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. 2008;60 Suppl 1(Suppl 1):51-60.
doi: 10.1080/01635580802381477.

Ayurvedic medicine constituent withaferin a causes G2 and M phase cell cycle arrest in human breast cancer cells

Silvia D Stan  1 , Yan Zeng, Shivendra V Singh
Affiliations

Ayurvedic medicine constituent withaferin a causes G2 and M phase cell cycle arrest in human breast cancer cells

Silvia D Stan et al. Nutr Cancer. 2008.

Abstract

Withaferin A (WA) is derived from the medicinal plant Withania somnifera that has been safely used for centuries in the Indian Ayurvedic medicine for treatment of various ailments. We now demonstrate that WA treatment causes G2 and mitotic arrest in human breast cancer cells. Treatment of MDA-MB-231 (estrogen-independent) and MCF-7 (estrogen-responsive) cell lines with WA resulted in a concentration- and time-dependent increase in G2-M fraction, which correlated with a decrease in levels of cyclin-dependent kinase 1 (Cdk1), cell division cycle 25C (Cdc25C) and/or Cdc25B proteins, leading to accumulation of Tyrosine15 phosphorylated (inactive) Cdk1. Ectopic expression of Cdc25C conferred partial yet significant protection against WA-mediated G2-M phase cell cycle arrest in MDA-MB-231 cells. The WA-treated MDA-MB-231 and MCF-7 cells were also arrested in mitosis as judged by fluorescence microscopy and analysis of Ser10 phosphorylated histone H3. Mitotic arrest resulting from exposure to WA was accompanied by an increase in the protein level of anaphase promoting complex/cyclosome substrate securin. In conclusion, the results of this study suggest that G2-M phase cell cycle arrest may be an important mechanism in antiproliferative effect of WA against human breast cancer cells.

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Figures

Figure 1
Figure 1
Representative histograms depicting cell cycle distribution in (A) MDA-MB-231 and (C) MCF-7 cultures following 24 h treatment with DMSO (control) or the indicated concentrations of withaferin A (WA). Percentage of cells in different phases of the cell cycle in (B) MDA-MB-231 and (D) MCF-7 cultures following 24 h treatment with DMSO (control) or the indicated concentrations of WA. Results are mean ± SE (n=3). *, P<0.05, significantly different compared with DMSO-treated control by one-way ANOVA followed by Dunnett's test. Similar results were observed in two independent experiments.
Figure 2
Figure 2
Percentage of G2-M fraction in (A) MDA-MB-231 and (B) MCF-7 cultures following treatment with DMSO (control) or 2 μM WA for the indicated time periods. C, left panels, cell cycle distribution in MDA-MB-231 cultures treated for 24 h with DMSO (control) or 2 μM WA; right panels, cell cycle distribution in MDA-MB-231 cells treated for 24 h with either DMSO (control) or 2 μM WA and then cultured in drug-free media for 24 h prior to the analysis of cell cycle distribution. Columns, mean (n=3); bars, SE. *, P<0.05, significantly different compared with the corresponding control by one-way ANOVA. Similar results were observed in two independent experiments.
Figure 3
Figure 3
Immunoblotting for cyclinB1, Cdk1, Cdc25B, Cdc25C, and Tyr15 phosphorylated Cdk1 using lysates from MDA-MB-231 and MCF-7 cells treated with 2 μM withaferin A (WA) for the indicated time periods. Membranes were stripped and re-probed with anti-actin antibody to ensure equal protein loading. Numbers on top of the bands represent changes in protein levels as determined by densitometric analysis of the immunoreactive bands and corrected for actin loading control. Immunoblotting for each protein was performed at least twice using independently prepared lysates.
Figure 4
Figure 4
(A) Percentage of 2′,7′-dichlorofluorescein (DCF) positive (a measure of reactive oxygen species production) MDA-MB-231 cells following treatment with DMSO (control) or 2 μM withaferin A (WA) for the indicated time periods. Columns, mean (n=3); bars, SE. *, P<0.05, significantly different compared with the corresponding control by one-way ANOVA. (B) Percentage of DCF fluorescence in MDA-MB-231 cells treated with 2 μM WA for 8 h with or without a 2 h pre-treatment with 30 μM EUK134. (C) Percentage of G2-M fraction in MDA-MB-231 cells treated with 2 μM WA for 8 h with or without a 2 h pretreatment with 30 μM EUK134. (D) Enrichment of G2-M fraction relative to corresponding DMSO-treated control in MDA-MB-231 cells transiently transfected with either empty-vector or vector encoding Cdc25C and then treated with DMSO (control) or 2 μM WA for 8 h. Inset, immunoblotting for Cdc25C protein using lysates from MDA-MB-231 cells transiently transfected with empty-vector (lane 1) or vector encoding Cdc25C (lane 2). In panels B-D, columns, mean (n=3); bars, SE; *, P<0.05, significantly different between the indicated groups by one-way ANOVA followed by Bonferroni's multiple comparison test.
Figure 5
Figure 5
(A) left panels, fluorescence microscopic images depicting α-tubulin (red) and DAPI (blue) staining in MDA-MB-231 cells treated for 24 h with either DMSO or 2 μM withaferin A (WA). Note abundance of mitotic figures with condensed chromatin (identified by arrows) in WA-treated MDA-MB-231 cells, which were rarely seen in DMSO-treated controls. Right panel, percentage of mitotic figures with condensed chromatin in MDA-MB-231 cells treated with DMSO (control) or 2 μM WA for 6, 12 or 24 h. Columns, mean (n= 2); error bars are included to indicate the range of values. (B) Immunoblotting for Ser10 phosphorylated histone H3 using lysates from MDA-MB-231 and MCF-7 cells treated with 2 μM WA for the indicated time intervals. (C) Percentage of mitotic cells, determined by flow-cytometric analysis of Ser10 phosphorylated histone H3, in MDA-MB-231 cells treated for 24 h with DMSO (control) or 2 μM WA or maintained in drug-free complete medium after DMSO or WA treatment for an additional 24 h. Columns, mean (n=3); bars, SE. *, P<0.05, significantly different between the indicated groups by one-way ANOVA followed by Bonferroni's multiple comparison test. (D) Immunoblotting for securin using lysates from MDA-MB-231 and MCF-7 cells treated with 2 μM WA for the indicated time periods. In panels B and D, membranes were stripped and re-probed with anti-actin antibody to ensure equal protein loading. Immunoblotting for each protein was performed at least twice using independently prepared lysates.
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