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Zinc Chelation Mediates the Lysosomal Disruption without Intracellular ROS Generation.

Matias AC, Manieri TM, Cerchiaro G - Oxid Med Cell Longev (2016)

Bottom Line: We found that TPEN is not responsible for ROS generation and the mechanism involves essentially lysosomal disruption caused by intracellular zinc depletion.We also observed a modest activation of Bax and no changes in the Bcl-2 proteins.As a result, we suggest that TPEN causes intracellular zinc depletion which can influence the breakdown of lysosomes and cell death without ROS generation.

View Article: PubMed Central - PubMed

Affiliation: Center for Natural Sciences and Humanities, Federal University of ABC, UFABC, Avenida dos Estados 5001, Bloco B, 09210-170 Santo André, SP, Brazil.

ABSTRACT
We report the molecular mechanism for zinc depletion caused by TPEN (N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine) in neuroblastoma cells. The activation of p38 MAP kinase and subsequently caspase 3 is not due to or followed by redox imbalance or ROS generation, though these are commonly observed in literature. We found that TPEN is not responsible for ROS generation and the mechanism involves essentially lysosomal disruption caused by intracellular zinc depletion. We also observed a modest activation of Bax and no changes in the Bcl-2 proteins. As a result, we suggest that TPEN causes intracellular zinc depletion which can influence the breakdown of lysosomes and cell death without ROS generation.

No MeSH data available.


Related in: MedlinePlus

Influence of the TPEN chelator in the expression levels of apoptotic proteins. SH-SY5Y cells were treated with 25 μM TPEN. At each time point, 100 μg of total proteins from the total cell lysates was loaded onto each lane for the detection of p-p38 (a), Bax (b), Bcl-2 (c), JNK (d), and p-JNK (e). β-actin and α-tubulin were used as a loading control. The Western blot images represent three independent experiments, and significant differences between untreated and treated cells were ∗p < 0.05 and ∗∗p < 0.001, respectively.
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fig6: Influence of the TPEN chelator in the expression levels of apoptotic proteins. SH-SY5Y cells were treated with 25 μM TPEN. At each time point, 100 μg of total proteins from the total cell lysates was loaded onto each lane for the detection of p-p38 (a), Bax (b), Bcl-2 (c), JNK (d), and p-JNK (e). β-actin and α-tubulin were used as a loading control. The Western blot images represent three independent experiments, and significant differences between untreated and treated cells were ∗p < 0.05 and ∗∗p < 0.001, respectively.

Mentions: Conversely, we analyzed the expression levels of another mitogen-activated protein kinase, p38 MAP kinase, and the results showed an increase compared with control experiments at all incubation times in the extracts obtained from cells (Figures 5(a) and 6(a)). We therefore used a p38 inhibitor to determine whether the activation of the cascade of apoptosis was first regulated by p38 MAP kinase. The compound SB202190 is a pyridinyl imidazole inhibitor that inhibits the activity of p38 MAP kinase through competition with ATP [46] and inhibits the phosphorylation of the protein [56]. When treating cells with an inhibitor of p38 MAP kinase, the highly selective and permeable membrane compound SB202190, it was observed that the compound completely inhibited the expression of both p38 MAP kinase and caspase 3; an increase in cell viability was also observed (Figure 5(d)), indicating the influence of p38 MAP kinase on the subsequent activation of caspase 3 (Figure 5(c)). Additionally, the expression levels of caspase 3 were analyzed, and the results showed that cells with zinc depletion also had an increased expression of this protein, particularly at 24 hours of incubation (Figure 5(b)).


Zinc Chelation Mediates the Lysosomal Disruption without Intracellular ROS Generation.

Matias AC, Manieri TM, Cerchiaro G - Oxid Med Cell Longev (2016)

Influence of the TPEN chelator in the expression levels of apoptotic proteins. SH-SY5Y cells were treated with 25 μM TPEN. At each time point, 100 μg of total proteins from the total cell lysates was loaded onto each lane for the detection of p-p38 (a), Bax (b), Bcl-2 (c), JNK (d), and p-JNK (e). β-actin and α-tubulin were used as a loading control. The Western blot images represent three independent experiments, and significant differences between untreated and treated cells were ∗p < 0.05 and ∗∗p < 0.001, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4829717&req=5

fig6: Influence of the TPEN chelator in the expression levels of apoptotic proteins. SH-SY5Y cells were treated with 25 μM TPEN. At each time point, 100 μg of total proteins from the total cell lysates was loaded onto each lane for the detection of p-p38 (a), Bax (b), Bcl-2 (c), JNK (d), and p-JNK (e). β-actin and α-tubulin were used as a loading control. The Western blot images represent three independent experiments, and significant differences between untreated and treated cells were ∗p < 0.05 and ∗∗p < 0.001, respectively.
Mentions: Conversely, we analyzed the expression levels of another mitogen-activated protein kinase, p38 MAP kinase, and the results showed an increase compared with control experiments at all incubation times in the extracts obtained from cells (Figures 5(a) and 6(a)). We therefore used a p38 inhibitor to determine whether the activation of the cascade of apoptosis was first regulated by p38 MAP kinase. The compound SB202190 is a pyridinyl imidazole inhibitor that inhibits the activity of p38 MAP kinase through competition with ATP [46] and inhibits the phosphorylation of the protein [56]. When treating cells with an inhibitor of p38 MAP kinase, the highly selective and permeable membrane compound SB202190, it was observed that the compound completely inhibited the expression of both p38 MAP kinase and caspase 3; an increase in cell viability was also observed (Figure 5(d)), indicating the influence of p38 MAP kinase on the subsequent activation of caspase 3 (Figure 5(c)). Additionally, the expression levels of caspase 3 were analyzed, and the results showed that cells with zinc depletion also had an increased expression of this protein, particularly at 24 hours of incubation (Figure 5(b)).

Bottom Line: We found that TPEN is not responsible for ROS generation and the mechanism involves essentially lysosomal disruption caused by intracellular zinc depletion.We also observed a modest activation of Bax and no changes in the Bcl-2 proteins.As a result, we suggest that TPEN causes intracellular zinc depletion which can influence the breakdown of lysosomes and cell death without ROS generation.

View Article: PubMed Central - PubMed

Affiliation: Center for Natural Sciences and Humanities, Federal University of ABC, UFABC, Avenida dos Estados 5001, Bloco B, 09210-170 Santo André, SP, Brazil.

ABSTRACT
We report the molecular mechanism for zinc depletion caused by TPEN (N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine) in neuroblastoma cells. The activation of p38 MAP kinase and subsequently caspase 3 is not due to or followed by redox imbalance or ROS generation, though these are commonly observed in literature. We found that TPEN is not responsible for ROS generation and the mechanism involves essentially lysosomal disruption caused by intracellular zinc depletion. We also observed a modest activation of Bax and no changes in the Bcl-2 proteins. As a result, we suggest that TPEN causes intracellular zinc depletion which can influence the breakdown of lysosomes and cell death without ROS generation.

No MeSH data available.


Related in: MedlinePlus