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Planta Med 2008; 74(14): 1701-1708
DOI: 10.1055/s-0028-1088316
Original Paper
Pharmacology
© Georg Thieme Verlag KG Stuttgart · New York

Effects of Leuzea carthamoides on Human Breast Adenocarcinoma MCF-7 Cells Determined by Gene Expression Profiling and Functional Assays

Friedemann Gaube1 , Stefan Wölfl2 , Larissa Pusch3 , Ulrike Werner1 , Torsten C. Kroll3 , Dieter Schrenk4 , Rolf W. Hartmann5 , Matthias Hamburger6
  • 1Institute of Pharmacy, University of Jena, Jena, Germany
  • 2Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Heidelberg, Germany
  • 3Clinic of Internal Medicine II, University of Jena, Jena, Germany
  • 4Department of Food Chemistry and Toxicology, University of Kaiserslautern, Kaiserslautern, Germany
  • 5Department of Pharmaceutical and Medicinal Chemistry, Saarland University, Saarbrücken, Germany
  • 6Department of Pharmaceutical Sciences, Institute of Pharmaceutical Biology, University of Basel, Basel, Switzerland; formerly Institute of Pharmacy, University of Jena, Jena, Germany
Further Information

Publication History

Received: April 3, 2008 Revised: July 25, 2008

Accepted: July 29, 2008

Publication Date:
29 October 2008 (online)

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Abstract

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 μg/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 17β-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 5α-reductase, type II. While the extract significantly modulates cellular activities, the phytoecdysteroids, are most likely not the active principles of L. carthamoides.

Abbreviations

AhR:aryl hydrocarbon receptor

AKR:aldo-keto reductase

CYP1A1:cytochrome P450, family 1, subfamily A, polypeptide 1

E2:17β-estratiol

ER:estrogen receptor

20-HE:20-hydroxyecdysone

Key words

Leuzea carthamoides - Asteraceae - phytoecdysteroids - expression profiling - CYP1A1 - aryl hydrocarbon receptor

References

  • 1 Varga E, Szendrei K, Hajdu Z, Hornok L, Csáki G. Study of the compounds contained in Hungarian-grown Leuzea carthamoides D.C., (Asteraceae), with special regard to ecdysteroids.  Herba Hung. 1986;  25 115-33
    PubMedGoogle Scholar
  • 2 Szendrei K, Reisch J, Varga E. Thiophene acetylenes from Leuzea roots.  Phytochemistry. 1984;  23 901-2
    CrossrefPubMedGoogle Scholar
  • 3 Chobot V, Vytlacilova J, Kubicova L, Opletal L, Jahodar L, Laakso I. et al . Phototoxic activity of a thiophene polyacetylene from Leuzea carthamoides. .  Fitoterapia. 2006;  77 194-8
    CrossrefPubMedGoogle Scholar
  • 4 Pavlík M, Laudová V, Gruner K, Vokac K, Harmatha J. High-performance liquid chromatographic analysis and separation of N-feruloylserotonin isomers.  J Chromatogr B. 2002;  770 291-5
    CrossrefPubMedGoogle Scholar
  • 5 Grimshaw J, Jaruszelski M, Lamerzarawska E, Rzadkowska-Bodalska H. Sterols and new triterpenoid alcohol from Leuzea carthamoides (Willd.) DC.  Pol J Chem. 1981;  55 2355-8
    PubMedGoogle Scholar
  • 6 Lafont R, Dinan L. Practical uses for ecdysteroids in mammals including humans: an update.  J Insect Sci. 2003;  3 7
    PubMedGoogle Scholar
  • 7 Le Bizec B, Antignac J P, Monteau F, Andre F. Ecdysteroids: one potential new anabolic family in breeding animals.  Anal Chim Acta. 2002;  473 89-97
    CrossrefPubMedGoogle Scholar
  • 8 Sláma K, Lafont R. Insect hormones – ecdysteroids: their presence and actions in vertebrates.  Eur J Entomol. 1995;  92 355-77
    PubMedGoogle Scholar
  • 9 Dinan L, Lafont R. Effects and applications of arthropod steroid hormones (ecdysteroids) in mammals.  J Endocrinol. 2006;  191 1-8
    CrossrefPubMedGoogle Scholar
  • 10 Vienonen A, Miettinen S, Manninen T, Altucci L, Wilhelm E, Ylikomi T. Regulation of nuclear receptor and cofactor expression in breast cancer cell lines.  Eur J Endocrinol. 2003;  148 469-79
    CrossrefPubMedGoogle Scholar
  • 11 Safe S, Wormke M, Samudio I. Mechanisms of inhibitory aryl hydrocarbon receptor-estrogen receptor crosstalk in human breast cancer cells.  J Mammary Gland Biol Neoplasia. 2000;  5 295-306
    CrossrefPubMedGoogle Scholar
  • 12 Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays.  J Immunol Methods. 1983;  65 55-63
    CrossrefPubMedGoogle Scholar
  • 13 Denizot F, Lang R. Rapid colorimetric assay for cellular growth and survival: Modifications to the tetrazolium dye procedure giving improved sensivity and reliability.  J Immunol Methods. 1986;  89 271-7
    CrossrefPubMedGoogle Scholar
  • 14 Kroll T C, Wolfl S. Ranking: a closer look on globalisation methods for normalisation of gene expression arrays.  Nucleic Acids Res. 2002;  30 e50
    CrossrefPubMedGoogle Scholar
  • 15 Bolstad B M, Irizarry R A, Astrand M, Speed T P. A comparison of normalization methods for high density oligonucleotide array data based on bias and variance.  Bioinformatics. 2003;  19 185-93
    CrossrefPubMedGoogle Scholar
  • 16 Irizarry R A, Bolstad B M, Collin F, Cope L M, Hobbs B, Speed T P. Summaries of Affymetrix GeneChip probe level data.  Nucleic Acids Res. 2003;  31 e15
    CrossrefPubMedGoogle Scholar
  • 17 Baumgart A, Schmidt M, Schmitz H J, Schrenk D. Natural furanocoumarins as inducers and inhibitors of cytochrome P450 1A1 in rat hepatocytes.  Biochem Pharmacol. 2005;  69 657-67
    CrossrefPubMedGoogle Scholar
  • 18 Reichert W, Hartmann R W, Jose J. Stable expression of the human 5alpha-reductase isoenzymes type I and type II in HEK293 cells to identify dual and selective inhibitors.  J Enzyme Inhib. 2001;  16 47-53
    PubMedGoogle Scholar
  • 19 Panter B U, Jose J, Hartmann R W. 5alpha-Reductase in human embryonic kidney cell line HEK293: evidence for type II enzyme expression and activity.  Mol Cell Biochem. 2005;  270 201-8
    CrossrefPubMedGoogle Scholar
  • 20 Villalobos M, Olea N, Brotons J A, Olea-Serrano M F, Ruiz de Almodovar J M, Pedraza V. The E-screen assay: a comparison of different MCF7 cell stocks.  Environ Health Perspect. 1995;  103 844-50
    CrossrefPubMedGoogle Scholar
  • 21 Tian C Y, Hu C Q, Xu G, Song H Y. Assessment of estrogenic activity of natural compounds using improved E-screen assay.  Acta Pharmacol Sin. 2002;  23 572-6
    PubMedGoogle Scholar
  • 22 Denison M S, Phelan D, Elferink C J. The Ah receptor signal transduction pathway. In: Denison MS, Helferich WG, editors. Toxicant-receptor-interactions.  Philadelphia: Taylor &. Francis;  1998 3-33
    Google Scholar
  • 23 Ma Q, Baldwin K T, Renzelli A J, McDaniel A, Dong L. TCDD-inducible poly (ADP-ribose) polymerase: a novel response to 2,3,7,8-tetrachlorodibenzo-p-dioxin.  Biochem Biophys Res Commun. 2001;  289 499-506
    CrossrefPubMedGoogle Scholar
  • 24 Hanlon P R, Zheng W, Ko A Y, Jefcoate C R. Identification of novel TCDD-regulated genes by microarray analysis.  Toxicol Appl Pharmacol. 2005;  202 215-28.
    CrossrefPubMedGoogle Scholar
  • 25 Potterat O, Hamburger M. Natural products in drug discovery – concepts and approaches for tracking bioactivity.  Curr Org Chem. 2006;  10 899-920
    CrossrefPubMedGoogle Scholar
  • 26 Gaube F, Wolfl S, Pusch L, Kroll T C, Hamburger M. Gene expression profiling reveals effects of Cimicifuga racemosa (L.) NUTT. (black cohosh) on the estrogen receptor positive human breast cancer cell line MCF-7.  BMC Pharmacol. 2007;  7 11
    CrossrefPubMedGoogle Scholar
  • 27 Dalton T P, Puga A, Shertzer H G. Induction of cellular oxidative stress by aryl hydrocarbon receptor activation.  Chem Biol Interact. 2002;  141 77-95
    CrossrefPubMedGoogle Scholar
  • 28 Delescluse C, Lemaire G, de Sousa G, Rahmani R. Is CYP1A1 induction always related to AHR signaling pathway?.  Toxicology. 2001;  153 73-82
    PubMedGoogle Scholar
  • 29 Parczyk K, Schneider M. The future of antihormone therapy: innovations based on anestablished principle.  J Cancer Res Clin Oncol. 1996;  122 383-96
    CrossrefPubMedGoogle Scholar
  • 30 Nawaz Z, Lonard D M, Dennis A P, Smith C L, O′Malley B W. Proteasome-dependent degradation of the human estrogen receptor.  Proc Natl Acad Sci USA. 1999;  96 1858-62
    CrossrefPubMedGoogle Scholar
  • 31 Wormke M, Stoner M, Saville B, Walker K, Abdelrahim M, Burghardt R. et al . The aryl hydrocarbon receptor mediates degradation of estrogen receptor alpha through activation of proteasomes.  Mol Cell Biol. 2003;  23 1843-55
    CrossrefPubMedGoogle Scholar
  • 32 Ohtake F, Takeyama K, Matsumoto T, Kitagawa H, Yamamoto Y, Nohara K. et al . Modulation of oestrogen receptor signalling by association with the activated dioxin receptor.  Nature. 2003;  423 545-50
    CrossrefPubMedGoogle Scholar
  • 33 Wiebe J P, Muzia D, Hu J, Szwajcer D, Hill S A, Seachrist J L. The 4-pregnene and 5alpha-pregnane progesterone metabolites formed in nontumorous and tumorous breast tissue have opposite effects on breast cell proliferation and adhesion.  Cancer Res. 2000;  60 936-43
    PubMedGoogle Scholar
  • 34 Wiebe J P. Progesterone metabolites in breast cancer.  Endocr Relat Cancer. 2006;  13 717-38
    CrossrefPubMedGoogle Scholar
  • 35 Wiebe J P, Lewis M J. Activity and expression of progesterone metabolizing 5alpha-reductase, 20alpha-hydroxysteroid oxidoreductase and 3alpha(beta)-hydroxysteroid oxidoreductases in tumorigenic (MCF-7, MDA-MB-231, T-47 D) and nontumorigenic (MCF-10A) human breast cancer cells.  BMC Cancer. 2003;  3 9
    CrossrefPubMedGoogle Scholar
  • 36 Lewis M J, Wiebe J P, Heathcote J G. Expression of progesterone metabolizing enzyme genes (AKR1C1, AKR1C2, AKR1C3, SRD5A1, SRD5A2) is altered in human breast carcinoma.  BMC Cancer. 2004;  4 27
    CrossrefPubMedGoogle Scholar
  • 37 Pawlak K J, Wiebe J P. Regulation of estrogen receptor (ER) levels in MCF-7 cells by progesterone metabolites.  J Steroid Biochem Mol Biol. 2007;  107 172-9
    CrossrefPubMedGoogle Scholar

Matthias Hamburger, PhD

Institute of Pharmaceutical Biology

Department of Pharmaceutical Sciences

University of Basel

Klingelbergstrasse 50

4053 Basel

Switzerland

Phone: +41-61-267-1475

Fax: +41-61-267-1474

Email: matthias.hamburger@unibas.ch

    www.thieme-connect.de/ejournals/toc/plantamedica