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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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Effects of zinc (II) chelators on p53-dependent radiation-induced cell death, the loss of ∆ψm, and caspase activationA. Dose-response of zinc (II) chelators on 10 Gy-irradiated MOLT-4 cell death. The percentage of cell death was assessed 18 h after IR by dye-exclusion test. B. Dose-response of zinc (II) chelators on the loss of ∆ψm in 10 Gy-irradiated MOLT-4 cells. The percentage of cells losing their ∆ψm was measured 12 h after IR by MitoTracker staining and flow-cytometry. Data shown in A and B are the means ± standard deviation (SD) from 3-5 independent experiments. C. Dose-response of zinc (II) chelators on caspase activation in 10 Gy-irradiated MOLT-4 cells. Cells were harvested 10 h after IR. Proteins were detected by immunoblotting.
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Figure 4: Effects of zinc (II) chelators on p53-dependent radiation-induced cell death, the loss of ∆ψm, and caspase activationA. Dose-response of zinc (II) chelators on 10 Gy-irradiated MOLT-4 cell death. The percentage of cell death was assessed 18 h after IR by dye-exclusion test. B. Dose-response of zinc (II) chelators on the loss of ∆ψm in 10 Gy-irradiated MOLT-4 cells. The percentage of cells losing their ∆ψm was measured 12 h after IR by MitoTracker staining and flow-cytometry. Data shown in A and B are the means ± standard deviation (SD) from 3-5 independent experiments. C. Dose-response of zinc (II) chelators on caspase activation in 10 Gy-irradiated MOLT-4 cells. Cells were harvested 10 h after IR. Proteins were detected by immunoblotting.

Mentions: The effect of the five chelators on intracellular p53 activity was examined with reference to p53-dependent apoptosis in irradiated MOLT-4 cells. The results of the dye-exclusion test as a method for determining cell death (Fig. 4A) and MitoTracker staining for measuring the loss of mitochondrial membrane potential (loss of Δψm; Fig. 4B) suggested that Bispicen potently suppressed apoptosis, while TPEN, cyclen, and BPA have negligible effects on apoptosis. TPA was a weak suppressor of cell death, and moderately suppressed the loss of Δψm (Fig. 4A, B), probably due to its cytotoxicity (Fig.4A; open circles). TPEN also failed to suppress apoptosis (remarkably, at 10 µM), possibly due to its cytotoxicity as well. Cyclen had no effect on apoptosis, despite its strong affinity for Zn2+, possibly because this molecule is hydrophilic and its membrane permeability is very low. BPA appeared to fail to suppress apoptosis due to its low zinc binding constant and low-affinity for 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)

Effects of zinc (II) chelators on p53-dependent radiation-induced cell death, the loss of ∆ψm, and caspase activationA. Dose-response of zinc (II) chelators on 10 Gy-irradiated MOLT-4 cell death. The percentage of cell death was assessed 18 h after IR by dye-exclusion test. B. Dose-response of zinc (II) chelators on the loss of ∆ψm in 10 Gy-irradiated MOLT-4 cells. The percentage of cells losing their ∆ψm was measured 12 h after IR by MitoTracker staining and flow-cytometry. Data shown in A and B are the means ± standard deviation (SD) from 3-5 independent experiments. C. Dose-response of zinc (II) chelators on caspase activation in 10 Gy-irradiated MOLT-4 cells. Cells were harvested 10 h after IR. Proteins were detected by immunoblotting.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Effects of zinc (II) chelators on p53-dependent radiation-induced cell death, the loss of ∆ψm, and caspase activationA. Dose-response of zinc (II) chelators on 10 Gy-irradiated MOLT-4 cell death. The percentage of cell death was assessed 18 h after IR by dye-exclusion test. B. Dose-response of zinc (II) chelators on the loss of ∆ψm in 10 Gy-irradiated MOLT-4 cells. The percentage of cells losing their ∆ψm was measured 12 h after IR by MitoTracker staining and flow-cytometry. Data shown in A and B are the means ± standard deviation (SD) from 3-5 independent experiments. C. Dose-response of zinc (II) chelators on caspase activation in 10 Gy-irradiated MOLT-4 cells. Cells were harvested 10 h after IR. Proteins were detected by immunoblotting.
Mentions: The effect of the five chelators on intracellular p53 activity was examined with reference to p53-dependent apoptosis in irradiated MOLT-4 cells. The results of the dye-exclusion test as a method for determining cell death (Fig. 4A) and MitoTracker staining for measuring the loss of mitochondrial membrane potential (loss of Δψm; Fig. 4B) suggested that Bispicen potently suppressed apoptosis, while TPEN, cyclen, and BPA have negligible effects on apoptosis. TPA was a weak suppressor of cell death, and moderately suppressed the loss of Δψm (Fig. 4A, B), probably due to its cytotoxicity (Fig.4A; open circles). TPEN also failed to suppress apoptosis (remarkably, at 10 µM), possibly due to its cytotoxicity as well. Cyclen had no effect on apoptosis, despite its strong affinity for Zn2+, possibly because this molecule is hydrophilic and its membrane permeability is very low. BPA appeared to fail to suppress apoptosis due to its low zinc binding constant and low-affinity for 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