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Epidermal growth factor prevents thallium(I)- and thallium(III)-mediated rat pheochromocytoma (PC12) cell apoptosis

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Abstract

We have reported recently that the proliferation of PC12 cells exposed to micromolar concentrations of Tl(I) or Tl(III) has different outcomes, depending on the absence (EGF− cells) or the presence (EGF+ cells) of epidermal growth factor (EGF) added to the media. In the current work, we investigated whether EGF supplementation could also modulate the extent of Tl(I)- or Tl(III)-induced cell apoptosis. Tl(I) and Tl(III) (25–100 μM) decreased cell viability in EGF− but not in EGF+ cells. In EGF− cells, Tl(I) decreased mitochondrial potential, enhanced H2O2 generation, and activated mitochondrial-dependent apoptosis. In addition, Tl(III) increased nitric oxide production and caused a misbalance between the anti- and pro-apoptotic members of Bcl-2 family. Tl(I) increased ERK1/2, JNK, p38, and p53 phosphorylation in EGF− cells. In these cells, Tl(III) did not affect ERK1/2 and JNK phosphorylation but increased p53 phosphorylation that was related to the promotion of cell senescence. In addition, this cation significantly activated p38 in both EGF− and EGF+ cells. The specific inhibition of ERK1/2, JNK, p38, or p53 abolished Tl(I)-mediated EGF− cell apoptosis. Only when p38 activity was inhibited, Tl(III)-mediated apoptosis was prevented in EGF− and EGF+ cells. Together, current results indicate that EGF partially prevents the noxious effects of Tl by preventing the sustained activation of MAPKs signaling cascade that lead cells to apoptosis and point to p38 as a key mediator of Tl(III)-induced PC12 cell apoptosis.

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Abbreviations

Ac-DEVD-pNA:

N-acetyl-Asp-Glu-Val-Asp p-nitroanilide

Ac-IETD-pNA:

N-acetyl-Ile-Glu-Thr-Asp p-nitroanilide

ASK-1:

Apoptosis signal-regulating kinase 1

Bax:

Bcl-2-associated X protein

Bcl-2:

B-cell lymphoma 2

DHR123:

Dihydrorhodamine 123

DMEM:

Dulbeccós modified Eagle medium

2,4-DNP:

2,4-dinitrophenol

EGF:

Epidermal growth factor

ERK:

Extracellular signal-regulated kinases

IOD:

Integrated optical density

JNK:

c-Jun N-terminal kinase

L-NAME:

L-NG-nitroarginine methyl ester

MAPK:

Mitogen-activated protein kinase

MAPKKK:

MAPK kinase kinase

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide

NEDA:

N-(1-naphtyl)ethylenediamine dihydrochloride

NO:

Nitric oxide

NOS:

Nitric oxide synthase

PARP:

Poly(AD ribose) polymerase

PI:

Propidium iodide

PBS:

Phosphate-buffered saline

PR:

Ponceau red

R123:

Rhodamine 123

ROS:

Reactive oxygen species

SA-β-Gal:

Senescence-associated-β-galactosidase

TBS:

Tris-buffered saline

X-Gal:

5-Bromo-4-chloro-3-indolyl β-d-galactoside

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Acknowledgments

This work was supported by grants of Universidad de Buenos Aires (B086 and 20020100100112) and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) (PICT 32273 and 2013–1018), Argentina. SVV is a career investigator of CONICET. Authors are grateful to Dr. Juan Pablo Carnevale, Dr. Ana M Adamo, Dr. Leonor Roguin and Dr. Johanna Miquet for the generous gift of etoposide, the antibodies against β-tubulin, ERK1/2, and p-p53, respectively.

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  1. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Instituto de Química y Fisicoquímica Biológicas (IQUIFIB), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina

    María Teresa Luján Pino, Clarisa Marotte & Sandra Viviana Verstraeten

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  1. María Teresa Luján Pino
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  2. Clarisa Marotte
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  3. Sandra Viviana Verstraeten
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Correspondence to Sandra Viviana Verstraeten.

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Suppl. Figure 1

Etoposide-mediated effects on EGF− and EGF+ cell viability. PC12 cells were incubated at 37ºC for 24 h in serum-free DMEM. After media replacement, EGF− (□) and EGF+ (■) cells (control) were further incubated for 24 h in the presence 50 μg/ml etoposide (Eto). Cell viability was evaluated from (A) MTT metabolization (B) lysosome ability to incorporate Neutral red, and (C) plasma membrane capacity to exclude PI. Results are shown as the mean ± SEM of at least four independent experiments. * denotes a significant difference from the value measured in control cells (P < 0.05). (TIFF 352 kb)

Suppl. Figure 2

Effects of 2,4-dinitrophenol (DNP) on mitochondrial potential and oxidant production in EGF− and EGF+ cells. PC12 cells were incubated at 37ºC for 24 h in serum-free DMEM. After media replacement, EGF− (□) and EGF+ (■) cells (control) were further incubated for 23.5 h in the presence of their respective culture media. After addition of 100 μM DNP, cells were incubated for 30 min at 37ºC and (A) mitochondrial potential, and (B) oxidant production were evaluated. Results are shown as the mean ± SEM of four independent experiments. * denotes a significant difference from the value measured in cells incubated in control cells (P < 0.05). # denotes a significant difference from the value measured in control EGF− cells (P < 0.05) (TIFF 281 kb)

Suppl. Figure 3

Etoposide promotion of apoptosis in EGF− and EGF+ cells. PC12 cells were incubated at 37ºC for 24 h in serum-free DMEM. After media replacement, EGF− (□) and EGF+ (■) cells (control) were further incubated for 24 h in the presence 50 μg/ml etoposide (Eto). Apoptotic cell content was evaluated in the samples by nuclear staining with PI and analysis by flow cytometry. Results are shown as the mean ± SEM of four independent experiments. * denotes a significant difference from the value measured in cells incubated in control cells (P < 0.05) (TIFF 281 kb)

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Pino, M.T.L., Marotte, C. & Verstraeten, S.V. Epidermal growth factor prevents thallium(I)- and thallium(III)-mediated rat pheochromocytoma (PC12) cell apoptosis. Arch Toxicol 91, 1157–1174 (2017). https://doi.org/10.1007/s00204-016-1793-9

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