UA dose-dependently induced cell death in human STS cells.

<p>Synovial sarcoma SW982 and leiomyosarcoma SK-UT-1 cells were treated with vehicle (DMSO) or UA (5–30 μM) for 24 h. DNA content of cells was measured by flow cytometry. Columns show percentage of apoptotic cells (sub-G1 phase) in the absence (vehicle) or presence of UA. Representative histograms of vehicle-treated and UA-treated (30 μM) SW982 cells are shown below.</p

UA induced intrinsic apoptosis in human STS cells.

<p>Synovial sarcoma SW982 and leiomyosarcoma SK-UT-1 cells were treated with vehicle (DMSO) or UA (5–30 μM) for 6, 9 and 24 h. <i>Upper panels</i> show immunoreactive bands of Bcl-2 and Bax proteins in representative immunoblots of synovial sarcoma SW982 cells treated with UA for 9 h. Columns represent the ratio of the Bax to Bcl-2 immunoreactivity in these cells treated with UA for 6 and 9 h. Each value represents mean ± SEM of 4 independent experiments normalized to vehicle-treated cells (taken as 100%). **<i>P</i> < 0.01 and ***<i>P</i> < 0.001 versus vehicle-treated cells. <i>Lower panels</i> show the immunoreactive bands of procaspase 9 and 3, caspase 9 and 3, and PARP fragmentation in representative immunoblots of synovial sarcoma SW982 (left) and leiomyosarcoma SK-UT-1 cells (right) treated with UA for 24 h.</p

UA down-regulated the AKT/GSK3β/β-catenin pathway in human STS cells.

<p>Synovial sarcoma SW982 cells were treated with vehicle (DMSO), TCN (30 μM) or UA (5–30 μM) for 6 and 9 h. <i>Upper panels</i> show representative immunoblots of SW982 cells treated with or without UA or TCN for 9 h. Columns represent the phosphorylated ratio of AKT and the ratio of P-GSK3β (ser9) or β-catenin to β-actin immunoreactivity. Each column represents mean ± SEM of 3 independent experiments normalized to vehicle-treated cells (taken as 100%). *<i>P</i> < 0.05, **<i>P</i> < 0.01 and ***<i>P</i> < 0.001 versus vehicle-treated cells.</p

UA enhanced the antitumoral effects of DXR in human STS cells while concomitantly down-regulating DXR-induced AKT phosphorylation and p21 protein levels.

<p>A) Synovial sarcoma SW982 (left) and leiomyosarcoma SK-UT-1 cells (right) were treated with vehicle (DMSO) or DXR (0.5, 1, 5 and 10 μM) alone or combined simultaneously with UA (10 μM for SW982 cells or 15 μM for SK-UT-1 cells) or TCN (30 μM) for 24 h. Cell viability was assessed as described in Materials and methods. Each value represent mean ± SEM of 3 independent experiments performed in triplicate and normalized to vehicle-treated cells (taken as 100%). *<i>P</i> < 0.05 and ***<i>P</i> < 0.001 versus UA-treated cells; ^###<i>P</i> < 0.001 versus DXR-treated cells. B) <i>Upper panels</i> show representative immunoblots of SW982 cells (left) or SK-UT-1 cells (right) treated with vehicle or DXR (1 and 10 μM) alone or in combination with UA for 9 h. Columns represent the phosphorylated ratio of AKT and the ratio of p21 to β-actin immunoreactivity. Each column represents mean ± SEM of 3 independent experiments normalized to vehicle-treated cells (taken as 100%). *<i>P</i> < 0.05, **<i>P</i> < 0.01 and ***<i>P</i> < 0.001 versus vehicle-treated cells; ^#<i>P</i> < 0.05, ^##<i>P</i> < 0.01 and ^###<i>P</i> < 0.001 versus DXR-treated cells.</p

UA down-regulated c-Myc and p21 protein levels in human STS cells.

<p>Synovial sarcoma SW982 and leiomyosarcoma SK-UT-1 cells were treated with vehicle (DMSO) or UA (5–30 μM) for 9 or 12 h, respectively. <i>Upper panels</i> show immunoreactive bands of c-Myc and p21 proteins in representative immunoblots of SW982 cells treated without or with UA for 9 h. Columns represent the ratio of the c-Myc to β-actin immunoreactivity in SW982 cells (left panel) or p21 to β-actin immunoreactivity in both cell types (right panel). Each value represents mean ± SEM of 3 independent experiments normalized to vehicle-treated cells (taken as 100%). **<i>P</i> < 0.01 and ***<i>P</i> < 0.001 versus vehicle-treated cells.</p

UA dose-dependently inhibited viability and proliferation in human STS cells.

<p>A) Fibrosarcoma HT-1080, leiomyosarcoma SK-UT-1 and synovial sarcoma SW982 cells were treated with vehicle (DMSO) or UA (0.1–50 μM) for 24 h. Cell viability was measured by MTT (upper left panel). SK-UT-1 and SW982 cells were cultured in soft agar in the absence (vehicle) or presence of UA (5–50 μM). Columns show only the number of soft agar colonies/field in SK-UT-1 cell cultures, as SW982 cells did not proliferate in semisolid medium (upper right panel). B) Representative images of SK-UT-1 and SW982 cells in the absence (0) or presence of UA (10–50 μM) after 24 h treatment. Magnification 200-fold. C) Representative images of soft agar colonies in SK-UT-1 and SW982 cell cultures in the absence (0) or presence of UA (10–50 μM) after 7 or 21 days, respectively. Magnification 40-fold. Inset bar: 100 μm.</p

Hazard Ratios (95% CI) for total mortality by quartiles of energy adjusted dietary glycemic index and dietary glycemic load assessed at baseline in non-diabetic subjects^a.

a<p> Abbreviations: Q, quartile; HR, hazard ratio; CI, confidence interval.</p>b<p> Adjusted for sex, age (years), recruitment center, intervention group (Med Diet with EVOO, Med Diet with Nuts, Low fat diet).</p>c<p> Adjusted for sex, age (years), recruitment center, intervention group (Med Diet + EVOO, Med Diet + Nuts, Low fat diet), smoking (never, past, current), education (low, middle, high, no data available), marital status (married, other), physical activity (continuous), BMI (continuous), self-reported history of cancer (yes, no), self-reported history of arterial hypertension (yes, no), total energy intake (continuous), alcohol intake (continuous) and dietary fiber intake (continuous, energy-adjusted).</p>d<p> Multivariate model with additional adjustment for intake of saturated fatty acids (continuous, energy-adjusted) and monounsaturated fatty acids (continuous, energy-adjusted).</p>e<p> Multivariate model with yearly updated measures of physical activity (continuous), BMI (continuous), total energy intake (continuous), alcohol intake (continuous), dietary fiber intake (continuous, energy-adjusted).</p>f<p> Multivariate model with yearly updated measures with additional adjustment for saturated fatty acids intake (continuous, energy-adjusted) and monounsaturated fatty acids intake (continuous, energy-adjusted).</p><p>Hazard Ratios (95% CI) for total mortality by quartiles of energy adjusted dietary glycemic index and dietary glycemic load assessed at baseline in non-diabetic subjects^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107968#nt104" target="_blank">a</a>}.</p

Fixed and random parameters from a multilevel linear regression model of GSH-Px activity.

<p>Abbreviations: EEPA, daily energy expenditure in leisure-time physical activity. PHCC, primary health care center.</p>a<p>Adjusted β, regression coefficient of the association between each variable in the model and GSH-Px activity, controlling for the other variables in the model.</p>b<p>Random parameters are multilevel measures of outcome variation. PHCC was considered as random.</p><p>Fixed and random parameters from a multilevel linear regression model of GSH-Px activity.</p

Participant characteristics by quintiles of serum glutathione peroxidase activity (U/L).

<p>Abbreviations: EEPA, daily energy expenditure in leisure-time physical activity. Data are shown as mean ±SD, median (interquartile range), or percentage.</p><p>* <i>P</i> value for trend adjusted for multiple testing using Holm correction <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105881#pone.0105881-Holm1" target="_blank">[24]</a>. One-factor analysis of variance, nonparametric Kruskal Wallis test, and chi-square test were used as appropriate.</p><p>Participant characteristics by quintiles of serum glutathione peroxidase activity (U/L).</p

Sensitivity analysis. Hazard Ratios (95% CI) for total mortality according to baseline quartiles of energy adjusted dietary glycemic index and dietary glycemic load^a.

a<p> Abbreviations: Q, quartile; HR, hazard ratio; CI, confidence interval; BMI, Body Mass Index.</p>b<p> Model adjusted for sex, age (years), recruitment center, intervention group (Med Diet + EVOO, Med Diet + Nuts, Low fat diet), smoking (never, past, current), education (low, middle, high, no data available), marital status (married, other), physical activity (continuous), BMI (continuous), self-reported history of cancer (yes, no), self-reported history of arterial hypertension (yes, no), total energy intake (continuous), alcohol (continuous), dietary fiber intake (continuous, energy-adjusted), saturated fatty acids intake (continuous, energy-adjusted) and monounsaturated fatty acids intake (continuous, energy-adjusted).</p>b<p> Dietary GI and GL quartiles were estimated for each specific population group.</p><p>Sensitivity analysis. Hazard Ratios (95% CI) for total mortality according to baseline quartiles of energy adjusted dietary glycemic index and dietary glycemic load^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107968#nt110" target="_blank">a</a>}.</p