Korean J Physiol Pharmacol.  2017 Mar;21(2):205-213. 10.4196/kjpp.2017.21.2.205.

Quercetin induces apoptosis and cell cycle arrest in triple-negative breast cancer cells through modulation of Foxo3a activity

• Nguyen LT
• Lee YH
• Sharma AR
• Park JB
• Jagga S
• Sharma G
• Lee SS
• Nam JS

• KMID: 2371039
• DOI: http://doi.org/10.4196/kjpp.2017.21.2.205

Abstract

Quercetin, a plant-derived flavonoid found in fruits, vegetables and tea, has been known to possess bioactive properties such as anti-oxidant, anti-inflammatory and anti-cancer. In this study, anti-cancer effect of quercetin and its underlying mechanisms in triple-negative breast cancer cells was investigated. MTT assay showed that quercetin reduced breast cancer cell viability in a time and dose dependent manner. For this, quercetin not only increased cell apoptosis but also inhibited cell cycle progression. Moreover, quercetin increased FasL mRNA expression and p51, p21 and GADD45 signaling activities. We also observed that quercetin induced protein level, transcriptional activity and nuclear translocation of Foxo3a. Knockdown of Foxo3a caused significant reduction in the effect of quercetin on cell apoptosis and cell cycle arrest. In addition, treatment of JNK inhibitor (SP 600125) abolished quercetin-stimulated Foxo3a activity, suggesting JNK as a possible upstream signaling in regulation of Foxo3a activity. Knockdown of Foxo3a and inhibition of JNK activity reduced the signaling activities of p53, p21 and GADD45, triggered by quercetin. Taken together, our study suggests that quercetin induces apoptosis and cell cycle arrest via modification of Foxo3a signaling in triple-negative breast cancer cells.

Keyword

Apoptosis; Cell cycle arrest; Foxo3a; Quercetin; Triple-negative breast cancer

MeSH Terms

Apoptosis*
Breast Neoplasms
Cell Cycle Checkpoints*
Cell Cycle*
Cell Survival
Fruit
Quercetin*
RNA, Messenger
Tea
Triple Negative Breast Neoplasms*
Vegetables
Quercetin
RNA, Messenger
Tea

Figure

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  Fig. 1 Quercetin reduced viability of MDA-MB-231 breast cancer cells.MDA-MB-231 cells were treated with various concentration of quercetin (2.5~80 µM) for 24 h, 48 h and 72 h. Cell viability was assessed by MTT assay. *p<0.001, significant difference compared to control.

• 

  Fig. 2 Quercetin induced apoptosis and cell cycle arrest in MDA-MB-231 breast cancer cells.(A) FACS analysis via Annexin V-FITC/PI staining was used to observe the induction of apoptosis in MDA-MB-231 by quercetin treatment. Representative images of the flow cytometry analysis are shown (left). Cells in the lower and upper right quadrant indicate cells with early and late apoptosis, respectively. Statistic graph presents apoptotic levels of quercetin-treated samples and untreated controls at 24 h and 48 h (right). (B) MDA-MB-231 cells were treated with quercetin for 48 h and stained with PI. Cell cycle was analyzed by FACS. M1, M2, M3 and M4 indicate sub G1, G0/G1, S and G2/M phases of cell cycle, respectively (up). The graph shows percentage of cells in each phase (down). (C) RT real-time PCR analysis for expression of FasL mRNA in MDA-MB-231 cells treated with 20 µM quercetin for 12 h, 24 h and 48 h. (D) MDA-MB-231 cells were transiently transfected with p53, p21 or GADD45-luciferase reporter for 24 h prior to treatment of 20 µM quercetin for another 24 h. Cells lysate were collected for Luciferase assay. Relative luciferase activity after normalized with Renilla luciferase reporter is shown as fold change from control. *p<0.05, **p<0.01, ***p<0.001.

• 

  Fig. 3 Quercetin induced Foxo3a activity.(A) MDA-MB-231 cells were transiently transfected with Foxo3a luciferase reporter for 24 h prior to treatment of 20 µM quercetin for another 24 h. Thereafter, cell lysates were collected for Luciferase assay. The graph shows relative luciferase activity. (B) RT real-time PCR analysis for expression of Foxo3a mRNA in MDA-MB-231 cells treated with 20 µM quercetin for 12 h, 24 h and 48 h. (C) MDA-MB-231 cells were treated with 20 µM quercetin for various time points from 0 to 48 h and total Foxo3a was detected by western blot. β-actin was used as loading control (left). Densitometric quantification of Foxo3a protein levels from western blot analysis (right). Foxo3a levels were compared to basal level at 0 h. (D) After treatment with quercetin for 24 h, cells were fixed and stained for Foxo3a, and DNA was counterstained with DAPI. Representative microcopy images are shown (E) Breast cancer cells were treated with quercetin for 6 h, 12 h and 24 h, and subcellular fractions were isolated and immunoblotted for Foxo3a. Lamin B1 and β-actin were used as loading controls of nucleus and cytoplasm respectively. Western blot analysis image (up) and densitometric quantification of Foxo3a protein expression from western blot analysis (down). *p<0.05, **p<0.01, ***p<0.001.

• 

  Fig. 4 Quercetin increased Foxo3a activity via activation of JNK signaling pathway.(A) MDA-MB-231 was treated with quercetin and levels of p-ERK, p-38, JNK, p-JNK and Foxo3a at 0~8 h were detected by western blot (left). Densitometric quantification of protein expression from western blot analysis (right). The protein levels were compared to basal level at 0 h (B) MDA-MB-231 cells were pre-treated for 30 mins with SP600125 prior to treatment of quercetin for 3 h. Samples were collected and levels of Foxo3a, JNK and p-JNK were determined by western blot. β-actin was used as loading control. (C) MDA-MB-231 cells were transiently transfected with Foxo3a luciferase reporter for 24 h prior to pre-treatment of SP600125 for 1 h and treatment of 20 µM quercetin for another 24 h. Luciferase assay was used to measure activity of Foxo3a. Relative luciferase activity after normalized with Renilla luciferase reporter is shown as fold change from control. *p<0.05, **p<0.01, ***p<0.001.

• 

  Fig. 5 Quercetin regulates apoptosis and cell cycle arrest through the JNK-Foxo3a axis.(A) Cancer cells were transfected with Foxo3a siRNA or scramble siRNA for control prior to treatment with 20 µM quercetin for 48 h. FACS analysis was applied to measure population of apoptotic cells. Representative images of the flow cytometry analysis are shown (left). Statistic graph presents apoptotic levels (right). (B) Cancer cells were transfected with Foxo3a siRNA or scramble siRNA for control prior to treatment with 20 µM quercetin for 48 h. Then, cells were collected and stained with PI, cell cycle was analyzed by FACS. M1, M2, M3, M4 indicate sub G1, G0/G1, S and G2/M phase, respectively (up). The graph shows the percentage of cells in each phase (down). (C) MDA-MB-231 cells were transiently co-transfected with Foxo3a shRNA and a plasmid containing p53, p21 or GADD45 luciferase reporter for 24 h prior to treatment of quercetin. Luciferase activity was measured after 24 h. (D) MDA-MB-231 cells were transfected with p53, p21 or GADD45 luciferase reporter for 24 h prior to pre-treatment of 1 µM SP600125 and treatment of 20 µM quercetin. Luciferase activity was measured after 24 h. *p<0.05, **p<0.01, ***p<0.001.

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