Effects of Leuzea carthamoides on human breast adenocarcinoma MCF-7 cells determined by gene expression profiling and functional assays.

Products derived from roots of Leuzea carthamoides (Maral root) are being promoted as dietary supplements with anti-aging, adaptogenic and anabolic activity, without much scientific evidence. We investigated the effects of a lipophilic Leuzea root extract and the major phytoecdysteroid, 20-hydroxyecdysone, in human breast adenocarcinoma MCF-7 cells. Cell proliferation was inhibited by the extract (IC50 = 30 microg/mL) but not by 20-hydroxyecdysone. Genome-wide expression profiling using Affymetrix HG U133 Plus 2.0 microarrays was carried out to analyse effects at the transcriptional level. 241 genes appeared to be differentially expressed after Leuzea treatment, more than after treatment with either 17beta-estradiol or tamoxifen. Transcripts linked to cell cycle regulation and DNA replication were highly over-represented and regulated in an anti-proliferative manner. Genes involved in apoptosis were regulated in a pro-apoptotic manner. Expression levels of several oxidoreductase transcripts were strongly induced, most prominent CYP1A1, known to be regulated via the aryl hydrocarbon receptor pathway. An XRE-dependent reporter gene assay confirmed the AhR-agonistic activity of the Leuzea root extract, whereas 20-hydroxyecdysone was not active. Leuzea extract also inhibited 5alpha-reductase, type II. While the extract significantly modulates cellular activities, the phytoecdysteroids, are most likely not the active principles of L. carthamoides.

Differential expression of cancer-related genes by single and permanent exposure to bone morphogenetic protein 2.

PURPOSE: Bone morphogenetic proteins (BMPs) are multifunctional regulators of various cell functions. The BMP-signalling network plays a pivotal role during embryogenesis and tumorigenesis. BMPs, e.g. BMP-2 exert their biological function in a time and concentration-dependent manner but also modulated by the context of the cellular microenvironment. In this study, we investigated the effect of a steady high level of BMP-2 versus a single application of BMP-2 on the breast cancer cell line MCF-7. METHODS: The effect of the incubation regimes was analysed by DNA microarray expression profiling. Data were verified by real-time PCR. The protein expression of apoptosis-related genes was studied by western blot analysis. RESULTS: We found a clear difference in the altered gene expression between the constant high level and the single application of BMP-2. After grouping the genes of interest into the biological processes of Gene Ontology, the group of apoptosis-related genes like BAX, BAG5 or PKR, was predominantly affected under the single-application regime of BMP-2. Among these protein kinase R was the most prominently regulated. Further studies on the protein level showed activation of PKR after 4 h with a subsequent enhanced phosphorylation of the PKR substrate eIF2alpha for several hours. CONCLUSIONS: The duration of treatment and the concentration of BMP-2 affect the global expression pattern of MCF-7 cells. Among the regulated cancer-related genes, the cohort of the apoptosis-related genes showed the pronounced alterations. Our data point to a novel role of BMP-2 in the regulation of the PKR pathway in tumorigenesis.

Gene expression profiling reveals effects of Cimicifuga racemosa (L.) NUTT. (black cohosh) on the estrogen receptor positive human breast cancer cell line MCF-7.

BACKGROUND: Extracts from the rhizome of Cimicifuga racemosa (black cohosh) are increasingly popular as herbal alternative to hormone replacement therapy (HRT) for the alleviation of postmenopausal disorders. However, the molecular mode of action and the active principles are presently not clear. Previously published data have been largely contradictory. We, therefore, investigated the effects of a lipophilic black cohosh rhizome extract and cycloartane-type triterpenoids on the estrogen receptor positive human breast cancer cell line MCF-7. RESULTS: Both extract and purified compounds clearly inhibited cellular proliferation. Gene expression profiling with the extract allowed us to identify 431 regulated genes with high significance. The extract induced expression pattern differed from those of 17beta-estradiol or the estrogen receptor antagonist tamoxifen. We observed a significant enrichment of genes in an anti-proliferative and apoptosis-sensitizing manner, as well as an increase of mRNAs coding for gene products involved in several stress response pathways. These functional groups were highly overrepresented among all regulated genes. Also several transcripts coding for oxidoreductases were induced, as for example the cytochrome P450 family members 1A1 and 1B1. In addition, some transcripts associated with antitumor but also tumor-promoting activity were regulated. Real-Time RT-PCR analysis of 13 selected genes was conducted after treatment with purified compounds - the cycloartane-type triterpene glycoside actein and triterpene aglycons - showing similar expression levels compared to the extract. CONCLUSION: No estrogenic but antiproliferative and proapoptotic gene expression was shown for black cohosh in MCF-7 cells at the transcriptional level. The effects may be results of the activation of different pathways. The cycloartane glycosides and - for the first time - their aglycons could be identified as an active principle in black cohosh.

Ranking: a closer look on globalisation methods for normalisation of gene expression arrays.

Data from gene expression arrays are influenced by many experimental parameters that lead to variations not simply accessible by standard quantification methods. To compare measurements from gene expression array experiments, quantitative data are commonly normalised using reference genes or global normalisation methods based on mean or median values. These methods are based on the assumption that (i) selected reference genes are expressed at a standard level in all experiments or (ii) that mean or median signal of expression will give a quantitative reference for each individual experiment. We introduce here a new ranking diagram, with which we can show how the different normalisation methods compare, and how they are influenced by variations in measurements (noise) that occur in every experiment. Furthermore, we show that an upper trimmed mean provides a simple and robust method for normalisation of larger sets of experiments by comparative analysis.