1α. The mean IC50 to ABT-737 calculated from 3 separate SRB assays is shown in normoxia and hypoxia for wild-type, nontarget siRNA–transfected, and HIF-1α siRNA–transfected SH-EP1 cells. The difference between the IC50 in SRB in normoxia and hypoxia was significant by Student t test (P < 0.006) for wild-type and nontarget controls, but no difference was seen between normoxia and hypoxia in HIF-1α siRNA transfected SH-EP1 cells. C, Sensitization of SH-EP1 cells to ABT-737–induced apoptosis is lost in SH-EP1 cells transiently transfected with siRNA to HIF-1α. Percentage of Annexin V/7-AAD–positive cells (mean of 3 independent experiments) in mock-treated, nontarget siRNA–transfected, and HIF-1α siRNA–transfected SH-EP1 cells is shown after 24-hour exposure to ABT-737 at the normoxic IC50 concentration in normoxia and hypoxia. The increase in apoptosis in hypoxia in mock and nontarget transfected SH-EP1 cells is significant by Student t test (P < 0.02), and the reduction in ABT-737–induced apoptosis in hypoxia in HIF-1α siRNA–transfected SH-EP1 cells in comparison with nontarget transfected cells is also significant by Student t test (P < 0.008). Ctrl, control; 737, ABT-737.

Figure 4.

Sensitization of neuroblastoma cells to ABT-737 in hypoxia is variably dependent on HIF-1α. A, SH-EP1 cells transiently transfected with siRNA to HIF-1α show no protein expression of HIF-1α over a 64-hour time course in hypoxia, and no hypoxia-induced upregulation of the known HIF-1 transcriptional targets CA IX and GLUT-1. Actin is shown as a loading control. Blots are representative of 3 independent experiments. B, Sensitization to ABT-737 by 48-hour exposure to hypoxia is lost in SH-EP1 cells transiently transfected with siRNA to HIF-1α. The mean IC50 to ABT-737 calculated from 3 separate SRB assays is shown in normoxia and hypoxia for wild-type, nontarget siRNA–transfected, and HIF-1α siRNA–transfected SH-EP1 cells. The difference between the IC50 in SRB in normoxia and hypoxia was significant by Student t test (P < 0.006) for wild-type and nontarget controls, but no difference was seen between normoxia and hypoxia in HIF-1α siRNA transfected SH-EP1 cells. C, Sensitization of SH-EP1 cells to ABT-737–induced apoptosis is lost in SH-EP1 cells transiently transfected with siRNA to HIF-1α. Percentage of Annexin V/7-AAD–positive cells (mean of 3 independent experiments) in mock-treated, nontarget siRNA–transfected, and HIF-1α siRNA–transfected SH-EP1 cells is shown after 24-hour exposure to ABT-737 at the normoxic IC50 concentration in normoxia and hypoxia. The increase in apoptosis in hypoxia in mock and nontarget transfected SH-EP1 cells is significant by Student t test (P < 0.02), and the reduction in ABT-737–induced apoptosis in hypoxia in HIF-1α siRNA–transfected SH-EP1 cells in comparison with nontarget transfected cells is also significant by Student t test (P < 0.008). Ctrl, control; 737, ABT-737.

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Changes in Mcl-1 protein level do not account for sensitization of neuroblastoma cells to ABT-737 in hypoxia

In colon carcinoma and non–small cell lung cancer (NSCLC) cell lines, Mcl-1 downregulation at the translational level accounts for sensitization to ABT-737 in hypoxia (31). In neuroblastoma cell lines, there was no detectable change in the protein level of Mcl-1 in hypoxia (Fig. 5A), nor of protein levels of other potential mediators of sensitization to ABT-737 (Bcl-2, Bcl-xL, Bid, and Noxa). In addition, there was no change in levels of these proteins after downregulating HIF-1α with siRNA, under which conditions hypoxic sensitization to ABT-737 is lost in SH-EP1 cells (Fig. 5B). Stable downregulation of Mcl-1 by shRNAi in SH-EP1 cells does lead to sensitization to ABT-737 in normoxia as might be expected given its importance as a resistance marker for ABT-737 (Fig. 5C–E). However, reduction in Mcl-1 protein levels by shRNAi has no effect on the response of SH-EP1 cells to ABT-737 in hypoxia measured either by SRB assay (Fig. 5E) or Annexin V assay (Fig. 5D) despite differences in levels of Mcl-1 protein between the nontarget and anti-Mcl-1–transfected cells being as great in hypoxia as in normoxia (Fig. 5C). Thus, although reduction of Mcl-1 levels in SH-EP1 cells can sensitize to ABT-737 in normoxia, the same degree of reduction in Mcl-1 protein level does not sensitize SH-EP1 cells to ABT-737 in hypoxia. In 3 other neuroblastoma cell lines, SH-SY5Y, LA-1-55n, and IMR-32, downregulation of Mcl-1 with siRNAi also sensitized cells to ABT-737 in normoxia but similarly failed to prevent sensitization to ABT-737 in hypoxia (Supplementary Fig. S4). Taken together with the lack of any change in Mcl-1 protein levels in hypoxia, or after loss of hypoxic sensitization after removal of functional HIF-1 in SH-EP1 cells, this suggests that, unlike in colon carcinoma and NSCLC cell lines, Mcl-1 downregulation is not the mechanism for hypoxic sensitization to ABT-737 in neuroblastoma cells.

Figure 5.

Changes in Mcl-1 protein level do not account for sensitization of neuroblastoma cells to ABT-737 in hypoxia. A, no changes in levels of Bcl-2, Bcl-xL, Mcl-1, Noxa, or Bid were seen in 4 neuroblastoma cell lines after 48-hour exposure to hypoxia (H). Actin is shown as a loading control. Blots are representative of 3 independent experiments. B, no changes in protein levels of Bcl-2, Mcl-1, Bid, and Noxa were seen over a 64-hour time course after transfection of SH-EP1 cells with either nontarget or HIF-1α siRNA, despite the dramatic loss of sensitization to ABT-737 in hypoxia following transfection with HIF-1α siRNA. Actin is shown as a loading control. Blots are representative of 3 independent experiments. C, reduction in protein expression of Mcl-1 in SH-EP1 cells over a 64-hour time course in SH-EP1 clone stably expressing shRNA against Mcl-1 in comparison with nontarget shRNA. Actin is shown as a loading control. Blots are representative of 3 independent experiments. D, reduction in protein level of Mcl-1 increases ABT-737–induced apoptosis in normoxia as shown by a significant increase in percentage of Annexin V/7-AAD–positive cells 24 hours after treatment with either 1 or 5 μmol/L ABT-737 (*, P < 0.02 at 1 μmol/L; **, P < 0.05 at 5 μmol/L by Student t test). No increase in ABT-737–induced apoptosis is seen in hypoxia. E, reduction in protein level of Mcl-1 sensitizes SH-EP1 cells to ABT-737 in normoxia, but not in hypoxia in SRB assay. Compare the dose–response curve for cell expressing nontarget shRNA (open diamonds) with cells stably expressing shRNA, which targets Mcl-1 (closed triangles). NT, nontreated.

Figure 5.

Changes in Mcl-1 protein level do not account for sensitization of neuroblastoma cells to ABT-737 in hypoxia. A, no changes in levels of Bcl-2, Bcl-xL, Mcl-1, Noxa, or Bid were seen in 4 neuroblastoma cell lines after 48-hour exposure to hypoxia (H). Actin is shown as a loading control. Blots are representative of 3 independent experiments. B, no changes in protein levels of Bcl-2, Mcl-1, Bid, and Noxa were seen over a 64-hour time course after transfection of SH-EP1 cells with either nontarget or HIF-1α siRNA, despite the dramatic loss of sensitization to ABT-737 in hypoxia following transfection with HIF-1α siRNA. Actin is shown as a loading control. Blots are representative of 3 independent experiments. C, reduction in protein expression of Mcl-1 in SH-EP1 cells over a 64-hour time course in SH-EP1 clone stably expressing shRNA against Mcl-1 in comparison with nontarget shRNA. Actin is shown as a loading control. Blots are representative of 3 independent experiments. D, reduction in protein level of Mcl-1 increases ABT-737–induced apoptosis in normoxia as shown by a significant increase in percentage of Annexin V/7-AAD–positive cells 24 hours after treatment with either 1 or 5 μmol/L ABT-737 (*, P < 0.02 at 1 μmol/L; **, P < 0.05 at 5 μmol/L by Student t test). No increase in ABT-737–induced apoptosis is seen in hypoxia. E, reduction in protein level of Mcl-1 sensitizes SH-EP1 cells to ABT-737 in normoxia, but not in hypoxia in SRB assay. Compare the dose–response curve for cell expressing nontarget shRNA (open diamonds) with cells stably expressing shRNA, which targets Mcl-1 (closed triangles). NT, nontreated.

Close modal

ABT-737 sensitizes neuroblastoma cell lines to conventional cytotoxic agents in normoxia and hypoxia

Neuroblastoma cell lines are relatively resistant to ABT-737 as a single agent, with IC50 values in the range of 0.58 to 15.3 μmol/L. However, it is likely that, in the clinic, ABT-737 would be used in combination with conventional cytotoxic agents. Neuroblastoma, Ewing sarcoma, and rhabdomyosarcoma cell lines are all resistant to conventional cytotoxic agents in hypoxia. Given the sensitization of neuroblastoma cell lines to ABT-737 in hypoxia, the ability of ABT-737 to sensitize to conventional cytotoxic agents in normoxia and hypoxia was evaluated using SRB assay, and formal combination analysis was carried out using the CI. Vincristine, etoposide, cisplatin, and doxorubicin were chosen as agents that have wide use in the clinical management of neuroblastoma. In general, the combination of ABT-737 and etoposide, when given simultaneously, was synergistic in all 4 cell lines in normoxia with CI values ranging from 0.122 for LA-15S cells to 0.91 for LA-155n cells (Table 2). Under hypoxia, the synergy between etoposide and ABT-737 was either maintained, in LA1-5S (0.12 in normoxia and 0.27 in hypoxia) or enhanced, in SH-EP1, SH-SY5Y, and LA1-55n cells (0.79–0.37; 038–015; and 0.91–0.82, respectively). Synergy was also observed between ABT-737 and doxorubicin in SH-EP1, SH-SY5Y, and LA1-5S cells, with additivity seen between ABT-737 and doxorubicin in LA-155n cells. Again in hypoxia, this was either maintained or improved, so that synergy between ABT-737 and doxorubicin was seen in all 4 cell lines in hypoxia, with the CI value in LA1-55n cells falling from 0.99 in normoxia to 0.51 in hypoxia. Synergy was seen between ABT-737 and cisplatin in SH-EP1, SH-SY5Y, and LA1-55n cells and additivity was seen between ABT-737 and cisplatin in LA-15S cells in normoxia. Under hypoxia, synergy was seen between cisplatin and ABT-737 in all 4 cell lines. Synergy between ABT-737 and vincristine was seen only in LA-15S cells in normoxia and slight antagonism was seen between vincristine and ABT-737 in SH-SY5Y and LA1-55n cells in normoxia. However, in all 4 cell lines in hypoxia the CI value was reduced and synergy was seen in SH-EP1, SH-SY5Y, and LA1-5S cells, and additivity in LA1-55n cells.

Table 2.

ABT-737 sensitizes neuroblastoma cell lines to conventional cytotoxics agents in both normoxia and hypoxia

Cell lineCombination with ABT-737IC50 normoxiaIC50 hypoxia
SH-EP1 Etoposide 0.79333 0.36945 
SH-EP1 Doxorubicin 0.8162 0.88215 
SH-EP1 Cisplatin 0.55949 0.66894 
SH-EP1 Vincristine 1.06206 0.60202 
SH-SY5Y Etoposide 0.38021 0.14637 
SH-SY5Y Doxorubicin 0.51517 0.17208 
SH-SY5Y Cisplatin 0.90007 0.20182 
SH-SY5Y Vincristine 1.22763 0.52357 
LA1-5s Etoposide 0.122 0.27131 
LA1-5s Doxorubicin 0.6305 0.76332 
LA1-5s Cisplatin 1.04588 0.61367 
LA1-5s Vincristine 0.73087 0.69199 
LA1-55n Etoposide 0.90738 0.81952 
LA1-55n Doxorubicin 0.99311 0.51199 
LA1-55n Cisplatin 0.89014 0.86183 
LA1-55n Vincristine 1.246 0.91284 
Cell lineCombination with ABT-737IC50 normoxiaIC50 hypoxia
SH-EP1 Etoposide 0.79333 0.36945 
SH-EP1 Doxorubicin 0.8162 0.88215 
SH-EP1 Cisplatin 0.55949 0.66894 
SH-EP1 Vincristine 1.06206 0.60202 
SH-SY5Y Etoposide 0.38021 0.14637 
SH-SY5Y Doxorubicin 0.51517 0.17208 
SH-SY5Y Cisplatin 0.90007 0.20182 
SH-SY5Y Vincristine 1.22763 0.52357 
LA1-5s Etoposide 0.122 0.27131 
LA1-5s Doxorubicin 0.6305 0.76332 
LA1-5s Cisplatin 1.04588 0.61367 
LA1-5s Vincristine 0.73087 0.69199 
LA1-55n Etoposide 0.90738 0.81952 
LA1-55n Doxorubicin 0.99311 0.51199 
LA1-55n Cisplatin 0.89014 0.86183 
LA1-55n Vincristine 1.246 0.91284 

NOTE: Combination index (CI) values are shown for the combination of ABT-737, at its IC50 dose in normoxia and hypoxia, and vincristine, cisplatin, etoposide, and doxorubicin in 4 neuroblastoma cell lines. CI values of 1 indicate additivity, values below 1 indicate synergy; the lower the value, the greater the synergy.

Hypoxia is a near universal feature of solid tumors and is associated with advanced stage and poor prognosis in a range of adult tumor types. Less is known about the significance of hypoxia in pediatric tumor types, although studies are emerging to suggest that hypoxia leads to the same drug resistance in childhood cancer cells that has been reported in adult tumor types (7–9). Data also suggest that hypoxia is a feature of neuroblastoma and contributes to poor prognosis and drug resistance (14, 15). Thus therapeutic strategies that target hypoxic areas of tumor, and are able to re-sensitize hypoxic tumor cells to clinically relevant cytotoxic agents would be of considerable interest in the treatment of this poor prognosis tumor.

Activity of the orally bioavailable ABT-737 analog, ABT-263, against neuroblastoma xenografts in the pediatric preclinical testing panel was limited (26), and in agreement with this we found that all 6 neuroblastoma cell lines studied were relatively resistant to ABT-737. However, all 6 biologically variable neuroblastoma cell lines were more sensitive to ABT-737 under 1% oxygen (Fig. 1), and this was because of increased ABT-737–induced apoptosis (Fig. 2). Protein levels of the known ABT-737 targets Bcl-2 and Bcl-xL, as well as those of the known resistance factor Mcl-1, have all been reported to be important in determining cellular sensitivity to ABT-737 (20, 22, 24, 25). A previous study has suggested that neuroblastoma cell lines can be classified according to their Bcl-2 family protein dependence, and that those that are Bcl-xL or Bcl-w dependent are very sensitive to ABT-737 (32). In our neuroblastoma cell line, panel no consistent relationship between the levels of Bcl-2, Bcl-xL, and Mcl-1 and the cellular response to ABT-737 in SRB assay could be observed, although it is noticeable that the 2 most sensitive cell lines were the 2 with the lowest protein levels of Mcl-1 (LA1-55n and IMR-32). We hypothesized that the relatively low protein level of the ABT-737 target Bcl-2 might account for the relative drug resistance of the 2 S-type neuroblastoma cell lines SH-EP1 and LA-15S. However, overexpression of mouse Bcl-2 in SH-EP1 cells had no effect on the response of SH-EP1 cells to ABT-737 in SRB assay in normoxia, nor on the induction of apoptosis by ABT-737 in normoxia and hypoxia (Fig. 3C, D). This data strongly suggests that protein levels of Bcl-2 are not a determinant of neuroblastoma cell response to ABT-737.

We have recently reported sensitization of SCLC and colorectal carcinoma (CRC) cell lines to ABT-737 in hypoxia in vitro and in xenograft (31) and in these cell types this sensitization was not dependent upon a functional HIF-1 pathway. In neuroblastoma cells, we have previously shown that hypoxia-induced drug resistance is dependent upon functional HIF-1 (7). In the current study, reduction in protein levels of HIF-1α by siRNA results in an inactive HIF-1 pathway in SH-EP1 neuroblastoma cells in hypoxia, and results in loss of hypoxia-induced sensitization to ABT-737, which is because of a reduction in ABT-737–induced apoptosis in hypoxia (Fig. 4B and C). However, loss of a functional HIF-1 pathway in SH-SY5Y, LA1-5S, and IMR-32 cells after siRNA to HIF-1α does not prevent sensitization to ABT-737 in hypoxia (Supplementary Fig. S3). Thus in some neuroblastoma cell lines, sensitization to ABT-737 in hypoxia in neuroblastoma cells is dependent upon a functional HIF-1 pathway, whereas in others, as in SCLC and CRC cell lines, it is not.

A number of Bcl-2 family proteins have been reported to be up- or downregulated in hypoxia, including Bid, Mcl-1, and Noxa (5, 33–35). Our previous work reported hypoxia-induced downregulation of Mcl-1 in SCLC and CRC cell lines, and suggested that this was because of reduced protein translation in hypoxia (31). In SCLC and CRC cell lines, this reduction in Mcl-1 protein levels is not dependent upon the presence of functional HIF-1, despite the mcl-1 promoter containing an HRE (31). However, changes in Mcl-1 protein levels in hypoxia seem to be variable between cell types. In hepatocellular carcinoma and tracheobronchial cells Mcl-1 is upregulated in hypoxia (34, 35), whereas in mouse embryonic fibroblasts hypoxia has no effect on Mcl-1 levels (36). In the current study, we did not observe any changes in the protein level of Mcl-1 in hypoxia in any of the 4 neuroblastoma cell lines studied (Fig. 5A). Furthermore, we also failed to observe any changes in the protein level of Mcl-1 after the HIF-1 pathway was inactivated with siRNA to HIF-1α, despite the loss of sensitization of SH-EP1 cells to ABT-737 in hypoxia in this setting (Fig. 5B). Reduction of protein levels of Mcl-1 with shRNA clearly sensitized SH-EP1, SH-SY5Y, LA1-55n, and IMR-32 cells to ABT-737 in normoxia (Fig. 5C–E; Supplementary Fig. S4), as would be expected given the importance of Mcl-1 in resistance to ABT-737 in other cell types and as previously reported in neuroblastoma (24, 32, 37). However, the degree of Mcl-1 protein reduction by shRNA in SH-EP1 cells was as marked in hypoxia as in normoxia, yet this change in Mcl-1 protein level failed to have any effect on ABT-737–induced apoptosis in SH-EP1 cells in hypoxia (Fig. 5B and C), and failed to prevent sensitization of the other 3 neuroblastoma cell lines to ABT-737 in hypoxia (Supplementary Fig. S4). This data, together with the lack of change in Mcl-1 protein levels in neuroblastoma cells in hypoxia, and the lack of change in Mcl-1 protein levels following siRNA-mediated loss of HIF-1α in SH-EP1 cells, indicates that Mcl-1 is not involved in hypoxia-induced sensitization of neuroblastoma cells to ABT-737.

Synergy between ABT-737 and conventional cytotoxic agents in normoxia has been reported in a range of adult tumor types (21), and ABT-737 is able to reverse 13-cis-retinoic acid-induced resistance to cytotoxic agents in neuroblastoma cell lines (38). Our previous work in SCLC and CRC had shown that this synergy was maintained, and occasionally enhanced, in hypoxia (31). In the current study, this enhancement of the interaction between ABT-737 and conventional clinically relevant cytotoxic agent was more pronounced. Thus although a degree of synergy between ABT-737 and cytotoxic agents was seen across all 4 neuroblastoma cell lines in normoxia, it was almost invariably more marked in hypoxia (Table 2). In particular in the situations where no synergy was observed between ABT-737 and cytotoxic agent in normoxia, such as with vincristine in SH-SY5Y cells, where the relationship was antagonistic (CI: 1.22), synergy was seen in hypoxia (CI: 0.52). Indeed in hypoxia, there was no combination between ABT-737 and cytotoxic with a CI value of above 0.91 in any of the neuroblastoma cell lines. Thus in addition to being more effective as a single agent against neuroblastoma cells in hypoxia, ABT-737 is also better able to sensitize neuroblastoma cell lines to conventional cytotoxic agents in hypoxia.

In conclusion, this study shows that ABT-737 is more effective against neuroblastoma cell lines under hypoxia than normoxia, and importantly is better able to synergize with conventional cytotoxic agents under hypoxia, when neuroblastoma cells are more resistant to these agents. This data have potential significance for the future use of Bcl-2 family targeting drugs against neuroblastoma suggesting that combination with conventional cytotoxics may offer a possible strategy for improving the survival of children with this poor prognosis disease. However, further work will be needed to investigate these combinations in animal models.

No potential conflicts of interest were disclosed.

The authors thank Dr. Ali Raoof for Supplementary Fig. S1.

This study was supported by Cancer Research UK core funding to the Paterson Institute (grant number: C147). M. Brandenburg received a CRUK PhD student stipend.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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