Hormone replacement therapy is contraindicated in women with breast cancer. Extracts from the rhizomes of Cimicifuga racemosa, have gained acceptance as a natural alternative for the treatment of menopausal symptoms. In the present study we investigated the antiproliferative activity of C. racemosa extracts (isopropanolic and ethanolic) on the estrogen receptor positive MCF-7 and estrogen receptor negative MDA-MB231 breast cancer cells by WST-1 assay. Down regulation of the proliferative activity and cell killing by isopropanolic and ethanolic extracts occurred in a clear dose-dependent response with a 50% growth inhibitory concentration of 54.1 +/- 11.4 and 80.6 +/- 17.7 micro g/ml in MCF-7 cells and of 29.5 +/- 3.0 and 58.6 +/- 12.6 microg/ml in MDA-MB231 cells, respectively. Further, the mode of cell death was identified as apoptosis by microscopic inspection and confirmed by light scatter characteristics and by detection of Annexin V adherence to phosphatidylserine by flow cytometry. In addition, the involvement of activated caspases was supported by the cleavage of cytokeratin 18 detected with M30 antibody. Increases in the level of M30-antigen of about 4-fold and 2-fold over untreated controls were observed in C. racemosa -treated MCF-7 and MDA-MB231 cells. These results indicate that C. racemosa extract exerts no proliferative activity, but kills the estrogen receptor positive MCF-7 as well as estrogen receptor negative MDA-MB231 cells by activation of caspases and induction of apoptosis.
We previously reported that the antiproliferative effect of an isopropanolic-aqueous extract of black cohosh (iCR) on MCF-7 estrogen-responsive breast cancer cell line was due to the induction of apoptosis. Here we address the question to what extent apoptosis induction can be ascribed to one of the two major fractions of iCR, the triterpene glycosides (TTG) or the cinnamic acid esters (CAE). Furthermore, as black cohosh is routinely administered orally, we studied whether its pharmacological effects would withstand simulated liver metabolism. The antiproliferative activity of TTG and CAE as well as of rat liver microsomal S9 fraction-pretreated iCR on MCF-7 cells were investigated by WST-1 assay. The features of cell death induced were tested for apoptosis by flow cytometry (light scatter characteristics, Annexin V binding). Irrespective of S9-pretreatment, 72 h iCR treatment induced a dose-dependent down regulation of cell proliferation with the same IC 50 of 55.3 m mg/ml dry residue which corresponds to 19.3 m mg/ml TTG and 2.7 m mg/ml CAE. The degree of apoptotic MCF-7 cells was also comparable. Both, isolated TTG and CAE fractions inhibited cell growth, the IC 50 being 59.3 m mg/ml and 26.1 m mg/ml, respectively. Interestingly, whereas IC 50 and apoptosis induction correspond well for the whole extract, TTG and CAE fractions induced apoptosis at concentrations (25 and 5 m mg/ml) well below those required for significant growth inhibition. Observation of this study firstly showed that the cell death induced by iCR withstood a metabolic activation system. In addition, TTG and CAE compounds significantly contributed to its apoptotic effect, CAE being the more potent inhibitor of proliferation and apoptosis inducer.
Hormone replacement therapy, which is a common menopausal treatment, is contraindicated in women with breast cancers due to concerns regarding the potential for breast cell proliferation. As such, there is a need for alternative methods for treating menopausal symptoms. To determine the influence of one such alternative, black cohosh (Cimicifuga racemosa [CR]), on estrogen-dependent mammary cancers, we conducted an in vitro investigation of the effect of an isopropanolic CR-extract on the proliferation of estrogen receptor-positive breast cancer cells. The experiments were performed using the human breast adenocarcinoma (MCF-7) cell test system, an established in vitro model for estrogen-dependent tumors. The influence of CR-extract on the proliferation of the MCF-7 cells was determined by measuring the incorporation of radioactively labeled thymidine. Under estrogen-deprived conditions, the CR-extract (10(-3)-10(-5) dilutions) significantly inhibited MCF-7 cell proliferation. Additionally, application of the CR-extract inhibited estrogen-induced proliferation of MCF-7 cells. Moreover, the proliferation-inhibiting effect of tamoxifen was enhanced by the CR-extract. Such data that suggest a non-estrogenic, or estrogen-antagonistic effect of CR on human breast cancer cells lead to the conclusion that CR treatment may be a safe, natural remedy for menopausal symptoms in breast cancer.
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