Validation of the GFP-P564 and Ca9-GFP hypoxia-inducible reporters.

<p>Hypoxic stimuli induced HIF-1 alpha accumulation in MDA-MB-231 cells and increased fluorescence in the GFP-P564 and Ca9-GFP cell models. Cells were treated with 200 µM cobalt (Co), 200 µM DFO, or 1 mM dimethyloxalylglycine (DMOG) or exposed to 1% O₂ for 24 H (hx). Untreated cells were incubated under normoxic conditions (nx) and used as controls. (A) Fluorescence was evaluated by flow cytometry, and the signal significantly increased (p<0.05) under hypoxic conditions. DFO induced (B) time- and (C) dose-dependent fluorescence, as assessed by flow cytometry in GFP-P564 and Ca9-GFP cells. (D) Total HIF-1 alpha was quantified in whole MDA-MB-231 cells using an ELISA assay. (E) HIF-1 alpha protein levels in MDA-MB-231 cells were analyzed by western blot. (F) HIF-1 alpha DNA-binding activity was analyzed in MDA-MB-231 nuclear extracts using a 96-well ELISA assay; the absorbance was assessed at 450 nm. Each well was coated with a dsDNA sequence containing the HIF-1 alpha response element. The data represent three independent experiments (average mean ± SEM).</p

Effect of anti-EGFR-targeted therapies on MDA-MB-231 cell migration and viability.

<p>(A) Cells were treated with 75 µg/mL cetuximab (cx), 10 µg/mL lapatinib (lp) or 10 µg/mL gefitinib (gf) and allowed to migrate through inserts (8 µm) under normoxia and hypoxia (1% O2) for 24 H. Cell migration is expressed as the percentage of unmigrated cells. Only gefitinib inhibited MDA-MB-231 cell migration under both conditions. (B) Cells were treated with increasing concentrations of cetuximab (C0:0, C1:25, C2:75, C3:100, C4:150 µg/mL), lapatinib and gefitinib (C0:0, C1:3, C2:10, C3:20, C4:40 µg/mL) under normoxic conditions. Viability was assessed using green calcein-AM labeling and fluorometry (ex: 490/em: 520 nm). Cetuximab and lapatinib did not affect cell viability, whereas gefitinib induced mortality in a dose-dependent manner. (C) A viability assay was performed in a 96-well plate by measuring drug effects under hypoxia in a CMV-GFP cell model that constitutively expressed EGFP. The fluorescence of each well was measured by fluorometry (ex: 488/em: 507 nm), and the signal intensity was proportional to the number of viable cells. Gefitinib strongly induced mortality. The results are representative of at least three independent experiments. Statistical significance was determined by unpaired t-test between treated cells and controls (* p<0.05).</p

Effect of EGFR inhibitors on hypoxia-induced responses in GFP-P564 and Ca9-GFP cells.

<p>Cells were treated with 75 µg/mL cetuximab (cx), 10 µg/mL lapatinib (lp) or 10 µg/mL gefitinib (gf) under hypoxic conditions and stimulated with 200 µM DFO, 5 µM MG132 or 1 mM DMOG or exposed to 1% O₂ (hx) for 24 H. Fluorescence intensity was assessed by flow cytometry. As controls (ct), cells were incubated under normoxic conditions and left untreated. (A) Cetuximab and lapatinib did not affect the induction of fluorescence by DFO, whereas gefitinib suppressed it in both models. (B-C) Gefitinib also inhibited the induction of fluorescence in GFP-P564 and Ca9-GFP cells in the presence of DMOG and under hypoxia. (D) MG132 is a proteasome inhibitor that induces the stabilization of HIF-1 alpha. Gefitinib diminished the MG132-mediated stabilization of HIF-1 alpha. (E) Cetuximab, lapatinib and gefitinib did not affect the constitutive fluorescence of MDA-MB-231 cells transfected with pEGFP-C1 (CMV-GFP model). Data are shown as the mean ± SEM (n = 3). Statistical differences were assessed using unpaired t-tests; p<0.05 was considered to be significant (* p<0.05).</p

Effect of EGFR inhibitors on HIF-1 alpha in MDA-MB-231 cells under hypoxic conditions.

<p>Cells were treated with 75 µg/mL cetuximab (cx), 10 µg/mL lapatinib (lp) or 10 µg/mL gefitinib (gf) under normoxia or hypoxia and stimulated with DFO or MG132 for 24 H. Controls (ct) were left untreated. (A) Total HIF-1 alpha was detected with a whole-cell ELISA assay. Only gefitinib prevented HIF-1 alpha induction by DFO or hypoxia. (B) HIF-1 alpha levels were quantified in nuclear extracts with a DNA-binding assay. Gefitinib reduced the HIF-1 alpha nuclear accumulation induced by hypoxia and MG132. The results are from three independent experiments, and the bars represent the SEM. Student's t-tests were performed to compare the drug responses according to the hypoxia conditions (* p<0.05).</p

Flow cytometric analysis of EGFP fluorescence in GFP-P564 and Ca9-GFP cells.

<p>Cells were treated with 250 µM desferioxamine (DFO) for 24 H or left untreated. R1 is the region of fluorescence induced by DFO. The percentage of cells in R1 and the mean fluorescence increased in response to hypoxic conditions.</p