Comparative Studies on a Standardized Subfraction of Red Onion Peel Ethanolic Extract (Plant Substance), Quercetin (Pure Compound), and Their Cell Mechanism and Metabolism on MDA-MB-231

Abstract

This study indicates the presence of quercetin in subfraction F1 and the standardized value of F1 derived from research using ultra-performance liquid chromatography (UPLC) and AlCl₃ colorimetric assays, which further proved that both F1 and quercetin are potential growth inhibitors in MDA-MB-231 cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In the process, staining of F1-treated cells with annexin/propidium iodide (PI) reduced cell proliferation and induced only S and G2 phases of cell cycle arrest in the treated cells by flow cytometry. Quercetin reduced cell proliferation by inducing apoptosis and S phase arrest. The 5'-bromo-2'-deoxyuridine (BrdU) incorporation of DNA synthesis in MDA-MB-231 cells was also inhibited after F1 and quercetin treatments. F1 and quercetin induced CYP1A1 and CYP1B1 gene expression, but only F1 induced CYP2S1 gene expression in the treated cells. Both F1 and quercetin inhibited the proliferation of MDA-MB-231 cells in different ways, but F1 is likely a better potential anticancer agent derived from the green approach towards breast cancer treatment.

Figure 1

Ultra-performance liquid chromatography (UPLC), 360 nm of phenolic compounds. (a) F1 (1 mg/mL; isolated from batch 1 of red onion peel), (b) F1 (1 mg/mL; isolated from batch 2 of red onion peel), (c) quercetin (200 μg/mL; standard), and (d) a standard curve of quercetin in the concentration range of 25 to 200 μg/mL. Quercetin was dissolved in 99.7% ethanol.

Figure 2

Dose-response curves of MDA-MB-231 following treatments with F1 for (a) 24 h, (b) 48 h, and (c) 72 h and with quercetin for (d) 24 h, (e) 48 h, and (f) 72 h. The maximal response (inhibition rate at the maximum dose concentration) identified hillslope value (slope at EC₅₀ value) and an EC₅₀ value of each test compound from the curve. The experiments were repeated several times to ensure the repeatability and reproducibility of the results. All values are expressed as the means ± SD.

Figure 3

Flow cytometry of treated MDA-MB-231 cells on cell cycle after 72 h treatment. (a) Histograms of the cell cycle distribution of MDA-MB-231 cells after treatment by flow cytometry. (A) MDA-MB-231 was treated with 0.1% DMSO for 24 h (control), (B) DMSO-treated for 48 h, (C) DMSO-treated for 72 h, (D) 50 μg/mL F1-treated for 24 h, (E) 50 μg/mL F1-treated for 48 h, (F) 50 μg/mL F1-treated for 72 h, (G) 60 μg/mL quercetin-treated for 24 h, (H) 60 μg/mL quercetin-treated for 48 h, and (I) 60 μg/mL quercetin-treated for 72 h. The vertical (y) axis represents cell number, whereas the horizontal (x) axis represents a channel. (b) Analyzed cell cycle profile of F1 (50 μg/mL) and quercetin (60 μg/mL) in MDA-MB-231 cells after (A) 24 h, (B) 48 h, and (C) 72 h of treatment. Each dataset represents the mean of two independent experiments with triplicate readings each. Significant differences were analyzed versus control using one-way ANOVA and Dunnett's multiple comparison test indicated as ^∗P < 0.05; ^∗∗P<0.01; and ^∗∗∗P<0.001. Control: DMSO-treated cells.

Figure 4

Flow cytometry of treated MDA-MB-231 apoptotic and necrotic cells after 72 h treatment. (a) Histograms of the apoptotic and necrotic distribution of MDA-MB-231 cells after treatment by flow cytometry. (A) MDA-MB-231 was treated with 0.1% DMSO for 24 h (control), (B) DMSO-treated for 48 h, (C) DMSO-treated for 72 h, (D) 50 μg/mL F1-treated for 24 h, (E) 50 μg/mL F1-treated for 48 h, (F) 50 µg/mL F1-treated for 72 h, (G) 60 μg/mL quercetin-treated for 24 h, (H) 60 μg/mL quercetin-treated for 48 h, and (I) 60 μg/mL quercetin-treated for 72 h. After staining with FITC-conjugated annexin and propidium iodide, the flow cytometer analyzed the cells. The lower left and upper left quadrants represent the percentage of viable and necrotic cells. In contrast, the early and late apoptosis events are shown in the lower and upper right quadrants. (b) Analyzed apoptotic and necrotic profiles of F1 (50 μg/mL) and quercetin (60 μg/mL) in MDA-MB-231 cells after (A) 24 h, (B) 48 h, and (C) 72 h of treatment. Each dataset represents the mean of two independent experiments with triplicate readings each. One-way ANOVA and Dunnett's multiple comparison test were used to interpret statistically significant differences between treated and untreated cells (control) as ^∗P < 0.05 and ^∗∗∗P<0.001. EA: early apoptosis, LA: late apoptosis.

Figure 5

Flow cytometry of BrdU-positive treated MDA-MB-231 cells after 72 h treatment. (a) Representative dot-plot analysis showing the proportion of BrdU-positive treated MDA-MB-231 cells by flow cytometry. (A) MDA-MB-231 cells were treated with 0.1% DMSO for 24 h (control), (B) DMSO-treated for 48 h, (C) DMSO-treated for 72 h, (D) 50 µg/mL F1-treated for 24 h, (E) 50 μg/mL F1-treated for 48 h, (F) 50 μg/mL F1-treated for 72 h, (G) 60 µg/mL quercetin-treated for 24 h, (H) 60 μg/mL quercetin-treated for 48 h, and (I) 60 μg/mL quercetin-treated for 72 h. The cells enclosed in the box gate labelled with R6 were used to calculate the percentage of BrdU-positive cells. (b) Percentage of BrdU-positive treated MDA-MB-231 cells. The percentage of BrdU-positive MDA-MB-231 cells treated with 0.1% DMSO (control), 50 μg/mL F1, and 60 μg/mL quercetin was determined by flow cytometry. The bar chart represents the mean ± SD of two independent experiments with significant differences indicated as ^∗P < 0.05, ^∗∗P<0.01, and ^∗∗∗P<0.001 compared to the controls. One-way ANOVA and Dunnett's multiple comparison test were used for the statistical analysis.

Figure 6

Effect of F1 (50 μg/mL) and quercetin (60 μg/mL) on the mRNA gene expression of (a) CYP1A1, (b) CYP1B1, and (c) CYP2S1 in MDA-MB-231 cells. The mRNA expression was normalized to the expression of GAPDH in each sample. The bar chart represents the mean ± SD of two independent experiments with significant differences indicated as ^∗P < 0.05, ^∗∗P<0.01, and ^∗∗∗P<0.001, compared to the controls. One-way ANOVA and Dunnett's multiple comparison test were used for the statistical analysis.