Figure 1.
Effect of various HIF inhibitory compounds.
(a) Hypoxia-dependent HIF-1α transcriptional activity was measured using HRE-dependent reporter assays as described under Materials and Methods. HEK293 cells were incubated at 1% O2 for 6 hours in the presence of the various inhibitors as indicated: mitomycin C (MC), 10 µg/ml; YC-1, 50 µM; S-trans,trans-farnesylthiosalicylic acid (FTS), 70 µM; SB203580, 20 µM; metavanadate, 50 µM; camptothecin (CPT), 2 µM; geldanamycin (GA), 10 µM. (a) Luminescence was measured and fold stimulation was obtained by normalizing the relative luciferase activity of cells cultured under hypoxic conditions to those of nontreated cells cultured under normoxic conditions. Results represent mean ± SEM of three independent experiments. Student's t test analysis of the ratio of fold stimulation obtained for cells transfected with pGL3-HRE to those transfected with the promoter-less pGL3 empty vector showed that treatment with MC, YC-1, CPT and GA significantly (p<0.05, denoted as an asterisk *, n = 3) reduced luciferase activity compared to untreated control. (b) HEK293 cells were transfected for two days with wild type or kinase-dead (K85R) GSK-3β expression plasmids, or treated with 20 µM SB-415286, 5 µM SB-216763, or 30 mM LiCl for 6 hours. In the upper panel, the cells were incubated at 1% O2 during the drug exposure. Cytosolic β-catenin concentrations were measured by lysing cells in hypotonic lysis buffer as previously described [26] In the bottom panel, 200 µM desferrioxamine (DFO) was used as a positive control for normoxic HIF-1α induction and a HIF-1α reactive non-specific (NS) band was used to demonstrate equal protein loading. (c,d) To study the effects of mitomycin C, YC-1, camptothecin and geldanamycin on HIF transcriptional activity in more detail, firefly and renilla luciferase activity of cells cotransfected with pGL3-HRE and pRL-CMV and incubated under hypoxic condition in the presence of MC, YC-1, CPT and GA (as in Fig. 1a) was measured. The renilla and firefly luciferase activities are shown in (c), the ratio of firefly to renilla luciferase activity in (d). The results are expressed as fold stimulation relative to the activity in nontreated cells under normoxic conditions. Statistical significance (p<0.05, n = 3) is indicated with an asterisk in the bottom panel.
Figure 2.
The effect of the mitomycin C, camptothecin and YC-1 is due to inhibition of HIF-1α protein synthesis.
(a–b) HEK293 cells were incubated for 10 hours in the presence of YC-1 (50 µM), mitomycin C (10 µg/ml), camptothecin (2 µM), and geldanamycin (10 µM) and cell lysates subjected to Western blotting using the indicated antibodies. Cells were incubated at 1% oxygen during the last three hours of inhibitor treatment (upper panel) or in the presence of 200 µM desferrioxamine (DFO) during the inhibitor treatment for 10 hours (middle panel), as indicated. In the bottom panel, stably transfected cells with tetracycline inducible expression of dnUbc12 (5) were treated with 1 µg/ml tetracycline to block VHL mediated HIF-1α degradation for 9 hours and cotreated with the indicated drugs for the last 5 hours, followed by Western blotting. The presence of drugs during the induction time is indicated. (b) HIF-1α mRNA concentrations after inhibitor treatment for 10 to 12 hours were measured as described under Material and Methods. A representative autoradiogram is shown in the top panel. The bottom panel shows the average mRNA levels for each inhibitor treatment expressed relative to those of DMSO control, as determined by densitometry of autoradiograms obtained from three independent experiments. Statistical analysis using one-way ANOVA test across all treatment groups showed that all inhibitors exerted an insignificant (p>0.05, n = 3) effect. (c) Cells were treated with 200 µM desferrioxamine and the indicated drugs in the absence or presence of 5 µM actinomycin D for 8 hours, followed by Western blotting. (d) HIF-1α protein accumulation was measured by Western blotting after cotreatment of cells with 25 µM MG-132 (added to cells for the last 4 hours) and the various inhibitors as indicated (added to cells for the last 9 hours). (e) To confirm the specificity of the effect of mitomycin C, YC-1 and camptothecin, Western blotting for the indicated proteins was carried out after drug treatment of cells for 11 hours and incubation at 1% oxygen during the last 4 hours, as indicated. Inhibitor concentrations in (b–e) were as in (a).
Figure 3.
Inhibition of HIF-1α protein accumulation by mitomycin C is not due to an effect on protein degradation.
(a) HEK293 cells were treated with 200 µM desferrioxamine (DFO) and 10 µg/ml mitomycin C for 10.5 hours, as indicated, in the absence or presence of the 26S proteasome inhibitors epoxomicin (1 µM) or lactacystin (5 µM). (b) The cells were pretreated with 200 µM desferrioxamine for three hours, followed by addition of 10 µg/ml mitomycin and 40 µM cycloheximide for 8 hours.
Figure 4.
The effect of mitomycin C, camptothecin and etoposide on HIF-1α protein concentrations in different cell lines.
The indicated cell types were treated with 200 µM desferrioxamine, 10 µg/ml mitomycin, 2 µM camptothecin and 50 µM etoposide, as specified, for 10 to 14 hours.
Figure 5.
Inhibition of HIF-1α by mitomycin C is independent of p53 and DNA damage-induced apoptosis.
(a) HEK293 cells were transfected with negative control or p53 siRNA for three days, followed by treatement with 200 µM desferrioxamine and 10 µg/ml mitomycin C for 10 hours and Western blotting of cell lysates with the indicated antibodies. (b,c) MCF7 cells were treated with 200 µM desferrioxamine and 10 µg/ml mitomycin C for 14 hours as indicated, followed by Western blotting of cell lysates. In (b) pifithrin-α (25 µM) was included, as indicated. In (c) MCF7 cells were retrovirally transduced with Bcl-xL or empty vector, as indicated. (d) Brca1 was knocked down using siRNA for three days, as described under Materials and Methods, followed by drug treatment (200 µM desferrioxamine, 10 µg/ml mitomycin, 2 µM camptothecin), as indicated.
Figure 6.
Mitomycin C and camptothecin mediated inhibition of HIF-1α protein synthesis is independent of the HIF-1α 5′UTR.
(a) HEK293 cells were transfected with HIF-1α-5′UTR-EGFP-HA plasmid for two days and then treated with 10 µg/ml mitomycin, 2 µM camptothecin, followed by Western blotting with HA antibody. (b,c) Cells were transfected with the indicated plasmids and treated with the indicated drugs. Myxothiazol, a mitochondrial complex III inhibitor and well established inhibitor of HIF-1α served as positive control in (b).
Figure 7.
The role of eIF2α phosphorylation in the mechanism of action of HIF-1α inhibitors.
(a) HEK293 cells treated with 10 µg/ml mitomycin or 2 µM camptothecin for 10 hours followed by Western blotting with antibodies specific for Ser51 phosphorylated and total eIF2α. (b–d) Cells were transfected with empty vector (control) or FLAG-GADD34 for two days followed by treatment with the indicated drugs: 1 µM thapsigargin, 200 µM desferrioxamine, 10 µg/ml mitomycin, 2 µM camptothecin, 1 µM NSC-652287. Treatment times were 10 hours. Western blotting of cell lysates was performed using the indicated antibodies.
Figure 8.
Inhibition of Cdk activity reverses HIF-1α inhibition by camptothecin.
(a) HEK293 cells were treated with 2 µM camptothecin and 50 µM DRB in hypoxia for 6 hours The lysates were used for Western blotting using HIF-1α antibody. A non-specific band detected with the HIF-1α antibody served as loading control. (b,c) As in (a), the drug used concentrations were CoCl2 (200 µM), mitomycin C (10 µg/ml), NSC-652287 (1 µM).