Short interfering RNA-mediated silencing of glutaredoxin 2 increases the sensitivity of HeLa cells toward doxorubicin and phenylarsine oxide

Abstract

Glutaredoxin (Grx) belongs to the thioredoxin fold superfamily and catalyzes glutathione-dependent oxidoreductions. The recently discovered mitochondrial and nuclear Grx (Grx2) differs from the more abundant cytosolic Grx (Grx1) by its higher affinity toward S-glutathionylated proteins and by being a substrate for thioredoxin reductase. Here, we have successfully established a method to silence the expression of Grx2 in HeLa cells by using short interfering RNA to study its role in the cell. Cells with levels of Grx2 <3% of the control were dramatically sensitized to cell death induced by doxorubicin/adriamycin and phenylarsine oxide but did not show signs of a general increase in oxidative damage with respect to carbonylation and glutathionylation. The ED(50) for doxorubicin dropped from 40 to 0.7 microM and for phenylarsine oxide from 200 to 5 nM. However, no differences were detected after treatment with cadmium, a known inhibitor of Grx1. These results indicate a crucial role of Grx2 in the regulation of the mitochondrial redox status and regulation of cell death at the mitochondrial checkpoint.

Fig. 1.

Specific knockdown of Grx2. (A) Levels of Grx2 in cells treated with different siRNAs specific for Grx2 (TS1, TS15, and TS28). Untreated HeLa cells (control), cells transfected with an unspecific siRNA (scr), and cells treated with only transfectant (oligofectamin) were used as controls. The levels were determined by ELISA; 100% corresponds to 2.9 mg·ml^–1.(B) Kinetic of the decline in Grx2 levels transfected with siRNA-TS28 as determined by ELISA. (C) Immunocytochemical detection of Grx2 in HeLa cells (images 1 and 3) and mitochondrial staining with MitoTracker red (images 2 and 4). Images 1 and 2 show HeLa transfected with the unspecific siRNA-scr. Images 3 and 4 show cells transfected with the Grx2-specific siRNA-TS28. (D) Relative levels of Grx1 in cells treated with siRNA-scr and siRNA-TS28; 100% Grx1 corresponds to 80 ng·mg^–1.

Fig. 2.

Mitochondrial localization of Grx2 in HeLa cells. (A) Immunocytochemical detection of Grx2 in HeLa cells. (B) Mitochondrial staining with MitoTracker red.

Fig. 3.

Viability of untreated and Grx2 knockdown HeLa cells after treatment with Cd. Cell viability was determined by using Roche's cell proliferation and viability kit II. The cells were seeded to a density of 50% in 96-well plates (≈4 × 10³ cells per well) 24 h before treatment with Cd for 24 h. The curves represent the averages of four different experiments. ○ and straight lines, untreated HeLa cells; ▪ and dotted lines, HeLa cells at 4 days after transfection with the human Grx2-specific siRNA-TS28.

Fig. 4.

Viability of untreated, control, and Grx2 knockdown HeLa cells after treatment with Dox (A) and PAO (B). Cell viability was determined by using Roche's cell proliferation and viability kit II, and the results were confirmed by the analysis of neutral red accumulation in the cells (Inset). The cells were seeded to a density of 50% in 96-well plates (≈4 × 10³ cells per well) 24 h before treatment with Dox or PAO for another 24 h. The curves represent the averages of four different experiments. ○ and straight lines, untreated HeLa cells. HeLa cells 4 days after transfection with an unspecific siRNA (• and dashed lines). HeLa cells 4 days after transfection with the human Grx2-specific siRNA-TS28 (▪ and dotted lines).

Fig. 5.

Levels of Grx2 in HeLa cells after treatment with PAO, Dox, BSO, and Cd. HeLa cells at a density of 50–70% were treated for 24 h with 0.1 nM PAO, 10 μM Dox, 100 μM BSO, or 20 μM Cd. The levels of Grx2 were determined by ELISA. The SDs are indicated. Significance of the difference between the means of the control group and the individual test group was calculated by using Student's t test. The following number of experiments were performed: n = 12, control group; n = 3, PAO, Dox, BSO, and Cd. No, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005.

Fig. 6.

Oxidative modifications of proteins in untreated, control, and Grx2 knockdown HeLa cells. Protein carbonylation (A) and glutathionylation (B and C) in HeLa cells, HeLa cells transfected with an unspecific siRNA (scr), and HeLa cells transfected with the human Grx2-specific siRNA-TS28 (TS28). (A) Dot blot of total cell extracts after treatment with 0.2 μM Dox for 24 h. (B) Western blot of cell extracts (40 μg per sample) by using antiglutathione moiety Abs specific for protein-mixed disulfides with glutathione. Lane 1, Untreated HeLa cells; lane 2, transfected with an unspecific siRNA; lane 3, lane 2 treated with 25 μM Dox for 1 h; lane 4, transfected with human Grx2-specific siRNA-TS28; lane 5, lane 4 treated with 25 μM Dox for 1 h, and lane 6, untreated HeLa cells. (C) Dot blot of total cell extracts (10 μg per sample). Treatment with 50 μM diamide for 1 h was used as positive control. The same extract treated with 10 mM DTT for 30 min was used as negative control. siRNA-TS28-transfected cells were additionally treated with 25 μM Dox for 1 h before harvesting.