Differential targeting of the cyclin-dependent kinase inhibitor, p21CIP1/WAF1, by chelators with anti-proliferative activity in a range of tumor cell-types

Chelators such as 2-hydroxy-1-napthylaldehyde isonicotinoyl hydrazone (311) and di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) target tumor cell iron pools and inhibit proliferation. These agents also modulate multiple targets, one of which is the cyclin-dependent kinase inhibitor, p21. Hence, this investigation examined the mechanism of action of these compounds in targeting p21. All the chelators up-regulated p21 mRNA in the five tumor cell-types assessed. In contrast, examining their effect on total p21 protein levels, these agents induced either: (1) down-regulation in MCF-7 cells; (2) up-regulation in SK-MEL-28 and CFPAC-1 cells; or (3) had no effect in LNCaP and SK-N-MC cells. The nuclear localization of p21 was also differentially affected by the ligands depending upon the cell-type, with it being decreased in MCF-7 cells, but increased in SK-MEL-28 and CFPAC-1 cells. Further studies assessing the mechanisms responsible for these effects demonstrated that p21 expression was not correlated with p53 status, suggesting a p53-independent mechanism. Considering this, we examined proteins that modulate p21 independently of p53, namely NDRG1, MDM2 and ΔNp63. These studies demonstrated that a dominant negative MDM2 isoform (p75(MDM2)) closely resembled p21 expression in response to chelation in three cell lines. These data suggest MDM2 may be involved in the regulation of p21 by chelators.