Sulforaphane causes epigenetic repression of hTERT expression in human breast cancer cell lines.
BACKGROUND
Sulforaphane (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.


RESULTS
We 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.


CONCLUSIONS
Collectively, 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.
Meeran
Syed M
SM
Department of Biology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
Patel
Shweta N
SN
Tollefsbol
Trygve O
TO
D000107:Acetylation / Q000187:drug effects; D017209:Apoptosis / Q000235:genetics / Q000502:physiology; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics* / Q000517:prevention & control; D000076246:CCCTC-Binding Factor; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D047369:Chromatin Immunoprecipitation; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000235:genetics; D006801:Humans; D017879:Isothiocyanates; D011401:Promoter Regions, Genetic / Q000235:genetics; D034741:RNA, Small Interfering; D012097:Repressor Proteins / Q000235:genetics / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013454:Sulfoxides; D019098:Telomerase / Q000235:genetics / Q000378:metabolism*; D013861:Thiocyanates / Q000627:therapeutic use*
D000076246:CCCTC-Binding Factor; C000619697:CTCF protein, human; C000717967:DNMT3A protein, human; D017879:Isothiocyanates; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D013454:Sulfoxides; D013861:Thiocyanates; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; C000619380:DNMT1 protein, human; C509186:TERT protein, human; D019098:Telomerase; C016766:sulforaphane