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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 inhibits SOD1 activity in JNK +/+ cells.A; PDTC represses SOD1 activity in U937 cells. B; SOD1 activity is not sensitive to PDTC in JNK deficient cells. Serum starved U937, JNK -/- and JNK +/+ cells were treated with 1 μM PDTC in RPMI1640 media supplemented with 1% serum. After 24 h of treatment, cells were lysed and SOD1 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.g008: PDTC inhibits SOD1 activity in JNK +/+ cells.A; PDTC represses SOD1 activity in U937 cells. B; SOD1 activity is not sensitive to PDTC in JNK deficient cells. Serum starved U937, JNK -/- and JNK +/+ cells were treated with 1 μM PDTC in RPMI1640 media supplemented with 1% serum. After 24 h of treatment, cells were lysed and SOD1 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: To determine the functional significance of JNK pathway in modulating SOD1 gene, we evaluated the effects of PDTC on SOD1 enzymatic activity in U937 cells. As shown in Fig 8A, PDTC treatment reduced significantly SOD1 activity. To confirm further that JNK pathway is required for the full action of PDTC on SOD1 activity, we used JNK deficient cells to evaluate SOD1 activity in the presence or absence of PDTC. Consistent with the results obtained with U937 cells, activity of SOD1 was markedly decreased in JNK+/+ mouse fibroblast cell line in the presence of PDTC (Fig 8B). In contrast, using JNK deficient cells the PDTC action was lost in the absence of JNK pathway (Fig 8B). As reported for SOD1 promoter activity (Fig 6), SOD1 basal enzymatic activity in JNK deficient cells was 3 fold higher than in wild type cells (Fig 8B). Taken together, these findings demonstrate that JNK pathway plays an important role in regulating SOD1 transcription and enzymatic activity.


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 inhibits SOD1 activity in JNK +/+ cells.A; PDTC represses SOD1 activity in U937 cells. B; SOD1 activity is not sensitive to PDTC in JNK deficient cells. Serum starved U937, JNK -/- and JNK +/+ cells were treated with 1 μM PDTC in RPMI1640 media supplemented with 1% serum. After 24 h of treatment, cells were lysed and SOD1 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

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getmorefigures.php?uid=PMC4440650&req=5

pone.0127571.g008: PDTC inhibits SOD1 activity in JNK +/+ cells.A; PDTC represses SOD1 activity in U937 cells. B; SOD1 activity is not sensitive to PDTC in JNK deficient cells. Serum starved U937, JNK -/- and JNK +/+ cells were treated with 1 μM PDTC in RPMI1640 media supplemented with 1% serum. After 24 h of treatment, cells were lysed and SOD1 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: To determine the functional significance of JNK pathway in modulating SOD1 gene, we evaluated the effects of PDTC on SOD1 enzymatic activity in U937 cells. As shown in Fig 8A, PDTC treatment reduced significantly SOD1 activity. To confirm further that JNK pathway is required for the full action of PDTC on SOD1 activity, we used JNK deficient cells to evaluate SOD1 activity in the presence or absence of PDTC. Consistent with the results obtained with U937 cells, activity of SOD1 was markedly decreased in JNK+/+ mouse fibroblast cell line in the presence of PDTC (Fig 8B). In contrast, using JNK deficient cells the PDTC action was lost in the absence of JNK pathway (Fig 8B). As reported for SOD1 promoter activity (Fig 6), SOD1 basal enzymatic activity in JNK deficient cells was 3 fold higher than in wild type cells (Fig 8B). Taken together, these findings demonstrate that JNK pathway plays an important role in regulating SOD1 transcription and enzymatic activity.

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