Hypoxic human cancer cells are sensitized to BH-3 mimetic–induced apoptosis via downregulation of the Bcl-2 protein Mcl-1 (original) (raw)
Cells were more sensitive to ABT-737 in hypoxia than normoxia. Hypoxia, prevalent in solid human tumors, causes drug resistance (23), and consistent with this hypoxic resistance was also observed with the conventional cytotoxic agents and cell lines used in this study (ref. 23 and Supplemental Table 1; supplemental material available online with this article; doi:10.1172/JCI43505DS1). The effect of hypoxia on the response of SCLC (H146 and H82) and CRC (HCT116) cells to ABT-737 was measured by resazurin or sulforhodamine B (SRB) assays. The concentration-response curves for the 3 cell lines are shown in Figure 1A, and resultant IC50 values are shown in Supplemental Table 2. In stark contrast to conventional cytotoxic agents, ABT-737 was significantly more potent in hypoxic compared with normoxic cells in all 3 cancer cell lines (P < 0.005, 2-way ANOVA; Figure 1A). In normoxic H82 and HCT116 cells, IC50 values for ABT-737 were similar, in the low micromolar range, and they were reduced 1.7- to 2.0-fold under hypoxia. The IC50 of ABT-737 for normoxic H146 cells was 82.1 nM, approximately 100-fold lower than for the other cell lines (consistent with previous assessments; ref. 9), and the degree of hypoxic sensitization was greatest for H526 cells: 21.5-fold more sensitive in hypoxia (Supplemental Table 1 and Figure 1A).
Effects of ABT-737 in cancer cell lines under normoxic and hypoxic conditions. (A) H146, H82 SCLC, and HCT116 CRC cells were incubated in normoxia or hypoxia (1% O2) for 18 hours, after which they were exposed to a range of ABT-737 concentrations under continuous normoxia or hypoxia for 72 hours prior to determination of IC50 values using the resazurin assay. (B) H146 SCLC and H82 SCLC cells were preincubated for 18 hours in normoxia or hypoxia (1% O2) before treatment with ABT-737 at 89.1 nM and 12.2 μM, respectively, for the indicated times. Control cells were exposed to an equivalent concentration of drug diluent (DMSO) in normoxia or hypoxia at selected time points. HCT116 CRC cancer cells were preincubated for 18 hours in normoxia or hypoxia and treated with the indicated ABT-737 concentrations or DMSO vehicle with maintained hypoxia or normoxia and assessed 24 hours later. The percentage of cells undergoing apoptosis was determined by DAPI staining and counting at least 100 cells per field in duplicate. Data are mean ± SEM of 3 independent experiments. *P < 0.05, **P < 0.01; (Student’s 2-tailed t test).
The generality of this observation is demonstrated in Supplemental Table 2, which reports increased efficacy under hypoxia versus normoxia in 3 additional CRC cell lines, DLD-1 (IC50 of 2.4 μM and 8.0 μM, respectively), HT29 (IC50 of 2.9 μM and 9.2 μM, respectively), and CaCo2 (IC50 of 2.7 μM and 8.5 μM, respectively), and 3 other SCLC cell lines, H526 (IC50 of 0.26 μM and 5.6 μM, respectively; see below), H1048 (IC50 of 0.23 μM and 1.3 μM, respectively), and H345 (IC50 of 0.049 μM and 0.85 μM, respectively). A similar hypoxic sensitization to ABT-737 was also seen in two neuroblastoma cell lines (SH-SY5Y and SHEP1; data not shown). Hypoxic sensitization to ABT-737 has been observed in every cell line investigated so far.
Hypoxic cells were sensitized to ABT-737–induced apoptosis. The sensitization in hypoxia to ABT-737 observed could have resulted from increased cell death and/or reduced cell proliferation. In the absence of ABT-737, hypoxia slowed the cell population growth kinetics in H82, H146, and HCT116 cell lines (Supplemental Figure 1) but did not alter basal levels of apoptosis per se (Figure 1B). Following 18 hours preincubation in normoxia or hypoxia, ABT-737–induced apoptosis was assessed by evaluation of nuclear morphology in H146, H82, and HCT116 cells maintained in hypoxic and normoxic conditions (Figure 1B). H146 and H82 cells exhibited a time-dependent induction of apoptosis in response to ABT-737 in both hypoxia and normoxia, with significantly increased apoptosis under hypoxia (P < 0.05, Student’s t test). Consistent with these data, Figure 2A (H146) and Figure 2B (H82) demonstrate the more rapid ABT-737–induced cleavage of PARP (CPARP) and caspase-3 (CC3) (biomarkers of apoptosis) in hypoxia compared with normoxia.
Western blot analysis of CC3 and CPARP in response to ABT-737 in cancer cell lines under hypoxic and normoxic conditions. Cells were preincubated in normoxia or hypoxia (1% O2) for 18 hours prior to treatment with ABT-737, after which cells were harvested for protein analysis by Western blot. (A) H146 cells (treated with 89.1 nM ABT-737). (B) H82 cells (treated with 12.2 μM ABT-737). “Time” indicates the number of hours exposed to ABT-737 or control (DMSO). (C) HCT116 cells were treated with ABT-737 for 24 hours; concentration of ABT-737 is shown. Actin was used as a protein loading control.
After 18 hours preincubation in hypoxia or normoxia, treatment of HCT116 cells with ABT-737 resulted in a concentration-dependent apoptotic response in both hypoxia and normoxia at 24 hours (Figure 1B). Significantly increased apoptosis was seen for hypoxic HCT116 cells treated with 5 μM ABT-737 compared with normoxic cells (P < 0.05, Student’s t test) (Figure 1B).
Increased CC3 was seen in hypoxic HCT116 cells treated with 0.5 μM and 1 μM ABT-737 compared with normoxic counterparts, although at higher concentrations no difference between CC3 levels was detectable (Figure 2C). Hypoxia alone did not induce apoptosis but decreased full-length PARP levels in HCT116, confounding interpretation of the comparisons of ABT-737 treatment on CPARP in hypoxia and normoxia; even so, at the lower ABT-737 concentrations (0.5 μM and 1 μM), full-length PARP was detected in normoxic but not hypoxic cells, again suggesting increased apoptosis in the latter. No full-length PARP available for cleavage was detected in hypoxic HCT116 cells treated with ABT-737. It was barely detectable in normoxic cells treated with ABT-737 concentrations below 2 μM and undetectable at higher concentrations where the increased cleaved PARP was observed. To investigate whether the decrease in full-length PARP seen in hypoxic HCT116 cells was a caspase-dependent event, we treated cells in the absence and presence of the pan-caspase inhibitor QVD (20 μM) and incubated them in normoxia or hypoxia for 24 hours. Supplemental Figure 2 shows that PARP was lower in hypoxia in comparison to normoxia regardless of whether QVD was present. As a control for apoptosis and activity of QVD, cells were also treated with ABT-737 (10 μM) for 24 hours; this caused cleavage of PARP, which was prevented by QVD.
Overall these data show that while hypoxic cells proliferate more slowly than normoxic cells (Supplemental Figure 1), they are also, in comparison to normoxic cells, more sensitive to ABT-737–induced apoptosis (Figures 1 and 2).
ABT-737–induced apoptosis in tumor spheroids. We have previously shown that hypoxic regions of HCT116 spheroids were less prone to apoptosis induced by the conventional cytotoxic agent oxaliplatin when compared with normoxic regions (24). Expression of a dominant negative (DN) HIF-1 prevents upregulation of the glucose transporter GLUT-1 in hypoxic regions of HCT116 spheroids (24). GLUT-1 and HIF1-α colocalized in these spheroids (Figure 3A).The same 3D culture model was used here to investigate further the hypoxic sensitization to ABT-737, where CC3 was used to report apoptosis and GLUT-1 was used to report hypoxia. Spheroids were treated with an IC20 (2.8 μM) or IC90 (14.4 μM) concentration of ABT-737 for 24 hours before serial sectioning and immunofluorescence analysis. GLUT-1 staining (green) revealed a hypoxic rim between the necrotic core and normoxic periphery (Figure 3B). Although sporadic apoptotic cells could be seen in the outermost layers of the spheroids, ABT-737 treatment resulted in a sharply defined “ring” of CC3 staining (red) several cell layers deep into the spheroid. This CC3-positive region overlapped the region that stained positively for the HIF-1 target GLUT-1. The spheroid data are consistent with Figure 1B and Figure 2 and show that ABT-737 is most potent at inducing apoptosis in an oxygen tension at which the HIF-1 target GLUT-1 is upregulated.
ABT -737–induced apoptosis in HCT116 tumor spheroids. (A) HCT116 cells were grown as spheroids, and serial sections were stained for GLUT1 or HIF-1α and then the appropriate secondary antibody or isotype negative as described in Methods. Scale bar: 500 μm. (B) Spheroids were treated with ABT-737 at IC20 (2.8 μM) or IC90 (14.4 μM) for 24 hours as described in Methods. DAPI: blue; GLUT-1: green; CC3: red. Data are representative of 3 independent experiments (n > 12 for both drug-treated and untreated spheroids). Scale bar: 500 μm.
Mcl-1 was downregulated in hypoxia. As Mcl-1 expression correlates with ABT-737 resistance (15) and increased potency of ABT-737 was observed in hypoxia, the impact of hypoxia in H146, H82, and HCT116 cells was investigated. Using the protocol used for the ABT-737 treatment studies, we found that Mcl-1 levels were consistently lower in hypoxic cells (which exhibited expression of the hypoxia marker, HIF-1α) compared to normoxic counterparts (Figure 4A and see below). Downregulation of Mcl-1 in hypoxia was observed in every cell line tested (including DLD-1 and CaCo2; Supplemental Figure 4). No other consistent changes in antiapoptotic Bcl-2 family members were observed across the cell line panel in hypoxia (data not shown). No consistent changes in proapoptotic family members were seen in normoxic or hypoxic cells before or after treatment with ABT-737, including the Mcl-1 expression modulating family member Noxa (data not shown).
Effect of hypoxia and HIF-1 on Mcl-1 protein expression levels. (A) Effect of hypoxia on Mcl-1 and HIF-1α protein levels after 18, 24, or 48 hours hypoxia (1% O2) or normoxia. (B) Validation of HCT116 cell line expressing DN HIF-1α protein. HCT116 EV control and HCT116 DN HIF-1α cells were incubated in hypoxia (1% O2) or normoxia for 18 hours, after which the fold induction of firefly luciferase in hypoxia was calculated over that of Renilla luciferase (see Methods). (C) HCT116 EV or HCT116 DN HIF-1α cells were incubated in normoxia or hypoxia (1% O2) for 18 hours, after which they were exposed to a range of ABT-737 concentrations under continuous normoxia or hypoxia for 72 hours prior to determination of IC50 values using the SRB assay. (D) Western blot analysis of Mcl-1 expression level in HCT116 EV and HCT116 DN cells after 18, 24, or 48 hours hypoxia (1% O2) or normoxia. (E) HCT116 cells were treated with HIF-1α or HIF-2α or NT siRNA for 24 hours, and then siRNA was removed and cells were incubated in normoxia or hypoxia for 24 hours, after which cells were harvested and levels of HIF-1α, HIF-2α, Mcl-1, and GAPDH were determined by Western blot. Data are mean ± SEM of 3 independent experiments.
Mcl-1 downregulation in hypoxia was caspase and HIF independent. The data so far demonstrate that increased sensitivity to ABT-737 in hypoxia was associated with decreased Mcl-1 level and that ABT-737–induced apoptosis in cells that upregulated the HIF-1 target GLUT-1. Two approaches were taken to determine whether hypoxic downregulation of Mcl-1 was HIF dependent (as previously noted with HIF-1 for the Bcl-2 family member Bid; ref. 16). First, Mcl-1 levels were examined in hypoxic and normoxic HCT116 cells containing stably overexpressed DN HIF-1α or empty vector (EV; ref. 25). Second, transient transfection of RNAi constructs to HIF-1α and HIF-2α was employed.
HCT116 DN cells express a truncated form of HIF-1α with a deleted oxygen-dependent degradation domain (ODDD) that is able to bind to HIF-1β and hypoxia-response elements (HREs) in target promoters; however, in contrast to wild-type HIF-1α, it cannot activate transcriptional machinery. These cells have been characterized previously (25). Hypoxic HCT116 EV control cells exhibited a 3-fold induction of firefly luciferase compared with that observed in normoxia, whereas hypoxia did not induce firefly luciferase in hypoxic HCT116 DN cells (Figure 4B), validating the model. Both HCT116 EV and HCT116 DN cells were significantly more sensitive to ABT-737 in hypoxia than normoxia, as evaluated by growth assay (P < 0.001 and P < 0.0001, respectively, 2-way ANOVA) (Figure 4C). Furthermore, Mcl-1 levels were downregulated in hypoxic compared with normoxic conditions regardless of HIF-1α function (Figure 4D). These data show that hypoxic sensitization to ABT-737 and Mcl-1 downregulation in hypoxia was a HIF-1–independent processes. To examine whether loss of Mcl-1 in hypoxia was due to either HIF-1α or HIF-2α, we knocked down these two proteins with RNAi in normoxia and hypoxia and measured levels of Mcl-1 by Western blot. Figure 4E shows that both HIF-1α and HIF-2α were stabilized in hypoxia and that their knockdown did not prevent Mcl-1 loss in hypoxia, indicating that Mcl-1 loss in hypoxia was a HIF-1α– and HIF-2α–independent effect.
Mcl-1 can be cleaved by caspase-3 for form two degradation products of 26 and 18 kDa (26). Only basal levels of apoptosis (<3% cells) were detected in hypoxia in HCT116 cells between 24 and 48 hours (Figure 1B), and no degradation products of Mcl-1 were observed when cells were incubated in hypoxia, suggesting that loss of Mcl-1 was not due to its cleavage by caspase-3 (Supplemental Figure 3A). To rule out the possibility that Mcl-1 loss in hypoxia was due to caspase-3 activation, cells were treated in the absence and presence of the pan-caspase inhibitor QVD (20 μM) and then incubated in normoxia or hypoxia for 24 hours before being harvested, and Mcl-1 levels were measured by Western blot. Mcl-1 levels were reduced in hypoxia in comparison to normoxia regardless of QVD exposure (Supplemental Figure 3B), confirming that Mcl-1 loss was a caspase-independent process.
Hypoxic sensitization to ABT-737 was Mcl-1 dependent. To examine whether hypoxic sensitization to ABT-737 was Mcl-1 dependent, we treated cells with siRNA targeted to Mcl-1. Figure 5A reconfirms the reduced expression of Mcl-1 in hypoxia compared with normoxia in HCT116 cells and demonstrates effective downregulation of Mcl-1 expression with targeted siRNA. Consistent with previous findings, cells treated with nontargeting (NT) siRNA showed significant hypoxic sensitization to ABT-737 (P < 0.001, 2-way ANOVA) (Figure 5B). When cells were treated with Mcl-1–targeted siRNA, two observations were made. First, both hypoxic and normoxic cell were more sensitive to ABT-737 when Mcl-1 was knocked down, indicating that decreased levels of Mcl-1 were sufficient to sensitize cells to ABT-737 (note different _x_-axis scales). Second, cells treated with Mcl-1 siRNA showed no significant sensitization to ABT-737 under hypoxic conditions (P = 0.74, 2-way ANOVA) (Figure 5B). An identical experiment performed in DLD-1 and CaCo2 cells gave identical results (Supplemental Figure 4), confirming that hypoxic sensitization was Mcl-1 dependent.
Role of Mcl-1 in hypoxic sensitization to ABT-737. (A) HCT116 cells were transfected with 100 nM Mcl-1–targeted siRNA or NT control for 24 hours, after which siRNA was removed and replaced with full growth medium. Cells were then incubated in normoxia or hypoxia (1% O2) for 24, 48, 72, or 96 hours, and cells were harvested for Western blot analysis of Mcl-1 and actin levels. (B) HCT116 cells were treated with Mcl-1 or NT siRNA as described above and then incubated in normoxia or hypoxia (1% O2) for 18 hours, after which they were exposed to a range of ABT-737 concentrations under continuous normoxia or hypoxia for 72 hours prior to determination of IC50 values using the SRB assay. Data are mean ± SEM of 3 independent experiments.
The converse experiment was also performed, where HCT116 cells were transfected with a vector containing MCL1 and GFP or GFP alone (control) and subsequently cultured in normoxia and hypoxia, and their ABT-737 sensitivity was determined by SRB assay (Supplemental Figure 5). Cells expressing GFP alone were sensitized to ABT-737 in hypoxia in comparison to normoxic GFP-expressing cells as expected. In the cells that had been transfected with Mcl-1 and GFP, Mcl-1 was maintained in hypoxia, and cells were more resistant to ABT-737 than GFP control (P < 0.05, Student’s t test at 5 μM ABT-737).
Together, these function testing experiments support the hypothesis that increased sensitization of cells to ABT-737 in hypoxia was due to decreased levels of Mcl-1.
Comparison of Mcl-1 synthesis and degradation in normoxia and hypoxia. Mcl-1 ubiquitin ligase E3 (MULE), an enzyme that directly ubiquitinylates Mcl-1, causing its degradation, is one of several proteins that regulate cellular levels of Mcl-1. MULE was increased in hypoxia, and this may have explained the decrease in Mcl-1; however, knockdown of MULE did not cause Mcl-1 levels to change and did not prevent loss of Mcl-1 in hypoxia (Figure 6A). In parallel, studies were performed to investigate whether hypoxia affected the rate of Mcl-1 synthesis or degradation. Before this was done, the kinetics of Mcl-1 loss in hypoxia was assessed initially by incubation of cells in hypoxia for up to 24 hours, during which cells were harvested at various time points and the relative amount of Mcl-1 was determined by densitometric analysis of Western blots. Mcl-1 levels did not change during the first 4 hours of hypoxia, but then decreased rapidly between 4 and 6 hours and remained at a low level from that point onward (Figure 6B). To investigate whether hypoxia increased the rate of Mcl-1 degradation, we added cycloheximide, which inhibits protein synthesis, to cells after 4 hours of hypoxia, and cells were harvested every 20 minutes for the next 2 hours (i.e., during the time when Mcl-1 levels become decreased in hypoxia). Mcl-1 levels were determined by densitometric analysis of Western blots, and rate of Mcl-1 loss was compared with that in normoxic counterparts. Hypoxia did not affect the rate of Mcl-1 degradation (Figure 6C), suggesting that Mcl-1 synthesis was decreased in hypoxia. To examine whether Mcl-1 synthesis was affected by hypoxia, we added the proteasome inhibitor MG132 to cells after 6 hours in hypoxia, harvested cells at short time points thereafter, and compared the Mcl-1 rate of accumulation with that in normoxic counterparts. Hypoxia decreased the rate of accumulation of Mcl-1, indicating a decrease in rate of synthesis of Mcl-1 (Figure 6D). To further illustrate this, we incubated cells that had been exposed to hypoxia or normoxia for 24 hours in the absence and presence of MG132 for 6 hours and then blotted them for levels of Mcl-1. Whereas normoxic cells treated with MG132 showed a clear increase in Mcl-1 upon addition of MG132, hypoxic cells showed a reproducibly smaller rise in Mcl-1 levels, confirming that Mcl-1 synthesis had been decreased (Figure 6D).
Mechanism of Mcl-1 loss in hypoxia. (A) HCT116 cells were treated with NT or MULE siRNA before incubation in either normoxia or hypoxia for 6 hours, after which cells were harvested and samples analyzed for expression of MULE, Mcl-1, and GAPDH by Western blot. (B) HCT116 cells were incubated in hypoxia for up to 24 hours and were harvested at various time points for measurement of Mcl-1 levels by Western blot followed by densitometry analysis of Mcl-1, results of which were then plotted as a function of time. (C) HCT116 cells were incubated in hypoxia or normoxia for 4 hours, after which cycloheximide (10 μg/ml) was added and cells harvested every 20 minutes for the next 2 hours. Samples were then analyzed as in B. (D) Cells were incubated in hypoxia or normoxia for 6 hours, after which MG132 (10 μM) was added and cells were harvested at various time points and analyzed as in B. (E) qRT-PCR analysis of MCL1 mRNA after 18 hours preincubation in normoxia and hypoxia; results were normalized to housekeeping genes. (F) Lysates from hypoxic and normoxic cells (3-hour incubation) were separated by density on a 10%–60% sucrose gradient before being fractionated into non-polysomal (fractions 1–9) or polysomal (fractions 10–18) fractions and subjected to OD254 measurement. Data are mean ± SEM of 3 independent experiments.
Quantitative RT-PCR (qRT-PCR) analysis was performed subsequently to determine whether Mcl-1 downregulation was mediated by decreased MCL1 transcription. When MCL1 mRNA levels were normalized to a panel of housekeeping genes, no significant difference could be detected between cells cultured in normoxia and hypoxia in any of the cell lines tested (P = 0.16, Student’s t test) (Figure 6E). To determine whether hypoxia affected the translation of MCL1, we incubated cells in normoxia or hypoxia for 3 hours, and cell lysates were centrifuged on a sucrose gradient and fractionated to separate free mRNA (non-polysomes) from the denser, ribosome-bound mRNA (polysomes — which represents the fraction of mRNA being actively translated into protein). Hypoxia caused a global decrease in translation after 3 hours (Figure 6F), one that was more marked after 24 hours and also observed in H82 cells (Supplemental Figure 6).
Hypoxic H526 SCLC cells were sensitized to ABT-737 in vitro and in vivo. To determine whether hypoxic sensitization to ABT-737 also occurs in vivo, we assessed the effect of ABT-737 using an H526 SCLC tumor xenograft model. H526 cells have an intermediate sensitivity to ABT-737 in vitro (9). H526 cells cultured in vitro in hypoxic conditions were 21.5-fold more sensitive to ABT-737 compared with cells cultured in normoxic conditions (Figure 7A, P < 0.001, 2-way ANOVA, Supplemental Table 2). This hypoxic sensitivity was associated with increased apoptotic cell death. Specifically, after 24 hours, 1 μM ABT-737 induced 12% apoptotic cell death in normoxic cells and 63% in hypoxic cells (P < 0.05, Student’s t test), as assessed by changes in nuclear morphology (Figure 7B). Furthermore, after 4 and 8 hours of 1 μM ABT-737 treatment, there were higher levels of CC3 in H526 cells cultured in hypoxic conditions than in cells cultured in normoxic conditions (Figure 7C). Consistent with the other cell lines investigated in this study, the level of Mcl-1 was lower in hypoxic compared with normoxic H526 cells (Figure 7D). Therefore, H526 cells exhibit increased sensitivity toward ABT-737 under hypoxic conditions in vitro, consistent with the other SCLC and CRC cell lines studied (Supplemental Table 2).
Hypoxic sensitization to ABT-737 in H526 SCLC cells in vitro and in vivo. (A–C) Hypoxic sensitization of H526 cells to ABT-737 in vitro. (A) H256 cell population growth (by resazurin assay) under continuous normoxia or hypoxia as in Figure 1A. (B) Apoptotic cell death (by DAPI staining and nuclear morphology) after 24 hours incubation in normoxia or hypoxia with or without ABT-737 (1 μM) or (C) as assessed by CC3 levels after 0, 4, or 8 hours incubation with ABT-737. CC3 and GAPDH protein level data shown are for noncontiguous lanes run on the same gel. (D) Expression levels of Mcl-1 in untreated H526 cells exposed to normoxia or hypoxia for up to 8 hours. GAPDH was used as a loading control. Data in A and B are mean ± SEM from 3 independent experiments. Data in C and D are representative of at least 3 independent experiments. (E–G) Hypoxic sensitization of H526 cells to ABT-737 in vivo. (E) Effect of ABT-737 (100 mg/kg/d) on tumor xenograft growth in SCID-bg mice (size-matched and dosed from day 17). Data represent the average of 6 mice per group. (F) Representative images of serial tumor sections showing staining for pimonidazole (hypoxia) and CC3 (apoptosis) from tumors harvested after 72 hours of ABT-737 treatment. Images are of identical magnification. Scale bar: 100 μm. (G) Percentage area of CC3-positive staining in 4 hypoxic or 4 normoxic tumor regions. Data are the average of 4 mice per time point and per treatment. *P < 0.05 (Student’s 2-tailed t test).
When male SCID-bg mice bearing H526 xenograft tumors were treated with 100 mg/kg/d ABT-737, there was a 26% reduction in tumor growth relative to vehicle-treated mice at 26 days (Figure 7E, P < 0.05, 2-way ANOVA). Animals bearing size-matched H526 tumors were treated with 100 mg/kg/d ABT-737 or vehicle and sacrificed 6, 24, or 72 hours after the first dose. Pimonidazole binds irreversibly to hypoxic cells and was administered to the animals 1 hour and 45 minutes prior to sacrifice to identify hypoxic tumor regions. Supplemental Figure 7 shows the overall distribution of hypoxic regions in a typical xenograft tumor. Serial sections of the tumors were analyzed (by an investigator blinded to mouse group) by immunohistochemistry (IHC) for pimonidazole binding (hypoxia) and CC3 expression (to report apoptosis). Increased apoptosis was observed in hypoxic regions of tumors from mice 72 hours after the start of ABT-737 dosing, but not those from vehicle-treated control mice (Figure 7F). To quantify this observation, we determined the percent area positive for CC3 staining in 4 hypoxic and 4 normoxic regions in each tumor (n = 4 tumors for each time point in each treatment group). The amount of CC3 staining was 3.2-fold higher in hypoxic regions of ABT-737–treated mice at 72 hours compared with the normoxic region of the same tumor, increasing from 4% to 12% (Figure 7G, P < 0.05, Student’s t test). There was no significant difference in CC3 staining between hypoxic and normoxic tumor regions from vehicle-treated mice. These proof-of-concept in vivo data demonstrate that tumor cells in a hypoxic microenvironment are preferentially killed by ABT-737.
Combination of ABT-737 with clinically relevant conventional cytotoxic agents in normoxia and hypoxia. Hypoxic tumor cells are typically resistant to conventional cytotoxic agents (ref. 26 and Supplemental Table 1). Many of these conventional cytotoxic agents are used in combination in the clinic; for example, in SCLC etoposide and cisplatin are generally combined (27). When cisplatin and etoposide were combined in H146 SCLC cells in vitro in normoxia, the combination was synergistic, with a combination index (CI) of 0.43 determined according to the method of Chou and Talalay (28). However, when etoposide and cisplatin were combined in hypoxia, an antagonistic CI value of 1.43 was obtained (Supplemental Figure 8).
A substantial body of preclinical evidence emerging from studies of several tumor types performed in normoxia suggests that Bcl-2 family–targeted therapeutics such as ABT-737 are additive or synergistic with conventional cytotoxic agents (11). The impact of combining ABT-737 with conventional cytotoxic agents relevant to the treatment of SCLC (etoposide, cisplatin) was investigated in H146 and H82 cells and compared in hypoxic and normoxic conditions. Selected concentration response curves are shown for ABT-737 in combination with etoposide (H146 cells, Figure 8A) and cisplatin (H82 cells, Figure 8B). ABT-737 was synergistic with cisplatin and with etoposide in both hypoxic and normoxic H146 SCLC cells. A synergistic effect was also seen for H82 SCLC cells when ABT-737 was combined with either cisplatin or etoposide in normoxia, with still greater synergy in hypoxia. CI values for these drug combinations in SCLC cells are reported in Supplemental Table 3.
Effect of ABT-737 in combination with conventional cytotoxic agents. (A) H146, (B) H82, and (C) HCT116 cells were treated with the indicated concentrations of ABT-737, the indicated cytotoxic drugs, or both drugs added simultaneously for 72 hours under normoxia or hypoxia, after which the effect on cell population growth was determined by resazurin assay (A and B) or SRB assay (C). Data are mean ± SEM from 3 independent experiments.
We reported previously conventional cytotoxic agent resistance in hypoxic HCT116 CRC cells (ref. 16 and Supplemental Table 1). Here, in HCT116 cells, ABT-737 was combined with fluorouracil (5FU), oxaliplatin, or SN-38 (the active metabolite of irinotecan) — drugs routinely used in the clinic to treat CRC. Synergy was seen in normoxic HCT116 when ABT-737 was combined with 5FU or SN-38, but not with oxaliplatin. However, all 3 cytotoxic drugs were synergistic with ABT-737 in hypoxic conditions, and for 5FU (as an example in Figure 8C) and SN-38 the synergistic interaction with ABT-737 was greater in hypoxia. CI values for these ABT-737 conventional cytotoxic combinations in CRC cells are reported in Supplemental Table 3.
Overall, in stark contrast to the hypoxic drug resistance profiles commonly observed for single or combined conventional cytotoxic agents, combinations of these drugs with ABT-737 show synergy in hypoxia.