Amentoflavone triggers cell cycle G2/M arrest by interfering with microtubule dynamics and inducing DNA damage in SKOV3 cells

Abstract

Ovarian cancer is the seventh most common cancer and the second most common cause of cancer-associated mortality among gynecological malignancies worldwide. The combination of antimitotic agents, such as taxanes, and the DNA-damaging agents, such as platinum compounds, is the standard treatment for ovarian cancer. However, due to chemoresistance, development of novel therapeutic strategies for the treatment of ovarian cancer remains critical. Amentoflavone (AMF) is a biflavonoid derived from the extracts of Selaginella tamariscina, which has been used as a Chinese herb for thousands of years. A previous study demonstrated that AMF inhibits angiogenesis of endothelial cells and induces apoptosis in hypertrophic scar fibroblasts. In order to check the influence of AMF on cell proliferation, the effects of AMF on cell cycle and DNA damage were measured by cell viability, flow cytometry, immunofluorescence and western blotting assays in SKOV3 cells, an ovarian cell line. In the present study, treatment with AMF inhibited ovarian cell proliferation, increased P21 expression, decreased CDK1/2 expression, interrupted the balance of microtubule dynamics and arrested cells at the G2 phase. Furthermore, treatment with AMF increased the expression levels of phospho-Histone H2AX (γ-H2AX; a variant of histone 2A, that belongs to the histone 2A family member X) and the DNA repair protein RAD51 homolog 1 (Rad51), indicating the occurrence of DNA damage since γ-H2AX and Rad51 are both key markers of DNA damage. Consistent with previous findings, the results of the present study suggest that AMF is a potential therapeutic agent for the treatment of ovarian cancer. In addition, the effects of AMF on cell cycle arrest and DNA damage induction may be the molecular mechanisms by which AMF might exert its potential therapeutic benefits in ovarian cancer.

Keywords: DNA damage; amentoflavone; cell cycle; microtubule dynamics.

Figure 1.

AMF decreases SKOV3 cell viability and induces cell cycle arrest. (A) SKOV3 cells were treated with different concentrations (0, 50 75, 100, 150 and 200 µmol/l) of AMF for 48 h and cell viability was assessed via the CellTiter 96 Aqueous One Solution Proliferation assay. The results demonstrated that AMF decreased SKOV3 cell viability in a dose-dependent manner. (B) Histograms showed the cell cycle distribution at G1, S and G2 phase. Data are presented as the mean ± SD (n=3). SKOV3 cells were treated with different concentrations of AMF (0, 100 and 150 µmol/l) for 48 h and cell cycle distribution was assessed via flow cytometric analysis. (C) Cell cycle analysis by flow cytometry. SKOV3 cells were treated with different concentrations of AMF (0, 100 and 150 µmol/l) for 48 h. (D) The expression levels of cyclin B, p-CDK1, CDK1, CDK2 and p21 were determined in SKOV3 cells treated with different concentrations of AMF (0, 100 and 150 µmol/l) for 48 h by western blot. GAPDH was used as the loading control. (E) Protein expression levels from the western blot in (D) relative to the GAPDH control. Data are presented as the mean ± SD (n=3). *P<0.05, **P<0.01 vs. control. AMF, amentoflavone; SD, standard deviation; CDK, cyclin-dependent kinase.

Figure 2.

AMF interferes with tubulin expression and spindle assembly. (A) Immunofluorescence staining of α-tubulin (green) in SKOV3 cells treated with different concentrations of AMF for 48 h. Nuclei (blue) were stained with DAPI (magnification, ×400; scale bar, 100 µm). (B) Immunofluorescence staining of β-tubulin (green) in SKOV3 cells treated with different concentrations of AMF for 48 h. Nuclei (blue) were stained with DAPI (magnification, ×400; scale bar, 100 µm). (C) The expression levels of stathmin and β-tubulin in SKOV3 cells treated with different concentrations of AMF, via western blot analysis. GAPDH was used as the loading control. (D) Protein expression levels from the western blot in (C) relative to the GAPDH control. Data are presented as the mean ± standard deviation (n=3). *P<0.05 vs. control. AMF, amentoflavone.

Figure 3.

AMF induces DNA damage in SKOV3 cells. (A) Immunofluorescence staining of nuclei (blue) and γ-H2AX (green) in SKOV3 cells treated with different concentrations of AMF for 48 h (magnification, ×400; scale bar, 100 µm). (B) Fluorescence intensities of γ-H2AX in SKOV3 cells treated with different concentrations of AMF for 48 h. Data are presented as the mean ± SD (n=3). (C) Expression levels of γ-H2AX and Rad51 in SKOV3 cells treated with different concentrations of AMF, via western blot analysis. GAPDH was used as the loading control. (D) Protein expression levels from the western blot in (C) relative to the GAPDH control. Data are presented as the mean ± SD (n=3). *P<0.05, **P<0.01 vs. control. AMF, amentoflavone; γ-H2AX, phospho-Histone H2AX; SD, standard deviation.