Effect of quercetin in CylinD1, twist and p38MAPK in MCF-7 cells.

<p>A. Expression of Cyclin D1, twist and Pp38 MAPK were assessed by western blot MCF-7 cells with quercetin. The significant values represent mean ± SD of three separate experiments, each consisting of duplicate cultures. B. Quantitative analysis of a.</p

Quercetin Suppresses Twist to Induce Apoptosis in MCF-7 Breast Cancer Cells

<div><p>Quercetin is a dietary flavonoid which exerts anti-oxidant, anti-inflammatory and anti-cancer properties. In this study, we investigated the anti-proliferative effect of quercetin in two breast cancer cell lines (MCF-7 and MDA-MB-231), which differed in hormone receptor. IC₅₀ value (37μM) of quercetin showed significant cytotoxicity in MCF-7 cells, which was not observed in MDA-MB-231 cells even at 100μM of quercetin treatment. To study the response of cancer cells to quercetin, with respect to different hormone receptors, both the cell lines were treated with a fixed concentration (40μM) of quercetin. MCF-7 cells on quercetin treatment showed more apoptotic cells with G1 phase arrest. In addition, quercetin effectively suppressed the expression of CyclinD1, p21, Twist and phospho p38MAPK, which was not observed in MDA-MB-231 cells. To analyse the molecular mechanism of quercetin in exerting an apoptotic effect in MCF-7 cells, Twist was over-expressed and the molecular changes were observed after quercetin administration. Quercetin effectively regulated the expression of Twist, in turn p16 and p21 which induced apoptosis in MCF-7 cells. In conclusion, quercetin induces apoptosis in breast cancer cells through suppression of Twist via p38MAPK pathway.</p></div

Cell pictures show the cell morphology and viability of MCF-7 and MDA-MB-231 control and quercetin treated cells.

<p>a(i) MCF-7 cells cultured with and without quercetin for 24h were examined for changes in cell morphology and photographed using a phase-contrast microscope. a(ii) MCF-7 cells with and without quercetin for 24h were examined for the viability of cells by dual staining. b(i) MDA-MB-231 cells cultured with and without quercetin for 24h were examined for changes in cell morphology and photographed using a phase-contrast microscope. b(ii) MDA-MB-231 cells with and without quercetin for 24h were examined for the viability of cells by dual staining. The green stained are viable cells and the yellowish orange stained are damaged/apoptotic cells.</p

Quercetin regulates cell cycle regulatory proteins.

<p>A. Protein expressions of Cyclin D1 and p21 were assessed by western blotting in MCF-7 cells treated with quercetin at different time intervals. β-actin was used as an internal control. c. Quantitative data of a. B. Protein expressions of Cyclin D1 and p21 were assessed by western blotting in MDA-MD-231 cells treated with quercetin at different time intervals. β-actin was used as an internal control. d. Quantitative data of b. The significant values represent mean ± SD of three separate experiments, each consisting of duplicate cultures.</p

Schematic representation of quercetin induced apoptosis through twist regulation.

<p>Quercetin being a potent anti-oxidant it reduces the cellular stress. This indirectly reduced the activation of p38MAPK in survival pathway which prevents the stabilization of twist. Quercetin reduced twist gene expression through suppression of Cyclin D1, and induced p16 and p21 to arrest the cells at G1 phase of cell cycle, thereby induced apoptosis. Our data thereby reveal the mechanism of quercetin induced apoptosis through twist regulation.</p

TEM picture represents the morphology and organelles of MCF-7 control and quercetin treated cells.

<p>A. TEM analysis showing the structural changes and damages occur on treatment with quercetin. Picture (i) shows a undamaged cell (blue arrow) in the middle along with the cells undergoing apoptotic necrosis (red arrow), chromatin condensation inside the nucleus (yellow arrow) and autophagosomes (green arrow). Magnification at 500nm (ii) shows the condensation of the chromatin (yellow arrow), autophagosomes (blue arrow) and ER vesiculation (red arrow). (iii) Represents cells at lower magnification showing plenty of chromatin condensation (yellow) and autophagosis (blue) in group. (iv) Represents a single damaged cell showing huge number of autophagosis (red arrow), nuclear membrane showing starting of hetero chromatin (yellow), large number of vesicles in the cytosol (green arrow). B. The picture shows a single cell with microvilli (blue arrow), nucleus (N) present at the center of the cell, nucleoli (Nu), mitochondria (red arrow) (shows active energy production), structured Golgi apparatus (green arrow) and endoplasmic reticulum (orange arrow) finely organised. All these events represent a cell in active state.</p

Inhibitory effect of quercetin post-transfection.

<p>A. Twist and Cyclin D1 expression levels were assessed by western blot in twist over-expressed MCF-7 cells and the same after Q treatment. c. quantitative analysis of a. B. Expression of p16, p21, p27 and p53 were analyzed by RT-PCR in twist over-expressed MCF-7 cells and the same after Q treatment. d. Quantitative analysis of b. The significant values represent mean ± SD of three separate experiments. Significance is indicated as *p<0.05.</p

Flow cytometry analysis showing the effect of quercetin on cell cycle.

<p>A. MCF-7 cells were treated with 40μM quercetin for 8h, 16h and 24h and then harvested for cell cycle analysis. The picture represents the distribution of MCF-7 cells in different phases of cell cycle. B. Bar graph represents the percentage distribution of MCF-7 cells in different phases of cell cycle. C. MDA-MB-231 cells were treated with 40μM quercetin for 24h and then harvested for cell cycle analysis. The picture represents the distribution of the cells in different phases of cell cycle. D. Bar graph represents the percentage distribution of MCF-7 cells in different phases of cell cycle. The cells were plated at a density of 1x10⁵ cells/dish and exposed to quercetin for various time periods. Cells were harvested and analysed for cell cycle. The cells with DMSO exposure served as control. The significant values represent mean ± SD of three separate experiments, each consisting of duplicate cultures.</p

Twist over-expression induced cell proliferation.

<p>A. Twist plasmid was transfected in MCF-7 cells to over-express twist. Levels of twist expression were assessed by immunofluorescence, in control and twist over-expressed MCF-7 cells. The twist over-expressed MCF-7 cells showed more number of dividing cells which was not observed in control MCF-7 cells. B. Number of highly positive stained cells in five random fields was counted by ImageJ. C. Viability of cells (%) assessed by MTT assay in control and twist over-expressed MCF-7 cells. The significant values represent mean ± SD of three separate experiments. Significance is indicated as *p<0.05.</p

Effect of quercetin on breast cancer cell lines.

<p>A. Cytotoxicity effect of various concentrations of quercetin on MCF-7 cells. Dose dependent cytotoxicity effect of quercetin was represented in the graph. The IC50 value was 37μM of quercetin at 24h exposure. B. Cytotoxicity effect of various concentrations of quercetin on MDA-MB-231 cells. Dose dependent cytotoxicity effect of quercetin was represented in the graph. Cells did not show significant cytotoxicity at lower dose of quercetin at 24h of exposure. The significant values represent mean ± SD of three separate experiments, each consisting of duplicate cultures.</p