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. 2015:2015:434825.
doi: 10.1155/2015/434825. Epub 2015 Mar 22.

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

Misbah Misdaq  1 , Sonia Ziegler  1 , Nicolas von Ahsen  2 , Michael Oellerich  1 , Abdul R Asif  1
Affiliations

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

Misbah Misdaq et al. Mediators Inflamm. 2015.

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.

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Figures

Figure 1
Figure 1
Schematic representation of experimental system. (a) Silver stained 2-DE gel (Jurkat wt DMSO), (b) phosphor stained 2-DE gel (Jurkat wt DMSO), and (c) Jurkat wt and kd cells were treated with IC60 doses of 6-MP, 6-TG, and vehicle (DMSO) for 48 h. Total protein lysates from DMSO, 6-MP, and 6-TG treated Jurkat wt and kd cells were separated by 2-DE, followed by staining with phosphor specific and silver stain. Consistently regulated (circled) spots were identified by Q-TOF MS/MS analysis. Densitometric analysis of Jurkat wt and kd groups (a); DMSO treated, silver stained, and phosphor stained representative gels of Jurkat wt ((b) and (c)) cell lysates.
Figure 2
Figure 2
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.
Figure 3
Figure 3
Differential expression of STMN1 as shown by silver stained gels (a) and Western blot analysis (b). (a) STMN1 spot in all six groups: significant downregulation in wt and kd 6-MP treated groups. Graphical representation of spot density (% volume) with mean ± SD of five independent experiments (* P ≤ 0.05). (b) 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 STMN1. Densitometric analyses were performed using LabImage 2.71 software. Beta-actin was used as a loading control. The error bars represent mean ± SD of four independent experiments (* P ≤ 0.05).
Figure 4
Figure 4
Thiopurine induced oxidative stress and proteome regulation 6-MP and 6-TG treatment induced phosphorylation changes in GSTM3 and PRDX3 (redox regulators of cell) which can consequently reduce their ROS neutralization activity [32, 36]. Similarly, TPMT knockdown and thiopurine treatment downregulated expression of SOD1 which together with altered activity of GSTM3 and PRDX3 might result in enhanced ROS accumulation. ROS assays showed an increase in ROS level after 6-MP and 6-TG treatment. Increased ROS levels may cause mitochondrial dysfunction [59]. Persistent and increasing mitochondrial dysfunction can induce cellular cytotoxicity and apoptosis [59]. On the other hand, to cope with increasing oxidative stress, cells could activate their cellular antioxidant mechanisms [59] as suggested by the increased expression of the antioxidant proteins THIO [60], SOD2 [61], and CAT [39]. ROS accumulation also affects cytoskeleton [62], suggested by the altered cytoskeleton regulator proteins (expression of COF1 and PROF1 and phosphorylation of p-ARP2, p-COR1A) [30, 50, 52, 53]. Oxidative stress influences the cell cycle [63], and the observed reduced expression of STMN1 (a regulator of microtubule dynamics during meiosis) and decreased phosphorylation of PRS10 (involved in ATP-dependent degradation of ubiquitinated proteins) may be indicative of this phenomenon [43, 46]. We hypothesize that 6-MP and 6-TG treatment affect the activity of antioxidant proteins which results in increased oxidative stress and consequently mitochondrial dysfunction, as well as cytoskeleton and cell cycle disturbances which collectively contribute to thiopurine induced cytotoxicity.
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