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

Gaube F, Wolfl S, Pusch L, Kroll TC, Hamburger M - BMC Pharmacol. (2007)

Bottom Line: 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.The effects may be results of the activation of different pathways.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Pharmacy, Department of Pharmaceutical Biology, University of Jena, Semmelweisstr, 10, 07743 Jena, Germany. Friedemann.Gaube@uni-jena.de

ABSTRACT

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.

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Overlap of expression profiles. Numbers within the 3 circles represent the genes that were differentially expressed according to our filters after 24 h treatment of MCF-7 cells with Cimicifuga racemosa (black cohosh) extract (CR), 17β-estradiol (E2) or tamoxifen (TAM). Numbers within the intersections represent genes regulated with both of the respective treatments or – in the middle – all 3 different treatments. For every intersection genes regulated in the same direction up or down (correlated) are marked with (+). Genes regulated in opposite directions (anti-correlated) are marked with (-).
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Figure 7: Overlap of expression profiles. Numbers within the 3 circles represent the genes that were differentially expressed according to our filters after 24 h treatment of MCF-7 cells with Cimicifuga racemosa (black cohosh) extract (CR), 17β-estradiol (E2) or tamoxifen (TAM). Numbers within the intersections represent genes regulated with both of the respective treatments or – in the middle – all 3 different treatments. For every intersection genes regulated in the same direction up or down (correlated) are marked with (+). Genes regulated in opposite directions (anti-correlated) are marked with (-).

Mentions: Expression profiles with 17β-estradiol and the estrogen receptor-antagonist tamoxifen were investigated in parallel to compare the patterns with the black cohosh extract. After treatment with 1 nM E2 146 transcripts met our selection criteria, among these known estrogen-regulated genes such as insulin-like growth factor binding protein 4 (IGFBP4) or GREB1 protein (GREB1). The latter transcript was strongly upregulated (microarray: 21.7 and 10.4 fold; RT-PCR: 26.6 ± 10.1, p < 0.05 and 15.4 ± 5.7, p < 0.05). With 10 μM tamoxifen 49 genes were observed to be regulated. Figure 7 shows the intersection of expression patterns of black cohosh, E2 and tamoxifen. Among a total of 39 genes that were regulated both under black cohosh and E2 treatment, 30 transcripts were affected in opposite directions (anti-correlated). In contrast, a comparison of black cohosh and tamoxifen revealed a correlated regulation of 32 transcripts. This is quite surprising given that only 49 genes were considered regulated in response to tamoxifen. Hence, the expression profile of black cohosh was more related to tamoxifen. Since E2 stimulated and tamoxifen inhibited proliferation of MCF-7 cells in our assay, it was not surprising that most genes in the intersections are related to cell cycle regulation and apoptosis. Among the genes associated with cell cycle arrest and apoptosis that were regulated in all treatments, the two cell cycle inhibitory transcripts cyclin G2 (Figure 8) and tumor protein p53 inducible nuclear protein 1 (TP53INP1) were both upregulated by black cohosh and tamoxifen and downregulated by E2. Apart from ESR1, which was downregulated with E2 and black cohosh treatment (Figure 8), and VEGF, which can be considered to be regulated via hypoxia response pathways, no gene affected by black cohosh is known to contain estrogen responsive elements (ERE) in the promoter region.


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

Gaube F, Wolfl S, Pusch L, Kroll TC, Hamburger M - BMC Pharmacol. (2007)

Overlap of expression profiles. Numbers within the 3 circles represent the genes that were differentially expressed according to our filters after 24 h treatment of MCF-7 cells with Cimicifuga racemosa (black cohosh) extract (CR), 17β-estradiol (E2) or tamoxifen (TAM). Numbers within the intersections represent genes regulated with both of the respective treatments or – in the middle – all 3 different treatments. For every intersection genes regulated in the same direction up or down (correlated) are marked with (+). Genes regulated in opposite directions (anti-correlated) are marked with (-).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2194763&req=5

Figure 7: Overlap of expression profiles. Numbers within the 3 circles represent the genes that were differentially expressed according to our filters after 24 h treatment of MCF-7 cells with Cimicifuga racemosa (black cohosh) extract (CR), 17β-estradiol (E2) or tamoxifen (TAM). Numbers within the intersections represent genes regulated with both of the respective treatments or – in the middle – all 3 different treatments. For every intersection genes regulated in the same direction up or down (correlated) are marked with (+). Genes regulated in opposite directions (anti-correlated) are marked with (-).
Mentions: Expression profiles with 17β-estradiol and the estrogen receptor-antagonist tamoxifen were investigated in parallel to compare the patterns with the black cohosh extract. After treatment with 1 nM E2 146 transcripts met our selection criteria, among these known estrogen-regulated genes such as insulin-like growth factor binding protein 4 (IGFBP4) or GREB1 protein (GREB1). The latter transcript was strongly upregulated (microarray: 21.7 and 10.4 fold; RT-PCR: 26.6 ± 10.1, p < 0.05 and 15.4 ± 5.7, p < 0.05). With 10 μM tamoxifen 49 genes were observed to be regulated. Figure 7 shows the intersection of expression patterns of black cohosh, E2 and tamoxifen. Among a total of 39 genes that were regulated both under black cohosh and E2 treatment, 30 transcripts were affected in opposite directions (anti-correlated). In contrast, a comparison of black cohosh and tamoxifen revealed a correlated regulation of 32 transcripts. This is quite surprising given that only 49 genes were considered regulated in response to tamoxifen. Hence, the expression profile of black cohosh was more related to tamoxifen. Since E2 stimulated and tamoxifen inhibited proliferation of MCF-7 cells in our assay, it was not surprising that most genes in the intersections are related to cell cycle regulation and apoptosis. Among the genes associated with cell cycle arrest and apoptosis that were regulated in all treatments, the two cell cycle inhibitory transcripts cyclin G2 (Figure 8) and tumor protein p53 inducible nuclear protein 1 (TP53INP1) were both upregulated by black cohosh and tamoxifen and downregulated by E2. Apart from ESR1, which was downregulated with E2 and black cohosh treatment (Figure 8), and VEGF, which can be considered to be regulated via hypoxia response pathways, no gene affected by black cohosh is known to contain estrogen responsive elements (ERE) in the promoter region.

Bottom Line: 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.The effects may be results of the activation of different pathways.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Pharmacy, Department of Pharmaceutical Biology, University of Jena, Semmelweisstr, 10, 07743 Jena, Germany. Friedemann.Gaube@uni-jena.de

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus