Low dose of kaempferol suppresses the migration and invasion of triple-negative breast cancer cells by downregulating the activities of RhoA and Rac1

Figures & data

Figure 1 Kaempferol inhibits the migration and invasion of TNBC cells.

Notes: Breast cancer cells were incubated with kaempferol at the indicated doses for 6 h. The cell motility rate was measured by wound healing assays. (A) Representative pictures of wound healing assays. Magnification ×100. (B, C) MDA-MB-231 and MDA-MB-453 TNBC cells were allowed to migrate in response to indicated doses of kaempferol (μmol/L) for 6 h. (D) MCF-7 ER⁺/PR⁺ breast cancer cells were allowed to migrate in response to indicated doses of kaempferol (μmol/L) for 6 h. (E) SK-BR-3 HER2⁺ breast cancer cells were allowed to migrate in response to indicated doses of kaempferol (μmol/L) for 6 h. Results are presented as mean ± SD of 5 independent experiments in (B–E). (F) The representative pictures of cell invasion assays. Magnification ×100. (G, H) MDA-MB-231 and MDA-MB-453 TNBC cells were allowed to invade in response to 20 μmol/L kaempferol for 6 h. Results are presented as mean ± SD of 3 independent experiments in (G and H).
Abbreviations: Ctrl, control; ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; PR, progesterone receptor; TNBC, triple-negative breast cancer; ns, no significance.

Figure 2 Kaempferol depresses the activations of RhoA and Rac1 in TNBC cells.

Notes: (A, B) TNBC cells (MDA-MB-231 and MDA-MB-453) were incubated with 20 μmol/L kaempferol for 1 h, and then subjected to the Rho activity assays. The relative levels of Rho activity were normalized to the average value of controls. Results are presented as mean ± SD of 3 independent experiments. (C, D) TNBC cells (MDA-MB-231 and MDA-MB-453) were incubated with 20 μmol/L kaempferol for 1 h, and then subjected to the pulldown assays and Western blot analysis.
Abbreviations: Ctrl, control; ns, no significance; TNBC, triple-negative breast cancer.

Figure 3 Constitutive activity RhoA and Rac1 rescue the migration and invasion of TNBC cells inhibited by kaempferol.

Notes: (A–C) MDA-MB-231 TNBC cells were transfected with constitutive activity RhoA (RhoA-V14) or vector, then subjected to the RhoA activity assays (A), wound healing assays (B), and cell invasion assays (C). (D–F) MDA-MB-453 TNBC cells were transfected with constitutive activity Rac1 (Rac1-V12) or vector, then subjected to the Rac1 activity assays (D), wound healing assays (E) and cell invasion assays (F). The relative levels of Rho activity and cell migration distance were normalized to the average value of controls. Results are presented as mean ± SD of 3 independent experiments in (A, C, D, F) and 5 independent experiments in (B, E).
Abbreviations: Ctrl, control; TNBC, triple-negative breast cancer.

Figure 4 Kaempferol and HER2 inhibitor suppress the migration and Rho activity of SK-BR-3 cells.

Notes: (A) SK-BR-3 HER2⁺ breast cancer cells were allowed to migrate in response to 20 μmol/L kaempferol and/or 10 μg/mL herceptin (HER2 inhibitor) for 6 h. Results are presented as mean ± SD of 5 independent experiments. (B, C) SK-BR-3 cells were incubated with 20 μmol/L kaempferol and/or 10 μg/mL herceptin for 1 h, and then subjected to the RhoA (B) and Rac1 (C) activity assays. The relative levels of Rho activity were normalized to the average value of controls. Results are presented as mean ± SD of 3 independent experiments.
Abbreviations: Ctrl, control; HER2, human epidermal growth factor receptor 2.

Figure 5 Overexpression of HER2 rescues the migration and Rho activity of TNBC cells inhibited by kaempferol.

Notes: (A) MDA-MB-231 TNBC cells were transfected with HA-HER2 or vector, and then subjected to Western blot analysis. β-actin as the internal control. (B) MDA-MB-231 TNBC cells were transfected with HA-HER2 or vector, then allowed to migrate in response to 20 μmol/L kaempferol for 6 h. Results are presented as mean ± SD of 5 independent experiments. (C, D) MDA-MB-231 TNBC cells were transfected with HA-HER2 or vector, then incubated with 20 μmol/L kaempferol for 1 h, and subsequently subjected to the RhoA (C) and Rac1 (D) activity assays. The relative levels of Rho activity were normalized to the average value of controls. Results are presented as mean ± SD of 3 independent experiments. (E) MDA-MB-231 TNBC cells were transfected with HA-HER2 or vector, then allowed to invade in response to 20 μmol/L kaempferol for 6 h. Results are presented as mean ± SD of 3 independent experiments.
Abbreviations: Ctrl, control; HER2, human epidermal growth factor receptor 2; ns, no significance; TNBC, triple-negative breast cancer.

Figure 6 Kaempferol and ER/PR inhibitor suppress the migration and Rho activity of MCF-7 cells.

Notes: (A) MCF-7 ER⁺/PR⁺ breast cancer cells were allowed to migrate in response to 20 μmol/L kaempferol and/or 0.1 nmol/L AZD9496 (ER inhibitor) and 200 μmol/L megestrol acetate (PR inhibitor) for 6 h. Results are presented as mean ± SD of 5 independent experiments. (B, C) MCF-7 cells were incubated with 20 μmol/L kaempferol and/or 0.1 nmol/L AZD9496 and 200 μmol/L MA for 1 h, and then subjected to the RhoA (B) and Rac1 (C) activity assays. The relative levels of Rho activity were normalized to the average value of controls. Results are presented as mean ± SD of 3 independent experiments.
Abbreviations: AZD, AZD9496; Ctrl, control; ER, estrogen receptor; MA, megestrol acetate; ns, no significance; PR, progesterone receptor.

Figure 7 Kaempferol downregulates the mRNA levels of EMT markers and MMPs in TNBC cells.

Notes: (A, B) MDA-MB-231 TNBC cells were incubated with 20 μmol/L kaempferol for 1 h, and then subjected to the qPCR assays. The relative mRNA levels of E-cadherin, Vimentin, Snail, Slug (A), and MMP2, MMP9 (B) were normalized to the average value of controls. Results are presented as mean ± SD of 3 independent experiments.
Abbreviations: EMT, epithelial–mesenchymal transition; MMP, matrix metalloproteinase; qRT-PCR, quantitative real-time polymerase chain reaction; TNBC, triple-negative breast cancer.