Evaluation of wild yam (dioscorea villosa) as a demethylating agent and its anti metastatic activity in human breast cancer cells

Aberrant epigenetic alterations in the genome are believed to be a potential cause of some forms of cancer. Due to their reversibility, epigenetic modifications are considered potentially useful in drug development (epi-drugs). Currently available synthetic epi-drugs have many adverse effects. Epigenetic pathways may represent targets for natural product utilized in cancer treatment. The present study was designed to evaluate the efficacy of a traditionally used product, wild yam root extract (WYRE), as a potential demethylating agent in breast cancer. Two breast cancer cell lines were used, MCF-7 (estrogen receptor positive, ER+) and MDA-MB-231 (ER negative, ER-), and the effect of WYRE on a gene, GATA3, a potential marker of breast cancer development was studied. GATA3 expression is controlled by methylation, and it is found to be expressed higher in ER+ cells with correlating promoter hypomethylation, and its expression is insignificant in ER- cells correlating with promoter hypermethylation. In the current study, cells were treated with WYRE (0-50 &mgr;g/mL) for 72h and then evaluated for changes in mRNA and in promoter methylation patterns. WYRE significantly reduced cell proliferation of both cell lines and increased transcription of GATA3 and methylating DNMT proteins (DNMT1, 3A, and 3B) in a concentration-dependent manner. Global DNA methylation as analyzed by 5'-methyl-2'-deoxycytidine (5-mC) and 5-hydroxymethylcytosine (5-hmC) showed that 5-mC was increased in MCF-7, whereas 5-hmC level was reduced in MDA-MB-231 cells. Since 5-hmC is generated from 5-mC by ten-eleven-translocation (TET) enzymes, their expression was examined as well following WYRE treatment. Of the TET proteins only TET3 mRNA expression was increased by WYRE (50 &mgr;g/mL) in MDA-MB-231 cells; no changes were noted in MCF-7 cells. Methylation analysis of the GATA3 promoter indicated demethylating activity of WYRE in MDA-MB-231 cells. Two compound isolates from WYRE considered responsible for the anti-cancer activity, dioscin and diosgenin, were also evaluated as described above for the root extract. Both dioscin and diosgenin were able to reduce in vitro cell viability of both ER+ (MCF-7) and ER- (MDA-MB-231) human breast cancer cells, with dioscin demonstrating greater potentcy than diosgenin in both cell lines. Dioscin at 5.76 &mgr;M showed upregulation of GATA3 mRNA expression in MDA-MB-231 cell only. GATA3 protein expression was also enhanced in a concentration- dependent manner in MDA-MB-231 cells treated with dioscin (0-5.76 &mgr;M)as seen by both western blot, and by immunocytochemistry followed by confocal microscopy. Interestingly, dioscin was founded to enhance the expression of GATA3 protein in both cell lines. Despite increased transcription in the MDA-MB-231 cells, an upstream (ZFPM2) and downstream (E-cadherin) markers of GATA3 function were examined. ZFPM2 mRNA expression significantly increased by dioscin at 5.76 &mgr;M in both cell lines, as did E-cadherin expression. Other cell transition markers were studied, such as MMP9 and VIM. We found that dioscin at 2.30 &mgr;M decreased expression of MMP9 transcription, and at 2.30 and 5.76 &mgr;M suppressed VIM mRNA expression in MDA-MB-231 cells. Notably, dioscin was found to have an inhibitory effect on migration of MDA-MB-231 cells in concentration- and time- dependent manner, as observed by a wound-healing assay and in migration/invasion assay in both breast cancer cell lines. These findings reveal a new therapeutic potential for anti-metastatic therapy targeting epigenome and in the future these plant extract as a whole or dioscin may be able to offer an efficacious adjuvant therapy to women with breast cancer.