Data were analyzed by Students t test. Results Proliferation assay We first performed a MTT based proliferation assay to explore how the black cohosh selleckbio extract and the selected compounds influence growth of MCF 7 cells. 17 estra diol, the positive control, significantly stimulated cell pro liferation after 120 h, reaching a plateau of maximal stimulation at a concentration of 1 nM. The magnitude was compara ble to effects observed in other studies with MCF 7 cells. At a concentration of 10M tamoxifen reduced cell proliferation to 45. 7 6. 8%. Exposure of MCF 7 cells for 120 h to different concentrations of black cohosh extract, the cycloartane glycoside actein and the cycloartane aglycon mixture all inhibited cell proliferation in a dose depend ent manner.
IC50 values the concentrations that caused 50% inhibition of growth were determined by extrapolation for black cohosh extract, actein and the aglycon mixture. These proliferation and cytotoxicity measure ments provide only a preliminary information of biologi cal activity. Gene expression microarray analysis with black cohosh extract General results For gene expression Inhibitors,Modulators,Libraries profiling MCF 7 cells were treated with 15g ml black cohosh extract, 1 nM 17 estradiol, 10M tamoxifen or DMSO control for 24 h in the pres ence of 10% charcoal stripped serum. After RNA extrac tion, gene expression profiles were recorded using Affymetrix Inhibitors,Modulators,Libraries HG U133 Plus 2. 0 GeneChip Arrays as described. Regulated genes were identified using the following selection criteria minimal signal intensity median and fold change vs. control 1. 5 in two independent experiments.
With these criteria we Inhibitors,Modulators,Libraries identified 544 probe sets representing 431 genes regulated by the extract. The false positive rate with the criteria used is about 5% based on random per mutation analysis of the gene expression results, showing a mean of 22 to 32 regulated probe sets. Of the genes regulated by the extract, 335 transcripts were upregulated and 96 genes were down regulated. These genes were grouped into functional cate gories according to Gene Ontology terms and gene description at the NetAffx Analysis Center and addi tion literature search. Most of the regulated genes could be clearly assigned to 5 larger groups of functionally related genes apoptosis, proliferation, general growth, signal ing transport and metabo lism.
Genes that could not be assigned to any of these groups were summarized as others. Furthermore, because of a striking presence of many transcripts linked to cellular stress response, we also created the functional category stress response, which is not directly linked to the other cat egories. Genes functionally connected to this group are already members Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries Wortmannin manufacturer of one of the 6 main categories. In most groups more genes were stimulated than inhibited. In the group proliferation, in contrast, a majority of genes appeared to be downregulated.