Evaluation of Zinc (II) chelators for inhibiting p53-mediated apoptosis

Abstract

In a previous study, we reported that sodium orthovanadate (vanadate) is the first known inhibitor that is capable of protecting mice from death from the radiation-induced gastrointestinal syndrome via its ability to block both transcription-dependent and transcription-independent p53 apoptotic pathways. In this paper, we report that vanadate has a unique activity for inducing the denaturation of p53 relative to other known radioprotective p53 inhibitors, pifithrin-α (PFTα) and pifithrin-µ (PFTµ). This potent radioprotective effect of vanadate prompted us to undertake a more extensive search for p53 inhibitors that can induce p53 denaturation. Based on the fact that p53 denaturation can be induced by the dissociation of a zinc ion, which is used as a structural factor of p53, we screened some zinc (II) chelators for the suppression of the DNA binding activity of p53 in vitro and the inhibition of radiation-induced p53-dependent apoptosis in MOLT-4 cells. The findings indicate that two of five zinc (II) chelators also suppressed apoptosis. Among the inhibitors tested, Bispicen (N,N'-Bis(2-pyridylmethyl)-1,2-ethanediamine) had the highest inhibition activity. A mechanistic study using cells bearing different p53 status or functions (i.e., p53-knockdown MOLT-4 transformant and its revertants, p53 mutant cells, p53-null cells), and p53-independent apoptotic stimuli revealed that the suppressive effect of Bispicen on apoptosis is specifically mediated through p53. Moreover, Bispicen, similar to vanadate, induces the denaturation of p53 as well as the blocking of both transcription-dependent and -independent apoptotic pathways. Our findings indicate that the use of zinc (II) chelators represent a new approach for protecting against radiation-induced p53-dependent apoptosis through the inhibition of p53-dependent apoptotic pathways.

AS-2, a novel inhibitor of p53-dependent apoptosis, prevents apoptotic mitochondrial dysfunction in a transcription-independent manner and protects mice from a lethal dose of ionizing radiation

“…Bispicen (N,N 0 -Bis(2-pyridylmethyl)-1,2-ethanediamine) was found to be highly efficacious in terms of inhibiting apoptosis, showing activity for p53 denaturation as well as on the inhibition of both the transcription-dependent and -independent apoptotic pathways [11,[18][19][20][21]. This is the first study to demonstrate that certain types of zinc(II) chelators can be used to inhibit p53-mediated apoptosis in irradiated cultured cells.…”

“…MOLT-4 cells and their derivative transformed cell lines (MOLT/ Nega, MOLT/p53KD-1, and MOLT/p53KD-1/R-p53-1), IM-9, KU812, CCRF-CEM, and U937 cells were cultured in RPMI 1640 medium (Wako) supplemented with 10% fetal bovine serum (FBS; Sigma) and antibiotics (100 U/ml penicillin and 0.1 mg/ml streptomycin (Nacalai Tesque)) [8,11]. The medium was also supplemented with 0.5 mg/ml G418 (Enzo Life Sciences) and/or 0.25 mg/ml Hygromycin B (Wako) in order to maintain stable transformants.…”

“…Three radioprotective p53 inhibitors have been reported, namely, pifithrin-a (PFTa), pifithrin-l (PFTl), and sodium orthovanadate (vanadate) [5][6][7][8][9][10][11]. Among the three radioprotective p53 inhibitors, the highest radioprotective efficacy in totalbody-irradiated mice was produced by vanadate, which has a unique activity in inducing the denaturation of p53 relative to PFTa and PFTl.…”

“…(>tridentate), and two tetradentate zinc(II) chelators were then found to suppress p53-dependent apoptosis in irradiated MOLT-4 cells [11]. Bispicen (N,N 0 -Bis(2-pyridylmethyl)-1,2-ethanediamine) was found to be highly efficacious in terms of inhibiting apoptosis, showing activity for p53 denaturation as well as on the inhibition of both the transcription-dependent and -independent apoptotic pathways [11,[18][19][20][21].…”

“…This is the first study to demonstrate that certain types of zinc(II) chelators can be used to inhibit p53-mediated apoptosis in irradiated cultured cells. However, because the highest dose of Bispicen tolerable in mice was substantially lower than the dose required to suppress p53-dependent apoptosis in cultured cells, it cannot be considered to be unsuitable for rescuing mice that had been exposed to a lethal dose of ionizing radiation [11]. In order to reduce the cytotoxic effects, we conducted a more extensive search for weaker zinc(II) chelators that are less toxic.…”