A134

Esculetin and daphnetin are coumarin derivatives contained in many plants which are commonly used in folk medicine. The antiproliferative effects at microM concentrations of both compounds have been reported in several cancer cell lines. In our laboratory we have observed a pronounced inhibitory activity of daphnetin in the MCF-7 cell line, whereas esculetin has a weak antiproliferative activity. The purpose of this study was the evaluation of the estrogenic activities of these coumarins, as well as the expression of cyclin D1 by quantitative real-time RT-PCR in the MCF-7 cell line. METHODS. The cells were cultured in high glucose DMEM medium (with and without phenol red and supplemented with 5% bovine calf serum) at 37°C and 5% of CO2. The cells were exposed for ten days to the test compounds at concentrations ranging from 1nM to 100 microM. 17beta-Estradiol was used as a positive control at concentrations ranging from 1 fM to 100 pM. Cell proliferation was estimated by the microculture Tetrazolium (MTT) assay. Total RNA was obtained using trizol reagent and processed according to manufacturer´s protocol (Invitrogen Corp). 100 ng of total RNA was incubated in presence of MLV reversetrancriptase, to obtain first strand cDNA. Later, 10 ng of cDNA was used as template and subjected to quantitative real-time PCR amplification using specific primer sets for human Cyclin D1 and CYBRGreen-Mix (Invitrogen Corp). Quantification and analysis of the data were performed with Rotorgene equipment and software from Corbett research platform. RESULTS: 17beta-Estradio,l at 1fM to 1pM concentrations, increased cell proliferation with respect to the control values. Esculetin had estrogenic effects at concentration of 10 pM to 10 microM, and inhibited cell proliferation at higher concentrations (100 microM). These effects were antagonized by tamoxifen. Daphnetin had no estrogenic effects and inhibited cell proliferation even at a 10 microM concentration. Esculetin increased the relative expression of cyclin D1 mRNA at concentrations of 10 pM to 10 microM but inhibited it at concentration of 100 microM. In contrast, daphnetin diminished the relative expression of ciclin D1 at a 1 microM and higher concentrations. These results suggest that esculetin might be useful as a phyto-estrogen in the treatment of postmenopausal women, whereas daphnetin might be useful in the treatment of breast cancer. Grants: PAPIIT/UNAM IN220606-2.

[Fifth AACR International Conference on Frontiers in Cancer Prevention Research, Nov 12-15, 2006]