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A Central Role for JNK/AP-1 Pathway in the Pro-Oxidant Effect of Pyrrolidine Dithiocarbamate through Superoxide Dismutase 1 Gene Repression and Reactive Oxygen Species Generation in Hematopoietic Human Cancer Cell Line U937.

Riera H, Afonso V, Collin P, Lomri A - PLoS ONE (2015)

Bottom Line: Ectopic NF-κB p65 subunit overexpression had no effect on SOD1 transcription.Indeed, using JNK deficient cells, PDTC effect was not observed nether on SOD1 transcription or enzymatic activity, nor on ROS production.Taken together, these results suggest that PDTC acts as pro-oxidant compound in JNK/AP-1 dependent-manner by repressing the superoxide dismutase 1 gene leading to intracellular ROS accumulation.

View Article: PubMed Central - PubMed

Affiliation: Unidad de Reumatología, Nivel plaza del Instituto Autónomo Hospital Universitario de Los Andes. Mérida, Venezuela.

ABSTRACT
Pyrrolidine dithiocarbamate (PDTC) known as antioxidant and specific inhibitor of NF-κB was also described as pro-oxidant by inducing cell death and reactive oxygen species (ROS) accumulation in cancer. However, the mechanism by which PDTC indices its pro-oxidant effect is unknown. Therefore, we aimed to evaluate the effect of PDTC on the human Cu/Zn superoxide dismutase 1 (SOD1) gene transcription in hematopoietic human cancer cell line U937. We herein show for the first time that PDTC decreases SOD1 transcripts, protein and promoter activity. Furthermore, SOD1 repression by PDTC was associated with an increase in oxidative stress as evidenced by ROS production. Electrophoretic mobility-shift assays (EMSA) show that PDTC increased binding of activating protein-1 (AP-1) in dose dependent-manner suggesting that the MAPkinase up-stream of AP-1 is involved. Ectopic NF-κB p65 subunit overexpression had no effect on SOD1 transcription. In contrast, in the presence of JNK inhibitor (SP600125), p65 induced a marked increase of SOD1 promoter, suggesting that JNK pathway is up-stream of NF-κB signaling and controls negatively its activity. Indeed, using JNK deficient cells, PDTC effect was not observed nether on SOD1 transcription or enzymatic activity, nor on ROS production. Finally, PDTC represses SOD1 in U937 cells through JNK/c-Jun phosphorylation. Taken together, these results suggest that PDTC acts as pro-oxidant compound in JNK/AP-1 dependent-manner by repressing the superoxide dismutase 1 gene leading to intracellular ROS accumulation.

No MeSH data available.


Related in: MedlinePlus

PDTC is inactive in JNK deficient cells.A; PDTC represses SOD1 activity in JNK +/+ cells. B; SOD1 activity is not sensitive to PDTC in JNK deficient cells. Serum starved JNK -/- & JNK +/+ cells were treated with various doses of PDTC in RPMI1640 media supplemented with 1% serum. After 24 h of treatment, transfected cells were lysed and SOD1 luciferase activity in cell lysates was assayed as described in material & methods. The results are presented as mean ± SE from 4 separate experiments. *P < 0.01 vs. control. NS, not significant.
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pone.0127571.g006: PDTC is inactive in JNK deficient cells.A; PDTC represses SOD1 activity in JNK +/+ cells. B; SOD1 activity is not sensitive to PDTC in JNK deficient cells. Serum starved JNK -/- & JNK +/+ cells were treated with various doses of PDTC in RPMI1640 media supplemented with 1% serum. After 24 h of treatment, transfected cells were lysed and SOD1 luciferase activity in cell lysates was assayed as described in material & methods. The results are presented as mean ± SE from 4 separate experiments. *P < 0.01 vs. control. NS, not significant.

Mentions: Because the use of commercial inhibitors may have some times second effects on cell function, we decided to use JNK deficient cells to confirm our finding with SP600125 known as specific inhibitor of JNK pathway. SOD1 basal transcriptional activity in immortalized mouse fibroblast cell line JNK1-/-–JNK2-/- was increased by 33 fold (Fig 6A) compared to mouse fibroblast cell line wild type (Fig 6B). Treatment of JNK deficient cells with PDTC did not affect SOD1 promoter activity, in contrast to wild type cells (Fig 6). These data demonstrate that active intracellular JNK pathway is necessary to PDTC action on SOD1 transcription.


A Central Role for JNK/AP-1 Pathway in the Pro-Oxidant Effect of Pyrrolidine Dithiocarbamate through Superoxide Dismutase 1 Gene Repression and Reactive Oxygen Species Generation in Hematopoietic Human Cancer Cell Line U937.

Riera H, Afonso V, Collin P, Lomri A - PLoS ONE (2015)

PDTC is inactive in JNK deficient cells.A; PDTC represses SOD1 activity in JNK +/+ cells. B; SOD1 activity is not sensitive to PDTC in JNK deficient cells. Serum starved JNK -/- & JNK +/+ cells were treated with various doses of PDTC in RPMI1640 media supplemented with 1% serum. After 24 h of treatment, transfected cells were lysed and SOD1 luciferase activity in cell lysates was assayed as described in material & methods. The results are presented as mean ± SE from 4 separate experiments. *P < 0.01 vs. control. NS, not significant.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440650&req=5

pone.0127571.g006: PDTC is inactive in JNK deficient cells.A; PDTC represses SOD1 activity in JNK +/+ cells. B; SOD1 activity is not sensitive to PDTC in JNK deficient cells. Serum starved JNK -/- & JNK +/+ cells were treated with various doses of PDTC in RPMI1640 media supplemented with 1% serum. After 24 h of treatment, transfected cells were lysed and SOD1 luciferase activity in cell lysates was assayed as described in material & methods. The results are presented as mean ± SE from 4 separate experiments. *P < 0.01 vs. control. NS, not significant.
Mentions: Because the use of commercial inhibitors may have some times second effects on cell function, we decided to use JNK deficient cells to confirm our finding with SP600125 known as specific inhibitor of JNK pathway. SOD1 basal transcriptional activity in immortalized mouse fibroblast cell line JNK1-/-–JNK2-/- was increased by 33 fold (Fig 6A) compared to mouse fibroblast cell line wild type (Fig 6B). Treatment of JNK deficient cells with PDTC did not affect SOD1 promoter activity, in contrast to wild type cells (Fig 6). These data demonstrate that active intracellular JNK pathway is necessary to PDTC action on SOD1 transcription.

Bottom Line: Ectopic NF-κB p65 subunit overexpression had no effect on SOD1 transcription.Indeed, using JNK deficient cells, PDTC effect was not observed nether on SOD1 transcription or enzymatic activity, nor on ROS production.Taken together, these results suggest that PDTC acts as pro-oxidant compound in JNK/AP-1 dependent-manner by repressing the superoxide dismutase 1 gene leading to intracellular ROS accumulation.

View Article: PubMed Central - PubMed

Affiliation: Unidad de Reumatología, Nivel plaza del Instituto Autónomo Hospital Universitario de Los Andes. Mérida, Venezuela.

ABSTRACT
Pyrrolidine dithiocarbamate (PDTC) known as antioxidant and specific inhibitor of NF-κB was also described as pro-oxidant by inducing cell death and reactive oxygen species (ROS) accumulation in cancer. However, the mechanism by which PDTC indices its pro-oxidant effect is unknown. Therefore, we aimed to evaluate the effect of PDTC on the human Cu/Zn superoxide dismutase 1 (SOD1) gene transcription in hematopoietic human cancer cell line U937. We herein show for the first time that PDTC decreases SOD1 transcripts, protein and promoter activity. Furthermore, SOD1 repression by PDTC was associated with an increase in oxidative stress as evidenced by ROS production. Electrophoretic mobility-shift assays (EMSA) show that PDTC increased binding of activating protein-1 (AP-1) in dose dependent-manner suggesting that the MAPkinase up-stream of AP-1 is involved. Ectopic NF-κB p65 subunit overexpression had no effect on SOD1 transcription. In contrast, in the presence of JNK inhibitor (SP600125), p65 induced a marked increase of SOD1 promoter, suggesting that JNK pathway is up-stream of NF-κB signaling and controls negatively its activity. Indeed, using JNK deficient cells, PDTC effect was not observed nether on SOD1 transcription or enzymatic activity, nor on ROS production. Finally, PDTC represses SOD1 in U937 cells through JNK/c-Jun phosphorylation. Taken together, these results suggest that PDTC acts as pro-oxidant compound in JNK/AP-1 dependent-manner by repressing the superoxide dismutase 1 gene leading to intracellular ROS accumulation.

No MeSH data available.


Related in: MedlinePlus