| Chemical agents can reduce cancer through chemoprevention or chemotherapy. Chemoprevention is the inhibition of the carcinogenic process. To discover inhibitors of estrogen signaling pathway, implicated during promotion of breast cancer, a series of in vitro assays were developed for high-throughput screening of natural products. Six extracts impeded (3H) -estradiol binding to the estrogen receptor; Erythrina rubinevria underwent bioassay-guided fractionation to isolate the known phytoestrogen coumestrol. Tamoxifen can bind at secondary receptor, the antiestrogen binding site, which acting as a molecular sponge can reduce the effective concentration of tamoxifen at the estrogen receptor. Utilizing (3H) -tamoxifen displacement assay, five extracts were found to inhibit tamoxifen from binding to this receptor; subsequent fractionation of Pachysandra procumbens led to the isolation of a series of steroidal alkaloids. However, isolated receptor assays cannot determine cellular responses; to that end the Ishikawa cell system was developed. Ishikawa cells, human endometrial cell line, have an estrogen-dependent alkaline phosphatase. Twelve extracts induced enzymatic activity, including the six active in the estrogen receptor assay. Anti-estrogenic activity was defined as reduction of phosphatase induction from exogenously added estradiol. Cerbera manghas, one of four extracts displaying anti-estrogenic activity at sub-toxic doses, was fractionated, and the known lignan cyclo-olivil was isolated.;Betulinic acid, isolated from high-throughput cytotoxic screening of natural products, displayed selective activity against human melanoma, MEL-2. In vivo analyses determined betulinic acid prevented tumor development and reduced tumor volume. DNA fragmentation and flow cytometric analyses concluded betulinic acid induced apoptosis. At sub-toxic doses, betulinic acid caused a G... |
