Withaferin A and sulforaphane regulate breast cancer cell cycle progression through epigenetic mechanisms

Abstract

Little is known about the effects of combinatorial dietary compounds on the regulation of epigenetic mechanisms involved in breast cancer prevention. The human diet consists of a multitude of components, and there is a need to elucidate how certain compounds interact in collaboration. Withaferin A (WA), found in the Indian winter cherry and documented as a DNA methyltransferase (DNMT) inhibitor, and sulforaphane (SFN), a well-known histone deacetylase (HDAC) inhibitor found in cruciferous vegetables, are two epigenetic modifying compounds that have only recently been studied in conjunction. The use of DNMT and HDAC inhibitors to reverse the malignant expression of certain genes in breast cancer has shown considerable promise. Previously, we found that SFN + WA synergistically promote breast cancer cell death. Herein, we determined that these compounds inhibit cell cycle progression from S to G2 phase in MDA-MB-231 and MCF-7 breast cancer. Furthermore, we demonstrate that this unique combination of epigenetic modifying compounds down-regulates the levels of Cyclin D1 and CDK4, and pRB; conversely, the levels of E2F mRNA and tumor suppressor p21 are increased independently of p53. We find these events coincide with an increase in unrestricted histone methylation. We propose SFN + WA-induced breast cancer cell death is attributed, in part, to epigenetic modifications that result in the modulated expression of key genes responsible for the regulation of cancer cell senescence.

Keywords: Adjuvant; Breast cancer; Chemotherapy; Combinatorial therapy; Sulforaphane; Withaferin A.

Fig. 1.. Combinatorial SFN and WA arrest cells most abundantly at G1 in breast cancer cells:

A. Combinatorial SFN and WA arrest cells at G1 phase and prevent transition into G2 in MCF-7 cells. B. In MDA-MB-231 breast cancer cells it can be noted that the incorporation of these compounds prevent transition into G2 phase (n=3).

Fig. 2.. Cell cycle genes are regulated by WA and SFN at the mRNA level in breast cancer cells:

A. Combinatorial SFN + WA down-regulate cyclin D1 in an extremely significant manner but not significantly more than the single dosages in MCF-7 breast cancer cells. B. Cyclin D1 is also down-regulated in MDA-MB-231 breast cancer cells yet the combination is not significantly different from the signal dosages. C. CDK4 in MCF-7 cells shows a downward trend with single treatments of SFN or WA although SFN + WA does not show greater significance than the single dosages. D. CDK4 in MDA-MB-231 cells is down-regulated. E. E2F gene expression in MCF-7 cells is significantly upregulated in comparison to the control with the introduction of WA and the combo. F. E2F mRNA expression in MDA-MB-231 cells is significantly increased in comparison to the control (n=3; p < 0.05 *, p < 0.01 **, p < 0.001 ***).

Fig. 3.. pRB protein is inhibited by SFN and WA in MCF-7 and MDA-MB-231 cells:

A. Combinatorial SFN and WA down-regulate pRB expression in MCF-7 breast cancer cells with the combination being the most effective. Densitometry was determined using ImageJ and bar graphs represent 3 replicates relative to B-actin. B. pRB protein is down-regulated by SFN + WA in MDA-MB-231. ImageJ was used to calculate densitometry (n=3; images are representative).

Fig. 4.. HDAC2 and HDAC3 protein levels are down-regulated by SFN and WA in breast cancer cells:

A. Combinatorial SFN and WA downregulate HDAC2 and HDAC3 expression in MCF-7 breast cancer cells with the combination appearing to be the most effective. Densitometry was determined using ImageJ and bar graphs represent 3 replicates relative to B-actin. B. HDAC2 and HDAC3 protein is downregulated by SFN + WA in MDA-MB-231. ImageJ was used to calculate densitometry (n=3; images are representative).

Fig. 5.. HMT enzymatic activity in breast cancer cells is down-regulated by natural compounds:

A. Combinatorial SFN and WA down-regulate HMT activity with SFN being most effective in MCF-7 breast cancer cells. B. Histone methyltransferase activity is down-regulated by SFN and WA in MDA-MB-231(n=3; p < 0.05*).

Fig. 6.. Effects of SFN and WA on HAT Activity in MCF-7 and MDA-MB-231 Cells:

A. Combinatorial SFN and WA show no significant change in HAT activity in MCF-7 breast cancer cells. B. Histone acetyltransferase activity is upregulated by WA and the combination appears more effective in MDA-MB-231 but not significantly (n=3; p < 0.05*, p < 0.01**).

Fig. 7.. Global methylation is increased in breast cancer cells by combinatorial WA and SFN:

A. SFN and WA alone have no significant effect on global methylation but when these two compounds are used together we report an increase in methylation in MCF-7 cells. B. Global methylation is significantly upregulated by combinatorial WA and SFN in MDA-MB-231 cells (n=3; p < 0.05 *, p < 0.01**).

Fig. 8.. Tumor suppressor proteins implicated in cell cycle progression are modulated by SFN and WA in breast cancer cells:

A. Combinatorial SFN and WA show no significant change in p53 protein expression in MCF-7 breast cancer cells; however, p21 is upregulated with the combination being most effective. B. p53 protein expression is down-regulated by SFN and the combination of SFN and WA where p21 is upregulated with the combination being the most effective (n=3; images are representative and bar graphs represent the densitometry results of 3 replicates).

Fig. 9.. Activation of p21 is mediated by transcriptional activator H3K4Me3 in MDA-MB-231 cells:

ChIP assay reveals a significant increase in the expression of H3K4Me3 at the p21 promoter of triple negative breast cancer cells. Values represent average +/− SEM. One exemplar shown (n=3; p < 0.05*, p < 0.01**).