related compounds present in black cohosh inhibit growth of human breast cancer cells ...... Herbert Irving Comprehensive Cancer Center, College of Physicians.
Breast Cancer Research and Treatment 83: 221–231, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands.
Report
Growth inhibitory activity of extracts and purified components of black cohosh on human breast cancer cells Linda Saxe Einbond1, Masahito Shimizu1 , Danhua Xiao1 , Paiboon Nuntanakorn2, Jin T. E. Lim1 , Masumi Suzui1 , Colette Seter1 , Thomas Pertel1 , Edward J. Kennelly2 , Fredi Kronenberg1 , and I. Bernard Weinstein1 1 Columbia
University College of Physicians and Surgeons, New York, NY; 2 Lehman College in the Graduate Center, The City University of New York, Bronx, NY, USA
Key words: Actaea racemosa, actein, Cimicifuga racemosa, cinnamic acid ester, cyclin D1, triterpene glycoside
Summary The purpose of this study was to determine whether black cohosh contains constituents that inhibit the growth of human breast cancer cells, and therefore might eventually be useful in the prevention or treatment of breast cancer. Black cohosh rhizomes were extracted with methanol/water and fractionated by solvent–solvent partitioning to yield three fractions: hexane, ethyl acetate and water. The ethyl acetate fraction displayed the highest potency in two cell-based assays, growth inhibition and cell cycle analysis. This fraction inhibited growth of both the ER+ MCF7 and ER− MDA-MB-453 human breast cancer cell lines with IC50 values of about 20 and 10 µg/ml, respectively. It also induced cell cycle arrest at G1 when tested at 30 µg/ml and at G2/M at 60 µg/ml in MCF7 cells. This suggests that the extract contains a mixture of components with the more active (or more abundant) causing G1 arrest and the less active causing G2/M arrest. We then examined specific components in this extract. The triterpene glycoside fraction obtained by polyamide column chromatography, and the specific triterpene glycosides actein, 23-epi-26-deoxyactein and cimiracemoside A, inhibited growth of the MCF7 human breast cancer cells and induced cell cycle arrest at G1. The most potent compound, actein, decreased the level of cyclin D1, cdk4 and the hyperphosphorylated form of the pRb protein and increased the level of p21cip1 in MCF7 cells, changes that may contribute to the arrest in G1. Further studies are in progress to identify the mechanisms by which actein and related compounds present in black cohosh inhibit growth of human breast cancer cells.
Introduction There is growing interest in the use of herbs to aid in the maintenance of women’s health. Plants contain a wide variety of chemicals that have potent biological effects, including anticancer activity [1]. Identification of the active components, their mechanisms of action, and their possible interactions is important in order to assess their potential for clinical use and possible adverse side effects. Black cohosh (Actaea racemosa L. syn. Cimicifuga racemosa {L.} Nutt) is a North American perennial in the buttercup family (Ranunculaceae) that has been in use in Europe for the last 50 years as
a natural alternative to hormone replacement therapy and for other gynecological conditions [2]. The plant grows in the understory of hardwood forests east of the Mississippi River, especially in Ohio and West Virginia [3]. Related Asian species have been employed in Traditional Chinese Medicine to treat infectious diseases [4]. The rhizome has been in use for hundreds of years by Native Americans for a variety of purposes that include inflammatory conditions, stimulation of menstrual flow, dysmenorrhea, suppression of cough, treatment of diarrhea, and rheumatism [5]. American women are increasingly turning to black cohosh as a ‘more natural’ alternative to estrogen replacement therapy, in the belief that it has the benefits,
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without the risks of estrogen therapy. A standardized black cohosh extract (Remifemin), developed in Germany, has been studied both in animals and in short-term clinical trials in menopausal women. These studies suggest that the extract appears to alleviate a variety of menopausal symptoms, most notably hot flashes [6, 7]. Although most studies report that black cohosh is free of significant side effects, these studies have not been sufficiently long to insure safety with respect to uterine function, nor the induction or stimulation of breast cancer growth. Therefore, research is needed to clarify whether black cohosh extract stimulates or inhibits breast cancer cells since the population using black cohosh (i.e., middle-aged females in developed countries) are at a higher risk for breast cancer. In addition, such studies could identify new approaches to breast cancer prevention and therapy. The constituents of the rhizome have been examined in several previous studies. The rhizome contains triterpene glycosides such as actein, 26deoxyactein, 23-epi-26-deoxyactein (formerly 27deoxyactein) [8], and cimifugoside, aromatic acids, cinnamic acid esters, sugars, tannins and long-chain fatty acids [5, 9]. Little is known about how these compounds are metabolized in vivo. Crude extracts and several components present in black cohosh exhibit biological activity. Fukinolic acid (2-E-caffeoylfukiic acid) inhibited the activity of neutrophil elastase, which is involved in the inflammatory process [10]. Bioactivity-guided fractionation of the methanolic extract resulted in the isolation of nine antioxidant compounds, Of these, methyl caffeate was the most active in reducing menadione-induced DNA damage in cultured S30 breast cancer cells [11]. None of the compounds were cytotoxic to S30 cells [11]. Extracts of black cohosh (ethanol extract, 0.1%, v/v) inhibited the growth of serum-stimulated T-47D breast cancer cells [12], and also inhibited growth of the 435 breast cancer cell line [13]. Bodinet et al. [14] found that isopropanolic extracts of black cohosh inhibited estrogen-induced proliferation of MCF7 cells and enhanced the inhibitory effect of tamoxifen. Watanabe et al. [15] have recently shown that cycloartane glycosides isolated from black cohosh inhibit the growth of human oral squamous cell carcinoma cells. All of these studies have, however, been limited in scope and have not addressed issues of specificity and mechanism of action. The purpose of the present study was to examine the effects of extracts of black cohosh and their specific constituents on human breast cancer cells,
including assays for growth inhibition, cell cycle progression and the expression of proteins involved in cell cycle control, utilizing both estrogen-dependent and estrogen-independent human breast cancer cell lines. The eventual goal of these studies is to identify specific inhibitory compounds, their modes of action and their specific cellular targets.
Methods Chemicals and reagents All solvents and reagents were reagent grade; water (H2 O) used was distilled and deionized. Polyamide resin SC6
