Sodium selenite induces apoptosis by generation of superoxide via the mitochondrial-dependent pathway in human prostate cancer cells

Abstract

Purpose: Studies have demonstrated that selenium supplementation reduces the incidence of cancer, particularly prostate cancer. Evidence from experimental studies suggests that apoptosis is a key event in cancer chemoprevention by selenium and reactive oxygen species play a role in induction of apoptosis by selenium compounds. The current study was designed to investigate the role of superoxide and mitochondria in selenite-induced apoptosis in human prostate cancer cells.

Methods: LNCaP cells were transduced with adenoviral constructs to overexpress four primary antioxidant enzymes: manganese superoxide dismutase (MnSOD), copper-zinc superoxide dismutase (CuZnSOD), catalase (CAT), or glutathione peroxidase 1 (GPx1). Cell viability, apoptosis, and superoxide production induced by sodium selenite were analyzed by the MTT assay, chemiluminescence, flow cytometry, western blot analysis, and Hoechst 33342 staining following overexpression of these antioxidant enzymes.

Results: Our study shows the following results: (1) selenite induced cancer cell death and apoptosis by producing superoxide radicals; (2) selenite-induced superoxide production, cell death, and apoptosis were inhibited by overexpression of MnSOD, but not by CuZnSOD, CAT, or GPx1; and (3) selenite treatment resulted in a decrease in mitochondrial membrane potential, release of cytochrome c into the cytosol, and activation of caspases 9 and 3, events that were suppressed by overexpression of MnSOD.

Conclusions: This study demonstrates that selenite induces cell death and apoptosis by production of superoxide in mitochondria and activation of the mitochondrial apoptotic pathway and MnSOD plays an important role in protection against prooxidant effects of superoxide from selenite. The data suggest that superoxide production in mitochondria is, at least in part, a key event in selenium-induced apoptosis in prostate cancer cells.

Fig. 1

Effect of selenite on viability and growth in LNCaP cells: a effect of selenite on cell viability. Percentage of survival is expressed as cell viability relative to control without selenite treatment. Cells were treated with selenite for 5 days and viability was measured by the MTT assay. Data represent mean ± SD, n = 3. * P < 0.05 compared with control without selenite. b effect of selenite on cell growth in LNCaP cells. Cell numbers were counted using a Coulter counter. Data represent mean ± SD, n = 3. * P < 0.05 compared with control at the corresponding time points.

Fig. 2

Adenovirus-mediated overexpression of MnSOD, CuZnSOD, CAT or GPx1 in LNCaP cells. Cells were transduced with adenoviral constructs for 48 h and cell lysates were harvested for western and activity gel analyses. 1 Control (no adenoviral transduction); 2 50 MOI AdEmpty; 3 100 MOI AdEmpty; 4 50 MOI AdMnSOD, AdCuZnSOD, AdCAT, or AdGPx1; 5 100 MOI AdMnSOD, AdCuZnSOD, AdCAT, or AdGPx1. a Western blot analysis of expression of MnSOD, CuZnSOD, CAT and GPx1. Twenty micrograms of total cellular protein were loaded per lane. b Native gel analysis of MnSOD, CuZnSOD, CAT or GPx1 enzymatic activities. A total amount of 200 μg of protein was loaded per lane. Data presented are one representative experiment of three independent experiments that showed similar results.

Fig. 3

Effect of overexpression of AEs on selenite-induced production of superoxide and viability in LNCaP cells. a Levels of selenite-induced superoxide production in LNCaP cells with overexpression of MnSOD, CuZnSOD, CAT, or GPx1. Cells were pretreated with 2.5 μM selenite for 6 min and then harvested for chemiluminescence assay using a luminometer after transduction with 50 MOI AdEmpty, AdMnSOD, AdCuZnSOD, AdCAT, or AdGPx1 for 48 h. The data were obtained from three independent experiments and shown as means ± SD. *P < 0.05 compared with control cells without selenite treatment. b Levels of superoxide in cells transduced with AdEmpty or AdMnSOD. After transduction with 50 MOI AdEmpty or AdMnSOD for 48 h, cells were harvested in suspension and then incubated with 2.5 μM selenite and 5 μM DHE for 5 min. DHE fluorescence was detected using a flow cytometer. c MTT assay of cell viability. Cells were plated in 96-well plates overnight, transduced with 50 MOI AdEmpty, AdMnSOD, AdCuZnSOD, AdCAT, or AdGPx1 for 48 h, and then treated with different concentrations of selenite for an additional 5 days. Data are presented as means ± SD of three independent experiments. * P < 0.05 compared with AdEmpty at the corresponding concentrations of selenite. d MTT assay of cell viability. Cells were grown in 96-well plates overnight, transduced with 50 MOI AdEmpty, AdMnSOD, or AdCAT for 48 h, and then treated with 50 μM H₂O₂ for 24 h. Data are presented as means ± SD of three independent experiments. * P < 0.05 compared with control cells without H₂O₂ treatment.

Fig. 4

Inhibition of selenite-induced apoptosis by overexpression of MnSOD. a Apoptosis detected by Hoechst 33342 staining. Cells were transduced with 50 MOI AdEmpty or AdMnSOD for 48 h and then treated with 2.5 μM selenite for 18 h. Cells were stained with Hoechst dye and apoptosis was analyzed using a fluorescence microscope. At least 300 nuclei were counted per sample in triplicate. Data are presented as means ± SD of three independent experiments. * P < 0.05 compared with cells without selenite treatment. ** P < 0.05 compared with control and AdEmpty-transduced cells with selenite treatment. b Sub-G₁ cell population measurement by flow cytometry for apoptosis. After transduction with 50 MOI AdEmpty or AdMnSOD for 48 h, cells were treated with 2.5 μM selenite for 24 h and harvested in suspension. After fixation, cell suspensions were stained with propidium iodide and sub-G₁ cell populations were measured using a flow cytometer.

Fig. 5

Effects of selenite on the mitochondrial membrane potential and caspases 9 and 3. a Fluorescence analysis of mitochondrial membrane potential. b Chemiluminescence analysis of caspase 9 activity. c Fluorescence analysis of caspase 3 activity. LNCaP cells were treated with 2.5 μM selenite for the times indicated. The mitochondrial membrane potential was measured using a flow cytometer. Casapases 9 and 3 were measured using a luminometer or a fluorescence microplate reader, respectively. Data are presented as means ± SD (n = 3). * P < 0.05 compared to cells without selenite treatment (time zero).

Fig. 6

Protection of mitochondrial damage from selenite by overexpression of MnSOD in LNCaP cells. a Fluorescence analysis of the mitochondrial membrane potential by flow cytometry. Cells were transduced with 50 MOI AdEmpty or AdMnSOD for 48 hr and then treated with 2.5 μM selenite for 18 h. * P < 0.05 compared with AdEmpty control and both AdMnSOD control and selenite. b Western blot analysis of cytochrome c release into the cytosol. Cells were transduced with 50 MOI AdEmpty or AdMnSOD for 48 h and then treated with 2.5 μM selenite for 18 h.

Fig. 7

Inhibition of activation of caspases 3 and 9 by selenite by overexpression of MnSOD in LNCaP cells. a Chemiluminescence analysis of caspase 9 activity. Cells were transduced with 50 MOI AdEmpty or AdMnSOD for 48 h and then treated with 2.5 μM selenite for 18 h. Twenty micrograms of total protein from LNCaP cells with or without AdEmpty or AdMnSOD transduction were mixed with the caspase-9 analysis reagent in 96-well plates. Chemiluminescence was measured using a luminometer. Each bar represents the average of three wells. Data are presented as means ± SD of three independent experiments. * P < 0.05 compared with cells without selenite treatment and AdMnSOD with selenite treatment. b Fluorescence analysis of caspase 3 activity. Cells were transduced with 50 MOI AdEmpty or AdMnSOD for 48 h and then treated with 2.5 μM selenite for 18 h. Fluorescence was measured using a fluorescence microplate reader. Data are presented as means ± SD of three independent experiments. * P < 0.05 compared with AdEmpty without selenite. ** P < 0.05 compared with AdEmpty without selenite and AdMnSOD with selenite.