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Supplementary material 2 (DOCX 19 kb

IC₅₀ values obtained for the selected flavonoids.

1<p>The IC₅₀ value represents the concentration of each compound that inhibits ACE activity by 50%.</p

HPLC analysis of the flavonoid samples used in the current study.

1<p>Absolute value as is.</p>2<p>The reference % assay is referred to the absolute content as is of the main flavonoid.</p

Intermolecular interactions between ACE inhibitors and the tACE binding site.

<p>Data used for lisinopril, enalaprilat, captopril, RXPA380, selenocaptopril, KAF, KAW, lisW-S and FII-A was obtained from the LigPlot+ diagrams that are available at the PDBsum resource (<a href="http://www.ebi.ac.uk/pdbsum/" target="_blank">http://www.ebi.ac.uk/pdbsum/</a>) for PDB files 1O86, 1UZE, 1UZF, 2OC2, 2YDM, 3BKK, 3BKL, 3L3N and 2XY9, respectively. Data for luteolin, quercetin, rutin and kaempferol was obtained by applying LigPlot+ to the structure of their predicted complexes with tACE. Hydrophobic contacts are indicated by a check mark whereas hydrogen bonds are indicated with the label of the protein atom that is involved.</p

Structural diagram which quantitatively assesses the effect of the addition or elimination of different structural elements from the flavonoid core on the ACEI activity of luteolin at 100 µM.

<p>According to these data, the significance order was: double bond C2 = C3 (absence: −91% activity by comparing naringenin <i>vs.</i> apigenin) >4′-O-methoxylation (presence: −78% by comparing diosmetin <i>vs.</i> luteolin) ≈ 4-carbonyl group (absence: −74% by comparing epicatechin <i>vs.</i> luteolin) >3′-hydroxylation (absence: −57% by comparing apigenin <i>vs.</i> luteolin) >3-hydroxylation (presence: −44% by comparing quercetin <i>vs.</i> luteolin) >3-O-glycosylation (presence: -36% by comparing rutin <i>vs.</i> luteolin).</p

Liver miR-122 and their target mRNA levels.

<p>Rats were fed a STD (STD group) or a STD plus CD for 10 weeks. After 10 weeks, rats fed a STD plus CD were orally treated with 25 mg GSPE/kg bw (CD-GSPE group), 515 mg ω-3 PUFAs/kg bw (CD- DHA-OR), 25 mg GSPE and 515 mg ω-3 PUFAs/kg bw (CD-GSPE- DHA-OR group) or vehicle (CD control group) for 3 weeks simultaneously with the CD. Each value is the mean ± s.e.m. of seven rats. Letters denotes a significant difference between groups (p<0.05; One-way ANOVA).</p

Plasma lipids levels of rats fed with STD or CD with or without a GSPE and/or DHA-OR in chronic treatments.

<p><i>Abbreviations: GSPE, grape seed proanthocyanidin extracts; CD, cafeteria-diet; STD, standard chow diet; DHA-OR, oil-rich in d^{ocosahexaenoic acid}; TG, triacylglyceride; TC, total cholesterol. Rats were fed with STD (STD group) or with STD plus CD for 10 weeks. After 10 weeks, rats fed a STD plus a CD were orally treated with 25 mg GSPE/kg bw (CD+GSPE), 515 mg PUFAs/kg bw (CD+DHA-OR), 25 mg GSPE and 515 mg ω-3 PUFAs/kg bw (CD+GSPE+DHA-OR group) or vehicle (CD control group) for 3 weeks simultaneously with the CD. Each value is the mean ± s.e.m. of seven rats. Letters denotes a significant difference between groups (p<0.05; One-way ANOVA)</i>.</p

(A) Effect of different flavonoids on Angiotensin Converting Enzyme (ACE) activity.

<p>Purified lung ACE was preincubated at 37°C for 30 min in the presence of 100 µM of flavonoids or DMSO as a control. The results are expressed as the percentage of ACE inhibition. The plot represents the mean result ± SD from three experiments. (<b>B</b>) Effect of different flavonoids on Angiotensin Converting Enzyme (ACE) activity. Purified lung ACE was preincubated at 37°C for 30 min in the presence of 500 µM of flavonoids or DMSO as control. The results are expressed as the percentage of ACE inhibition. The plot represents the mean ± SD from three experiments.</p

Time-dependent effects of increasing RSV concentrations on XBP1 splicing, CHOP expression and viability.

<p>HepG2 cells were exposed to vehicle (0 µM) or increasing RSV concentrations (5, 10, 25, 50 and 100 µM) and harvested at specific time points (8, 4 and 24 h). <b>A</b>) RSV exerts a time- and concentration-dependent activation of XBP1 splicing (XBP1 unspliced-197 bp amplicon; XBP1 spliced-171 bp amplicon). Representative image of three independent experiments <b>B</b>) RSV exerts a time- and concentration-dependent activation of CHOP expression. <b>C</b>) RSV decreases HepG2 viability at higher doses and incubation times. The viability was evaluated using a MTT assay. The data are shown as the mean ± SD of three independent experiments. Significant differences relative to the control (vehicle) were analyzed by one-way ANOVA followed by the Bonferroni post hoc test: ***p<0.001 and *p<0.05.</p

Resveratrol Enhances Palmitate-Induced ER Stress and Apoptosis in Cancer Cells

<div><p>Background</p><p>Palmitate, a saturated fatty acid (FA), is known to induce toxicity and cell death in various types of cells. Resveratrol (RSV) is able to prevent pathogenesis and/or decelerate the progression of a variety of diseases. Several <i>in vitro</i> and <i>in vivo</i> studies have also shown a protective effect of RSV on fat accumulation induced by FAs. Additionally, endoplasmic reticulum (ER) stress has recently been linked to cellular adipogenic responses. To address the hypothesis that the RSV effect on excessive fat accumulation promoted by elevated saturated FAs could be partially mediated by a reduction of ER stress, we studied the RSV action on experimentally induced ER stress using palmitate in several cancer cell lines.</p><p>Principal Findings</p><p>We show that, unexpectedly, RSV promotes an amplification of palmitate toxicity and cell death and that this mechanism is likely due to a perturbation of palmitate accumulation in the triglyceride form and to a less important membrane fluidity variation. Additionally, RSV decreases radical oxygen species (ROS) generation in palmitate-treated cells but leads to enhanced X-box binding protein-1 (XBP1) splicing and C/EBP homologous protein (CHOP) expression. These molecular effects are induced simultaneously to caspase-3 cleavage, suggesting that RSV promotes palmitate lipoapoptosis primarily through an ER stress-dependent mechanism. Moreover, the lipotoxicity reversion induced by eicosapentaenoic acid (EPA) or by a liver X receptor (LXR) agonist reinforces the hypothesis that RSV-mediated inhibition of palmitate channeling into triglyceride pools could be a key factor in the aggravation of palmitate-induced cytotoxicity.</p><p>Conclusions</p><p>Our results suggest that RSV exerts its cytotoxic role in cancer cells exposed to a saturated FA context primarily by triglyceride accumulation inhibition, probably leading to an intracellular palmitate accumulation that triggers a lipid-mediated cell death. Additionally, this cell death is promoted by ER stress through a CHOP-mediated apoptotic process and may represent a potential anticancer strategy.</p></div