Effect of combined RQC or individual quercetin on breast cancer cell apoptosis.

<p>Quiescent MDA-MB-231 (<b>A</b>) or MDA-MB-435 (<b>B</b>) cells in 5% serum and phenol red-free media were treated with vehicle, combined RQC at 5μM each, or Quercetin 15μM for 48h. Trypsinized cells were incubated with Annexin V conjugate and propidium iodide, and analyzed by flow cytometry, using a four-color flow cytometer (FACSCalibur, BD Biosciences, San Jose, CA). Representative dot plots of 20,000 events/ treatment (N = 3), collected using Cell Quest software 3.3 (BD Biosciences, San Jose, CA) and analyzed using Flow Jo software vX.0.7 (BD Biosciences, San Jose, CA). Cell size and granularity were determined on forward versus side scatter (FSC vs. SSC). Annexin-V fluorescence was measured at 530/30 nm and Propidium Iodide at 585/42nm. The average percentage of early and late apoptotic cells obtained from Annexin V vs. Propidium Iodide dot plots are shown below. <b>(C)</b> Effect of combined RQC or individual quercetin on caspase 3/7 activity. Quiescent MDA-MB-231 cells in 96 well plate at 5% serum and phenol red-free media were treated with vehicle, combined RQC at 5μM each or quercetin 15μM for 48hr or 96hr. Then Caspase-Glo®3/7 reagent were added to each treatment, and after 1hr of incubation the luminescence was measured in a plate-reader luminometer. Relative luminescence to vehicle is shown, N = 3±SEM, asterisk indicates statistical significance (p≤0.05) when compared to vehicle.</p

Effect of individual or combined RQC on Akt activity in breast cancer cells.

<p>Quiescent MDA-MB-231 cells were treated with vehicle, or 1, 3, 5, 9, or 15μM of resveratrol (Res), quercetin (Quer), catechin (Cat), or combined Res, Quer, and Cat (RQC) at 3μM total (1μM each), 9μM total (3μM each), or 15μM total (5μM each). MDA-MB-435 cells were treated only with vehicle or 15μM quercetin. Cells were lysed immediately following treatment for 15min, and western blotted for total or active (phospho-Akt^Ser473) Akt. <b>(A)</b> Relative Akt activity (phospho-Akt/Akt) of MDA-MB-231 cells following RQC or individual Res, Quer, or Cat at the indicated concentrations. <b>(B)</b> Relative Akt activity of MDA-MB-231 cells following vehicle or 15μM quercetin. <b>(C)</b> Relative Akt activity of MDA-MB-435 cells following vehicle or 15μM quercetin. For <b>(B)</b> and <b>(C)</b>, representative western blots from 3 separate experiments are shown. Graphs show the analyses of the integrated densities of positive bands relative to vehicle, as quantified from image J analysis. An asterisk indicates statistical significance (<i>p≤</i>0.05) when compared to vehicle.</p

Effect of quercetin on the growth of MDA-MB-231 mammary fat pad tumors.

<p>GFP-MDA-MB-231 cells (0.5x10⁶) were inoculated at the mammary fat pad of SCID mice. One week after inoculation, mice were treated with vehicle, or quercetin at 15 or 45mg/kg BW three times a week by intraperitoneal injection. Fluorescence images of the mammary fatpad tumors were acquired once a week. <b>(A)</b> Average relative tumor area (N = 12) as a function of days following injection. Relative tumor area was calculated as the area of fluorescence, measured by fluorescence intensity on each day of imaging as a function of the fluorescence intensity of the same tumor on day 1. <b>(B)</b> Representative digital images of MDA-MB-231 tumors following vehicle, or quercetin at 15 or 45mg/kg BW at 13 weeks, followed by the quantification of tumor growth. N = 12±SEM, asterisk indicates statistical significance (<i>p≤</i>0.05) when compared to vehicle. <b>(C)</b> Relative SCID mice weight following vehicle, or quercetin at 15 or 45mg/kg BW treatments.</p

Effect of individual or combined RQC on AMPK activity in breast cancer cells.

<p>Quiescent MDA-MB-231 cells were treated with vehicle, or 1, 3, 5, 9, or 15μM of resveratrol (Res), quercetin (Quer), catechin (Cat), or combined RQC at 3μM total (1μM each), 9μM total (3μM each), or 15μM total (5μM each). MDA-MB-435 cells were treated only with vehicle and 15μM quercetin. Cells were treated for 15 min, lysed immediately, and western blotted for total or active (phospho-AMPK^Thr172) AMPK. <b>(A)</b> Relative AMPK activity (phospho-AMPK/AMPK) of MDA-MB-231 cells following RQC or individual Res, Quer, or Cat at the indicated concentrations. Relative AMPK activity of <b>(B)</b> MDA-MB-231 cells following vehicle or 15μM quercetin, or <b>(C)</b> MDA-MB-435 cells following vehicle or 15μM quercetin. For <b>(B)</b> and <b>(C)</b>, representative western blots from 3 separate experiments are shown. Graphs show the analyses of the integrated densities of positive bands relative to vehicle as quantified from image J analysis. An asterisk indicates statistical significance (<i>p≤</i>0.05) when compared to vehicle.</p

Effect of combined RQC or individual quercetin on breast cancer cell proliferation.

<p>MDA-MB-231 and MDA-MB-435 cells in 5% serum and phenol red-free media were treated with vehicle, combined resveratrol, quercetin, and catechin (RQC) at 5μM each or 15μM quercetin for 48h. Cells were fixed, nuclei stained with Propidium Iodide (PI), and intact (non-apoptotic) nuclei quantified. Percentage of viable cells ± SEM for 30 microscopic fields/triplicate treatments (N = 3) is presented. <b>A)</b> Average cell viability of MDA-MB-231 cells treated with RQC or quercetin relative to vehicle. <b>(B)</b> Average cell viability of MDA-MB-435 cells treated with RQC or quercetin relative to vehicle.</p

Effect of combined RQC or individual quercetin on breast cancer cell migration.

<p>Quiescent MDA-MB-231 and MDA-MB-435 cells were placed in the top of a transwell chamber with the bottom well containing: vehicle, combined resveratrol, quercetin, and catechin (RQC) at 5μM each, or 15μM quercetin in serum- and phenol red- free media. After 8h incubation, cells that migrated through the 8μM pore membrane were fixed, nuclei stained with Propidium Iodide and quantified. Percentage of migrated cells ± SEM for 15 microscopic fields/duplicate treatments for 3 independent experiments is presented. (<b>A)</b> Average cell migration of MDA-MB-231 cells treated with RQC or quercetin relative to vehicle. <b>(B)</b> Average cell migration of MDA-MB-435 cells treated with RQC or quercetin relative to vehicle.</p

Effect of quercetin on mTOR signaling in breast cancer cells.

<p>Quiescent MDA-MB-231 and MDA-MB-435 cells were treated with vehicle or quercetin at 15μM for 15min, lysed immediately, and western blotted for total or active (phospho-p70S6K^Thr389) p70S6K or (phospho-4EBP-1^Thr37/46) 4EBP-1. <b>(A)</b> Relative p70S6K activity (phospho-p70S6K/p70S6K) from MDA-MB-231 cells. <b>(B)</b> Relative p70S6K activity from MDA-MB-435 cells. <b>(C)</b> Relative 4EBP-1 activity (phospho-4EBP-1/4EBP-1) from MDA-MB-231 cells. <b>(D)</b> Relative 4EBP-1 activity from MDA-MB-435 cells. Representative western blots from 3 separate experiments are shown. Graphs show the analyses of the integrated densities of positive bands relative to vehicle as quantified from image J analysis. An asterisk indicates statistical significance (<i>p≤</i>0.05) when compared to vehicle.</p

Membrane insulin receptor (mIR), insulin receptor substrate 1 (IRS-1), and IRS-1 tyrosine phosphorylation levels in the CSF white cell pellet (CSF WCP) stratified by HAND.

a<p>For the mIR (MFI) and IRS-1 we analyzed 11 women with normal cognition, 8 with asymptomatic impairment, and 15 with symptomatic impairment. For IRS-1 tyrosine phosphorylation 5 women per group were analyzed;</p>b<p>significant p value<0.05^*;</p>c<p>MFI = Median Fluorescence Intensity; median (interquartile range [25^th and 75^th percentile]).</p

Soluble Insulin Receptor Levels (MFI) and HIV-seropositive women stratified by HAND.

a<p>MFI  =  Median Fluorescence Intensity; median (interquartile range [25^th and 75^th percentile]),</p>b<p>significant p value<0.05^*.</p

Soluble insulin receptor full-length (sIRαβ) stratified by HAND.

<p>Soluble insulin receptor (sIR) intact or full-length (αβ) subunit was measured by ELISA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037358#pone.0037358-The1" target="_blank">[47]</a> in plasma (A) and CSF (B) of HIV-seropositive women (HIV+) (n = 34) stratified by HAND into normal cognition (n = 11), asymptomatic impairment (n = 8), and symptomatic impairment (n = 15); and 10 HIV-negative controls (HIV−) (5 plasma were different women from 5 CSF). In plasma (A), levels of full-length sIR were significantly increased from controls in all HIV-seropositive women and it correlated with the severity of HAND (normal cognition [p = 0.003], asymptomatic impairment [p<0.001], and symptomatic impairment [p<0.001]). Also, women with symptomatic impairment had significant higher levels of full-length sIR when compared to those with normal cognition (p = 0.009). A similar trend was observed in CSF samples (B), although the only significant increased was observed in the women with symptomatic impairment when compared to controls. (MFI = Median Fluorescence Intensity).</p