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Thiopurines induce oxidative stress in T-lymphocytes: a proteomic approach.

Misdaq M, Ziegler S, von Ahsen N, Oellerich M, Asif AR - Mediators Inflamm. (2015)

Bottom Line: We used a TPMT knockdown (kd) model of human Jurkat T-lymphocytes cells to study the effects of treatment with 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) on proteome and phosphoproteome.Three proteins (THIO, TXD17, and GSTM3) with putative functions in cellular oxidative stress responses were altered by 6-TG treatment and another protein PRDX3 was differentially phosphorylated in TPMT kd cells.Immunoblot analyses showed treatment altered expression of key antioxidant enzymes (i.e., SOD2 and catalase) in both wt and kd groups, while SOD1 was downregulated by 6-TG treatment and TPMT knockdown.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Chemistry/UMG Laboratories, University Medical Centre Goettingen, 37075 Goettingen, Germany.

ABSTRACT
Thiopurines are extensively used immunosuppressants for the treatment of inflammatory bowel disease (IBD). The polymorphism of thiopurine S-methyltransferase (TPMT) influences thiopurine metabolism and therapy outcome. We used a TPMT knockdown (kd) model of human Jurkat T-lymphocytes cells to study the effects of treatment with 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) on proteome and phosphoproteome. We identified thirteen proteins with altered expression and nine proteins with altered phosphorylation signals. Three proteins (THIO, TXD17, and GSTM3) with putative functions in cellular oxidative stress responses were altered by 6-TG treatment and another protein PRDX3 was differentially phosphorylated in TPMT kd cells. Furthermore, reactive oxygen species (ROS) assay results were consistent with a significant induction of oxidative stress by both TPMT knockdown and thiopurine treatments. Immunoblot analyses showed treatment altered expression of key antioxidant enzymes (i.e., SOD2 and catalase) in both wt and kd groups, while SOD1 was downregulated by 6-TG treatment and TPMT knockdown. Collectively, increased oxidative stress might be a mechanism involved in thiopurine induced cytotoxicity and adverse effects (i.e., hepatotoxicity) and an antioxidant cotherapy might help to combat this. Results highlight the significance of oxidative stress in thiopurines' actions and could have important implications for the treatment of IBD patients.

No MeSH data available.


Related in: MedlinePlus

ROS assay after 6-MP or 6-TG treatment (a) and differential expression of ROS related SOD1, SOD2, and CAT proteins (b). (a) Cells were treated with vehicle DMSO or IC60 doses of 6-MP or 6-TG for 48 h. After treatment media were removed and the cells were resuspended in PBS containing 10 μmol/L DCFDA. Fluorescence intensity was measured after 1 h incubation at 37°C. The error bars represent mean ± SD of four independent experiments (in triplicate format each). *P ≤ 0.05; ***P ≤ 0.0005. For expressional regulation of ROS related proteins, total protein lysates from DMSO or 6-MP or 6-TG treated Jurkat wt and kd cells were separated by 1D gel electrophoresis and immunoblotted with antibody against SOD1, SOD2, and CAT (b). Beta-actin was used as a loading control.
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fig2: ROS assay after 6-MP or 6-TG treatment (a) and differential expression of ROS related SOD1, SOD2, and CAT proteins (b). (a) Cells were treated with vehicle DMSO or IC60 doses of 6-MP or 6-TG for 48 h. After treatment media were removed and the cells were resuspended in PBS containing 10 μmol/L DCFDA. Fluorescence intensity was measured after 1 h incubation at 37°C. The error bars represent mean ± SD of four independent experiments (in triplicate format each). *P ≤ 0.05; ***P ≤ 0.0005. For expressional regulation of ROS related proteins, total protein lysates from DMSO or 6-MP or 6-TG treated Jurkat wt and kd cells were separated by 1D gel electrophoresis and immunoblotted with antibody against SOD1, SOD2, and CAT (b). Beta-actin was used as a loading control.

Mentions: Based on these findings, we further assessed cellular oxidative stress using ROS assays (Figure 2(a)). Intensity of fluorescence generated by incubating treated and control cells with the fluorescence dye DCFDA was measured and normalized to total cell number. The results showed significantly increased fluorescence intensity in wt and kd cells as a result of 6-MP and 6-TG treatment compared to their DMSO treated controls. All TPMT knockdown groups showed higher ROS levels compared to their wild-type counterparts. In order to see the effects of ROS accumulation on cellular antioxidant machinery, we then measured the alterations in the expression of three important antioxidant enzymes, that is, superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), and catalase (CAT) by Western blot analysis (Figure 2(b)).


Thiopurines induce oxidative stress in T-lymphocytes: a proteomic approach.

Misdaq M, Ziegler S, von Ahsen N, Oellerich M, Asif AR - Mediators Inflamm. (2015)

ROS assay after 6-MP or 6-TG treatment (a) and differential expression of ROS related SOD1, SOD2, and CAT proteins (b). (a) Cells were treated with vehicle DMSO or IC60 doses of 6-MP or 6-TG for 48 h. After treatment media were removed and the cells were resuspended in PBS containing 10 μmol/L DCFDA. Fluorescence intensity was measured after 1 h incubation at 37°C. The error bars represent mean ± SD of four independent experiments (in triplicate format each). *P ≤ 0.05; ***P ≤ 0.0005. For expressional regulation of ROS related proteins, total protein lysates from DMSO or 6-MP or 6-TG treated Jurkat wt and kd cells were separated by 1D gel electrophoresis and immunoblotted with antibody against SOD1, SOD2, and CAT (b). Beta-actin was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4385670&req=5

fig2: ROS assay after 6-MP or 6-TG treatment (a) and differential expression of ROS related SOD1, SOD2, and CAT proteins (b). (a) Cells were treated with vehicle DMSO or IC60 doses of 6-MP or 6-TG for 48 h. After treatment media were removed and the cells were resuspended in PBS containing 10 μmol/L DCFDA. Fluorescence intensity was measured after 1 h incubation at 37°C. The error bars represent mean ± SD of four independent experiments (in triplicate format each). *P ≤ 0.05; ***P ≤ 0.0005. For expressional regulation of ROS related proteins, total protein lysates from DMSO or 6-MP or 6-TG treated Jurkat wt and kd cells were separated by 1D gel electrophoresis and immunoblotted with antibody against SOD1, SOD2, and CAT (b). Beta-actin was used as a loading control.
Mentions: Based on these findings, we further assessed cellular oxidative stress using ROS assays (Figure 2(a)). Intensity of fluorescence generated by incubating treated and control cells with the fluorescence dye DCFDA was measured and normalized to total cell number. The results showed significantly increased fluorescence intensity in wt and kd cells as a result of 6-MP and 6-TG treatment compared to their DMSO treated controls. All TPMT knockdown groups showed higher ROS levels compared to their wild-type counterparts. In order to see the effects of ROS accumulation on cellular antioxidant machinery, we then measured the alterations in the expression of three important antioxidant enzymes, that is, superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), and catalase (CAT) by Western blot analysis (Figure 2(b)).

Bottom Line: We used a TPMT knockdown (kd) model of human Jurkat T-lymphocytes cells to study the effects of treatment with 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) on proteome and phosphoproteome.Three proteins (THIO, TXD17, and GSTM3) with putative functions in cellular oxidative stress responses were altered by 6-TG treatment and another protein PRDX3 was differentially phosphorylated in TPMT kd cells.Immunoblot analyses showed treatment altered expression of key antioxidant enzymes (i.e., SOD2 and catalase) in both wt and kd groups, while SOD1 was downregulated by 6-TG treatment and TPMT knockdown.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Chemistry/UMG Laboratories, University Medical Centre Goettingen, 37075 Goettingen, Germany.

ABSTRACT
Thiopurines are extensively used immunosuppressants for the treatment of inflammatory bowel disease (IBD). The polymorphism of thiopurine S-methyltransferase (TPMT) influences thiopurine metabolism and therapy outcome. We used a TPMT knockdown (kd) model of human Jurkat T-lymphocytes cells to study the effects of treatment with 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) on proteome and phosphoproteome. We identified thirteen proteins with altered expression and nine proteins with altered phosphorylation signals. Three proteins (THIO, TXD17, and GSTM3) with putative functions in cellular oxidative stress responses were altered by 6-TG treatment and another protein PRDX3 was differentially phosphorylated in TPMT kd cells. Furthermore, reactive oxygen species (ROS) assay results were consistent with a significant induction of oxidative stress by both TPMT knockdown and thiopurine treatments. Immunoblot analyses showed treatment altered expression of key antioxidant enzymes (i.e., SOD2 and catalase) in both wt and kd groups, while SOD1 was downregulated by 6-TG treatment and TPMT knockdown. Collectively, increased oxidative stress might be a mechanism involved in thiopurine induced cytotoxicity and adverse effects (i.e., hepatotoxicity) and an antioxidant cotherapy might help to combat this. Results highlight the significance of oxidative stress in thiopurines' actions and could have important implications for the treatment of IBD patients.

No MeSH data available.


Related in: MedlinePlus