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.
Highlights
In this study, we examined the effect of vanadate on the denaturation of p53 [6], and found that vanadate has a unique activity in inducing a p53 denaturation relative to the other two radioprotective p53 inhibitors, PFTα and PFTμ
Our findings indicate that the use of zinc (II) chelators represent a potentially viable approach for inhibiting p53-dependent apoptosis
We initially investigated the effect of three radioprotective p53 inhibitors on the conformation of p53 by means of immunoprecipitation with an anti-p53 PAb 240 monoclonal antibody as a specific probe for the conformationally inactivated form of p53 [18, 19, 21, 22]
Summary
We examined the effect of vanadate on the denaturation of p53 [6], and found that vanadate has a unique activity in inducing a p53 denaturation relative to the other two radioprotective p53 inhibitors, PFTα and PFTμ. We postulated that the activity should be responsible for the potent radioprotective effect of vanadate, and initiated a search for a zinc (II) chelator that is capable of suppressing p53-dependent apoptosis and inducing p53 denaturation. We evaluated five zinc (II) chelators as candidates for novel p53 inhibitors. Two zinc (II) chelators were found to suppress p53-dependent apoptosis in irradiated MOLT-4 cells. Our findings indicate that the use of zinc (II) chelators represent a potentially viable approach for inhibiting p53-dependent apoptosis
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