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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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Only vanadate induces the denaturation of p53 among the three tested radioprotective p53 inhibitorsImmunoprecipitation (IP) of p53 using anti-p53 PAb 240 mAb (upper panel) and DO-1 mAb (lower panel). PAb 240 mAb recognized p53 in 10 Gy-irradiated MOLT-4 cells treated with 800 µM vanadate (V) but not in the cells exposed to γ-rays alone (6 h after IR) or cells treated with IR plus 50 µM PFTα (α) or 7.5 µM PFTµ (µ). 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 7) were visualized by immunoblotting using anti-p53 DO-1 mAb
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Figure 1: Only vanadate induces the denaturation of p53 among the three tested radioprotective p53 inhibitorsImmunoprecipitation (IP) of p53 using anti-p53 PAb 240 mAb (upper panel) and DO-1 mAb (lower panel). PAb 240 mAb recognized p53 in 10 Gy-irradiated MOLT-4 cells treated with 800 µM vanadate (V) but not in the cells exposed to γ-rays alone (6 h after IR) or cells treated with IR plus 50 µM PFTα (α) or 7.5 µM PFTµ (µ). 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 7) were visualized by immunoblotting using anti-p53 DO-1 mAb

Mentions: 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 (mAb) as a specific probe for the conformationally inactivated form of p53 [18, 19, 21, 22]. Under nondenaturing conditions, PAb 240 mAb recognized the inactivated form of p53 but not the normal form, whereas the DO-1 mAb recognizes both forms [6]. For the immunoprecipitation experiment, we used 800 µM, 50 µM, and 7.5 µM concentrations of vanadate, PFTα, and PFTµ, respectively. Each concentration was an optimal dose, as determined in a previous study, for suppressing apoptosis in 10 Gy-irradiated MOLT-4 cells [7]. As shown previously [6], PAb240 mAb immunoprecipitated the p53 from irradiated MOLT-4 cells that had been treated with vanadate (Fig. 1). However, PFTα and PFTµ did not induce any detectable conformational change. These data indicate that only vanadate is able to induce the denaturation of p53. We therefore postulated that the activity might be responsible for the potent radioprotective effect of vanadate, and then began a search for a zinc (II) chelator capable of suppressing p53-dependent apoptosis and inducing p53 denaturation.


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)

Only vanadate induces the denaturation of p53 among the three tested radioprotective p53 inhibitorsImmunoprecipitation (IP) of p53 using anti-p53 PAb 240 mAb (upper panel) and DO-1 mAb (lower panel). PAb 240 mAb recognized p53 in 10 Gy-irradiated MOLT-4 cells treated with 800 µM vanadate (V) but not in the cells exposed to γ-rays alone (6 h after IR) or cells treated with IR plus 50 µM PFTα (α) or 7.5 µM PFTµ (µ). 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 7) were visualized by immunoblotting using anti-p53 DO-1 mAb
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Only vanadate induces the denaturation of p53 among the three tested radioprotective p53 inhibitorsImmunoprecipitation (IP) of p53 using anti-p53 PAb 240 mAb (upper panel) and DO-1 mAb (lower panel). PAb 240 mAb recognized p53 in 10 Gy-irradiated MOLT-4 cells treated with 800 µM vanadate (V) but not in the cells exposed to γ-rays alone (6 h after IR) or cells treated with IR plus 50 µM PFTα (α) or 7.5 µM PFTµ (µ). 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 7) were visualized by immunoblotting using anti-p53 DO-1 mAb
Mentions: 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 (mAb) as a specific probe for the conformationally inactivated form of p53 [18, 19, 21, 22]. Under nondenaturing conditions, PAb 240 mAb recognized the inactivated form of p53 but not the normal form, whereas the DO-1 mAb recognizes both forms [6]. For the immunoprecipitation experiment, we used 800 µM, 50 µM, and 7.5 µM concentrations of vanadate, PFTα, and PFTµ, respectively. Each concentration was an optimal dose, as determined in a previous study, for suppressing apoptosis in 10 Gy-irradiated MOLT-4 cells [7]. As shown previously [6], PAb240 mAb immunoprecipitated the p53 from irradiated MOLT-4 cells that had been treated with vanadate (Fig. 1). However, PFTα and PFTµ did not induce any detectable conformational change. These data indicate that only vanadate is able to induce the denaturation of p53. We therefore postulated that the activity might be responsible for the potent radioprotective effect of vanadate, and then began a search for a zinc (II) chelator capable of suppressing p53-dependent apoptosis and inducing p53 denaturation.

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