Fisetin Inhibited Growth and Metastasis of Triple-Negative Breast Cancer by Reversing Epithelial-to-Mesenchymal Transition via PTEN/Akt/GSK3β Signal Pathway

Abstract

Triple negative breast cancer (TNBC), characterized by its highly aggressive and metastatic features, is associated with poor prognosis and high mortality partly due to lack of effective treatment. Fisetin, a natural flavonoid compound, has been demonstrated to possess anti-cancer effects in various cancers. However, the effects and mechanisms of fisetin on metastasis of TNBC remain uncovered. In this study, we found that fisetin dose-dependently inhibited cell proliferation, migration and invasion in TNBC cell lines MDA-MB-231 and BT549 cells. In addition, fisetin reversed epithelial to mesenchymal transition (EMT) as evaluated by cell morphology and EMT markers in MDA-MB-231 and BT549 cells. Furthermore, fisetin suppressed phosphoinositol 3-kinase (PI3K)-Akt-GSK-3β signaling pathway but upregulated the expression of PTEN mRNA and protein in a concentration-dependent manner. Further, silence of PTEN by siRNA abolished these benefits of fisetin on proliferation and metastasis of TNBCs. In vivo, using the metastatic breast cancer xenograft model bearing MDA-MB-231 cells, we found that fisetin dramatically inhibited growth of primary breast tumor and reduced lung metastasis, meanwhile, the expression of EMT molecules and PTEN/Akt/GSK-3β in primary and metastatic tissues changed in the same way as those in vitro experiments. In conclusion, all these results indicated that fisetin could effectively suppress proliferation and metastasis of TNBC and reverse EMT process, which might be mediated by PTEN/Akt/GSK-3β signaling pathway.

Keywords: AKT; EMT; PTEN; fisetin; triple negative breast cancer.

FIGURE 1

Fisetin suppresses the proliferation, migration and invasion of TNBC cells in vitro. Triple-negative breast cancer (TNBC) cell lines MDA-MB-231 and BT549 were treated with various concentrations of fisetin (10, 30, and 100 μM) for indicated time. (A) Chemical structure of fisetin. (B) The cell proliferation was determined by MTT assay at 72 h after fisetin treatment. (C) The cell migration was determined by wound-healing assay. (D) Quantification of the migrated cells. (E) The cell invasion was determined by transwell invasion assay. (F) Quantification of the invasive cells. The results are shown as the mean ± SD of three experiments, ^∗P < 0.05, ^∗∗P < 0.01 compared with control.

FIGURE 2

Fisetin reverses EMT in TNBC cells in vitro. TNBC cell lines MDA-MB-231 and BT549 were treated with vehicle or fisetin for 24 h. (A) The morphology of the cells treated with vehicle or 30 μM fisetin was observed by phase-contrast microscopy. (B) E-cadherin and (C) Vimentin and F-actin expression were evaluated by immunofluorescence in TNBC cells treated by vehicle or 30 μM fisetin. The cells pretreated with vehicle or various concentrations of fisetin (10, 30, and 100 μM, respectively) were subjected to western blot for the indicated proteins (D) and qRT-PCR for the indicated mRNA (E). The results are shown as the mean ± SD of three experiments, ^∗P < 0.05, ^∗∗P < 0.01 compared with control.

FIGURE 3

Fisetin suppresses PI3K-Akt-GSK-3β signal pathway but upregulates PTEN expression in vitro. TNBC cell lines MDA-MB-231 and BT549 were treated with vehicle or 30 μM fisetin for immunofluorescence assay and with vehicle or various concentrations of fisetin (10, 30, and 100 μM) for western blot and qRT-PCR. (A) The expression of p-AKT was evaluated by immunofluorescence. (B) The expression of PTEN was evaluated by immunofluorescence. (C) The expression of PTEN protein as well as p-AKT and p-GSK-3β was determined by western blot. (D) The expression of PTEN mRNA was determined by qRT-PCR. The results are shown as the mean ± SD of three experiments, ^∗P < 0.05, ^∗∗P < 0.01.

FIGURE 4

Silencing of PTEN abrogates the effects of fisetin on TNBC cells proliferation and metastasis as well as EMT. TNBC cell line MDA-MB-231 cells were transfected with Ad-RFP or Ad-siPTEN, and subsequently treated with fisetin (100 μM). (A) The expression of PTEN and p-AKT and p-GSK-3β protein was determined by western blot. (B) EMT molecule markers were determined by western blot. (C) The cell proliferation was determined by MTT assay. (D) The cell migration was determined by wound-healing assay. (E) The cell invasion was determined by transwell invasion assay. The results are shown as the mean ± SD of three experiments, ^∗P < 0.05, ^∗∗P < 0.01 compared with control.

FIGURE 5

Fisetin inhibits the growth and metastasis of TNBC in vivo. Xenograft breast cancer model was established by subcutaneous injection of MDA-MB-231 cells in the presence or absence of fisetin (100 mg/kg). (A) Tumor growth curve was recorded. (B) Tumor weight was measured. (C) The expression of Ki67 in the primary tumor tissues was evaluated by immunohistochemistry staining. (D) Metastatic tumor nodules on the surface of lungs were counted. (E) Representative images of HE staining in the metastatic nodules of lungs. The results are shown as the mean ± SD of six experiments, ^∗P < 0.05, ^∗∗P < 0.01 compared with control.

FIGURE 6

Fisetin inhibits PI3K/AKT/GSK-3β signaling pathway and reverses EMT in vivo. Xenograft tumor metastasis was established by subcutaneous injection of MDA-MB-231 cells in the presence or absence of fisetin (100 mg/kg). (A) AKT activation was evaluated by immunohistochemistry staining. (B) Representative images of PTEN stained section of tumor tissues isolated from nude mice bearing breast cancer. (C) Vimentin and Snail in primary tumor tissues were examined by immunofluorescence assay. (D) PTEN and p-Akt and p-GSK-3β protein in the primary tumor tissues was examined by western blot. (E) EMT molecule markers were determined by western blot. The results are shown as the mean ± SD of six experiments, ^∗P < 0.05, ^∗∗P < 0.01 compared with control.