The effect of potent iron chelators on the regulation of p53: examination of the expression, localization and DNA-binding activity of p53 and the transactivation of WAF1

Abstract

Iron (Fe) chelators induce a G1/S arrest and several of these are undergoing clinical trials as anticancer agents. Despite this, little is known concerning the precise function of Fe in cell cycle progression and the role of p53 in this process. The aim of this study was to assess the effect of Fe chelators on p53 and the mechanism involved in the chelator-mediated increase in mRNA levels of the universal cyclin-dependent kinase inhibitor p21CIP1/WAF1. Cells were incubated with the potent Fe chelator 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) and the results compared with those from cells treated with actinomycin D (Act D), which induces p53. Following incubation with 311, a 3- to 5-fold increase in nuclear p53 protein was observed in cells with wild-type p53. In addition, 311 increased p53 DNA-binding activity 2-fold, while Act D increased it 3- to 5-fold in cells with native p53. To determine the role of p53 in WAF1 transcription, a reporter construct was used consisting of a WAF1 promoter containing the p53-binding site. In cells with wild-type p53, chelators had no effect on luciferase activity, while the positive control, Act D, caused a significant increase. Hence, despite increased p53 protein expression and p53 DNA-binding activity following chelation, these latter results suggested it had no role in up-regulating WAF1 mRNA. Our experiments demonstrated: (i) that the elevated WAF1 mRNA expression after Fe chelation was due to increased transcription and also to a post-transcriptional mechanism that was sensitive to cycloheximide; and (ii) that Fe-chelation increased WAF1 expression through a p53-independent pathway.

Rice Bran Phytic Acid Induced Apoptosis Through Regulation of Bcl-2/Bax and p53 Genes in HepG2 Human Hepatocellular Carcinoma Cells

“…Our findings correlated with the previous findings in which iron-chelator compounds have been reported for multiple mechanisms in the antitumor activity (Richardson et al, 2009). Iron-chelators induced down regulation of Bcl-2, up-regulation of the pro-apoptotic protein Bax and p53 and increases Caspase-3,-8, and-9 activities in cancerious cells (Liang and Richardson, 2003). Also, reported that the iron depletion alters expression of many molecules that cause cell-cycle arrest (Fu and Richardson, 2007).…”

Tuning Cell Cycle Regulation with an Iron Key

“…101,104,105,132 A number of studies have reported elevated levels of p53 protein expression following Fe-depletion. 105,106,133 This increase appears to be at the post-transcriptional level as there is no change in p53 mRNA after Fe-depletion. 101,105 In some studies, Fe-depletion resulted in the upregulation of p53 expression only in cells expressing the wild-type molecule, 106 while in other investigations, p53 protein levels increased after Fe chelation irrespective of whether it was mutated or not.…”

“…105,106,133 This increase appears to be at the post-transcriptional level as there is no change in p53 mRNA after Fe-depletion. 101,105 In some studies, Fe-depletion resulted in the upregulation of p53 expression only in cells expressing the wild-type molecule, 106 while in other investigations, p53 protein levels increased after Fe chelation irrespective of whether it was mutated or not. 105 In whole cell systems, Fe-depletion was able to induce p53-transactivational activity and sequence-specific DNA binding in a dose-and time-dependent manner.…”

“…105 In whole cell systems, Fe-depletion was able to induce p53-transactivational activity and sequence-specific DNA binding in a dose-and time-dependent manner. 106,133 Further studies have revealed that several mechanisms may be involved in the activation of p53 by Fe chelation. These include: (1) an increase in p53 protein expression; 106 (2) increased conversion of latent p53 to its active DNA-binding form; 104 (3) phosphorylation of p53 at serine-15 which increases its stability and prevents proteasomal degradation by mdm-2.…”

Novel di-2-pyridyl–derived iron chelators with marked and selective antitumor activity: in vitro and in vivo assessment

“…23,24,45 Some of these ligands, such as 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311; Figure 1) inhibit RR activity 9 and affect the expression of molecules vital for cell cycle control. [46][47][48] One mechanism by which chelators and other factors (eg, oxidative stress) cause tumor cell death may be through the induction of apoptosis. 22,24,[49][50][51][52][53][54][55][56] However, the precise mechanisms involved in chelator-mediated apoptosis remain unclear, particularly for aroylhydrazone ligands.…”