Fig 1.
Chemical structures of damsin, ambrosin, coronopolin, and dindol-01.
Fig 2.
Damsin, ambrosin, coronopilin, and dindol-01 inhibit TNFα-induced p65/NF-κB nuclear translocation.
MCF-7 and JIMT-1 cells were treated with 5 μM damsin, ambrosin, coronopilin, or dindol-01 for 60 minutes, and cells were then stimulated with 25 ng/ml TNF-α for 40 minutes. Control-only cells received compound vehicle and TNF-α vehicle. Control-TNF-α cells received compound vehicle for 60 minutes and then TNF-α. The cells were fixed and stained to visualize p65/NF-κB expression (green). Images were taken with a 100x oil immersion objective using an Olympus/Nikon epifluorescence microscope. The scale bars denote 20 μm. Representative images from three independent experiments are shown.
Table 1.
IC50 values for damsin, ambrosin, coronopilin, and dindol-01 in MCF-10A, MCF-7, JIMT-1, and HCC1937 cells.a
Fig 3.
Damsin and ambrosin inhibit cell proliferation of MCF-10A normal-like breast epithelial cells and of MCF-7, JIMT-1, and HCC1937 breast cancer cells in a time- and dose-dependent manner.
Cells were seeded day 0, and the compounds were added 1 day after seeding. The cell number was determined by counting in a haemocytometer after cell detachment. ○: control. ●: 1 μM. ■: 2.5 μM. ▲: 5 μM. Data are presented as the mean of 3 experiments, and bars indicate SD.
Fig 4.
Damsin and ambrosin treatment results in altered cell cycle phase distribution, and ambrosin treatment also induces cell death.
The cell cycle phase distribution (panels A-D and I-L) and the % debris (sub-G1 region) (panels E-H and M-P) related to cell death was deduced from DNA histograms obtained by flow cytometry of cells treated with damsin or ambrosin for 72 hours. C: control. D: damsin. A: ambrosin. The numbers 1, 2.5, and 5 denote micromolar concentration. Column definition for cell cycle phase distribution: Black, G1 phase; White, S phase; Grey, G2 phase. Data are presented as the mean ± SD for n = 3.
Fig 5.
Damsin and ambrosin treatment affect cell cycle regulatory proteins.
Panels A-E show the results of densitometric scanning, and panels F-H are representative Western blots used for the scanning. A. Cyclin D1 expression in JIMT-1 cells. B. CDK2 expression in JIMT-1 cells. C. p53 expression in MCF-7 cells. D. p21 expression in MCF-7 cells. E. p53 expression in JIMT-1 cells. Representative blots used for densitometric scanning. F. Cyclin D1 and CDK2 expression in JIMT-1 cells. G. p53 and p21 expression in MCF-7 cells. H. p53 expression in JIMT-1 cells. The cells were treated with 1 (D 1 and A 1) or 5 (D 5 and A 5) μM concentrations of damsin (D) or ambrosin (A) for 72 hours. The data in A-E are expressed in % of control and presented as the mean ± SE for n = 3. D and E. *, p < 0.05 compared to control.
Fig 6.
Treatment of MCF-10A (A), MCF-7 (B), and JIMT-1 (C) cells with damsin (D) or ambrosin (A) increases the number of micronuclei. The cells were treated with 1 (D 1 and A 1) or 5 (D 5 and A 5) μM concentrations for 72 hours. The cells were then fixed, and the nuclei were stained with bisbenzimide. Micronuclei were counted in the fluorescence microscopy images. Data are presented as the mean ± SE for n = 6 experiments.
Fig 7.
Effect of damsin and ambrosin treatment on the expression of proteins in the NF-κB pathway.
Panels A-D. MCF-7 cells. Panels E-H. JIMT-1 cells. Panels D and H show the representative Western blots used to derive the data. D: damsin. A: ambrosin. The numbers 1 and 5 denote micromolar concentration for 72 hours of treatment. The data are expressed in % of control and presented as the mean ± SE for n = 3. *, p < 0.05 compared to control.
Fig 8.
Damsin (D) and ambrosin (A) treatment decreases the CSC population.
The cells were treated with 1 (D 1 and A 1) or 5 (D 5 and A 5) μM concentrations for 72 hours. A. The CD44+/CD24- population and B. the ALDH+ populations were evaluated by flow cytometry. C. Colony forming efficiency was evaluated using a serum-free soft agar assay. JIMT-1 cells were treated for 72 hours and then reseeded at cloning density. The colonies were counted after two weeks of incubation. The numbers denote treatment concentrations (μM). Data are presented as the mean ± SE for n = 3–6. Significantly different compared to control: *: p < 0.05.
Fig 9.
Damsin (D) and ambrosin (A) treatment inhibits wound healing evaluated with a wound-healing assay using JIMT-1 cells.
Panels A and B: Treatment with a 1 μM concentration. Panels C and D: Treatment with a 5 μM concentration. Data in A and C are presented as the mean ± SE for n = 6. Panels B and D: Representative phase-contrast images 72 hours after wounding. The bars denote 100 μm. The numbers in B and D denote treatment concentrations (μM). Panel A. Significantly different compared to control: A: *: p < 0.05. Panel C. Ambrosin is significantly different compared to control and damsin: *: p < 0.05.