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

Morita A, Ariyasu S, Ohya S, Takahashi I, Wang B, Tanaka K, Uchida T, Okazaki H, Hanaya K, Enomoto A, Nenoi M, Ikekita M, Aoki S, Hosoi Y - Oncotarget (2013)

Bottom Line: This potent radioprotective effect of vanadate prompted us to undertake a more extensive search for p53 inhibitors that can induce p53 denaturation.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- 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiological Science, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan.

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- 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.

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Bispicen induces the denaturation of p53 in a dose-dependent mannerA. Immunoprecipitation (IP) was performed as in Figure 1 using anti-p53 PAb 240 mAb (upper panel) and DO-1 mAb (lower panel). MOLT-4 cells were 10 Gy-irradiated and treated with the indicated concentrations of chelators or vanadate (Vana), and then harvested at 6 h after IR. Loading of IP samples from the irradiated cells was normalized for the equal amount of DO-1-immunoprecipitated p53. Whole cell lysate (WCL) from unirradiated (1st lane) or 10 Gy-irradiated (2nd lane) MOLT-4 cells cultured for 6 h were used, respectively, as the negative and positive controls for p53. The p53 from WCLs (lanes 1 and 2) and the immunoprecipitated p53 (lanes 3 to 11) was visualized by immunoblotting using anti-p53 DO-1 mAb. B. CD spectra of recombinant FLAG-p53 (20 nM) in PBS buffer (pH 7.4) in the absence and presence of zinc (II) chelators or vanadate (2 µM each) and Zn2+ (20 µM) at 25 °C.
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Figure 7: Bispicen induces the denaturation of p53 in a dose-dependent mannerA. Immunoprecipitation (IP) was performed as in Figure 1 using anti-p53 PAb 240 mAb (upper panel) and DO-1 mAb (lower panel). MOLT-4 cells were 10 Gy-irradiated and treated with the indicated concentrations of chelators or vanadate (Vana), and then harvested at 6 h after IR. Loading of IP samples from the irradiated cells was normalized for the equal amount of DO-1-immunoprecipitated p53. Whole cell lysate (WCL) from unirradiated (1st lane) or 10 Gy-irradiated (2nd lane) MOLT-4 cells cultured for 6 h were used, respectively, as the negative and positive controls for p53. The p53 from WCLs (lanes 1 and 2) and the immunoprecipitated p53 (lanes 3 to 11) was visualized by immunoblotting using anti-p53 DO-1 mAb. B. CD spectra of recombinant FLAG-p53 (20 nM) in PBS buffer (pH 7.4) in the absence and presence of zinc (II) chelators or vanadate (2 µM each) and Zn2+ (20 µM) at 25 °C.

Mentions: To study the inhibitory mechanism of the Bispicen against p53, we examined its effect on the conformation of p53 by immunoprecipitation with PAb 240 mAb as was used in Figure 1, in comparison with vanadate and other zinc (II) chelators that can inhibit caspase activation (TPA and TPEN). As expected, Bispicen induced p53 denaturation in a dose dependent manner, the level of which was comparable to that for vanadate and other chelators (Fig. 7). These data indicate the zinc (II) chelators induce the denaturation of p53.


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

Morita A, Ariyasu S, Ohya S, Takahashi I, Wang B, Tanaka K, Uchida T, Okazaki H, Hanaya K, Enomoto A, Nenoi M, Ikekita M, Aoki S, Hosoi Y - Oncotarget (2013)

Bispicen induces the denaturation of p53 in a dose-dependent mannerA. Immunoprecipitation (IP) was performed as in Figure 1 using anti-p53 PAb 240 mAb (upper panel) and DO-1 mAb (lower panel). MOLT-4 cells were 10 Gy-irradiated and treated with the indicated concentrations of chelators or vanadate (Vana), and then harvested at 6 h after IR. Loading of IP samples from the irradiated cells was normalized for the equal amount of DO-1-immunoprecipitated p53. Whole cell lysate (WCL) from unirradiated (1st lane) or 10 Gy-irradiated (2nd lane) MOLT-4 cells cultured for 6 h were used, respectively, as the negative and positive controls for p53. The p53 from WCLs (lanes 1 and 2) and the immunoprecipitated p53 (lanes 3 to 11) was visualized by immunoblotting using anti-p53 DO-1 mAb. B. CD spectra of recombinant FLAG-p53 (20 nM) in PBS buffer (pH 7.4) in the absence and presence of zinc (II) chelators or vanadate (2 µM each) and Zn2+ (20 µM) at 25 °C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3926839&req=5

Figure 7: Bispicen induces the denaturation of p53 in a dose-dependent mannerA. Immunoprecipitation (IP) was performed as in Figure 1 using anti-p53 PAb 240 mAb (upper panel) and DO-1 mAb (lower panel). MOLT-4 cells were 10 Gy-irradiated and treated with the indicated concentrations of chelators or vanadate (Vana), and then harvested at 6 h after IR. Loading of IP samples from the irradiated cells was normalized for the equal amount of DO-1-immunoprecipitated p53. Whole cell lysate (WCL) from unirradiated (1st lane) or 10 Gy-irradiated (2nd lane) MOLT-4 cells cultured for 6 h were used, respectively, as the negative and positive controls for p53. The p53 from WCLs (lanes 1 and 2) and the immunoprecipitated p53 (lanes 3 to 11) was visualized by immunoblotting using anti-p53 DO-1 mAb. B. CD spectra of recombinant FLAG-p53 (20 nM) in PBS buffer (pH 7.4) in the absence and presence of zinc (II) chelators or vanadate (2 µM each) and Zn2+ (20 µM) at 25 °C.
Mentions: To study the inhibitory mechanism of the Bispicen against p53, we examined its effect on the conformation of p53 by immunoprecipitation with PAb 240 mAb as was used in Figure 1, in comparison with vanadate and other zinc (II) chelators that can inhibit caspase activation (TPA and TPEN). As expected, Bispicen induced p53 denaturation in a dose dependent manner, the level of which was comparable to that for vanadate and other chelators (Fig. 7). These data indicate the zinc (II) chelators induce the denaturation of p53.

Bottom Line: This potent radioprotective effect of vanadate prompted us to undertake a more extensive search for p53 inhibitors that can induce p53 denaturation.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- 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiological Science, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan.

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- 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.

Show MeSH
Related in: MedlinePlus