EFFECTS OF HISTONE DEACETYLASE INHIBITORS ON VITAMIN D ACTIVITY IN HUMAN BREAST CANCER CELLS

ABSTRACT

Breast cancer is one of the leading causes of death among cancer cases in women worldwide. Ultimately, cancer is the result of a combination of environmental and genetic factors that contribute to alterations in cellular control of proliferation, differentiation and programmed cell death. Vitamin D had been emerging as a potentially important nutrient in the prevention and treatment of cancer due to its ability to modulate proliferation, apoptosis, invasion and metastasis in vivo in animal cancer models and in vitro cell culture studies. To accomplish these cellular effects, Vitamin D exerts its biological activity via the binding of its hormonal metabolite 1,25(OH) 2D3 to a specific, high-affinity, intracellular vitamin D receptor (VDR).

Although VDR expression can be identified in mammary cancer cell lines, levels are often reduced compared to non-cancerous cells, which could limit vitamin D-induced gene expression and function in these cells. In addition, changes in chromatin structure, associated with epigenetic modifications of nuclear histone proteins, can lead to changes in gene expression, including the suppression of important “tumor suppressor” genes, thereby promoting carcinogenesis. Our study investigated the extent to which two compounds with histone deacetylase inhibitor (HDACI) activity, trichostatin A (TSA), a well-known and potent HDACI, and sulforaphane (SFN), a bioactive food component with HDACI activity, can influence the expression of some important genes involved in vitamin D action and metabolism in cells. The genes investigated were: CYP24A1, which codes for a 24-hydroxylase that deactivates 1,25(OH) 2D3; CYP27B1, which codes for a 1α-hydroxylase that activates 25(OH)D to the 1,25(OH) 2D3 hormone; VDR, the nuclear receptor transcription factor that is activated by 1,25(OH) 2D3; and TRPV6, which codes for a calcium-specific channel implicated in breast cancer cell invasiveness.

 

We found that in MCF-7 breast cancer cells, 1,25(OH)2D3 treatment alone induced the expression of VDR mRNA (in 1 of 2 studies), CYP24A1 mRNA (in both studies) and CYP27B1(in 1 of 1 study where measured). Surprisingly, TRPV6 mRNA expression was not evident in MCF-7 cells in either the presence or absence of 1,25(OH)2D3. The HDACI TSA alone increased expression of VDR mRNA, but SFN alone had no effect. Importantly, VDR mRNA was increased by co-treatment of 1,25(OH)2D3 and TSA compared to 1,25(OH)2D3 alone in Experiment 1 with a similar, but not statistically significant, trend in Experiment 2. As expected, 1,25(OH)2D3 markedly increased CYP24A1 mRNA. Unexpectedly, treatment with either TSA or SFN alone increased CYP24A1 mRNA, suggesting that endogenous expression of CYP24A1 may be normally suppressed in MCF-7 cells by epigenetic mechanisms involving histone acetylation status. An apparent increase in CYP24A1 mRNA following co-treatment of TSA with 1,25(OH)2D3 in both experiments was not statistically significant. SFN co-treatment with 1,25(OH)2D3 also did not cause a further increase in CYP24A1 mRNA. 1,25(OH)2D3 treatment increased CYP27B1 expression, but co-treatment with TSA had no additional effect. In contrast, co-treatment of SFN and 1,25(OH)2D3 inhibited much of the positive effects of vitamin D. TSA is already being used as a co-treatment in cancer therapy. Vorinostat, a drug with the same chemical structure as TSA, is being investigated in hundreds of clinical trials. (Marks & Breslow, 2007) Our findings support further study of the effects of the HDACI TSA in breast cancer, and suggest that this HDACI may be beneficial in augmenting vitamin D cellular responsiveness.