Therapy Effects of Wogonin on Ovarian Cancer Cells

Abstract

Background: Wogonin is a plant monoflavonoid and has been reported to induce apoptosis of cancer cells and show inhibitory effect on cancer cell growth. However, the detailed and underlying molecular mechanisms are not elucidated. In this study, we investigated the molecular and biological effects of wogonin in human ovarian A2780 cancer cells.

Materials and methods: We determined the effects of wogonin on the changes of cell cycling and apoptotic responses of cells. Western blot analysis was used to measure the effects of wogonin on protein expressions.

Results: Our results showed that treatment with wogonin inhibited the cancer cell proliferation, decreased the percentage of G0/G1 subpopulation, and reduced invasiveness of A2780 cells. Exposure to wogonin also resulted in downregulated protein levels of estrogen receptor alpha (ER-α), VEGF, Bcl-2, and Akt and increased expressions of Bax and p53. In addition, exposure to wogonin increased caspase-3 cleavage and induced apoptosis in A2780 cells. Our study further showed that MPP, a specific ER-α inhibitor, significantly enhanced antitumor effects of wogonin in A2780 cells.

Conclusion: Our results suggest a potential clinical impact of wogonin on management of ovarian cancer.

Figure 1

Wogonin inhibits cancer cell growth of A2780 ovarian cancer cells. (a) The chemical structure of the active form of 5,7-dihydroxy-8-methoxyflavone (named as wogonin). (b) Graphs showing the inhibitory effects of wogonin on proliferation of A2780 cells. Data present average results from three independent experiment; SD means the standard deviation (n = 3).

Figure 2

Combination effects of wogonin and MPP on cancer cell invasion and clonogenic survival in A2780 ovarian cancer cells. ((a) and (b)) Wogonin inhibits A2780 ovarian cancer cells migration. Representative images of wound scratch assay showing the effect of wogonin on cell migration. Graph showing the change of inhibition ratio for wound scratch rehealing. (c) Invasion assay. Representative images showing the effect of wogonin on cancer cell invasiveness. (d) Combined effects of wogonin and MPP on clonogenic survival of A2780 ovarian cancer cells. Representative images showing the surviving colonies. (e) Graphs showing the changes of clonogenic survival fraction. The error bars represent the standard error, ^∗P < 0.05 versus control group and ^#P < 0.05 versus wogonin + MPP group, n = 3. (f) Western blot analysis showing the effects of wogonin and MPP on ER-α, VEGF, and p53 protein expression in A2780 ovarian cancer cells. β-Actin was included as a loading control. Data present average results from three independent experiments; SD means the standard deviation (n = 3).

Figure 3

Wogonin treatment induces apoptosis in A2780 ovarian cancer cells. (a) Representative images showing apoptosis detected with Annexin V-FITC and PI staining in A2780 cells. Percentage of the bottom right quadrant and the top right quadrant showing the representative events of early and late apoptosis, respectively. (b) Representative images of fluorescence microscopy analysis showing the effects of treatment with wogonin, MPP, and the combination on nuclei apoptosis and cell necrosis in A2780 cells. Cells were stained with Hoechst and PI, and images were taken under magnification of ×200. (c) The protein expression of Akt, Bcl-2,Bax, cytochrome C, caspase-3, and cleaved-caspase-3. β-Actin was included as a loading control.

Figure 4

Effects of wogonin on cell cycling distribution of A2780 ovarian cancer cells. (a) Representative results of the flow cytometry analysis with A2780 cells treated with or without wogonin combined with MPP. (b) Graphs showing the changes of the percentage for each cell cycle in A2780 cells. (c) Representative results of Western blot analysis showing the effects of wogonin on the expression of cyclin D1, CDK4, and CDK6. Data present average results from three independent experiments; SD means the standard deviation (n = 3). ^∗P < 0.05 and ^∗∗P < 0.01 versus control group.