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Clinical Cancer Research 13, 1010-1018, February 1, 2007. doi: 10.1158/1078-0432.CCR-06-2301
© 2007 American Association for Cancer Research
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Cancer Therapy: Preclinical

Cell Type–Specific, Topoisomerase II–Dependent Inhibition of Hypoxia-Inducible Factor-1{alpha} Protein Accumulation by NSC 644221

Mark Creighton-Gutteridge1, John H. Cardellina, II1, Andrew G. Stephen2, Annamaria Rapisarda2, Badarch Uranchimeg2, Karen Hite2, William A. Denny3, Robert H. Shoemaker1 and Giovanni Melillo2

Authors' Affiliations: 1 Screening Technologies Branch, Developmental Therapeutics Program and 2 Developmental Therapeutics Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland and 3 Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand

Requests for reprints: Giovanni Melillo, Developmental Therapeutics Program-Tumor Hypoxia Laboratory, National Cancer Institute at Frederick, Building 432, Room 218, Frederick, MD 21702. Phone: 301-846-5050; Fax: 301-846-6081; E-mail: melillog{at}ncifcrf.gov.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: The discovery and development of small-molecule inhibitors of hypoxia-inducible factor-1 (HIF-1) is an attractive, yet challenging, strategy for the development of new cancer therapeutic agents. Here, we report on a novel tricyclic carboxamide inhibitor of HIF-1{alpha}, NSC 644221.

Experimental Design: We investigated the mechanism by which the novel compound NSC 644221 inhibited HIF-1{alpha}.

Results: NSC 644221 inhibited HIF-1–dependent, but not constitutive, luciferase expression in U251-HRE and U251-pGL3 cells, respectively, as well as hypoxic induction of vascular endothelial growth factor mRNA expression in U251 cells. HIF-1{alpha}, but not HIF-1ß, protein expression was inhibited by NSC 644221 in a time- and dose-dependent fashion. Interestingly, NSC 644221 was unable to inhibit HIF-1{alpha} protein accumulation in the presence of the proteasome inhibitors MG132 or PS341, yet it did not directly affect the degradation of HIF-1{alpha} as shown by experiments done in the presence of cyclohexamide or pulse-chase labeling using [35S]methionine. In contrast, NSC 644221 decreased the rate of HIF-1{alpha} translation relative to untreated controls. Silencing of topoisomerase (topo) II{alpha}, but not topo I, by specific small interfering RNA completely blocked the ability of NSC 644221 to inhibit HIF-1{alpha}. The data presented show that topo II is required for the inhibition of HIF-1{alpha} by NSC 644221. Furthermore, although NSC 644221 induced p21 expression, {gamma}H2A.X, and G2-M arrest in the majority of cell lines tested, it only inhibited HIF-1{alpha} in a distinct subset of cells, raising the possibility of pathway-specific "resistance" to HIF-1 inhibition in cancer cells.

Conclusions: NSC 644221 is a novel HIF-1 inhibitor with potential for use as both an analytic tool and a therapeutic agent. Our data provide a strong rationale for pursuing the preclinical development of NSC 644221 as a HIF-1 inhibitor.

Inhibition of dysregulated signaling pathways that contribute to the growth of cancer cells is a promising avenue for the development of novel therapeutic agents that target unique features of the cancer phenotype. Hypoxia (a decrease in oxygen concentration) has long been recognized as a potential target for therapeutic intervention given its frequent detection in solid tumors, its involvement in the selection of more malignant and metastatic cancer cells, and its contribution to the resistance to chemotherapy and radiation therapy (1). Recently, the transcription factor hypoxia-inducible factor-1 (HIF-1) has been identified as a master regulator of the transcriptional response to oxygen deprivation (2, 3) and, accordingly, has attracted considerable interest as a potential target for the development of therapeutics. HIF-1 is a basic helix-loop-helix heterodimeric transcription factor composed of a constitutively expressed ß-subunit and an inducible {alpha}-subunit, which is rapidly degraded by the proteasome in the presence of oxygen, iron, and 2-oxoglutarate. Hydroxylation of HIF-1{alpha} at proline residues 402 and 564 by a family of enzymes known as prolyl hydroxylases, which are inhibited under hypoxic conditions, forms the recognition component for the von Hippel-Lindau (VHL) protein, an endogenous E3 ubiquitin ligase that targets HIF-1{alpha} for proteasomal degradation (4). In addition, nonhypoxic signaling pathways activated by growth factors and cytokines can induce HIF-1{alpha} accumulation by increasing the rate of protein translation (5). Thus, HIF-1{alpha} steady state is controlled at the level of both protein synthesis and protein degradation.

Over the last few years, several HIF-1 inhibitors have been described in the literature, most of which are nonselective and target redundant signaling pathways implicated in HIF-1 activation. Examples include small molecules that target the chaperone protein Hsp90 (6, 7), microtubules (8), soluble guanylate cyclase (9), topoisomerase (topo) I (10), or HIF-1 DNA binding (11). We have previously reported on the identification of small-molecule inhibitors of HIF-1 using a cell-based high-throughput screen of the National Cancer Institute (NCI) Diversity Set, a collection of ~2,000 compounds (10). We now describe a novel HIF-1{alpha} inhibitor (i.e., NSC 644221), which emerged from a screen of ~140,000 compounds from the open synthetic repository of the NCI. NSC 644221 inhibited HIF-1{alpha}, but not HIF-1ß, protein accumulation irrespective of the VHL status of the cells. NSC 644221 was unable to inhibit HIF-1{alpha} in the presence of inhibitors of the proteasome yet did not affect HIF-1{alpha} degradation but inhibited HIF-1{alpha} protein synthesis. topo II{alpha} was required for the inhibition of HIF-1{alpha} by NSC 644221 but not for the hypoxic induction of HIF-1{alpha} protein. Finally, inhibition of HIF-1{alpha}, but not induction of p21 or G2-M arrest, by NSC 644221 occurred in a cell line–specific fashion, raising the possibility of intrinsic "resistance" of the HIF-1 pathway to inhibition by small molecules.

These findings provide novel insight on mechanisms implicated in the inhibition of HIF-1{alpha} in cancer cells and identify NSC 644221 as a potential candidate for further preclinical development as a HIF-1 inhibitor.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and reagents. U251, MCF-7, SNB-75, U251-HRE, and U251-pGL3 were cultured in RPMI 1640 with 5% heat-inactivated fetal bovine serum, 50 IU/mL penicillin, 50 µg/mL streptomycin, and 2 mmol/L glutamine. HCT116 cells were cultured in RPMI 1640 with 10% fetal bovine serum, and RCC4 cells were cultured in DMEM with 10% fetal bovine serum. Cells were grown at 37°C, 21% oxygen and 5% carbon dioxide (normoxia). Hypoxic conditions were achieved in an Invivo2 400 hypoxic workstation (Ruskinn Technologies, Cincinnati, OH) at 1% oxygen (hypoxia). Bis-cyclohexamide oxaldihydrazone (CHX) and carbobenzoxy-leucyl-leucyl-leucinal-ZLLal (MG132) were from Sigma (St. Louis, MO), and PS341 was from the NCI chemical repository. NSC 644221 was dissolved in water and stored in aliquots at –70°C. NSC 644221 was routinely added to cells 30 min before incubation under hypoxia or addition of other agents, unless otherwise indicated in the figure legends.

Immunoblot analysis. Cells were lysed with radioimmunoprecipitation assay buffer [0.5 mol/L Tris-HCl (pH 7.4), 1.5 mol/L NaCl, 2.5% deoxycholic acid, 10% NP40, 10 mmol/L EDTA, 1 mmol/L NaF, 1 mmol/L NaVa] with a protease inhibitor tablet (Roche, Mannheim, Germany). Total protein (80 µg) was separated on a 4% to 20% Tris-glycine gel (Invitrogen, Carlsbad, CA), transferred to a polyvinylidene difluoride membrane (Invitrogen), and probed with a monoclonal HIF-1{alpha} antibody (BD Biosciences, San Jose, CA). Other antibodies used included an actin antibody (Chemicon International, Temecula, CA), HIF-1ß (Novus Biologicals, Littleton, CO), {gamma}H2A.X (Cell Signaling, Inc.), p21 (BD Transduction Laboratories), topo I (a gift from Yves Pommier, NCI, Frederick, MD), and topo II{alpha} (Cell Signaling, Danvers, MA).

Small interfering RNA transfection. Cells were transfected with 100 nmol/L of small interfering RNA (siRNA). Transfection was done using Oligofectamine reagent according to the manufacturer's instructions. The siRNA used was either topo I SMARTpool, topo II{alpha} SMARTpool (Dharmacon, Inc., Lafayette, CO), or control (nonsilencing) siRNA (Qiagen, Inc., Valencia, CA). Following transfection, cells were allowed to recover for 48 h before treatment.

HIF-1{alpha} pulse labeling. Cells were grown for 1 h in medium lacking methionine and then pulse labeled with 150 µCi/mL of [35S]methionine (Trans Label; ICN Biomedicals, Irvine, CA). Total cell lysates were obtained, and HIF-1{alpha} protein was immunoprecipitated using a HIF-1{alpha} antibody (Novus Biologicals). Proteins were eluted from the beads and separated on a 4% to 20% Tris-glycine gel. The gel was dried using the HydroTech gel drying apparatus (Bio-Rad Laboratories, Hercules, CA) and autoradiographed.

Real-time PCR. U251 cells were lysed, and total RNA was extracted using a RNA Mini kit (Qiagen). RNA (1 µg) was reverse transcribed using a RT-PCR kit (Roche, Molecular Systems Inc., Branchburg, NJ) as described previously (10). Human vascular endothelial growth factor (VEGF) expression was measured with specific VEGF primers and probes as described previously (10). Additionally, HIF-1{alpha} mRNA was measured using specific primers as described previously (10). Lastly, 18S rRNA was used as an internal control and assessed using premixed reagents from Applied Biosystems (Foster City, CA). Values are expressed as fold increases relative to the reference sample (medium).

HRE-luciferase activity. Stably transfected U251-HRE and U251-pGL3 cells were generated as described previously (10). U251-HRE and U251-pGL3 cells were seeded at 1 x 104 in 96-well optiplates. Cells were lysed using Bright-Glo luciferase assay reagent (Promega, Inc., Madison, WI), and activity was measured using a Top Count NXT (Perkin Elmer, Shelton, CT).

Cytochrome P450IA1 luciferase activity. MCF-7 cells were transiently transfected with a xenobiotic response element–luciferase reporter plasmid (kindly provided by Dr. F.J. Gonzalez, NCI, NIH, Bethesda, MD) using Effectine reagent (Qiagen). Twenty-four hours following transfection, cells were treated and then lysed. Changes in xenobiotic response element–luciferase activity were measured with Bright-Glo luciferase assay reagent using a Top Count NXT. Luciferase activity was normalized according to the protein expression for each condition.

Cell cycle analysis. Cells were grown overnight and then treated with NSC 644221 for 24 h. Cell cycle status was measured by staining the nuclei of harvested cells with propidium iodide. Briefly, after trypsinization, cells were washed with PBS and then fixed by adding 5 mL of 70% ethanol solution. After storage at –20°C overnight, cells were washed with cold PBS, counted, and resuspended in 400 µL PBS. Cells were then treated with 65 µg/mL RNase A (Worthington Biochemicals, Lakewood, NJ) and 50 µg/mL of propidium iodide solution (BD Biosciences, San Jose, CA) for 1 h in the absence of light. The distribution of cells between phases of the cell cycle was determined using a FACScan (Becton Dickinson, Franklin Lakes, NJ).

DNA thermal melting analysis. Thermal transition curves were obtained with a Perkin-Elmer Lambda 20 spectrophotometer (Perkin-Elmer, Boston, MA) using a DBP Peltier System. Absorption changes at 260 nm were collected as a function of temperature. Oligonucleotide (0.5 µmol/L) was prepared in the presence or absence of 5 µmol/L NSC 644221 in 10 mmol/L Tris (pH 8.0) and 1 mmol/L EDTA. DMSO concentrations were 0.1% and had no effect on the Tm of the oligonucleotides. Melting temperatures were calculated using TempLAB software provided by Perkin-Elmer. The sense strand sequence of the double-stranded oligonucleotide used in this assay was GCGGAATTCGCCCCATGCCCGGGCGTTGGATCCGCGC.

topo II assay. U251 or HCT116 cells were detached from plates and then pelleted at 800 x g for 3 min and resuspended in 5 mL of TEMP buffer [10 mmol/L Tris-HCl (pH 7.5), 1 mmol/L EDTA, 4 mmol/L MgCl2, 0.5 mmol/L phenylmethylsulfonyl fluoride]. Supernatants were assayed for topo II activity using the Topoisomerase II assay kit (TopoGEN, Inc., Port Orange, FL).


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
NSC 644221 decreases HIF-1–dependent, but not constitutive, luciferase expression and hypoxic induction of VEGF mRNA expression. NSC 644221 (structure shown in Table 1 ) was initially discovered in a screen that used U251-HRE cells, in which luciferase expression is under the control of three copies of a canonical HRE sequence (10). Hypoxia increased luciferase expression in U251-HRE cells by 9.7-fold (100% in Fig. 1A ) relative to cells cultured under normoxic conditions. The addition of NSC 644221 decreased hypoxic induction of luciferase expression in a dose-dependent fashion, with an EC50 of ~1 µmol/L (Fig. 1A). In contrast, concentrations up to 10 µmol/L NSC 644221 did not significantly affect constitutive luciferase expression in the control cell line U251-pGL3, showing that the inhibition of luciferase activity was HRE specific. Because NSC 644221 contains a core dioxin structure, we next did experiments to rule out that NSC 644221 acted as a ligand for the dioxin receptor aryl hydrocarbon receptor, which dimerizes with, and might potentially limit the abundance of, HIF-1ß/aryl hydrocarbon receptor nuclear translocator (12). A classic aryl hydrocarbon receptor ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin, strongly activated a luciferase reporter plasmid containing the xenobiotic response element from the cytochrome P450IA1 promoter in a dose-dependent fashion (Fig. 1B). In contrast, NSC 644221 up to 10 µmol/L had no activity, ruling out that NSC 644221 acted as a ligand for aryl hydrocarbon receptor.


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  		Table 1. Subtle analogues of NSC 644221 lose topo II activity and their capacity to inhibit HIF-1{alpha}

 

Figure 1
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  		Fig. 1. NSC 644221 decreases HIF-1–dependent luciferase expression and transcriptional activity. A, U251-HRE and U251-pGL3 cells were cultured under normoxic or hypoxic conditions in the presence or absence of the indicated concentrations of NSC 644221 for 18 h. Luciferase activity was measured as described in Materials and Methods and is expressed as percentage induction relative to hypoxia alone, which is arbitrarily considered equal to 100% (hypoxic induction/normoxia = 100%). Columns, mean of four independent experiments; bars, SE. B, MCF-7 cells were transiently transfected with a xenobiotic response element–luciferase reporter plasmid. Twenty-four hours following transfection, cells were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or NSC 644221 at the indicated concentrations for 18 h. Luciferase expression was measured as relative light units, and changes in xenobiotic response element–luciferase activity were normalized according to protein expression. Columns, mean of three independent experiments; bars, SE. C, U251 cells were cultured under normoxia or hypoxia for 18 h in the presence or absence of the indicated concentrations of NSC 644221. Total RNA was isolated, and changes in VEGF mRNA expression were analyzed using real-time PCR and normalized to 18S rRNA expression. Results are expressed as fold change induced under hypoxic conditions relative to normoxia. Columns, mean of three independent experiments; bars, SE. Statistical analysis was done using ANOVA test (two-way factor with replication). *, P < 0.01, statistically significant difference.

 
To further validate the ability of NSC 644221 to inhibit endogenous HIF-1 transcriptional activity, we tested its effects on the mRNA expression of VEGF, a classic HIF-1 target gene. Hypoxia (18 h) increased VEGF mRNA expression by 3.6-fold in U251 cells (Fig. 1C). NSC 644221 inhibited hypoxic induction of VEGF mRNA expression in a dose-dependent fashion with an EC50 of ~3 µmol/L, consistent with inhibition of HIF-1–dependent reporter activity. Taken together, these data show that NSC 644221 exerts specific and biologically relevant inhibition of HIF-1 transcriptional activity in U251 cells.

NSC 644221 inhibits HIF-1{alpha}, but not HIF-1ß, protein accumulation in a dose- and time-dependent manner. We next determined if HIF-1{alpha} protein levels were inhibited by the addition of NSC 644221. In U251 cells, hypoxia induced a rapid accumulation of HIF-1{alpha} protein, which was already detectable at 2 h and was sustained up to 18 h. NSC 644221 inhibited HIF-1{alpha} protein accumulation in a time-dependent manner. A slight decrease was already detectable at 4 h, whereas significant inhibition was observed at 6 to 8 h and was maximal at 18 h (Fig. 2A ). Furthermore, after 8 h, NSC 644221 inhibited hypoxic induction of HIF-1{alpha} protein expression in a dose-dependent fashion with an EC50 of ~2.1 µmol/L (Fig. 2B). NSC 644221 also decreased the level of HIF-1{alpha} induced by the iron chelator desferrioxamine and by cobalt chloride (data not shown). In contrast to HIF-1{alpha}, HIF-1ß expression did not change in cells treated with NSC 644221 (Fig. 2B) under either normoxic or hypoxic conditions. In conclusion, these data show that NSC 644221 decreases the expression of HIF-1{alpha}, but not HIF-1ß, in a time- and dose-dependent manner in U251 cells.


Figure 2
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  		Fig. 2. NSC 644221 decreases HIF-1{alpha} protein levels, which is abrogated in the presence of proteasome blockade. A, U251 cells were cultured under normoxia for 4 or 18 h (lane 1 of each blot) or under hypoxia (lanes 2-5 of each blot) in the presence or absence of NSC 644221 (5 µmol/L) for the indicated times. Immunoblot analysis was done using an anti-HIF-1{alpha} antibody. Actin as a loading control. B, U251 cells were cultured under normoxic (lane 1) or hypoxic (lanes 2-7) conditions for 8 h in the presence or absence of increasing concentrations of NSC 644221. Immunoblot analysis was done using anti-HIF-1{alpha} and anti-HIF-1ß (shown as loading control) antibodies. C, U251 cells were cultured under normoxic conditions in the presence or absence of the proteasome inhibitors MG132 (20 µmol/L) or PS341 (5 µmol/L) and NSC 644221 (5 µmol/L) or topotecan (TPT; 0.75 µmol/L) for 7 h. Cells were pretreated for 1 h with MG132 and PS341 to allow functional inhibition of the proteasome. Cells were then lysed, and immunoblot analysis was done using anti-HIF-1{alpha} antibodies. Actin as loading control. D, RCC4 and RCC4-VHL cells were cultured under normoxic or hypoxic conditions in the presence or absence of NSC 644221 (5 µmol/L) for 8 h. Protein was analyzed using immunoblot analysis for HIF-1{alpha} and actin.

 
NSC 644221 does not inhibit HIF-1{alpha} protein expression in the presence of proteasome inhibitors. To probe the mechanism of HIF-1{alpha} inhibition by NSC 644221, we tested whether proteasome activity was required. Inhibition of the proteasome causes an accumulation of HIF-1{alpha} under normoxia as shown in Fig. 2C following addition of MG132 (lane 4) or PS341 (lane 7). Addition of NSC 644221 for 7 h could not inhibit HIF-1{alpha} expression if the proteasome was blocked by the addition of either MG132 (lane 5) or PS341 (lane 8). In contrast, the addition of topotecan resulted in the inhibition of HIF-1{alpha} despite the presence of proteasome inhibitors (Fig. 2A, lanes 6 and 9) as previously reported (13). This result suggested that, unlike topotecan, NSC 644221 requires the presence of a functional proteasome pathway to inhibit HIF-1{alpha}. To further investigate the involvement of HIF-1{alpha} degradation pathway in the effect of NSC 644221, we used renal cancer cell lines that were lacking (RCC4) the VHL protein or in which VHL had been reintroduced (RCC4-VHL). RCC4 cells, deficient in the VHL protein, display constitutively high levels of HIF-1{alpha}, which is expressed in an oxygen-independent fashion (Fig. 2D, lane 1). NSC 644221 (5 µmol/L) inhibited constitutive HIF-1{alpha} protein expression in RCC4 cells and both constitutive and hypoxia-inducible HIF-1{alpha} accumulation in RCC-VHL, providing evidence that a functional VHL protein is not required for the inhibition of HIF-1{alpha} by NSC 644221. Taken together, these results indicate that a functional proteasome pathway, but not the endogenous pathway of HIF-1{alpha} degradation, is required for the inhibition of HIF-1{alpha} by NSC 644221.

NSC 644221 does not affect HIF-1{alpha} half-life but decreases the rate of HIF-1{alpha} translation. Data shown in Fig. 2C were consistent with the possibility that NSC 644221 inhibited HIF-1{alpha} protein accumulation by increasing its proteasome-dependent degradation. To address this question, we did experiments in the presence of cyclohexamide (40 µg/mL), which prevents de novo protein synthesis, and estimated the half-life of HIF-1{alpha} in U251 cells cultured under hypoxic conditions in the presence or absence of NSC 644221. U251 cells were cultured under hypoxic conditions for 6 h in the presence or absence of NSC 644221, at which point CHX was added and subsequent samples were harvested at 60, 90, and 120 min after addition of CHX to estimate HIF-1{alpha} half-life. As shown in Fig. 3A (top) NSC 644221 caused a 50% decrease of hypoxic induction of HIF-{alpha} accumulation after 6 h (Fig. 3A, compare lane 2 with lane 6). Densitometry analysis of the blot shown in Fig. 3A relative to the actin loading control indicated that HIF-1{alpha} half-life in cells cultured under hypoxia was 84.5 min in the absence of NSC 644221 and 78.5 min in the presence of NSC 644221, suggesting that NSC 644221 does not significantly affect HIF-1{alpha} degradation. This conclusion was further supported by pulse-chase analysis of HIF-1{alpha} degradation done in RCC4 cells using [35S]methionine, which also showed that addition of NSC 644221 did not significantly affect the rate of degradation of HIF-1{alpha} relative to untreated control (Supplementary Fig. S1). To further rule out that NSC 644221 was affecting HIF-1{alpha} degradation, we also showed that addition of NSC 644221 neither affected the amount of endogenous ubiquitylated HIF-1{alpha} (data not shown) nor decreased the levels of exogenously transfected HA-tagged HIF-1{alpha} (data not shown), consistent with the lack of effect on the degradation pathway of HIF-1{alpha}.


Figure 3
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  		Fig. 3. NSC 644221 does not affect the degradation of HIF-1{alpha} and decreases the rate of HIF-1{alpha} translation. A, U251 cells were cultured under normoxia (lane 1) or hypoxia in the absence (lanes 2-5) or presence (lane 6-9) of NSC 644221 (5 µmol/L) for 6 h, at which time CHX (40 µg/mL) was added. Cells cultured under hypoxia in the absence (lane 2) or presence (lane 6) of NSC 644221 were harvested immediately before addition of CHX and used to calculate the decay of HIF-1{alpha} in cells untreated (lanes 2-5) or treated (lanes 6-9) with NSC 644221. The displayed protein lanes have been rearranged to enable the comparison of NSC 644221 untreated and treated samples against the respective controls without CHX. Bottom, densitometry analysis was done by normalizing the levels of HIF-1{alpha} expression to the corresponding levels of actin. Values for lane 2 (hypoxia alone) and lane 6 (hypoxia plus NSC 644221) were arbitrarily considered equal to 100% and used to calculate the rate of degradation of samples treated in hypoxia alone (lanes 3-5) or hypoxia plus NSC 644221 (lanes 7-9). B, RCC4 cells were treated with NSC 644221 (5 µmol/L) for 18 h under normoxia in complete medium. Cells were then placed in cysteine- and methionine-free DMEM for 2 h in the presence or absence of NSC 644221. 35S-labeled methionine was then added for the indicated times. Cell extracts were immunoprecipitated with a HIF-1{alpha} antibody, and autoradiography was used to visualize 35S-labeled HIF-1{alpha}. C, U251 cells were cultured under normoxic conditions in the presence or absence of NSC 644221 for 18 h. Total RNA was isolated and analyzed for HIF-1{alpha} mRNA expression using real-time PCR. Changes in mRNA were normalized using 18S rRNA expression, and results are expressed as fold increase relative to untreated control arbitrarily considered equal to 1.

 
HIF-1{alpha} protein steady state is the result of a balance between protein translation and its degradation. Because NSC 644221 did not affect HIF-1{alpha} degradation (Fig. 3A), we determined if NSC 644221 altered its translation. Accordingly, pulse-labeling experiments were done in RCC4 cells in the presence or absence of NSC 644221 (5 µmol/L) for 18 h and the rate of newly synthesized HIF-1{alpha} protein was measured by using [35S]methionine. At 0 min following pulsing with [35S]methionine, no HIF-1{alpha} protein was detectable. However, after 15 min of 35S labeling, HIF-1{alpha} was clearly detectable in lysates from RCC4 cells in the absence, but not in the presence, of treatment with NSC 644221, suggesting an inhibition of HIF-1{alpha} protein synthesis (Fig. 3B). Accordingly, significantly more HIF-1{alpha} protein had accumulated in untreated versus treated cells after 30 and 60 min. Importantly, NSC 644221 did not affect total synthesis of new proteins in U251 cells as indicated by experiments measuring [35S]methionine incorporation in trichloroacetic acid–precipitated proteins (data not shown). Finally, we investigated whether NSC 644221 affected the mammalian target of rapamycin pathway, which has been implicated in the regulation of HIF-1 translation, and we found that both p4EBP-1 and p70S6 kinase were unaffected by NSC 644221 (data not shown). These results, along with the finding that NSC 644221 up to 10 µmol/L did not affect HIF-1{alpha} mRNA expression in U251 cells cultured under either normoxia or hypoxia (Fig. 3C), show that NSC 644221 decreases the rate of HIF-1{alpha} protein synthesis without significantly affecting its degradation.

The inhibition of HIF-1{alpha} by NSC 644221 is cell line specific. The ability of NSC 644221 to inhibit HIF-1{alpha} protein accumulation in different human cancer cell lines revealed striking cell line specificity. Figure 4A shows a representative immunoblot of HIF-1{alpha} expression in cells sensitive (U251) or resistant (HCT116) to HIF-1{alpha} inhibition. Hypoxia induced HIF-1{alpha} accumulation in both cell lines to a similar extent. However, addition of NSC 644221 completely inhibited HIF-1{alpha} accumulation in U251 but had no effect in HCT116 even at concentrations of NSC 644221 as high as 50 µmol/L (data not shown). To rule out the possibility that the lack of effect of NSC 644221 in HCT116 was due to a general phenomenon of drug resistance, we tested whether other cellular effects of NSC 644221 were detectable in these cells. Preliminary experiments had suggested that NSC 644221 induced genes involved in cell cycle regulation and DNA damage signaling. We then tested the ability of NSC 644221 (5 µmol/L) to induce p21 and H2A.X phosphorylation ({gamma}H2A.X, an early marker of DNA damage) in cells sensitive and resistant to the HIF-1{alpha} inhibition. As shown in Fig. 4A, the addition of NSC 644221 induced p21 expression and {gamma}H2A.X in both U251 and HCT116 cells under either normoxic or hypoxic conditions, suggesting that the lack of HIF-1{alpha} inhibition in HCT116 did not simply reflect a general cellular resistance to NSC 644221. Similar results were observed in several human cancer cell lines, such as HeLa and MCF-7, in which we found that NSC 644221 induced p21 and/or {gamma}H2A.X but did not significantly inhibit HIF-1{alpha} protein. Overall, these data suggest that the pattern of HIF-1{alpha} inhibition displayed by NSC 644221 reflects more an "intrinsic" resistance to HIF-1 inhibition rather than a general phenomenon of cellular resistance. In addition, our data also indicate that activation of a DNA damage signaling pathway is neither sufficient for nor is associated with the inhibition of HIF-1{alpha} by NSC 644221.


Figure 4
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  		Fig. 4. Inhibition of HIF-1{alpha} by NSC 644221, but not induction of p21, {gamma}H2A.X, and G2-M arrest, is cell line specific. A, U251 and HCT116 cell lines were cultured under normoxic or hypoxic conditions for 8 h in the presence or absence of NSC 644221 (5 µmol/L). Total cellular protein was isolated, and the specific proteins p21, {gamma}H2A.X, HIF-1{alpha}, and actin were analyzed by immunoblot analysis. B, HCT116 and U251 cells were treated with NSC 644221 (1 µmol/L) for 24 h under normoxic conditions. Cell cycle was assessed by flow cytometry analysis. 2N, G0-G1; 4N, G2-M. Data were obtained from the measurement of 10,000 cells.

 
NSC 644221 causes a G2-M arrest and a cytostatic effect. To further investigate the existence of differential cellular responses in cells sensitive or resistant to the inhibition of HIF-1{alpha}, we tested the effects of NSC 644221 on cell cycle and cell viability in U251 and HCT116 cells. We found that NSC 644221 was capable of causing a G2-M arrest (Fig. 4B) in both U251 and HCT116 cells. NSC 644221 caused an increase of the G2-M fraction from 15.3% to 77.9% in HCT116 cells (Fig. 4B, top) and from 8.1% to 82.3% in U251 (Fig. 4B, bottom) after 24 h of treatment. Consistent with these results, NSC 644221 caused a dose-dependent decrease in cell number relative to untreated control in both U251 and HCT116 cells after 72 h under normoxic or hypoxic conditions as indicated by formazan production measured to estimate viable cells (data not shown). However, NSC 644221 had minimal, if any, cytotoxic effect as indicated by the total number of cells remaining at the end of the experiment and by the lack of increased trypan blue uptake in cells treated with NSC 644221 relative to untreated control (data not shown). Taken together, these data further show that NSC 644221 mediates similar cellular responses in both cells sensitive (U251) or resistant (HCT116) to the inhibition of HIF-1{alpha} and that NSC 644221 exerts predominantly a cytostatic activity.

topo II{alpha} expression is required for HIF-1{alpha} inhibition by NSC 644221 but not for the hypoxic induction of HIF-1{alpha}. Data shown above and published earlier (14) were consistent with the possibility that some of the cellular effects caused by NSC 644221 might be mediated by inhibition of topo II. To explore whether topo II was required for HIF-1{alpha} inhibition by NSC 644221, we tested the hypoxic induction of HIF-1{alpha} protein in the presence or absence of NSC 644221 in U251 cells that were either nontransfected or transfected with siRNA targeting topo II{alpha} or topo I. As shown in Fig. 5A , transfection of topo II{alpha} siRNA in U251 cells completely silenced the expression of topo II{alpha} and had little, if any, effect on the hypoxic induction of HIF-1{alpha} (compare lane 3 with lane 5). Interestingly, NSC 644221 could not inhibit HIF-1{alpha} expression when cells were transfected with siRNA targeting topo II{alpha} (compare lane 4 with lane 7), showing that topo II{alpha} expression was required for the inhibition of HIF-1{alpha} by NSC 644221. In contrast, transfection of topo I siRNA, which completely abrogated the expression of topo I, had no effect on the ability of NSC 644221 to decrease hypoxic induction of HIF-1{alpha} protein (compare lane 4 with lane 8) yet effectively prevented inhibition of HIF-1{alpha} by topotecan (data not shown). These results show that expression of topo II{alpha}, but not topo I, is required for HIF-1{alpha} inhibition induced by NSC 644221.


Figure 5
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  		Fig. 5. topo II{alpha} expression is required for HIF-1{alpha} inhibition by NSC 644221. A, U251 cells were nontransfected or transfected with topo I or topo II{alpha} siRNA. Forty-eight hours following transfection, cells were placed under normoxic or hypoxic conditions in the presence or absence of NSC 644221 for an additional 18 h. Expression of topo I, topo II{alpha}, HIF-1{alpha}, or actin was then measured by immunoblot analysis. B, U251, SNB-75, MCF-7, and HCT116 cells were cultured under normoxia or hypoxia for 18 h in the presence or absence of NSC 644221 (5 µmol/L). Total cellular protein was isolated, and HIF-1{alpha} and actin were measured by immunoblot analysis. Right, additionally, cellular protein from the untreated control of each cell line was analyzed separately to measure the levels of topo II{alpha} expression. C, U251 cells were cultured under normoxia (lane 1) or hypoxia in the absence (lane 2) or presence of NSC 644221 (lane 3), NSC 644219 (lane 4), or NSC 644220 (lane 5) for 18 h. HIF-1{alpha}, p21, {gamma}H2A.X, and actin expression was measured by immunoblot analysis.

 
To rule out that cell line–dependent variations in topo II{alpha} levels were associated with the cell line–specific inhibition of HIF-1{alpha} by NSC 644221, topo II{alpha} protein levels were measured in cell lines sensitive (U251 and SNB-75) or resistant (HCT116 and MCF-7) to HIF-1{alpha} inhibition. topo II{alpha} levels were high in U251 and MCF-7 cells and substantially lower in both SNB-75 and HCT116 cells (Fig. 5B, right). However, NSC 644221 caused a pronounced inhibition of HIF-1{alpha} in U251 and SNB-75 cell lines but did not affect hypoxic induction of HIF-1{alpha} protein in MCF-7 and HCT116 cell lines. In contrast, NSC 644221 induced p21 and {gamma}H2A.X in both HCT116 (Fig. 4A) and MCF-7 (data not shown), indicating that NSC 644221 was active in these cells.

Together, these results show that topo II expression is required for HIF-1{alpha} inhibition by NSC 644221 and that cellular topo II{alpha} levels do not contribute to the cell type–specific resistance to HIF-1{alpha} inhibition by NSC 644221.

Analogues of NSC 644221 lose their capacity to inhibit HIF-1{alpha}. To assess structure-activity relationships for NSC 644221, we used two analogues of NSC 644221 in which the nitro group is at position C6 (NSC 644219) or at position C3 (NSC 644220; Table 1). As shown in Fig. 5C, the parent compound NSC 644221 inhibited hypoxic induction of HIF-1{alpha} expression and induced p21 expression and {gamma}H2A.X in U251 cells. In contrast, NSC 644219 and NSC 644220, up to 50 µmol/L, did not inhibit HIF-1{alpha} expression and did not induce p21 or {gamma}H2A.X, suggesting that these compounds were inactive and incapable of inducing DNA damage (Fig. 5C). In addition, both analogues were unable to inhibit topo II–mediated decatenation of kDNA (Table 1). Because published results indicated that NSC 644221 could weakly bind to DNA (14), we assessed the DNA-binding properties of NSC 644221 and its analogues by doing DNA melting temperature analysis, in which intercalation of small molecules into the double helix is reflected by an increase of the helix melting temperature (Tm). NSC 644221, but not NSC 644219 or NSC 644220, significantly increased the Tm relative to untreated control (from 65°C to 72°C; Table 1), indicating that NSC 644221, unlike the analogues, was capable of binding to DNA. Overall, these data show that the nitro group at C9 is required for the biological activities of NSC 644221, including inhibition of HIF-1{alpha} and topo II activity.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
There is overwhelming evidence to support the continued discovery and development of small-molecule inhibitors of HIF-1 as potential antiangiogenic and anticancer agents (2, 15–17). However, the best strategy to effectively target HIF-1 activity has yet to be determined (18). NSC 644221 is a novel HIF-1 inhibitor characterized by several unique features, which are as follows: (a) inhibition of HIF-1{alpha} translation by a pathway that requires an intact proteasome function, (b) a requirement for the {alpha} subunit of topo II, and (c) distinct cell type specificity. NSC 644221 is a novel and attractive candidate for further preclinical development as a HIF-1 inhibitor.

A screen of ~140,000 synthetic compounds from the open synthetic repository of the NCI using a previously described cell-based HRE-luciferase screen (10) yielded many HIF-1 inhibitors that were DNA-interacting agents, including but not limited to the topo I poisons described previously (10, 13). This observation could not be simply explained by increased toxicity displayed by these compounds, as the screen was designed to carefully exclude overtly cytotoxic compounds or those that inhibited luciferase expression in a nonspecific and/or HIF-1–independent manner (10). Results presented herein and published previously are rather consistent with the hypothesis that complex and distinct biochemical pathways link DNA-interacting agents, including but not limited to topo-targeting compounds, to HIF-1{alpha} inhibition.

NSC 644221 was originally identified as a specific inhibitor of HIF-1–dependent, but not constitutive, luciferase expression in U251-HRE cells. Despite early experiments indicating that a functional proteasome was required for NSC 644221 to inhibit HIF-1{alpha}, we found that NSC 644221 did not affect HIF-1{alpha} degradation. This conclusion was supported by experiments conducted in the presence of CHX (Fig. 3A) to estimate the half-life of HIF-1{alpha} degradation in the presence or absence of NSC 644221 and by pulse-chase experiments using [35S]methionine (data not shown). Both experiments indicated that NSC 644221 did not significantly affect the half-life of HIF-1{alpha} relative to hypoxia, thus showing that NSC 644221 did not affect HIF-1{alpha} degradation. Furthermore, experiments done in VHL-deficient cells, in which HIF-1{alpha} is constitutively expressed in an oxygen-independent fashion, indicated that an intact VHL-dependent degradation pathway was dispensable for the inhibition of HIF-1{alpha} by NSC 644221. In contrast, pulse-labeling experiments showed that the rate of HIF-1{alpha} translation relative to untreated control was markedly decreased in the presence of NSC 644221. Notably, NSC 644221 did not affect global protein synthesis but actually increased the expression of several short-lived proteins, including p21 (Fig. 4A) and p53 (data not shown). These results are consistent with the possibility that NSC 644221 might target a novel pathway that links the proteasome function with HIF-1{alpha} translation (e.g., by increasing proteasomal degradation of a putative protein that is required for HIF-1{alpha} translation). Alternatively, the degradation of a direct target of NSC 644221 by the proteasome may be required for its optimal activity. However, further experiments will be required to fully elucidate the requirement of a functional proteasome pathway for the inhibition of HIF-1{alpha} translation by NSC 644221.

To identify the molecular mechanism by which NSC 644221 inhibits HIF-1{alpha}, we focused our attention on the possible involvement of topo II. This hypothesis was based, at least in part, on the knowledge that NSC 644221 had been originally designed as a weakly DNA-binding agent and topo II inhibitor (14). Our preliminary results, indicating that NSC 644221 induced {gamma}H2A.X as well as G2-M arrest, were consistent with this possibility. topo II is a nuclear protein expressed as two homodimeric isoenzymes ({alpha} and ß), which are coded on separate chromosomes (19, 20). Targeting topo II{alpha} was the preferred strategy because the {alpha} form relative to the ß isoform proved indispensable, was more sensitive to topo II agents, and possessed a more defined role within cells (19, 21–23). Interestingly, silencing of topo II{alpha} expression by specific siRNA completely abrogated the ability of NSC 644221 to inhibit HIF-1{alpha} protein accumulation but did not affect hypoxic induction of HIF-1{alpha}. In contrast, silencing of topo I had no effect on the ability of NSC 644221 to inhibit HIF-1{alpha} yet completely blocked its inhibition by topotecan, a topo I poison (13). These results not only implicate topo II{alpha} in the mechanism of inhibition of HIF-1{alpha} by NSC 644221 but also provide strong evidence that this pathway of HIF-1{alpha} inhibition is biochemically distinct from the one previously shown to involve topo I. Consistent with topo II inhibition, NSC 644221 also induced G2-M arrest and {gamma}H2A.X in cells both sensitive (U251) and resistant (HCT116) to HIF-1{alpha} inhibition, suggesting that inhibition of topo II was required but not sufficient per se to mediate HIF-1{alpha} inhibition. Importantly, the ability of NSC 644221 to induce p21, {gamma}H2A.X, and G2-M arrest even in cells in which it did not inhibit HIF-1{alpha} expression provided evidence of a biochemical distinction between DNA damage signaling pathways and inhibition of HIF-1{alpha}. This conclusion is further supported by results showing that blocking the proteasome function enhanced the cytotoxic activity of topo II–directed drugs (24) yet blocked the ability of NSC 644221 to inhibit HIF-1{alpha} expression, showing the existence of distinct downstream pathways mediated by topo II{alpha} inhibition.

Despite the finding that both NSC 644221 and topotecan inhibited HIF-1{alpha} translation, these pathways are clearly distinct. (a) A functional proteasome is required for the inhibition of HIF-1{alpha} by NSC 644221; in contrast, topotecan inhibits HIF-1{alpha} also in the presence of proteasome inhibitors. This difference cannot be simply explained by the more rapid kinetic of HIF-1{alpha} inhibition exhibited by topotecan, as proteasome blockade also prevented NSC 644221 from inhibiting HIF-1{alpha} after 16 h of incubation (data not shown). (b) NSC 644221 and topotecan have distinct cell line specificity. This is best exemplified by HCT116 cells in which NSC 644221 does not inhibit HIF-1{alpha} whereas topotecan does (data not shown). (c) Inhibition of HIF-1{alpha} by NSC 644221 requires expression of topo II{alpha}, but not topo I, which is required for the activity of topotecan, as evidenced by siRNA studies. Although these results raise the possibility of achieving more profound inhibition of HIF-1{alpha} by combining agents belonging to these two classes of compounds, we did not observe any additive effect on HIF-1{alpha} inhibition when NSC 644221 and topotecan were used in combination (data not shown). This is perhaps because, although these agents have distinct modes of action, they both affect the translation of HIF-1{alpha} and their inhibitory activity may overlap.

A striking feature of NSC 644221 was the cell line specificity of HIF-1{alpha} inhibition. It was important to establish whether this pattern of activity was restricted to HIF-1 or was a general phenomenon. By surveying a large number of human cancer cell lines, we found that cellular responses to NSC 644221, such as induction of p21 expression, {gamma}H2A.X, and G2-M arrest, were still present in cell lines in which HIF-1{alpha} was not inhibited even at concentrations as high as 50 µmol/L. These results have several implications. (a) They rule out that the lack of HIF-1{alpha} inhibition was due to cellular resistance to NSC 644221 or more general resistance to topo II targeting agents. Accordingly, NSC 644221 was capable of inhibiting HIF-1{alpha} in cell lines expressing multidrug resistance, such as UO-31, which are highly resistant to topo II inhibitors, and irrespective of the p53 status of the cells (25). (b) They raise, for the first time, the intriguing concept of "drug resistance" relative to the HIF-1 pathway. This conclusion is supported by the fact that NSC 644221 induced p21 expression and other cellular responses in HCT116 but did not inhibit HIF-1{alpha}. Although these findings need to be further expanded and confirmed in larger surveys, they do warn against simply translating a general concept of HIF-1 inhibition to genetically diverse cancer cells. Accordingly, future consideration should be given to better understanding how to translate these concepts to the clinical setting where inhibition of HIF-1 is now being attempted and represents an attractive therapeutic strategy.

NSC 644221 was originally developed as a weakly DNA-binding topo II inhibitor and as an alternative to highly toxic and poorly bioavailable acridine compounds (14, 26, 27). However, this compound showed little or no activity as a cytotoxic agent when analyzed either in the NCI 60 cell line screen or in an in vivo P388 leukemia cell model (14). We also found that NSC 644221 exerts predominantly a cytostatic but not cytotoxic activity in several cancer cell lines irrespective of its ability to inhibit HIF-1{alpha}. The fact that NSC 644221 is only moderately cytotoxic may be a desirable feature for its potential application as a HIF-1 inhibitor, where chronic schedules of administration may be anticipated. In addition, cancer cells in hypoxic areas of solid tumors are resistant to the cytotoxic activity of chemotherapeutic agents, and modulation of HIF-1–dependent responses under these circumstances might have a beneficial therapeutic effect.


   Acknowledgments
 
We thank Shawn Clopper, Paul Klausmeyer, Dominic Scudiero, Michael Currens, Anne Monks, Erik Harris, Rene Delosh, Yili Yang, and Gillian Whittaker for their valuable input and assistance.


   Footnotes
 
Grant support: National Cancer Institute, NIH, funded by contract N01-CO-12400 and Developmental Therapeutics Program in the Division of Cancer Treatment and Cancer Diagnosis of the National Cancer Institute.

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.

Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

The content of this publication does not necessarily reflect the views of policies of the Department of Health and Human Services nor does mention of trade terms, commercial products, or organizations imply endorsement by the U.S. government.

Received 9/18/06; accepted 11/ 9/06.


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 Materials and Methods
 Results
 Discussion
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