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Ets-1 regulates intracellular glutathione levels: key target for resistant ovarian cancer.

Verschoor ML, Singh G - Mol. Cancer (2013)

Bottom Line: Overexpression of Ets-1 was associated with decreased intracellular ROS, concomitantly with increased intracellular GSH, GPX antioxidant activity, and Sx(c)- transporter activity.Under basal conditions, inhibition of the transsulfuration pathway resulted in decreased GSH levels and GPX activity in all cell lines, whereas inhibition of Sx(c)- by sulfasalazine decreased GPX activity in Ets-1-expressing cells only.However, under oxidative stress the intracellular GSH levels decreased significantly in correlation with increased Ets-1 expression following sulfasalazine treatment.

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Affiliation: Department of Pathology and Molecular Medicine, McMaster University, 1280 Main Street W, Hamilton, Ontario 12943, Canada. singhg@mcmaster.ca.

ABSTRACT

Background: Ovarian cancer is characterized by high rates of metastasis and therapeutic resistance. Many chemotherapeutic agents rely on the induction of oxidative stress to cause cancer cell death, thus targeting redox regulation is a promising strategy to overcome drug resistance.

Methods: We have used a tetracycline-inducible Ets-1 overexpression model derived from 2008 ovarian cancer cells in the present study. To examine the role of Ets-1 in glutathione regulation we have measured intracellular reactive oxygen species and glutathione levels, as well as glutathione peroxidase enzyme activity. Glutathione synthesis was limited using transsulfuration or Sx(c)- pathway blocking agents, and glutamate release was measured to confirm Sx(c)- blockade. Cell viability following drug treatment was assessed via crystal violet assay. Oxidative stress was induced through glucose oxidase treatment, which produces hydrogen peroxide by glucose oxidation. The protein expressions of redox-related factors were measured through western blotting.

Results: Overexpression of Ets-1 was associated with decreased intracellular ROS, concomitantly with increased intracellular GSH, GPX antioxidant activity, and Sx(c)- transporter activity. Under basal conditions, inhibition of the transsulfuration pathway resulted in decreased GSH levels and GPX activity in all cell lines, whereas inhibition of Sx(c)- by sulfasalazine decreased GPX activity in Ets-1-expressing cells only. However, under oxidative stress the intracellular GSH levels decreased significantly in correlation with increased Ets-1 expression following sulfasalazine treatment.

Conclusions: In this study we have identified a role for proto-oncogene Ets-1 in the regulation of intracellular glutathione levels, and examined the effects of the anti-inflammatory drug sulfasalazine on glutathione depletion using an ovarian cancer cell model. The findings from this study show that Ets-1 mediates enhanced Sx(c)- activity to increase glutathione levels under oxidative stress, suggesting that Ets-1 could be a promising putative target to enhance conventional therapeutic strategies.

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Ets-1 decreases intracellular ROS, while increasing intracellular GSH and GPX activity. A) Basal intracellular ROS levels were measured in 2008, 2008-Ets1 (Ets-1), and tetracycline-induced 2008-Ets1 (Ets-1 + Tet) cells using CM2-H2DCFDA. There is a trend for decreased ROS levels in correlation with increased Ets-1 expression (n = 3). B) Total intracellular amounts of GSH were measured using an enzymatic recycling method as described by Rahman et al. (12). Metabolite extracts from 2008, Ets-1 and induced Ets-1 cells contained increased GSH levels concomitantly with increased Ets-1 expression (n = 4). C) Glutathione peroxidase activity also displayed a trend towards increased activity in association with higher levels of Ets-1 expression (n = 4).
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Figure 1: Ets-1 decreases intracellular ROS, while increasing intracellular GSH and GPX activity. A) Basal intracellular ROS levels were measured in 2008, 2008-Ets1 (Ets-1), and tetracycline-induced 2008-Ets1 (Ets-1 + Tet) cells using CM2-H2DCFDA. There is a trend for decreased ROS levels in correlation with increased Ets-1 expression (n = 3). B) Total intracellular amounts of GSH were measured using an enzymatic recycling method as described by Rahman et al. (12). Metabolite extracts from 2008, Ets-1 and induced Ets-1 cells contained increased GSH levels concomitantly with increased Ets-1 expression (n = 4). C) Glutathione peroxidase activity also displayed a trend towards increased activity in association with higher levels of Ets-1 expression (n = 4).

Mentions: To examine the role of Ets-1 in the regulation of cellular antioxidant capacity, we measured intracellular ROS levels, total cellular GSH, and GPX enzyme activity. Intracellular ROS was measured using CM2-H2DCFDA in 2008, 2008-Ets1, and induced 2008-Ets1 cells (Figure 1A). The amount of ROS present in induced 2008-Ets1 cells (1264.4 AFU) was significantly less than their parental 2008 cells (1885.5 AFU), while non-induced 2008-Ets1 cells were not significantly different. Quantitative total GSH was measured via colourimetric assay under basal conditions, and both 2008-Ets1 and induced 2008-Ets1 cells showed increased amounts of total GSH relative to 2008 cells (11.11- and 22.14-fold respectively) (Figure 1B). GPX enzyme activity was assessed by measure of the oxidation of NADPH, and induced 2008-Ets1 cells were found to have a higher GPX activity rate (1.96-fold) than 2008 cells (Figure 1C).


Ets-1 regulates intracellular glutathione levels: key target for resistant ovarian cancer.

Verschoor ML, Singh G - Mol. Cancer (2013)

Ets-1 decreases intracellular ROS, while increasing intracellular GSH and GPX activity. A) Basal intracellular ROS levels were measured in 2008, 2008-Ets1 (Ets-1), and tetracycline-induced 2008-Ets1 (Ets-1 + Tet) cells using CM2-H2DCFDA. There is a trend for decreased ROS levels in correlation with increased Ets-1 expression (n = 3). B) Total intracellular amounts of GSH were measured using an enzymatic recycling method as described by Rahman et al. (12). Metabolite extracts from 2008, Ets-1 and induced Ets-1 cells contained increased GSH levels concomitantly with increased Ets-1 expression (n = 4). C) Glutathione peroxidase activity also displayed a trend towards increased activity in association with higher levels of Ets-1 expression (n = 4).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3842663&req=5

Figure 1: Ets-1 decreases intracellular ROS, while increasing intracellular GSH and GPX activity. A) Basal intracellular ROS levels were measured in 2008, 2008-Ets1 (Ets-1), and tetracycline-induced 2008-Ets1 (Ets-1 + Tet) cells using CM2-H2DCFDA. There is a trend for decreased ROS levels in correlation with increased Ets-1 expression (n = 3). B) Total intracellular amounts of GSH were measured using an enzymatic recycling method as described by Rahman et al. (12). Metabolite extracts from 2008, Ets-1 and induced Ets-1 cells contained increased GSH levels concomitantly with increased Ets-1 expression (n = 4). C) Glutathione peroxidase activity also displayed a trend towards increased activity in association with higher levels of Ets-1 expression (n = 4).
Mentions: To examine the role of Ets-1 in the regulation of cellular antioxidant capacity, we measured intracellular ROS levels, total cellular GSH, and GPX enzyme activity. Intracellular ROS was measured using CM2-H2DCFDA in 2008, 2008-Ets1, and induced 2008-Ets1 cells (Figure 1A). The amount of ROS present in induced 2008-Ets1 cells (1264.4 AFU) was significantly less than their parental 2008 cells (1885.5 AFU), while non-induced 2008-Ets1 cells were not significantly different. Quantitative total GSH was measured via colourimetric assay under basal conditions, and both 2008-Ets1 and induced 2008-Ets1 cells showed increased amounts of total GSH relative to 2008 cells (11.11- and 22.14-fold respectively) (Figure 1B). GPX enzyme activity was assessed by measure of the oxidation of NADPH, and induced 2008-Ets1 cells were found to have a higher GPX activity rate (1.96-fold) than 2008 cells (Figure 1C).

Bottom Line: Overexpression of Ets-1 was associated with decreased intracellular ROS, concomitantly with increased intracellular GSH, GPX antioxidant activity, and Sx(c)- transporter activity.Under basal conditions, inhibition of the transsulfuration pathway resulted in decreased GSH levels and GPX activity in all cell lines, whereas inhibition of Sx(c)- by sulfasalazine decreased GPX activity in Ets-1-expressing cells only.However, under oxidative stress the intracellular GSH levels decreased significantly in correlation with increased Ets-1 expression following sulfasalazine treatment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Molecular Medicine, McMaster University, 1280 Main Street W, Hamilton, Ontario 12943, Canada. singhg@mcmaster.ca.

ABSTRACT

Background: Ovarian cancer is characterized by high rates of metastasis and therapeutic resistance. Many chemotherapeutic agents rely on the induction of oxidative stress to cause cancer cell death, thus targeting redox regulation is a promising strategy to overcome drug resistance.

Methods: We have used a tetracycline-inducible Ets-1 overexpression model derived from 2008 ovarian cancer cells in the present study. To examine the role of Ets-1 in glutathione regulation we have measured intracellular reactive oxygen species and glutathione levels, as well as glutathione peroxidase enzyme activity. Glutathione synthesis was limited using transsulfuration or Sx(c)- pathway blocking agents, and glutamate release was measured to confirm Sx(c)- blockade. Cell viability following drug treatment was assessed via crystal violet assay. Oxidative stress was induced through glucose oxidase treatment, which produces hydrogen peroxide by glucose oxidation. The protein expressions of redox-related factors were measured through western blotting.

Results: Overexpression of Ets-1 was associated with decreased intracellular ROS, concomitantly with increased intracellular GSH, GPX antioxidant activity, and Sx(c)- transporter activity. Under basal conditions, inhibition of the transsulfuration pathway resulted in decreased GSH levels and GPX activity in all cell lines, whereas inhibition of Sx(c)- by sulfasalazine decreased GPX activity in Ets-1-expressing cells only. However, under oxidative stress the intracellular GSH levels decreased significantly in correlation with increased Ets-1 expression following sulfasalazine treatment.

Conclusions: In this study we have identified a role for proto-oncogene Ets-1 in the regulation of intracellular glutathione levels, and examined the effects of the anti-inflammatory drug sulfasalazine on glutathione depletion using an ovarian cancer cell model. The findings from this study show that Ets-1 mediates enhanced Sx(c)- activity to increase glutathione levels under oxidative stress, suggesting that Ets-1 could be a promising putative target to enhance conventional therapeutic strategies.

Show MeSH
Related in: MedlinePlus