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. 2020 Jan 3;25(1):207.
doi: 10.3390/molecules25010207.

Isoflavones Isolated From the Seeds of Millettia ferruginea Induced Apoptotic Cell Death in Human Ovarian Cancer Cells

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Free PMC article

Isoflavones Isolated From the Seeds of Millettia ferruginea Induced Apoptotic Cell Death in Human Ovarian Cancer Cells

Yi-Yue Wang et al. Molecules. .
Free PMC article

Abstract

The seeds of Millettia ferruginea are used in fishing, pesticides, and folk medicine in Ethiopia. Here, the anti-cancer effects of isoflavones isolated from M. ferruginea were evaluated in human ovarian cancer cells. We found that isoflavone ferrugone and 6,7-dimethoxy-3',4'-methylenedioxy-8-(3,3-dimethylallyl)isoflavone (DMI) had potent cytotoxic effects on human ovarian cancer cell A2780 and SKOV3. Ferrugone and DMI treatment increased the sub-G1 cell population in a dose-dependent manner in A2780 cells. The cytotoxic activity of ferrugone and DMI was associated with the induction of apoptosis, as shown by an increase in annexin V-positive cells. Z-VAD-fmk, a broad-spectrum caspase inhibitor, and z-DEVD-fmk, a caspase-3 inhibitor, significantly reversed both the ferrugone and DMI-induced apoptosis, suggesting that cell death stimulated by the isoflavones is mediated by caspase-3-dependent apoptosis. Additionally, ferrugone-induced apoptosis was found to be caspase-8-dependent, while DMI-induced apoptosis was caspase-9-dependent. Notably, DMI, but not ferrugone, increased the intracellular levels of reactive oxygen species (ROS), and antioxidant N-acetyl-L-cysteine (NAC) attenuated the pro-apoptotic activity of DMI. These data suggest that DMI induced apoptotic cell death through the intrinsic pathway via ROS production, while ferrugone stimulated the extrinsic pathway in human ovarian cancer cells.

Keywords: 6,7-dimethoxy-3’,4’-methylenedioxy-8-(3,3-dimethylallyl) isoflavone; Millettia ferruginea; apoptosis; caspase; ferrugone; ovarian cancer.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effect of ferrugone and DMI on cell cycle distribution in human ovarian cancer cells. A2780 cells were treated with ferrugone (A) or DMI (B) (0.25, 0.5, and 1 µM) for 48 h and then stained with propidium iodide (PI). The cell-cycle distribution profiles of the cells were determined by flow cytometry. The graph indicates the percentages of cells in the sub-G1, G0/G1, S, and G2/M phases of the cell cycle. The data are representative of three independent experiments.
Figure 2
Figure 2
Effect of ferrugone and DMI on apoptotic cell death in human ovarian cancer cells. A2780 cells were treated with ferrugone (A) and DMI (B) (0.25, 0.5, and 1 µM) for 48 h and co-stained with PI and Annexin V-FITC (fluorescein isothiocyanate). The translocation of phosphatidyl serine was detected by flow cytometry. The graph indicates the percentages of annexin V-positive apoptotic cells in the right quadrants of flow cytometry graphs. The data are representative of three independent experiments. Data were analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test. * p < 0.05 as compared with untreated group.
Figure 3
Figure 3
Involvement of caspases in ferrugone and DMI-induced cell death in human ovarian cancer cells. A2780 cells were pretreated with broad caspase inhibitor z-VAD-fmk (50 µM) for 2 h, and then treated with ferrugone (A) and DMI (B) (0.5 µM) for 48 h. Annexin/PI V-FITC staining assay was performed to determine apoptosis. The graph indicates the percentages of annexin V-positive apoptotic cells in the right quadrants of flow cytometry graphs. The data are representative of three independent experiments. Student’s t-test (two-tailed) was applied to evaluate the significance. # p < 0.05 as compared with the untreated group. * p < 0.05 as compared with the ferrugone or DMI only-treated group.
Figure 4
Figure 4
Involvement of caspase-3, -8, and -9 in ferrugone and DMI-induced cell death in human ovarian cancer cells. (A,B) A2780 cells were pretreated with caspase-3 inhibitor z-DEVD-fmk (75 µM), caspase-8 inhibitor z-IETD-fmk (50 µM), and caspase-9 inhibitor z-LEHD-fmk (75 µM) for 2 h, and then treated with ferrugone (A) and DMI (B) (0.5 µM) for 24 h. MTT assay was performed to determine cell death. (C,D) A2780 cells were treated with ferrugone (C) and DMI (B) (0.25, 0.5, and 1 µM) for 48 h. Cleaved caspase-3 and caspase-8 levels were determined by Western blot assay. β-actin was used as an internal control. The data are representative of three independent experiments. Student’s t-test (two-tailed) was applied to evaluate the significance. # p < 0.05 as compared with the untreated group. * p < 0.05 as compared with the ferrugone or DMI only-treated group.
Figure 5
Figure 5
Involvement of ROS (reactive oxygen species) in DMI-induced cell death in human ovarian cancer cells. (A) A2780 cells were treated with 0.5 µM of ferrugone for the indicated times (1, 2, and 4 h). The cells were stained with DCF-DA (2’,7’–dichlorofluorescin diacetate), and analyzed by flow cytometry. (B) A2780 cells were pretreated with N-acetyl-L-cysteine (NAC, 5 mM) for 30 min, then treated with ferrugone (0.5 µM) for 48 h. MTT assay was performed to examine the cell death. (C) A2780 cells were treated with 0.5 µM of DMI for the indicated times (1, 2, and 4 h). The cells were stained with DCF-DA and analyzed by flow cytometry. (D) A2780 cells were pretreated with NAC (5 mM) for 30 min, then treated with DMI (0.5 µM) for 48 h. MTT assay was performed to examine the cell death. Student’s t-test (two-tailed) was applied to evaluate the significance. # p < 0.05 as compared with the untreated group. * p < 0.05 as compared with the ferrugone or DMI only treated group.

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