Figure 1.
HIF1αCTAD interacts with p300.
A. Deletion structure of HIF1α. The schematic diagram indicates their length and location compared with full-length HIF1α. B. Fragments of HIF1α were cloned and expressed as His-GST fusion proteins and were analyzed by SDS-PAGE (4–20% gradient gel, BioRad). Proteins were visualized by Coomassie Blue staining. C. Deletion HIF1α 425 and HIF1α 776 bind to endogenous p300: ∼10 µg of bacterially expressed and GST purified HIF1α 425 and HIF1α 776 (CTAD) were mixed with 100 µl of HeLa nuclear extract and 15 µl of glutathione-sepharose 4 Fast Flow beads (Amersham Biosciences) in binding buffer containing 40 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.2% (v/v) Triton X-100. Protein mixtures were incubated for 4 hours at 4°C and bound proteins were eluted with elution buffer containing 25 µl of 30 mM glutathione, 40 mM Tris-HCl (pH 7.5), 10% (v/v) glycerol, and 150 mM NaCl. Pulled-down p300 was confirmed by Western blotting with anti-p300 antibody. D. Overexpressed full length (FL) HIF1α interacts with endogenous p300 in the presence or absence of CoCl2. HeLa cells were transiently transfected with 1 µg of HPC4-HIF1α. Hypoxia conditions were induced by adding CoCl2 (Sigma, 150 µM). Proteasome inhibitor MG132 (50 µM) was added to block protein degradation and assess the total amount of HIF1α 3 hours before harvest. Then, 72 hours after HPC4-HIF1α transfection, cells were harvested and lysed for immunoblot.
Figure 2.
Novobiocin directly disrupts the HIF1αCTAD/p300 CH1 complex.
A. Novobiocin inhibits His-HIF1α FL protein pulling down p300 CH1. Insect cell expressed and purified His-HIF1α FL (∼5 µg), Flag-p300 CH1, 400 µM of antibiotic novobiocin or DMSO (control), and 15 µl of Ni2+-agarose were mixed in binding buffer containing 40 mM Hepes-NaOH (pH 7.9), 250 mM NaCl, 10% glycerol, and 40 mM imidazol, and incubated at 4°C for 2 hours. Bound proteins were eluted with 20 µl of elution buffer containing 300 mM imidazol and were then confirmed by Western blotting with anti-HIF1α and anti-Flag antibodies (upper panel). Western blot images were quantified using Quantity One software (BioRad) (lower panel). B. Novobiocin inhibited GST-p300 CH1 protein pulling down the HIF1α FL. GST-p300 CH1 protein (∼10 µg GST only as a negative control) was mixed with His-HIF1α FL, 15 µl of glutathione sepharose 4 Fast Flow beads, and novobiocin (100–400 µM). Bound proteins were then detected by Western blotting with anti-HIF1α antibody. Quantified HIF1α is depicted in the graph (lower panel). Percentages given above each bar represent bound HIF1α protein compared to lane 2. C. Novobiocin directly disrupts the HIF1α CTAD/p300 CH1 complex. The GST pull-down assay was performed by mixing (as indicated in the figure) ∼10 µg GST fusion proteins, 5 µg Flag-p300 CH1, and 100 or 400 µM novobiocin. Pulled-down p300 CH1 was detected by Western blotting with anti-Flag antibody. D. Novobiocin inhibited the interaction between overexpressed HIF1α FL and endogenous p300 in the presence or absence of CoCl2. HPC4-HIF1α FL was overexpressed in HeLa cells in the presence or absence of novobiocin (100 or 400 µM). Hypoxic conditions were induced by the addition of CoCl2 (150 µM) for 8 hours. Then, 50 µM MG132 was added 3 hours before harvest. At 72 hours after HPC4-HIF1α transfection, cells were harvested and an HPC4 immunoprecipitation assay was performed by mixing 250 µl whole-cell lysate, 2 µg HPC4 antibody, and 15 µl of glutathione sepharose. Bound p300 was then detected by Western blotting with anti-p300 antibody.
Figure 3.
Novobiocin specifically inhibits the interaction between HIF1αCTAD and p300 CH1.
A. Cisplatin did not disrupt the HIF1α CTAD/p300 CH1 complex. A GST pull-down assay was performed by mixing GST fusion HIF1α CTAD, Flag-p300 CH1, and novobiocin (300 µM) or cisplatin (10–30 µM). HIF1α CTAD and p300 CH1 were detected by Western blotting with anti-GST and anti-Flag antibodies (upper panel). Quantified proteins are depicted (lower panel). B. Galdanamycin, an inhibitor of Hsp90, does not affect the interaction between HIF1α and p300 CH1. His pull-down was carried out by mixing purified proteins (combinations indicated in the figure). Bound p300 CH1 protein was detected by Western blotting with anti-Flag antibody. Western blot images were quantified using Quantity One software (lower panel). Percentages given above each bar represents the bound p300 CH1 protein compared to lane 1.
Figure 4.
Novobiocin directly binds to HIF1α CTAD.
A. Novobiocin-sepharose pulled down HIF1α CTAD. Immobilized novobiocin-sepharose was prepared as described under §Experimental Procedures. GST fusion HIF1α CTAD (∼5 µg) was mixed with novobiocin-sepharose or novobiocin-free sepharose in a buffer containing 40 mM Tris-HCl (pH 7.4), 1% NP-40, 2 mM EDTA, 100 mM NaCl, and 1 mM sodium orthovanadate, and a protease inhibitor cocktail (Sigma). Protein mixtures were incubated at 4°C for 2 hours, and the beads were washed with ice-cold buffer. Bound proteins were eluted by boiling in 4×SDS loading buffer and were separated by SDS-PAGE, followed by Western blotting with anti-GST antibody. B. Novobiocin specifically binds to HIF1α CTAD. Protein mixtures of GST-HIF1α CTAD, novobiocin-sepharose and free novobiocin from 0.25 to 8 mM were incubated at 4°C for 2 hours, and the beads were washed with the same ice-cold buffer. Bound proteins were confirmed by Western blotting with anti-GST antibody (upper panel). Quantified proteins are shown in the bar graph (lower panel). Percentages above each bar represents bound HIF1α 776 protein compared to lane 2. C. HIF1α CTAD and p300 CH1 cannot compete for binding to novobiocin. Pre-equiliblited novobiocin-sepharose (∼20 µl) was mixed with ∼5 µg GST-HIF1α CTAD and Flag-p300 CH1 (5, 10, and 15 µg) in combinations indicated in the figure. Bound proteins were detected by Western blotting with indicated antibodies.
Figure 5.
Novobiocin represses the transcriptional activity of HIF1α CTAD and down regulates HIF1α target genes.
A. Novobiocin inhibits the activation of GAL4-dependent luciferase reporter in 293T cells. 293T cells were co-transfected with luciferase reporter carrying GAL4-Luc and a Renilla luciferase plasmid (HIF1α DBD and HIF1α CTAD). After 24 hours transfection, cells were treated with 50, 100, and 200 µM novobiocin with or without CoCl2. After 48 hours transfection, dual luciferase activities were measured and firefly values were normalized with Renilla values. B. 293T cells were co-transfected with GAL4-Luc and VP16 plasmid. After 24 hours transfection, cells were treated with 50 and 100 µM novobiocin. C. Novobiocin down-regulates HIF1α target genes. A549 cells were cultured with or without 200 µM novobiocin for 24 hours in the presence or absence of CoCl2 (150 µM). Selective HIF1α target or non-target gene expressions were measured by RT-PCR (left panel). Western blot analysis was performed with indicated antibodies (right panel). D. Novobiocin affects mTOR gene expression in MCF-7 cells. MCF-7 cells were treated with 200 µM novobiocin in the presence or absence of CoCl2 (150 µM). Indicated gene expressions including Akt1and mTOR were measured by RT-PCR (upper panel). HIF1α protein is shown in the lower panel. E. mRNA was quantified by densitometry using Quantity One software (BioRad) (right panel). Error bars represent the standard error of the mean of 2∼3 independent experiments. The Student’s t-test was performed to compare difference between with or without novobiocin treatment (significance: *p<0.05 and **p<0.01).
Figure 6.
Inhibitory effects of novobiocin on cell proliferation and colony formation in A549 and MCF-7 cells.
A. Cell growth pattern of MCF-7. MCF-7 cells were transiently transfected with pcDNA3.1 0.8 µg (control) or HIF1α CTAD plasmid 0.8 µg. Later (24 hours), cells were seeded in 12-well plates and grown in medium with (200 µM) or without novobiocin for various days. Cell numbers were counted on a hemacytometer in triplicate each day of the culture, up to the seventh day. B. Cell growth numbers were not affected in HIF1α siRNA knockdown MCF-7 cells. MCF-7cells were transfected with 20 nM HIF1α siRNA and non-targeting siRNA (as control) and cultures were treated as described above. As an example, HIF1α protein at Day3 is shown in the bottom panel. C. Soft-agar formation assay. Three independent soft agar colony formation assays were performed. Colonies numbers produced by A549 and MCF-7 cells with/without 200 µM novobiocin are shown as bar graphs. D. HIF1α CTAD partially rescued the inhibitory effects of novobiocin on colony formation. MCF-7 cells were transiently transfected with pcDNA3.1 plasmid 0.8 µg (control) or HIF1α CTAD plasmid at 0.4 and 0.8 µg. Then (24 hours later), a colony formation assay was performed with or without 100 µM novobiocin. Error bars indicate SE; (significance: *p<0.05 and **p<0.01).
Figure 7.
Potential therapeutic model of novobiocin on cancer.
Under hypoxia, HIF1α dimerises with HIF1β, recruits transcriptional coactivator p300/CBP, and binds to the HRE, thereby leading to activation of hypoxically regulated genes such as VEGF and CA9 (upper panel). Novobiocin directly binds to HIF1α CTAD, inhibits the recruitment of transcriptional co-activator p300/CBP, leading to reduction of hypoxically regulated genes. Ultimately, this suppresses tumor well growth (lower panel).