Fig 1.
Structure of ramentaceone (5-hydroxy-7-methyl-1,4-naphthoquinone).
Fig 2.
Effects of ramentaceone on the viability of breast cancer cells.
(A) Cytotoxic activity of ramentaceone toward HER2-overexpressing (BT474 and SKBR3) and HER2-negative (MCF-7 and MDA-MB-231) breast cancer cells. Cells were treated with ramentaceone (0–15 μM) for 24 h and cell survival was assessed with the MTT assay (n = 3). (B) Influence of ramentaceone on clonogenicity of HER2-positive breast cancer cells, SKBR3 and BT474. Cells were treated with ramentaceone (0–15 μM) for 3 h and allowed to grow for 16 days to form colonies. Values represent mean ± SD of three independent experiments. p < 0.05 (*) indicates differences between control and ramentaceone-treated cells.
Fig 3.
Effects of ramentaceone on the expression levels of PI3K/Akt signaling proteins.
(A) Concentration-dependent inhibition of PI3K/Akt signaling. Breast cancer cells were treated with ramentaceone (0–15 μM) for 24 h and the levels of PI3K p85, phospho-PI3K p85 (Tyr458)/p55 (Tyr199), phospho-Akt (Ser 473), and Akt were assessed by Western blot analysis. (B) Time-dependent inhibition of PI3K/Akt signaling. BT474 and SKBR3 cells were treated with ramentaceone (5 μM) for the indicated time periods and the levels of PI3K p85, phospho-Akt (Ser 473), and Akt were assessed by Western blot analysis. Protein levels were determined by densitometric analysis. Values represent mean ± SD of three independent experiments. p < 0.05 (*) indicates differences between control and ramentaceone-treated cells.
Fig 4.
Inhibition of phosphoinositide-3-kinase (PI3-kinase) activity by ramentaceone in breast cancer cells.
The AlphaScreen technology was used to determine enzyme activity. The AlphaScreen assay was performed with ramentaceone or LY294002, added at concentrations ranging from 100 μM to 0.01 nM, 25 ng PI3-kinase, 5 μM PIP2, 10 μM ATP and with an incubation time of 1 h. Values represent mean ± SD of three independent experiments.
Fig 5.
Induction of apoptosis by ramentaceone in breast cancer cells.
Apoptotic changes in plasma membrane induced by ramentaceone. Cells were treated with ramentaceone (0–15 μM) for 24 h, stained with Annexin V-PE/7-AAD, and analyzed by flow cytometry. Values represent mean ± SD of three independent experiments. p < 0.05 (*) indicates differences between control and ramentaceone-treated cells.
Fig 6.
Effects of ramentaceone on expression levels Bcl-2 family proteins and caspase activity in breast cancer cells.
(A) Effects of ramentaceone on the expression levels of Bax, Bak and Bcl-2 in SKBR3 and BT474 cells. Cells were treated with ramentaceone (0–15 μM) for 24 h, and the levels of Bax, Bak, Bcl-2 were assessed by Western blot analysis. Protein levels were determined by densitometric analysis (B) Induction of caspase activity in BT474 and SKBR3 cells. Cells were treated with ramentaceone (0–15 μM) for 12 h, and enzyme activity was determined by flow cytometry with the use of a caspase inhibitor, FAM-VAD-FMK (C) Activation of caspase-3 and cleavage of PARP by ramentaceone in BT474 and SKBR3 cells. Cells were treated with ramentaceone (0–15 μM) for 24 h, and the levels of cleaved caspase-3 and PARP were assessed by Western blot analysis. Protein levels were determined by densitometric analysis. Values represent mean ± SD of three independent experiments. p < 0.05 (*) indicates differences between control and ramentaceone-treated cells.
Fig 7.
The role of PI3K inhibition in ramentaceone-mediated apoptosis induction.
BT474 and SKBR3 cells were pre-treated with the PI3K inhibitor LY294002 (5 μM) after which cells were treated with ramentaceone (5 μM) for 24 h. Bax, Bak, Bcl-2 levels were assessed by Western blot analysis. Protein levels were determined by densitometric analysis. Values represent mean ± SD of three independent experiments. p < 0.05 (*) indicates differences between control and ramentaceone-treated cells. p < 0.05 (#) indicates differences between ramentaceone-treated and LY294002 pre-treated samples.
Fig 8.
The role of Akt inhibition in ramentaceone-mediated apoptosis induction.
(A) Silencing of Akt by siRNA in BT474 and SKBR3 cells. Cells were transiently transfected with Akt siRNA and 24 after transfection, Akt silencing was confirmed with Western blot analysis. (B) Induction of apoptosis by ramentaceone in cells transfected with Akt siRNA and control siRNA. 24 h after transfection cells were treated with ramentaceone (5 μM) for 24 h, stained with Annexin V-PE/7-AAD, and analyzed by flow cytometry. Representative of three independent experiments. p < 0.05 (*) indicate differences between control siRNA and Akt siRNA transfected cells treated with ramentaceone.