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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

Gel-shift analysis of the effects of PDTC on nuclear factors bound to the AP-1 probe.EMSA experiments were performed using nuclear extracts from control (lane 1) and PDTC-treated U937 cells for 2 (lane 2) and 6 h (lane 3) and a 32P-labeled double-stranded AP-1 oligonucleotide as a probe. Competition experiments were performed by pre-incubating nuclear extracts from control cells with 100 x excess cold AP-1 double-stranded oligonucleotides (lane 4) or anti-AP-1 antibody (lane 5 & 6 are duplicates) for supershift prior incubation with labeled AP-1 probe addition. Results are representative of three independent experiments. Unspecific bands (ns)
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pone.0127571.g003: Gel-shift analysis of the effects of PDTC on nuclear factors bound to the AP-1 probe.EMSA experiments were performed using nuclear extracts from control (lane 1) and PDTC-treated U937 cells for 2 (lane 2) and 6 h (lane 3) and a 32P-labeled double-stranded AP-1 oligonucleotide as a probe. Competition experiments were performed by pre-incubating nuclear extracts from control cells with 100 x excess cold AP-1 double-stranded oligonucleotides (lane 4) or anti-AP-1 antibody (lane 5 & 6 are duplicates) for supershift prior incubation with labeled AP-1 probe addition. Results are representative of three independent experiments. Unspecific bands (ns)

Mentions: SOD1 expression is regulated by many transcription factors that can act as activators or inhibitors of SOD1 transcription. In fact, SOD1 proximal promoter (−157) contains several motifs that are homologous to known cis-acting regulatory elements. In particular, we have reported [30] that Sp1 transcription factor is an activator of SOD1 transcription, whereas AP-1 is involved in SOD1 repression. In particular, AP-1 inhibits SOD1 transcription by sequestrating essential co-activators, rather than binding to the SOD1 gene promoter [33]. Therefore, we investigated the binding activity of AP-1 by EMSA in nuclear protein extracts. Incubation of nuclear extracts from PDTC treated cells (Fig 3, lanes 2 and 3) with consensus AP-1 oligonucleotide resulted in a significant increase of retarded DNA-protein complex with respect to control cells (Fig 3, lane 1). The specificity of the binding was demonstrated using the appropriate competition assay with excess of AP-1 unlabeled probe (Fig 3, lane 4). In addition, AP-1 complex was completely supershifted by anti-AP-1 antibody (Fig 3, lanes 5 and 6), confirming the specificity of the binding. This result suggests that AP-1 is involved in PDTC negative effect on SOD1 promoter activity by interfering with the binding of other factors as we have previously suggested [30].


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)

Gel-shift analysis of the effects of PDTC on nuclear factors bound to the AP-1 probe.EMSA experiments were performed using nuclear extracts from control (lane 1) and PDTC-treated U937 cells for 2 (lane 2) and 6 h (lane 3) and a 32P-labeled double-stranded AP-1 oligonucleotide as a probe. Competition experiments were performed by pre-incubating nuclear extracts from control cells with 100 x excess cold AP-1 double-stranded oligonucleotides (lane 4) or anti-AP-1 antibody (lane 5 & 6 are duplicates) for supershift prior incubation with labeled AP-1 probe addition. Results are representative of three independent experiments. Unspecific bands (ns)
© Copyright Policy
Related In: Results  -  Collection

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

pone.0127571.g003: Gel-shift analysis of the effects of PDTC on nuclear factors bound to the AP-1 probe.EMSA experiments were performed using nuclear extracts from control (lane 1) and PDTC-treated U937 cells for 2 (lane 2) and 6 h (lane 3) and a 32P-labeled double-stranded AP-1 oligonucleotide as a probe. Competition experiments were performed by pre-incubating nuclear extracts from control cells with 100 x excess cold AP-1 double-stranded oligonucleotides (lane 4) or anti-AP-1 antibody (lane 5 & 6 are duplicates) for supershift prior incubation with labeled AP-1 probe addition. Results are representative of three independent experiments. Unspecific bands (ns)
Mentions: SOD1 expression is regulated by many transcription factors that can act as activators or inhibitors of SOD1 transcription. In fact, SOD1 proximal promoter (−157) contains several motifs that are homologous to known cis-acting regulatory elements. In particular, we have reported [30] that Sp1 transcription factor is an activator of SOD1 transcription, whereas AP-1 is involved in SOD1 repression. In particular, AP-1 inhibits SOD1 transcription by sequestrating essential co-activators, rather than binding to the SOD1 gene promoter [33]. Therefore, we investigated the binding activity of AP-1 by EMSA in nuclear protein extracts. Incubation of nuclear extracts from PDTC treated cells (Fig 3, lanes 2 and 3) with consensus AP-1 oligonucleotide resulted in a significant increase of retarded DNA-protein complex with respect to control cells (Fig 3, lane 1). The specificity of the binding was demonstrated using the appropriate competition assay with excess of AP-1 unlabeled probe (Fig 3, lane 4). In addition, AP-1 complex was completely supershifted by anti-AP-1 antibody (Fig 3, lanes 5 and 6), confirming the specificity of the binding. This result suggests that AP-1 is involved in PDTC negative effect on SOD1 promoter activity by interfering with the binding of other factors as we have previously suggested [30].

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