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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

ROS Analysis. (a) Intracellular fluorescence of 2′,7′-dichlorodihydrofluorescein (DCF) in SH-SY5Y cells that were treated or not with 5 or 25 μM TPEN for 12, 24, and 48 hours measured by FACS (a1) flow cytometric data compiled on a single graphic showing no change in DCF fluorescence in different incubation time and concentration of TPEN (a2). Viable cells were labeled with PI to exclude dead cells and only viable cells were analyzed. (b) Dihydroethidium (DHE) in SH-SY5Y cells treated with 5 or 25 μM TPEN for 12 (b1), 24 (b2), and 48 hours (b3). Flow cytometric data compiled on a single graphic showing no change in DHE fluorescence in different treatment time and concentration of TPEN (b4). Data represent the mean values ± standard deviations (n = 3). Significant differences between positive control (DMNQ) and treated cell were ∗∗p < 0.001. There were no significant differences between untreated and treated cells.
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fig3: ROS Analysis. (a) Intracellular fluorescence of 2′,7′-dichlorodihydrofluorescein (DCF) in SH-SY5Y cells that were treated or not with 5 or 25 μM TPEN for 12, 24, and 48 hours measured by FACS (a1) flow cytometric data compiled on a single graphic showing no change in DCF fluorescence in different incubation time and concentration of TPEN (a2). Viable cells were labeled with PI to exclude dead cells and only viable cells were analyzed. (b) Dihydroethidium (DHE) in SH-SY5Y cells treated with 5 or 25 μM TPEN for 12 (b1), 24 (b2), and 48 hours (b3). Flow cytometric data compiled on a single graphic showing no change in DHE fluorescence in different treatment time and concentration of TPEN (b4). Data represent the mean values ± standard deviations (n = 3). Significant differences between positive control (DMNQ) and treated cell were ∗∗p < 0.001. There were no significant differences between untreated and treated cells.

Mentions: To analyze the influence of reactive oxygen species (ROS) on zinc depletion, cells were incubated with the probe DCFH-DA and PI and analyzed by flow cytometry [47]. Cells were treated with PI to discriminate dead cells from the viable ones. Viable cells were gated and the results indicated that both untreated cells (control) and those treated with TPEN exhibited the same fluorescence profile that remained unchanged throughout the incubation period of 12, 24, or 48 hours, indicating that there is no ROS generation to cause probe oxidation (Figures 3(a) and 3(b)). The results of the indirect quantification of ROS suggest that the activation of the observed cell death related to TPEN is not involved in redox imbalance, in contrast to many cell death processes.


Zinc Chelation Mediates the Lysosomal Disruption without Intracellular ROS Generation.

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

ROS Analysis. (a) Intracellular fluorescence of 2′,7′-dichlorodihydrofluorescein (DCF) in SH-SY5Y cells that were treated or not with 5 or 25 μM TPEN for 12, 24, and 48 hours measured by FACS (a1) flow cytometric data compiled on a single graphic showing no change in DCF fluorescence in different incubation time and concentration of TPEN (a2). Viable cells were labeled with PI to exclude dead cells and only viable cells were analyzed. (b) Dihydroethidium (DHE) in SH-SY5Y cells treated with 5 or 25 μM TPEN for 12 (b1), 24 (b2), and 48 hours (b3). Flow cytometric data compiled on a single graphic showing no change in DHE fluorescence in different treatment time and concentration of TPEN (b4). Data represent the mean values ± standard deviations (n = 3). Significant differences between positive control (DMNQ) and treated cell were ∗∗p < 0.001. There were no significant differences between untreated and treated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: ROS Analysis. (a) Intracellular fluorescence of 2′,7′-dichlorodihydrofluorescein (DCF) in SH-SY5Y cells that were treated or not with 5 or 25 μM TPEN for 12, 24, and 48 hours measured by FACS (a1) flow cytometric data compiled on a single graphic showing no change in DCF fluorescence in different incubation time and concentration of TPEN (a2). Viable cells were labeled with PI to exclude dead cells and only viable cells were analyzed. (b) Dihydroethidium (DHE) in SH-SY5Y cells treated with 5 or 25 μM TPEN for 12 (b1), 24 (b2), and 48 hours (b3). Flow cytometric data compiled on a single graphic showing no change in DHE fluorescence in different treatment time and concentration of TPEN (b4). Data represent the mean values ± standard deviations (n = 3). Significant differences between positive control (DMNQ) and treated cell were ∗∗p < 0.001. There were no significant differences between untreated and treated cells.
Mentions: To analyze the influence of reactive oxygen species (ROS) on zinc depletion, cells were incubated with the probe DCFH-DA and PI and analyzed by flow cytometry [47]. Cells were treated with PI to discriminate dead cells from the viable ones. Viable cells were gated and the results indicated that both untreated cells (control) and those treated with TPEN exhibited the same fluorescence profile that remained unchanged throughout the incubation period of 12, 24, or 48 hours, indicating that there is no ROS generation to cause probe oxidation (Figures 3(a) and 3(b)). The results of the indirect quantification of ROS suggest that the activation of the observed cell death related to TPEN is not involved in redox imbalance, in contrast to many cell death processes.

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