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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

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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fig4: 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.

Mentions: This is the first proteomics based study to investigate the effects of both thiopurine and differential TPMT activity on a cellular proteome. In both wild-type and knockdown cells, 6-MP and 6-TG treatment resulted in significant protein regulation. The regulated proteins identified were directly or indirectly involved in cellular responses to oxidative stress. Similarly, significant influence on cell cycle regulation and cytoskeleton reorganization mechanisms was identified. Taken together, these results suggest that thiopurine treatment can induce cytotoxicity by both DNA damage (a known mechanism of thiopurine action) and increased oxidative stress. Cell responses to these stresses include regulation of the expression and activity of important targets in the cell cycle and cytoskeleton systems. Both 6-MP and 6-TG affected the activity of antioxidant proteins which resulted in signs of increased oxidative stress and mitochondrial dysfunction, as well as cytoskeleton and cell cycle disturbances. These effects might contribute to thiopurine induced cytotoxicity in patients including myelotoxicity and hepatotoxicity (Figure 4). Protein targets with possible role in thiopurine induced oxidative stress and cytotoxicity were identified for the first time using proteomic and phosphoproteomic methods in both wild-type and knockdown human cell lymphocyte cell lines. Although the exact mechanism of these alterations is not known, further investigation may help to understand the complex mechanism of action of thiopurines or their prevention with antioxidant cotreatment in thiopurine treated patients.


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

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

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig4: 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.
Mentions: This is the first proteomics based study to investigate the effects of both thiopurine and differential TPMT activity on a cellular proteome. In both wild-type and knockdown cells, 6-MP and 6-TG treatment resulted in significant protein regulation. The regulated proteins identified were directly or indirectly involved in cellular responses to oxidative stress. Similarly, significant influence on cell cycle regulation and cytoskeleton reorganization mechanisms was identified. Taken together, these results suggest that thiopurine treatment can induce cytotoxicity by both DNA damage (a known mechanism of thiopurine action) and increased oxidative stress. Cell responses to these stresses include regulation of the expression and activity of important targets in the cell cycle and cytoskeleton systems. Both 6-MP and 6-TG affected the activity of antioxidant proteins which resulted in signs of increased oxidative stress and mitochondrial dysfunction, as well as cytoskeleton and cell cycle disturbances. These effects might contribute to thiopurine induced cytotoxicity in patients including myelotoxicity and hepatotoxicity (Figure 4). Protein targets with possible role in thiopurine induced oxidative stress and cytotoxicity were identified for the first time using proteomic and phosphoproteomic methods in both wild-type and knockdown human cell lymphocyte cell lines. Although the exact mechanism of these alterations is not known, further investigation may help to understand the complex mechanism of action of thiopurines or their prevention with antioxidant cotreatment in thiopurine treated patients.

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