Structures of the chelators used and microarray data showing that DFO and Dp44mT significantly alter the expression of a large number of genes involved in the MAPK signaling transduction pathway. A, chemical structures of DFO and Dp44mT. B, functional pathway annotation of genes significantly altered (p < 0.05) by DFO (250 μm) or Dp44mT (25 μm) after a 24 h/37 °C incubation of DMS-53 lung cancer cells as assessed by a human genome gene array (Affymetrix U133 plus 2.0 array). Only genes with a greater than log₂ value of 1.0 as compared with the control were included in the analysis. Genes in the category of cellular processes (C) and environmental information processing (D) were further sub-categorized according to their functions. E, various signaling transduction pathways affected. Lists of genes were assessed using DAVID. Pathway annotation was obtained through the public data base, KEGG pathway mapping.

DFO and Dp44mT increase the expression of MAPK phosphatases and the phosphorylation of JNK and p38. mRNA expression of MAPK phosphatases, dual specificity phosphatases (DUSP) (identified from the microarray) (A), and MAPKs, ERK1/2, JNK, and p38 (B) in DMS-53 cells after incubation with DFO (250 μm) or Dp44mT (25 μm) over 24 h/37 °C. C–E, effect of DFO (2.5 and 250 μm) or Dp44mT (0.25 and 25 μm) after a 24 h/37 °C incubation of DMS-53 cells on the protein (phosphorylated and nonphosphorylated) expression of MAPKs as follows. C, ERK1/2 (phospho-Thr-202/Tyr-204); D, JNK (phospho-Thr-183/Tyr-185); and E, p38 (phospho-Thr-180/Tyr-182). Experiments were performed using Western blotting implementing rabbit anti-human phospho-specific and nonphospho-specific antibodies for ERK1/2, JNK, or p38. Sorbitol (Sor; 500 mm) and anisomycin (Ani; 10 μg/ml) were included as positive controls. It should be noted that semi-quantitative RT-PCR may not necessary reflect direct fold change in gene expression but was used to validate the up- and down-regulated expression observed in the microarray. Densitometry of phospho- and nonphospho-JNK includes both bands. RT-PCR and Western blot panels are typical experiments from three performed, and the densitometry is the mean ± S.E. of at least three experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Effect of chelator-iron complexes on the phosphorylation of JNK and p38. The expression of JNK (phospho-Thr-183/Tyr-185) (A) and p38 (phospho-Thr-180/Tyr-182) (B) in DMS-53 cells after incubation with either chelator alone (250 μm DFO or 25 μm Dp44mT) or chelator-iron(III) complexes at the same concentrations for 24 h/37 °C. The hexadentate DFO-iron complex was examined at a 1:1 molar ratio (chelator-iron(III) ratio; 250 μm). Tridentate Dp44mT was assessed at 1:1 molar ratio (chelator/iron(III) ratio; 25 μm) or 2:1 molar ratio (chelator/iron(III) ratio; 25:12.5 μm). The iron(III) complexes were pre-formed using ferric chloride (FeCl₃). FeCl₃ at 12.5, 25, or 250 μm was included as a relevant control as it is a component of the complexes. Western blotting was performed using rabbit anti-human phospho- and nonphospho-specific antibodies for JNK or p38. Densitometry of phospho- and nonphospho-JNK includes both bands. The panels and the densitometric analysis are a typical experiment from three performed or mean ± S.E. of at least three experiments, respectively. **, p < 0.01.

Effect of DFO and Dp44mT on phospho-ASK1 and ASK1-Trx complex. A, expression of phospho-ASK1 (Ser-83) and ASK1 in DMS-53 cells after incubation with either chelator alone (250 μm DFO or 25 μm Dp44mT) or chelator-iron(III) complexes at 1:1 (chelator/iron) molar ratio as determined by Western blotting using phospho- and nonphospho-specific antibodies for ASK1. B, expression of Trx in DMS-53 cells after incubation as in A. C, co-immunoprecipitation (Co-IP) examining the ASK1-Trx interaction as determined in DMS-53 cells after incubation as in A using mouse anti-human Trx antibody. The levels of ASK1 and Trx in the immunoprecipitates (IP) were determined using rabbit anti-human ASK1 and Trx antibodies. The panels and the densitometric analysis are either a typical experiment from three performed or the mean ± S.E. of at least three experiments, respectively. **, p < 0.01; ***, p < 0.001. WB, Western blot. D, schematic summary of the effect of iron chelation on the MAPK signaling transduction pathway. The chelators, DFO and Dp44mT increase phosphorylation of ASK1 because of the dissociation of the ASK1-Trx complex that is caused by the oxidation of Trx.⁴ This results in elevation of phospho-ASK1, which can modulate signaling cascades to increase phosphorylation of JNK and p38. A number of dual specificity phosphatases (DUSPs), including DUSP1, DUSP10, and DUSP16, which directly de-phosphorylate JNK and p38, were also increased by the chelators. This latter effect could be a counteractive response to the increased phosphorylation of JNK and p38. Hyperphosphorylation of p53 at multiple sites was also prominent as it is directly targeted by the JNK and p38 pathways. JNK, p38, ASK1, and p53 are pro-apoptotic molecules, and their increased phosphorylation could serve as important signals leading to apoptosis after iron depletion mediated by chelators.

Validation of MAPK pathway genes identified in the microarray. RT-PCR confirmation of genes that are part of the MAPK pathway from the microarray analysis as described in Fig. 1 and detailed in Table 2. Shown are the genes that were up-regulated (A) or down-regulated (B) by either DFO (250 μm) or Dp44mT (25 μm) after a 24 h/37 °C incubation with DMS-53 cells. It should be noted that semi-quantitative RT-PCR may not necessarily reflect direct fold change in gene expression, but it was used to validate the up- and down-regulated expression observed in the microarray. RT-PCR panels and densitometric analysis are from a typical experiment of three performed or the mean ± S.E. of at least three independent experiments, respectively. *, p < 0.05; ***, p < 0.001.

Iron chelators increase phosphorylation of p53 and ATF2 that are downstream targets of JNK and p38 kinases. A, p53 at multiple phosphorylation sites; B, ATF2 (Thr-71) that was significantly increased upon incubation of DMS-53 cells with DFO (250 μm) or Dp44mT (25 μm) over 24 h/37 °C. These targets were identified using the phospho-MAPK antibody array (Table 3) and validated here by Western blotting. The panels and the densitometric analysis are typical experiments from three performed or mean ± S.E. of at least three experiments, respectively. *, p < 0.05; **, p < 0.01; ***, p < 0.001. HU, hydroxyurea; Ani, anisomycin.

Effect of iron or NAC supplementation on the DFO- or Dp44mT-induced phosphorylation of JNK and p38. The expression of JNK (phospho-Thr-183/Tyr-185) (A) and p38 (phospho-Thr-180/Tyr-182) (B) after DMS-53 cells were incubated (i.e. first incubation) with either DFO (250 μm) or Dp44mT (25 μm) for 24 h/37 °C and then re-incubated (i.e. second incubation) with either ferric ammonium citrate (100 μg/ml), DFO (250 μm), or Dp44mT (25 μm) for another 24 h/37 °C. The expressions of JNK (phospho-Thr-183/Tyr-185) (C) and p38 (phospho-Thr-180/Tyr-182) (D) after DMS-53 cells were incubated the same way as in A but using NAC (5 mm) in the second incubation rather than FAC. Western blotting was performed using anti-rabbit phospho- and nonphospho-specific antibodies for JNK or p38. Densitometry of phospho- and nonphospho-JNK includes both bands. The panels and the densitometric analysis are either a typical experiment from three performed or the mean ± S.E. of at least three experiments, respectively. **, p < 0.01.