Sulforaphane Causes Epigenetic Repression of hTERT Expression in Human Breast Cancer Cell Lines

Abstract

BackgroundSulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, is a common dietary component that has histone deacetylase inhibition activity and exciting potential in cancer prevention. The mechanisms by which SFN imparts its chemopreventive properties are of considerable interest and little is known of its preventive potential for breast cancer.Principal FindingsWe found that SFN significantly inhibits the viability and proliferation of breast cancer cells in vitro while it has negligible effects on normal breast cells. Inhibition of telomerase has received considerable attention because of its high expression in cancer cells and extremely low level of expression in normal cells. SFN treatment dose- and time-dependently inhibited human telomerase reverse transcriptase (hTERT), the catalytic regulatory subunit of telomerase, in both MCF-7 and MDA-MB-231 human breast cancer cells. DNA methyltransferases (DNMTs), especially DNMT1 and DNMT3a, were also decreased in SFN-treated breast cancer cells suggesting that SFN may repress hTERT by impacting epigenetic pathways. Down-regulation of DNMTs in response to SFN induced site-specific CpG demethylation occurring primarily in the first exon of the hTERT gene thereby facilitating CTCF binding associated with hTERT repression. Chromatin immunoprecipitation (ChIP) analysis of the hTERT promoter revealed that SFN increased the level of active chromatin markers acetyl-H3, acetyl-H3K9 and acetyl-H4, whereas the trimethyl-H3K9 and trimethyl-H3K27 inactive chromatin markers were decreased in a dose-dependent manner. SFN-induced hyperacetylation facilitated the binding of many hTERT repressor proteins such as MAD1 and CTCF to the hTERT regulatory region. Depletion of CTCF using siRNA reduced the SFN-induced down-regulation of hTERT mRNA transcription in these breast cancer cells. In addition, down-regulation of hTERT expression facilitated the induction of cellular apoptosis in human breast cancer cells.SignificanceCollectively, our results provide novel insights into SFN-mediated epigenetic down-regulation of telomerase in breast cancer prevention and may open new avenues for approaches to SFN-mediated cancer prevention.

Sulforaphane Inhibits Growth of Human Breast Cancer Cells and Augments the Therapeutic Index of the Chemotherapeutic Drug, Gemcitabine

“…The effective concentration (EC 50 ) was found to be 25μM SFN for duration of 24h (Figure 1). Other studies also showed that SFN treatment significantly reduces cell viability of various cancer cells i.e., human leukemia T-cells, breast, pancreatic, lymphoblastoid and glioma cancer cells in a dose-dependent manner (Fimognari et al, 2002;Misiewicz et al, 2004;Meeran et al, 2010;Huang et al, 2012;Li et al, 2012). Interestingly, SFN has a safe cytotoxicity profile towards the normal cells thus it can be utilized in the development of safe cancer treatment strategies (Sharma et al, 2012).…”

Cucurbitacin B Alters the Expression of Tumor-Related Genes by Epigenetic Modifications in NSCLC and Inhibits NNK-Induced Lung Tumorigenesis

“…RNA isolation and RT-PCR were performed as described previously (6,8,21,22). The primer sequences and annealing temperatures (T A ) are given in Supplementary Table S2.…”

“…The procedures have been described previously (6,21). Briefly, equal amounts of protein were electrophoresced and transferred onto a 0.45-mm polyvinylidene difluoride (PVDF) membrane.…”

“…The HDACs and DNMTs activities were analyzed using the colorimetric assay kits (Active Motif) as described previously (21,22).…”

“…Chromatin immunoprecipitation (ChIP) assays were performed using the EZ-ChIP Kit according to the manufacturer's protocol (Millipore) as described previously (21,22). The antibodies used in the ChIP assays were ChIP-validated anti-acetyl histone H3 (06-599), anti-acetyl histone H4 (06-598), anti-acetyl histone H3K9 (07-352), anti-histone H3K9me3 (07-442), antihistone H3K27me3 (07-449; Millipore) and anti-MeCP2 (ab2828; Abcam).…”