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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 repression of the SOD1 promoter from a 5′ deletion series linked to the luciferase gene in U937 cells.A; The 5′ boundaries of plasmids containing various truncations of the SOD1 promoter are shown with the C/EBP, Egr-1/Sp1 sites indicated. B; SOD1 constructs (1 μg) or promoter-less pGL2-Basic control vectors were transiently transfected into U937 cells as described under Experimental procedures. The relative luciferase activity expressed by each plasmid in the absence or presence of 1 μM PDTC for 24 h is shown with SD. Statistical analysis was performed by using Student's t test (*P < 0.01) by comparing values from PDTC-treated cells with control values for each SOD deletion. Results are representative of four independent experiments.
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pone.0127571.g002: PDTC repression of the SOD1 promoter from a 5′ deletion series linked to the luciferase gene in U937 cells.A; The 5′ boundaries of plasmids containing various truncations of the SOD1 promoter are shown with the C/EBP, Egr-1/Sp1 sites indicated. B; SOD1 constructs (1 μg) or promoter-less pGL2-Basic control vectors were transiently transfected into U937 cells as described under Experimental procedures. The relative luciferase activity expressed by each plasmid in the absence or presence of 1 μM PDTC for 24 h is shown with SD. Statistical analysis was performed by using Student's t test (*P < 0.01) by comparing values from PDTC-treated cells with control values for each SOD deletion. Results are representative of four independent experiments.

Mentions: In order to confirm the real PCR data and to localize the DNA element involved in the transcriptional repression of the SOD1 gene by PDTC, we used different SOD1 truncated reporter constructs linked to luciferase gene (Fig 2A) to transfect the U937 cells. As shown in Fig 2B, constructs pGLS-1499 to -157 bp displayed basal promoter activity, which was inhibited by PDTC. Removal of the -157 to -71 region reduced drastically promoter basal activity (Fig 2B). PDTC effect was abolished when the promoter was deleted to -29 bp. This region contains binding sites for multiple transcription factors, including C/EBP, E2F, and Sp1/Egr-1 as previously reported [30]. The luciferase expression level from cell transfected with deletion -29 was similar to that obtained with the promoterless pGL2-Basic control vector, which was barely detectable. These data were confirmed using the human U2OS osteosarcoma cell line (data not shown).


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 repression of the SOD1 promoter from a 5′ deletion series linked to the luciferase gene in U937 cells.A; The 5′ boundaries of plasmids containing various truncations of the SOD1 promoter are shown with the C/EBP, Egr-1/Sp1 sites indicated. B; SOD1 constructs (1 μg) or promoter-less pGL2-Basic control vectors were transiently transfected into U937 cells as described under Experimental procedures. The relative luciferase activity expressed by each plasmid in the absence or presence of 1 μM PDTC for 24 h is shown with SD. Statistical analysis was performed by using Student's t test (*P < 0.01) by comparing values from PDTC-treated cells with control values for each SOD deletion. Results are representative of four independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0127571.g002: PDTC repression of the SOD1 promoter from a 5′ deletion series linked to the luciferase gene in U937 cells.A; The 5′ boundaries of plasmids containing various truncations of the SOD1 promoter are shown with the C/EBP, Egr-1/Sp1 sites indicated. B; SOD1 constructs (1 μg) or promoter-less pGL2-Basic control vectors were transiently transfected into U937 cells as described under Experimental procedures. The relative luciferase activity expressed by each plasmid in the absence or presence of 1 μM PDTC for 24 h is shown with SD. Statistical analysis was performed by using Student's t test (*P < 0.01) by comparing values from PDTC-treated cells with control values for each SOD deletion. Results are representative of four independent experiments.
Mentions: In order to confirm the real PCR data and to localize the DNA element involved in the transcriptional repression of the SOD1 gene by PDTC, we used different SOD1 truncated reporter constructs linked to luciferase gene (Fig 2A) to transfect the U937 cells. As shown in Fig 2B, constructs pGLS-1499 to -157 bp displayed basal promoter activity, which was inhibited by PDTC. Removal of the -157 to -71 region reduced drastically promoter basal activity (Fig 2B). PDTC effect was abolished when the promoter was deleted to -29 bp. This region contains binding sites for multiple transcription factors, including C/EBP, E2F, and Sp1/Egr-1 as previously reported [30]. The luciferase expression level from cell transfected with deletion -29 was similar to that obtained with the promoterless pGL2-Basic control vector, which was barely detectable. These data were confirmed using the human U2OS osteosarcoma cell line (data not shown).

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