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.
Highlights
Increases in reactive oxygen species (ROS) production and defects in the ROS-removing enzymatic system can cause serious damage to cells [1]
We investigated whether Pyrrolidine dithiocarbamate (PDTC)-induced ROS generation could be linked to a decrease in the anti-oxidant gene superoxide dismutase 1 (SOD1)
U937 human myeloid leukemia cells were treated with different doses of PDTC and SOD1 mRNA levels were determined by real- time PCR analysis
Summary
Increases in reactive oxygen species (ROS) production and defects in the ROS-removing enzymatic system can cause serious damage to cells [1]. Over-expression of Cu/Zn-SOD in mice blunted ethanol-induced activation of redox-sensitive transcription factor AP-1 and production of TNF-α and interleukin-6 [5]. SOD expression suppressed JNK ( called stress-activated protein kinase, SAPK) and p38 phosphorylation and attenuated intracellular superoxide anion production and NADPH oxidase activity in TNF-α-treated cells [6]. JNK family members belong to the mitogen-activated protein kinase (MAPK) super family including extracellular signal-regulated kinases (ERKs) and the p38-MAPK family [7, 8]. The JNK pathway responds to diverse stimuli including mitogens, pro-inflammatory cytokines and environmental stresses [7,8,9]. Activation of JNK signal pathway by IL-1β and TNF-α in inflammatory conditions has been shown to require ROS as a signaling intermediate [10]
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