1, 9 PA downregulates HIF-1α independent of its JNK inhibitory function.

<p>(A) Temporal order and correlation of 1, 9 PA-induced HIF-1α downregulation and JNK inhibition, and the treatment-induced compensatory activation of Akt and Erk. A431 cells were treated with 5 µM 1, 9 PA in 0.5% FBS culture medium for the indicated time intervals. Cell lysates were then prepared for Western blotting with the antibodies shown. (B) Dose-dependent effects of 1, 9 PA on downregulating HIF-1α, inhibiting JNK, and activating Erk. A431 cells were exposed to increasing concentrations of 1, 9 PA for 16 h in 0.5% FBS culture medium at 37°C. Cell lysates were then prepared for Western blotting with the antibodies shown. (C) Independence of 1, 9 PA-induced HIF-1α downregulation from its JNK inhibitory function. A431 cells were treated with increasing concentrations of 1, 9 PA or 1-methyl-1, 9 PA for 1 h in 0.5% FBS culture medium. Cell lysates were then prepared for Western blotting with the antibodies shown. (D) No changes in HIF-1α level after activation or inhibition of JNK. A431 cells wert transiently transfected with a control vector or one of the constructs containing a constitutively active SEK1 S220E/T224D mutant (SEK1-CA), a dominant-negative SEK1 K129R mutant (SEK1-DN), or a dominant-negative MEKK1 K432M mutant (MEKK1-DN) overnight. Cell lysates were then prepared for Western blotting with the antibodies shown. (E) Compensatory activation of Erk after HIF-1α silencing in comparison with treatment by 1, 9 PA or 1-methyl-1, 9 PA. A431 cells were subjected to knockdown of HIF-1α with specific or control siRNA, or treated with 10 µM 1, 9 PA or 1-methyl-1, 9 PA for 16 h. Cell lysates were then prepared for Western blotting with the antibodies shown. (F) Downregulation of HIF-1α by 1, 9 PA in various cancer cell lines. Indicated cell lines were exposed to 10 or 40 µM 1, 9 PA for 1 h in 0.5% FBS culture medium. Cell lysates were then prepared for Western blotting with the antibodies shown.</p

1, 9 PA enhances HIF-1α ubiquitination.

<p>(A). A431 cells were treated with 10 µM 1, 9 PA for indicated time period. Cell lysates were prepared for HIF-1α immunoprecipitation, followed by Western blotting of the immunoprecipitates with antibodies directed against ubiquitin (top), HIF-1α, and β-actin. (B) A431 cells were untreated or treated with 10 µM 1, 9 PA in the presence of 10 µM MG132 for 1 h in 0.5% FBS medium. HIF-1α was immunoprecipitated followed by Western blot analysis with an anti-HIF-1α antibody.</p

Dose- and time-dependent responses of wild-type EGFR and tyrosine kinase domain-mutated EGFR cells to cetuximab and gefitinib treatment

<p><b>Copyright information:</b></p><p>Taken from "Responses of cancer cells with wild-type or tyrosine kinase domain-mutated epidermal growth factor receptor (EGFR) to EGFR-targeted therapy are linked to downregulation of hypoxia-inducible factor-1α"</p><p>http://www.molecular-cancer.com/content/6/1/63</p><p>Molecular Cancer 2007;6():63-63.</p><p>Published online 11 Oct 2007</p><p>PMCID:PMC2117021.</p><p></p> () Absolute cell numbers in each treatment group (control [DMSO]), 5 nM cetuximab, and 0.5 μM gefitinib, all in 0.5% FBS medium) were plotted against the duration of treatment. () The inhibition of cell proliferation after treatment with cetuximab was measured by an MTT assay and is shown as a percentage of the optical density value of control cells (untreated) for each concentration tested. () The inhibition of cell proliferation after treatment with gefitinib was measured as in () and is shown as a percentage of the optical density value of vehicle-treated cells (DMSO) for each concentration tested. Results are shown as the mean of five independent measurements, plus or minus the standard deviation (SD). The magnitude of some SDs was smaller than the symbol size; thus some bars do not appear in the figure

Effects of cetuximab and gefitinib treatment on the levels of total and phosphorylated EGFR and EGFR substrates in cell lines with wild-type and mutated EGFR

<p><b>Copyright information:</b></p><p>Taken from "Responses of cancer cells with wild-type or tyrosine kinase domain-mutated epidermal growth factor receptor (EGFR) to EGFR-targeted therapy are linked to downregulation of hypoxia-inducible factor-1α"</p><p>http://www.molecular-cancer.com/content/6/1/63</p><p>Molecular Cancer 2007;6():63-63.</p><p>Published online 11 Oct 2007</p><p>PMCID:PMC2117021.</p><p></p> Cells from the indicated lines were simultaneously switched to a culture medium containing 0.5% FBS and were either left untreated or treated with cetuximab (2 and 10 nM), vehicle, or gefitinib (0.1 and 0.5 μM) overnight (16 hours). A master medium containing either cetuximab or gefitinib was used in all cell lines. After treatment, the cells were lysed, and equal amounts of cell lysates were subjected to Western blot analysis using antibodies directed against total and phosphorylated EGFR (Y-1068) () and antibodies directed against phosphorylated ERK, Akt, and STAT3 as indicated (). The levels of β-actin and total ERK served as internal controls for equal protein loading in each lane in () and (), respectively. The numeric values under each gel were derived from a densitometric analysis of the signals

Expression of HIF-1α/ΔODD mutant leads to cellular resistance to cetuximab without affecting cellular sensitivity to cetuximab-induced inhibition of EGFR signaling

<p><b>Copyright information:</b></p><p>Taken from "Responses of cancer cells with wild-type or tyrosine kinase domain-mutated epidermal growth factor receptor (EGFR) to EGFR-targeted therapy are linked to downregulation of hypoxia-inducible factor-1α"</p><p>http://www.molecular-cancer.com/content/6/1/63</p><p>Molecular Cancer 2007;6():63-63.</p><p>Published online 11 Oct 2007</p><p>PMCID:PMC2117021.</p><p></p> () A431neo and A431/HIF-1α/ΔODD cells were treated as indicated for 16 hours (overnight). Cell lysates were prepared and subjected to Western blot analysis with the indicated antibodies. () Cells were left untreated or treated with the indicated concentrations of cetuximab in culture with 0.5% FBS for 5 days. Relative cell numbers were measured by the MTT assay and are presented as a percentage of the untreated control. () A431neo and A431/HIF-1α/ΔODD cells (300 cells/dish) were cultured, with or without cetuximab (2 nM), for 9 days. After treatment, cells were fixed and the colonies were counted, as described in the Methods section

Downregulation of HIF-1α protein levels in cell lines with wild-type or mutated EGFR after treatment with cetuximab or gefitinib

<p><b>Copyright information:</b></p><p>Taken from "Responses of cancer cells with wild-type or tyrosine kinase domain-mutated epidermal growth factor receptor (EGFR) to EGFR-targeted therapy are linked to downregulation of hypoxia-inducible factor-1α"</p><p>http://www.molecular-cancer.com/content/6/1/63</p><p>Molecular Cancer 2007;6():63-63.</p><p>Published online 11 Oct 2007</p><p>PMCID:PMC2117021.</p><p></p> Cells from the indicated cell lines were treated with cetuximab or gefitinib overnight as described in Figure 4. After treatment, the cells were lysed, and equal amounts of cell lysates were subjected to Western blot analysis using antibodies directed against HIF-1α, as indicated. The level of β-actin served as the internal control for equal protein loading in each lane. The numeric values shown under each gel were derived from a densitometric analysis of the signals

Combination of 1, 9 PA and cetuximab induces apoptosis through downregulation of HIF-1α.

<p>(A) Induction of PARP cleavage by the combination of 1, 9 PA and cetuximab. A431, HN5, and DiFi cells were untreated or treated with cetuximab (10 nM for A431 and HN5 cells and 2 nM for DiFi cells for 16 h), 1, 9 PA (10 µM or 40 µM added the last hour before cell lysis), or both in 0.5% FBS culture medium. Cell lysates were prepared and analyzed by Western blotting with the antibodies shown. (B) Increased induction of apoptosis by the combination of 1, 9 PA and cetuximab. A431 cells were treated as described in (A). Cell lysates were prepared and analyzed by apoptosis ELISA. The relative absorbance values are plotted. The <i>p</i> value was <0.01 when comparing the level of apoptosis by 1, 9 PA alone (10 or 40 µM) or cetuximab alone with that of apoptosis by combination of the 2 agents (note: only the <i>p</i> values comparing 10 µM 1, 9 PA alone and in combination with cetuximab are shown). (C) Dependence of induction of apoptosis by the combination of 1, 9 PA and cetuximab on HIF-1α downregulation. A431neo and A431/HIF-1α-ΔODD cells were treated as indicated, and the cell lysates were prepared and analyzed as described in (A).</p

1, 9 PA enhances responses of cancer cells expressing an oncogenic Ras mutant to cetuximab.

<p>(A) Effect of 1, 9 PA and cetuximab, either alone or in combination, on the HIF-1α level and induction of apoptosis. A431neo and A431/RasG12V cells were untreated or treated with cetuximab (10 nM for 16 h), 10 µM 1, 9 PA (added the last hour before cell lysis), or both in 0.5% FBS culture medium. Cell lysates were prepared and analyzed by Western blotting with the antibodies shown. (B) 1, 9 PA-mediated sensitization to cetuximab-induced growth inhibition. A431neo and A431/RasG12V cells were treated with increasing concentrations of cetuximab ±5 µM 1, 9 PA in 0.5% FBS culture medium for 5 days. After treatment, the cells were subjected to an MTT assay. The optical density values of the treated groups were normalized to the values of the control groups (with or without 1, 9 PA treatment) and expressed as a percentage of respective control. The percentage of surviving cells was plotted as a function of treatment with increasing concentrations of cetuximab. The differences in cell survival between the two groups were statistically significant (<i>p</i><0.01) when the concentrations of cetuximab were greater than 0.625 nM in A431neo cells and 1.25 nM in A431/RasG12V cells. (C) 1, 9 PA-mediated sensitization to cetuximab-induced inhibition of VEGF production. A431neo and A431/RasG12V cells were untreated or treated with 10 nM cetuximab, 10 µM 1, 9 PA, or both in 0.5% FBS culture medium for 16 h. The VEGF secreted into the conditioned media by the cells was measured by ELISA. The <i>p</i>-values for indicated comparisons were shown. (D) Comparison of A431 and GEO cells to treatment with 1, 9 PA and cetuximab, either alone or in combination. A431 and GEO cells were treated as described in (A). Cell lysates were prepared and analyzed by Western blotting with the antibodies shown. (E). 1, 9 PA-mediated sensitization GEO cells to cetuximab-induced growth inhibition. GEO cells were treated with increasing concentrations of cetuximab ±5 µM 1, 9 PA in 0.5% FBS culture medium for 5 days. After treatment, the cells were subjected to an MTT assay. The data were processed as described in (B). The differences in cell survival between the two groups were statistically significant (<i>p</i><0.01) at all concentrations of cetuximab tested.</p

Induction of apoptosis in cancer cells with wild-type EGFR or tyrosine kinase domain-mutated EGFR by cetuximab and gefitinib

<p><b>Copyright information:</b></p><p>Taken from "Responses of cancer cells with wild-type or tyrosine kinase domain-mutated epidermal growth factor receptor (EGFR) to EGFR-targeted therapy are linked to downregulation of hypoxia-inducible factor-1α"</p><p>http://www.molecular-cancer.com/content/6/1/63</p><p>Molecular Cancer 2007;6():63-63.</p><p>Published online 11 Oct 2007</p><p>PMCID:PMC2117021.</p><p></p> Cells from each line were left untreated or were treated with vehicle (DMSO), 5 nM cetuximab, or 0.5 μM gefitinib in a medium containing 0.5% FBS. After 16 hours of treatment, the cells were harvested and lysed for quantitative apoptosis measurement by () an enzyme-linked immunosorbent assay, as described in the Methods section, and () Western blot analysis with anti-PARP antibodies. *P < 0.05, **P < 0.01 compared with corresponding controls

Cancer cell lines with wild-type or tyrosine kinase domain-mutated EGFR

<p><b>Copyright information:</b></p><p>Taken from "Responses of cancer cells with wild-type or tyrosine kinase domain-mutated epidermal growth factor receptor (EGFR) to EGFR-targeted therapy are linked to downregulation of hypoxia-inducible factor-1α"</p><p>http://www.molecular-cancer.com/content/6/1/63</p><p>Molecular Cancer 2007;6():63-63.</p><p>Published online 11 Oct 2007</p><p>PMCID:PMC2117021.</p><p></p> () PCR fragments from the indicated cell lines were compared with the wild-type sequence of EGFR. The black arrows indicate the codons E746 to A750, which are present in the EGFR in DiFi cells but have been deleted in HCC827 and HCC2279 cells. Codon L858R substitution in H1975 and H3255 cells is indicated by arrows. () Lysates from the indicated cell lines maintained in regular culture medium were prepared for Western blot analysis using antibodies directed against EGFR, HER2, and HER3, and antibodies directed against total and activation-specific phosphorylated downstream signaling molecules (ERK, Akt, and STAT3). The level of β-actin was used as a reference of lysate protein loading control of each cell line