Hypoxia-Activated Small Molecule-Induced Gene Expression

<div><div><div><p>Hypoxia, conditions of reduced oxygen, occur in a wide variety of biological contexts, including solid tumours and bacterial biofilms, which are relevant to human health. Consequently, the development of chemical tools to study hypoxia is vital. Here we report a hypoxia-activated small molecule-mediated gene expression system using a bioreductive prodrug of the inducer isopropyl 1-thio-β-D-galactopyranoside (IPTG). As a proof-of-concept we have placed the production of a green fluorescent protein under the control of hypoxia. Our system has the potential to be extended to regulate the production any given protein of choice.</p></div></div></div

Inhibition of HIF-1α expression by DNA damaging agents.

<p>(<b>A</b>) Western blot showing the protein levels of HIF-1α, phosphorylated ERK 1/2 (P-MAPK) and ERK 1/2 (MAPK) in HCT116 cells treated with 1.25 µM U0126 and 0.4 µg/ml doxorubicin, and cultured at 20% or 5% O₂ for 2 days. Cells treated with 500 µM CoCl₂ for 16 hours were used as a positive control for the induction of HIF-1α (H). (<b>B</b>) Western blot showing the protein levels of HIF-1α in HCT116 and HCT116 p53^−/− in lysates collected 24 hours after treatment with 1 µg/ml Actinomicyn D (ActD) or 200 µM tert-Butyl Hydroperoxyde (tBH) for 2hours (<b>C</b>) Western blot showing the protein levels of HIF-1α, p53 and p21 in HCT116 and MCF-7 cells treated with 0.4 µg/ml doxorubicin or 10 Gy γ-radiation for 24 hours at 20% or <1% O₂ (hypoxia).</p

The role of the HIF-1α transcription factor in increased cell division at physiological oxygen tensions

HIF-1 is a transcription factor that mediates the cellular responses to low oxygen environments, mainly as a result of having an oxygen-labile subunit, HIF-1α. HIF-1α has been carefully studied in the context of severe hypoxic stresses (<1% O[subscript 2]), but it is also known to be present at oxygen tensions commonly found in normal tissues in vivo (∼1-13% O[subscript 2]), albeit at much lower levels. Its role under these physiological conditions is not fully understood. Here, we show that a transcriptionally active HIF-1α was up-regulated at 5% O[subscript 2], both in normal and cancer cells, but only some of its target genes were elevated as a result. HIF-1α induction was in part dependent on the activation of the ERK1/2 MAPK signalling pathway, which we have previously shown is active at 5% O[subscript 2]. We also found that HIF-1α does not contribute to the protection against DNA damage that can be observed in low oxygen environments, and that there are certain DNA damaging agents, such as doxorubicin and actinomycin D, that prevent HIF-1α induction independently of p53. Moreover, absence of HIF-1α significantly reduced the growth advantage of cells cultured at 5% O[subscript 2]. In view of these data, we conclude that HIF-1α can be induced and activated at physiological oxygen tensions in a MAPK-dependent manner and that, although this does not lead to pro-survival responses to stress, it determines the increased cell proliferation rates that are common under these conditions

HIF-1α contributes to increased proliferation of cells at physiological oxygen tensions.

<p>(<b>A</b>) Western blot showing the protein levels of HIF-1α in HCT116 HIF^+/+ and HIF^−/−cultured either at 20% O₂ or under hypoxic stress (<0.1% O₂) for 16 hours. (<b>B</b>) Proliferation curves of HIF^+/+ and HIF^−/− HCT116 cells cultured at 20% or 5% O₂ from 2 to 8 days. Values represent ratio of cell numbers normalized to the initial seeded cells (10⁶). (<b>C</b>) Representative colony formation assay for HCT116 HIF^+/+ and HIF^−/−cultured at 20% or 5% O₂. 200 cells were seeded in each plate and 14 days later they were stained with Giemsa. Media was not changed during the process. (<b>D</b>) Percentage of EdU positive HCT116 HIF^+/+ and HIF^−/− cells as assessed by immunofluorescence (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097938#pone.0097938.s002" target="_blank">Figure S2</a>). Cells were incubated with EdU for 30 minutes in the corresponding oxygen tensions. Results represent means of two independent experiments. Two microscope fields were scored in each experiment. Error bars represent standard error. P value (unpaired t-test): 0.0127 (*), (<b>E</b>) Proposed model of the roles of HIF-1 at different oxygen concentrations.</p

HIF-1α has no effect on the activation of MAPK at physiological oxygen tensions.

<p>(<b>A</b>) Western blot showing the protein levels of HIF-1α, p53, phosphorylated ERK 1/2 (P-MAPK) and ERK 1/2 (MAPK) in HCT116 cells transfected with 200 pmol of siRNA against HIF-1α and treated with doxorubicin (0.4 µg/ml). Control cells were transfected with a luciferase siRNA instead. Cells treated with 500 µM CoCl₂ for 16 hours were used as a positive control for the induction of HIF-1α (H). (<b>B</b>) Western blot showing the protein levels of HIF-1α, phosphorylated ERK 1/2 (P-MAPK) and ERK 1/2 (MAPK) in HCT116 treated with 40 µM YC-1 and/or 0.4 µg/ml doxorubicin and cultured at 20% or 5% O₂ for 24 hours. Cells treated with 500 µM CoCl₂ for 16 hours were used as a positive control for the induction of HIF-1α (H). (<b>C</b>) Representative FACS plots of HCT116 cells stained with PI. Cells were transfected with 50 pmol of siRNA against HIF-1α and treated with 0.4 µg/ml doxorubicin. Cells were cultured at 20% or 5% O₂ for 24 hours. Percentages indicate number of subG₁ events (dead cells).</p

Chemical inhibition of MAPK reduces the activation of HIF-1α.

<p>Western blot showing the protein levels of HIF-1α and phosphorylated (active) ERK 1/2 MAPK in HCT116 cultured at 20% or 5% O₂for 12 to 48 hours, in the presence of 1.25 µM U0126. U0126 was added at the same time cells were transferred to 5%O₂. Total MAPK levels are provided as loading control.</p

Physiological oxygen tensions induce HIF-1α expression and activity.

<p>(<b>A</b>) Western blot showing the protein levels of HIF-1α in HCT116. Cells were cultured at 20% or 5% O₂ for 72 hours. (<b>B</b>) Western blot showing the protein levels of HIF-1α in normal human keratinocytes cultured for 1 to 4 days at 5% O₂ or treated 500 µM of chemical hypoximimetic CoCl₂ for 16 hours (H). (<b>C</b>) Luciferase assay showing HIF-1α in HCT116 cells. HCT116 were transfected with a PGK-1 luciferase reporter plasmid and a β-galactosidase control plasmid and then cultured for 48 hours at 5% O₂. β-galactosidase activity was used to normalize luciferase activity. Luciferase activity is expressed as a ratio to 20% O₂ levels. Results show mean values of 3 independent experiments and error bars represent standard deviation (<b>D</b>) qRT-PCR showing mRNA levels of Glut-1, PGK-1 and VEGF in HCT116 cells cultured at 20% and 5% O₂ for 24 hours. Results show mean values of 3 independent experiments and error bars represent standard deviation. P values (unpaired t-tests): 0.03 (*), 0.001 (**), 0.7 (ns). (E) Western blot of lysates of HCT116 cultured at 20% or 5% O₂ for 72 hours, showing expression of Glut-1 and PHD2.</p

CH-01 is a Hypoxia-Activated Prodrug That Sensitizes Cells to Hypoxia/Reoxygenation Through Inhibition of Chk1 and Aurora A

The increased resistance of hypoxic cells to all forms of cancer therapy presents a major barrier to the successful treatment of most solid tumors. Inhibition of the essential kinase Checkpoint kinase 1 (Chk1) has been described as a promising cancer therapy for tumors with high levels of hypoxia-induced replication stress. However, as inhibition of Chk1 affects normal replication and induces DNA damage, these agents also have the potential to induce genomic instability and contribute to tumorigenesis. To overcome this problem, we have developed a bioreductive prodrug, which functions as a Chk1/Aurora A inhibitor specifically in hypoxic conditions. To achieve this activity, a key functionality on the Chk1 inhibitor (CH-01) is masked by a bioreductive group, rendering the compound inactive as a Chk1/Aurora A inhibitor. Reduction of the bioreductive group nitro moiety, under hypoxic conditions, reveals an electron-donating substituent that leads to fragmentation of the molecule, affording the active inhibitor. Most importantly, we show a significant loss of viability in cancer cell lines exposed to hypoxia in the presence of CH-01. This novel approach targets the most aggressive and therapy-resistant tumor fraction while protecting normal tissue from therapy-induced genomic instability

Optimization of 3,5-Dimethylisoxazole Derivatives as Potent Bromodomain Ligands

The bromodomain protein module, which binds to acetylated lysine, is emerging as an important epigenetic therapeutic target. We report the structure-guided optimization of 3,5-dimethylisoxazole derivatives to develop potent inhibitors of the BET (bromodomain and extra terminal domain) bromodomain family with good ligand efficiency. X-ray crystal structures of the most potent compounds reveal key interactions required for high affinity at BRD4(1). Cellular studies demonstrate that the phenol and acetate derivatives of the lead compounds showed strong antiproliferative effects on MV4;11 acute myeloid leukemia cells, as shown for other BET bromodomain inhibitors and genetic BRD4 knockdown, whereas the reported compounds showed no general cytotoxicity in other cancer cell lines tested