Increased SBP1 protein levels and SBP1-dependent retention of intracellular selenium content after selenomethionine treatment in HeLa cells.

<p>(A) Intracellular selenium concentration was determined in scrambled and SBP1 shRNA HeLa cells treated with a gradient concentration of selenomethionine (0–5 μM, 3 h). Values are means ± SEM (n = 3). *, p< 0.05, compared to SBP1 shRNA HeLa cells. (B) Western analyses of SBP1 and β-tubulin in HeLa cells treated with a gradient concentration of selenomethionine (0–5 μM, 24 h).</p

Knockdown of SBP1 desensitized HeLa cells to three clastogens and increased GPX1 protein levels.

(A) Efficacy of SBP1 shRNA knockdown in two clones of HeLa cells as evidenced by Western analysis. (B) Effect of SBP1 knockdown on the protein levels of GPX1 and GPX4 by Western analysis. (C-E) Clonogenic assays were employed to determine sensitivity of scrambled and SBP1 shRNA HeLa cells to hydrogen peroxide (2–15 μM, C), paraquat (20–100 μM, D), and camptothecin (CPT, 5–200 μM, E) for 24 h, followed by a 7-day recovery and then crystal violet staining for detection of viable colonies. Values are means ± SEM (n = 3). *, p< 0.05 and **, p< 0.01, compared to scrambled shRNA HeLa cells.</p

Synergistic effect of MSeA and carboplatin on the killing of OVCA429/NICD3 cells.

<p>OVCA429/pCEG and OVCA429/NICD3 cancer cells were treated with a gradient concentration of MSeA (<i>A</i>) or carboplatin (<i>B</i>) for 2 days. *, <i>p</i> < 0.05, compare to OVCA429/pCEG cells. OVCA429/pCEG cells (<i>C</i>) and OVCA429/NICD3 cells (<i>D</i>) were treated with carboplatin (0–25 µmol/L) in the absence or presence of MSeA (2 µmol/L) for 2 days. Values are mean ± S.E.M. (n  =  3). Dashed lines predict the additive effect of MSeA and carboplatin.</p

Knockdown of SBP1 promotes HeLa cell migration.

<p>(A) Cells cultured in DMEM supplemented with 0.2% FBS were made the wound on cell culture dishes and visualized at 0–72 h. (B) The wound distance was semi-quantified by randomly selecting the shortest distance between the two patches of cells. Values are means ± SEM (n = 3).*, <i>p</i>< 0.05, compared to scrambled shRNA cells.</p

Knockdown of SBP1 decreases ROS levels in HeLa cells before and after treatment with hydrogen peroxide.

<p>Scrambled and SBP1 shRNA HeLa cells were cultured in the presence or absence of hydrogen peroxide (100 μM, 3 h), followed by incubation with CM-H2DCFDA (5 μM, 15 min, 37°C) and acquisition of FITC signal through a fluorescence microscope. (A) Representative pictures as observed under a fluorescence microscope. (B) FITC signals were quantitated using the intensity tool of the Zeiss AxioObserver 100 software. Values are means ± SEM (n = 3) and differ when not sharing a common letter (p< 0.05).</p

Combination index (CI) values for MSeA and carboplatin treatment in OVCA429/pCEG and OVCA429/NICD3 ovarian cancer cells.

<p>Based on a refined description made by an inventor of the theorem of Chou-Talalay, the following descriptions of CI values are employed: <0.3, strong synergism; 0.3–0.7, synergism; 0.7–0.9, moderate or slight synergism; 0.9–1.1, nearly additive; 1.1–1.45, slight or moderate antagonism; 1.45–3.3, antagonism; >3.3, strong antagonism <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101664#pone.0101664-Chou1" target="_blank">[37]</a>. Cell viability and treatment are as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101664#pone-0101664-t001" target="_blank">Table 1</a>.</p

Sensitivity of OVCA429/pCEG and OVCA429/NICD3 ovarian cancer cells to MSeA and carboplatin treatment.

<p>Cells were cultured in 96-well plates and treated with MSeA and carboplatin at the indicated concentration for 2 days. Cell viability was determined by SRB assay. The condition without MSeA or carboplatin treatment was set as 100%. Values are mean ± S.E.M. (n  =  3). ^#, <i>p</i> < 0.05, compared to no MSeA treatment. *, <i>p</i> < 0.05, compared to no carboplatin treatment.</p

Role for p53 in Selenium-Induced Senescence

The tumor suppressor p53 and the ataxia-telangiectasia mutated (ATM) kinase play important roles in the senescence response to oncogene activation and DNA damage. It was previously shown that selenium-containing compounds can activate an ATM-dependent senescence response in MRC-5 normal fibroblasts. Here, the shRNA knockdown approach and other DNA damage assays are employed to test the hypothesis that p53 plays a role in selenium-induced senescence. In MRC-5 cells treated with methylseleninic acid (MSeA, 0–10 μM), depletion of p53 hampers senescence-associated expression of β-galactosidase, disrupts the otherwise S and G2/M cell cycle arrest, desensitizes such cells to MSeA treatment, and increases genome instability. Pretreatment with KU55933, an ATM kinase inhibitor, or NU7026, an inhibitor of DNA-dependent protein kinase, desensitizes MSeA cytotoxicity in scrambled but not p53 shRNA MRC-5 cells. These results suggest that p53 is critical for senescence induction in the response of MRC-5 noncancerous cells to selenium compounds

The effect of NAC on the sensitivity of OVCA429/pCEG and OVCA429/NICD3 cells to MSeA and carboplatin co-treatment.

<p>OVCA429/pCEG (<i>A</i>, <i>C</i>) and OVCA429/NICD3 (<i>B</i>, <i>D</i>) cells were treated with MSeA and a gradient concentration of carboplatin (<i>A</i>, <i>B</i>) or carboplatin and a gradient concentration of MSeA (<i>C</i>, <i>D</i>) in the presence or absence of NAC. Cell viability was determined by SRB assay. Viability of the cells without carboplatin (<i>A</i>, <i>B</i>) or MSeA (<i>C</i>, <i>D</i>) treatment was set as 100%. Values are mean ± S.E.M. (n  =  3). *, <i>p</i> < 0.05, compared to MSeA or carboplatin only treatment.</p

Flow cytometric analyses of the percent G1, S, and G2/M OVCA429/pCEG and OVCA429/NICD3 cells co-treated with MSeA (2 µmol/L) and carboplatin (5 µmol/L) for 1 or 2 days.

<p>Values are mean ± S.E.M. (n  =  3). *, <i>p</i> < 0.05, compared to OVCA429/NICD3 cells. ^#, <i>p</i> < 0.05, compared to Day 0.</p