Cellular iron depletion stimulates the JNK and p38 MAPK signaling transduction pathways, dissociation of ASK1-thioredoxin, and activation of ASK1
ABSTRACT The role of signaling pathways in the regulation of cellular iron metabolism is becoming increasingly recognized. Iron chelation is used for the treatment of iron overload but also as a potential strategy for cancer therapy, because iron depletion results in cell cycle arrest and apoptosis. This study examined potential signaling pathways affected by iron depletion induced by desferrioxamine (DFO) or di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT). Both chelators affected multiple molecules in the mitogen-activated protein kinase (MAPK) pathway, including a number of dual specificity phosphatases that directly de-phosphorylate MAPKs. Examination of the phosphorylation of major MAPKs revealed that DFO and Dp44mT markedly increased phosphorylation of stress-activated protein kinases, JNK and p38, without significantly affecting the extracellular signal-regulated kinase (ERK). Redox-inactive DFO-iron complexes did not affect phosphorylation of JNK or p38, whereas the redox-active Dp44mT-iron complex significantly increased the phosphorylation of these kinases similarly to Dp44mT alone. Iron or N-acetylcysteine supplementation reversed Dp44mT-induced up-regulation of phospho-JNK, but only iron was able to reverse the effect of DFO on JNK. Both iron chelators significantly reduced ASK1-thioredoxin complex formation, resulting in the increased phosphorylation of ASK1, which activates the JNK and p38 pathways. Thus, dissociation of ASK1 could serve as an important signal for the phosphorylation of JNK and p38 activation observed after iron chelation. Phosphorylation of JNK and p38 likely play an important role in mediating the cell cycle arrest and apoptosis induced by iron depletion.
- SourceAvailable from: Robert Trondl
[Show abstract] [Hide abstract]
- "In good accordance with our results, mRNA expression studies with the thiosemicarbazone Dp44mT (di-2-pyridylketone-4,4,- dimethyl-3-thiosemicarbazone) suggested that iron depletion is responsible for elevated levels of DITT3 (a synonym for the gene encoding CHOP) (Yu and Richardson, 2011). Indeed, investigations regarding the stability of metal complexes of 3-AP and 3-AP-Me with iron, zinc, and copper revealed that terminally dimethylated thiosemicarbazones, such as 3-AP-Me, form metal complexes with higher stability (Enyedy et al., 2010, 2011). "
ABSTRACT: Triapine (3-AP), a ribonucleotide reductase inhibitor, has been extensively evaluated in clinical trials in the last decade. This study addresses the role of ER stress in the anticancer activity of 3-AP and the derivative 3-AP-Me, differing from 3-AP only by dimethylation of the terminal nitrogen. Treatment of colon cancer cells with 3-AP or 3-AP-Me activated all three ER stress pathways (PERK, IRE1a and ATF6) by phosphorylation of eIF2α and upregulation of ATF4 and ATF6 gene expression, and particularly 3-AP-Me lead to an upregulation of the alternatively spliced mRNA variant XBP1s (16-fold). Moreover, 3-AP and 3-AP-Me activated the cellular stress kinases JNK and p38 MAPK, and inhibition of JNK activity antagonized the cytotoxic effect of both compounds. Subsequent to induction of the unfolded protein response (UPR), a significant upregulation of pro-apoptotic proteins was detected, including the transcription factor CHOP and the BH3-only member protein Bim, an essential factor for ER stress-related apoptosis. In correlation with the higher degree of ER stress after 3-AP-Me treatment, also a more potent depolarization of mitochondrial membranes was found. These data suggest that 3-AP and 3-AP-Me induce apoptosis via ER stress. This was further corroborated by showing that inhibition of protein biosynthesis with cycloheximide prior to 3-AP and 3-AP-Me treatment leads to a significant reduction of the anti-proliferative properties of both compounds. Taken together, this study demonstrates that induction of ER stress contributes to the mode of action of 3-AP and that terminal methylation leads to an even more pronounced manifestation of this effect.Molecular pharmacology 12/2013; 85(3). DOI:10.1124/mol.113.090605 · 4.12 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Iron plays a crucial part in proliferation while iron deficiency results in G(1)/S arrest, DNA damage, and apoptosis. However, the precise role of iron in cell cycle control remains unclear. We showed that iron depletion using the iron chelators, desferrioxamine (DFO), or 2-hydroxy-1-napthylaldehyde isonicotinoyl hydrazone (311), increased the mRNA levels of the growth arrest and DNA damage 45α gene, GADD45α (Darnell, G. and Richardson, D. R. (1999) Blood 94, 781-792). In this study, we examined the effect of iron depletion on up-regulating GADD family members involved in growth control, including cell cycle arrest, apoptosis, and DNA repair, making them therapeutic targets for tumor suppression. We showed the GADD family members were up-regulated by cellular iron depletion. Further, up-regulation of GADD45α after iron deprivation was independent of hypoxia-inducible factor-1α (HIF-1α), octamer-1 (Oct-1), p53 and early growth response 1 (Egr1). We then analyzed the regulatory elements responsible for iron depletion-mediated regulation of GADD45α and identified the specific transcription factor/s involved. This region was within -117 bp and -81 bp relative to the start codon where the consensus sequences of three transcription factors are located: the CCAAT-binding factor/nuclear factor-Y (NF-Y), the stabilizing molecule v-MYB and the enhancer, CCAAT enhancer-binding protein (CEBPα). Mutation analysis, shRNA studies, Western blotting, and electrophoretic mobility shift assays led to the identification of NF-Y in the transcriptional up-regulation of GADD45α after iron depletion. Furthermore, like GADD45α, NF-YA was up-regulated after iron chelation and down-regulated by iron supplementation. These results are important for understanding the mechanisms of iron depletion-mediated cell cycle arrest, DNA damage repair, and apoptosis.Journal of Biological Chemistry 08/2011; 286(41):35396-406. DOI:10.1074/jbc.M111.273060 · 4.60 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Fanconi anemia, complementation group C (FANCC)-deficient hematopoietic stem and progenitor cells are hypersensitive to a variety of inhibitory cytokines, one of which, TNFα, can induce BM failure and clonal evolution in Fancc-deficient mice. FANCC-deficient macrophages are also hypersensitive to TLR activation and produce TNFα in an unrestrained fashion. Reasoning that suppression of inhibitory cytokine production might enhance hematopoiesis, we screened small molecules using TLR agonist-stimulated FANCC- and Fanconi anemia, complementation group A (FANCA)-deficient macrophages containing an NF-κB/AP-1-responsive reporter gene (SEAP). Of the 75 small molecules screened, the p38 MAPK inhibitor BIRB 796 and dasatinib potently suppressed TLR8-dependent expression of the reporter gene. Fanconi anemia (FA) macrophages were hypersensitive to the TLR7/8 activator R848, overproducing SEAP and TNFα in response to all doses of the agonist. Low doses (50nM) of both agents inhibited p38 MAPK-dependent activation of MAPKAPK2 (MK2) and suppressed MK2-dependent TNFα production without substantially influencing TNFα gene transcription. Overproduction of TNFα by primary FA cells was likewise suppressed by these agents and involved inhibition of MK2 activation. Because MK2 is also known to influence production and/or sensitivity to 2 other suppressive factors (MIP-1α and IFNγ) to which FA hematopoietic progenitor cells are uniquely vulnerable, targeting of p38 MAPK in FA hematopoietic cells is a rational objective for preclinical evaluation.Blood 03/2012; 119(9):1992-2002. DOI:10.1182/blood-2011-06-354647 · 10.43 Impact Factor