Article Figures & Data
Figures
- Fig. 1.
Results of the diversity set HTS. U251-HRE cells were inoculated into 384-well white flat-bottomed plates at 3000 cells/well in a volume of 25 μl and incubated for 24 h at 37°C, 5% CO2, and ambient O2. Experimental agents (1 μm) were added in a volume of 25 μl. After a 20-h incubation in the hypoxia chamber at 37°C, 5% CO2, and 1% O2, the plates were removed and incubated at room temperature and ambient O2 for 1.5 h before luminescence was measured. Compound toxicity was assayed using the SRB assay. Data from the HTS are plotted as percentage induction of luciferase expression (relative to hypoxia alone, equal to 100%) on the X axis and percentage toxicity (relative to untreated cells, equal to 100%) on the Y axis for each individual compound.
- Fig. 2.
Summary of the SI of 26 compounds identified in the primary HIF-1 HTS. U251-HRE cells were treated as described in Fig. 1 <$REFLINK> in the presence of an increasing concentration of the indicated compounds under normoxic or hypoxic conditions. U251-pGL3 cells were treated identically, except that they were treated at 37°C, 5% CO2, and ambient O2. EC50s were calculated for each compound, and a SI was determined by dividing EC50 U251-pGL3/EC50 U251-HRE. Compound toxicity was assayed using the SRB assay.
- Fig. 3.
Inhibition of hypoxic induction of VEGF mRNA expression in U251 cells. U251 cells (2 × 105 cells/well) were seeded in 6-well plates and incubated for 8 h under normoxic or hypoxic conditions, in the presence or absence of the indicated compounds (0.5 μm). Total RNA was harvested and tested for VEGF mRNA expression by real-time PCR, as described in “Materials and Methods.” Results are expressed as fold increase relative to VEGF mRNA levels under normoxic conditions (equal to 1) in the absence of drugs. 18S rRNA was tested in parallel as internal control for input RNA. Results are the average ± SE of three independent experiments. Statistical analysis was performed using ANOVA (two-factor with replication) test (P < 0.05).
- Fig. 4.
Chemical structures of positive hits. A, NSC-609699, topotecan. B, NSC-606985, camptothecin, 20-ester(S). C, NSC-639174, 9-glycineamido-20(S)-camptothecin HCl. D, NSC-607097, DX-52-1, quinocarmycin analogue. E, NSC-359449, 2-(5,11-dimethyl-6H-2λ5-pyrido[4,3-b]carbazol-2-yl)-N,N-diethylethanamine acetate. F, NSC-254681, 3-acetyl-3,5,12-trihydroxy-11-imino-10-methoxy-6-oxo-1,2,3,4,6,11-hexahydro-1-naphthacenyl 3-amino-2,3,6-trideoxyhexopyranoside. G, NSC-675865, 1-(7-aminoisothiazolo[4,5-d]pyrimidin-3-yl)-1,4-anhydropentitol.
- Fig. 5.
NSC-609699 specifically inhibits hypoxic induction of luciferase expression in U251-HRE, but not in U251-pGL3. U251-HRE and U251-pGL3 cells (1 × 104 cells/well) were seeded in 96-well optiplates and incubated under normoxic or hypoxic conditions in the presence or absence of the indicated concentration (μM) of NSC-609699. SRB assay was performed on parallel plates to monitor toxicity. Cells were treated for 24 h and then tested for cell viability and luciferase expression. Results are expressed as percentage of luciferase levels (normalized to SRB data) induced under hypoxic conditions (equal to 100%). Data are presented as the average ± SE of six independent experiments. Statistical analysis was performed using ANOVA (two-factor with replication) test (P < 0.01). U251-HRE cells, ▵; U251-pGL3 cells, ▴.
- Fig. 6.
NSC-609699 differentially regulates VEGF mRNA and COX-2 mRNA expression in U251 cells. U251 cells (2 × 105 cells/well) were seeded in 6-well plates and incubated under normoxic or hypoxic conditions for 8 h, in the presence or absence of increasing concentrations (μM) of NSC-609699 as indicated. Total RNA was harvested and processed for expression of VEGF and COX-2 mRNA by real-time PCR as described. 18S rRNA was tested in parallel to control for input RNA. Results are expressed as fold increase relative to levels of mRNA under normoxic conditions in the absence of drug (equal to 1). Data are presented as the average ± SE of four independent experiments. Statistical analysis was performed using ANOVA (two-factor with replication) test (P < 0.01).
- Fig. 7.
NSC-609699 specifically inhibits VEGF mRNA expression at the transcriptional level. A, U251-p7 and U251-p11 cells (1 × 104 cells/well) were seeded in 96-well optiplates and incubated under normoxic or hypoxic conditions in the presence or absence of the indicated concentration (μM) of NSC-609699. SRB assay was performed on parallel plates to monitor toxicity. Cells were treated for 24 h and then tested for cell viability and luciferase expression. Results are expressed as percentage of luciferase levels (normalized to SRB data) induced under hypoxic conditions (equal to 100%). Data are presented as the average ± SE of four independent experiments. Statistical analysis was performed using ANOVA (two-factor with replication) test (P < 0.01). U251-p7 cells, ♦; U251-p11 cells, □. B, U251 cells (2 × 105 cells/well) were seeded in 6-well plates and incubated under normoxic conditions for 6 h, in the presence or absence of NSC-609699 (0.1 μm) and ActD (5 μg/ml). Total RNA was harvested and processed for expression of COX-2 mRNA by real-time PCR as described. 18S rRNA was tested in parallel to control for input RNA. Results are expressed as fold increase relative to levels of mRNA present under normoxic conditions in the absence of drugs (equal to 1). Data presented are representative of three independent experiment performed.
- Fig. 8.
NSC-609699 specifically inhibits HIF-1α protein accumulation and DNA binding activity. A, U251 cells were incubated under normoxic or hypoxic conditions in the presence or absence of NSC-609699 (0.1 μm) for 6 and 24 h, and then nuclear extracts were prepared as described in “Materials and Methods.” Fifty μg of protein were separated on an 8% Tris-glycine gel. Specific monoclonal antibodies for detection of HIF-1α and HIF-1β were used as described. Results shown are from one representative experiment of four performed. B, U251 cells were cultured under normoxic or hypoxic conditions as described in A) for 6 and 24 h, and then nuclear extracts were prepared as described. Electrophoretic mobility shift assay was performed with radiolabeled AB.2 probe as described. Binding activities are labeled as follows: C, constitutive; and I, inducible. Results shown are from one experiment of at least three performed.
Volume 62, Issue 15
