ANTICANCER RESEARCH 27: 697-712 (2007)

Gene Expression Analysis of the Mechanisms whereby Black Cohosh Inhibits Human Breast Cancer Cell Growth

LINDA SAXE EINBOND1, TAO SU2*, HSAN-AU WU1*, RICHARD FRIEDMAN2, XIAOMEI WANG2, BEI JIANG1, TIMOTHY HAGAN1, EDWARD J. KENNELLY3, FREDI KRONENBERG1 and I. BERNARD WEINSTEIN2

1Department of Rehabilitation Medicine, 2Herbert Irving Comprehensive Cancer Centers, Columbia University College of Physicians and Surgeons, New York, NY 10032; 3Lehman College in the Graduate Center, The City University of New York, Bronx, NY 10468, U.S.A.

Abstract. Background: Previous studies indicate that glycosides actein and 23-epi-26-deoxyactein constitute about specific extracts and the pure triterpene glycoside actein 5.6% of a methanolic (MeOH) extract of black cohosh obtained from black cohosh inhibit growth of human breast (Figure 1A, B). cancer cells. Our aim is to identify alterations in Several recent studies indicate that black cohosh does not expression induced by treatment with a methanolic extract promote breast cancer cell growth, either in vitro or in vivo (MeOH) of black cohosh. Materials and Methods: We treated (1). A review of Borrelli et al. (3) suggested that the in vivo MDA-MB-453 human breast cancer cells with the MeOH effects of black cohosh are central rather than hormonal. extract at 40 Ìg/ml and collected RNA at 6 and 24 h; we Extracts of black cohosh have been shown to inhibit the in confirmed the microarray results with real-time RT-PCR for vitro growth of breast cancer cells (4-8). Isopropanolic 18 . Results: At 6 h after treatment there was significant extracts of black cohosh enhanced the inhibitory effect of increase in expression of ER stress (GRP78), apoptotic tamoxifen, and inhibited MCF7 cell proliferation and (GDF15), biosynthetic (INSIG1 and HSD17B7) and -induced proliferation of MCF7 cells (6). The Phase 1 (CYP1A1) genes and, at 24 h, decrease in expression proliferation of ER+ MCF7 and ER– MDA-MB-231 human of (HELLS and PLK4) genes. Conclusion: Since breast cancer cells was inhibited by isopropanolic and the MeOH extract activated genes that enhance apoptosis and ethanolic extracts of black cohosh via induction of apoptosis repressed cell cycle genes, it may be useful in the prevention through activation of caspases (7). Further, fractions of black and therapy of breast cancer. cohosh enriched in triterpene glycosides or cinnamic acid esters, the more potent fraction, inhibited cell growth and The North American perennial black cohosh (Actaea induced apoptosis (8). Garita-Hernandez et al. (9) found that racemasa) has been used for centuries by Native Americans an ethanolic extract inhibited the activity of the cyclin D1 as an anti-inflammatory agent (1). It has recently been used promoter and increased the activity of the p21cip1 promoter as an alternative therapy for female conditions, particularly in the ER-negative human breast cancer cell line T-47D. In menopause (2). The plant parts used are the roots and previous studies (10), we found that the ethyl acetate rhizomes, which contain 2 major classes of compounds, fraction of black cohosh inhibited growth of MCF7 (ER+, triterpene glycosides and phenylpropanoids. Of the more Her2 low) cells and induced cell cycle arrest at G1 after than 50 triterpene glycosides in black cohosh, the triterpene treatment with 30 Ìg/ml and at G2/M after treatment with 60 Ìg/ml. This suggests that the fraction contains a mixture of components with the more active, or more abundant, *Both authors contributed equally to this manuscript. component causing G1 arrest and the less active causing G2/M arrest and/or individual component(s) in the fraction Correspondence to: Linda Saxe Einbond, The Herbert Irving exert different effects at different concentrations. Therefore, Comprehensive Cancer Center, College of Physicians and it is possible that at high concentrations the fraction affects Surgeons, Columbia University, HHSC-1509, 701 W. 168th Street, that regulate later phases in the cell cycle. New York, NY 10032, U.S.A. Tel: +1 212 305 6924, Fax: +1 212 The purpose of this study was to use gene expression 305 6889, e-mail: [email protected] analysis to examine the molecular effects of the MeOH Key Words: Actaea racemosa, triterpene glycoside, actein, microarray, (100%) extract of black cohosh when compared to those of black cohosh. the pure compound actein on MDA-MB-453 human breast

0250-7005/2007 $2.00+.40 697 ANTICANCER RESEARCH 27: 697-712 (2007)

Figure 1. HPLC analysis of black cohosh extracts, schematic of cellular stress responses and effects of black cohosh extracts on cell proliferation. HPLC analysis of the amounts of constituents in the MeOH extract of black cohosh: A, triterpene glycosides; B, polyphenols; C, Cellular responses to diverse stresses including the Integrated Stress Response (ISR), Unfolded Response (UPR), (ER) Stress Response, and NF-κB Pathways. The responses to stress are integrated in this diagram; the ISR is adapted from Wek et al. (15), the UPR from Benjamin and Xu et al. (17, 18), the Cholesterol and NF-κB pathway from Harding et al. (33) and the ATF3 pathway from Jiang et al. (16). The underlined genes are those whose expression was significantly altered in the RT-PCR analyses, when the cells were treated with the MeOH extract. D, Effects of extracts of black cohosh on cell proliferation in MDA-MB-453 cells. MDA-MB-453 cells were exposed to increasing concentrations of extracts for 96 hours and the percent inhibition of cell proliferation determined using the Coulter Counter assay. These results were corroborated by an additional experiment.

698 Einbond et al: Effects of Black Cohosh on Gene Expression in Breast Cancer Cells

Figure 1. continued

cancer cells. We found significant effects of the MeOH (Figure 1C) (12-14). BiP (GRP78, HSPA5, a calcium extract on the expression of genes involved in the response dependent molecular chaperone) associates with the of cells to diverse cellular stresses. The integrated stress transmembrane receptors, PERK, IRE1 and ATF6, in their response {ISR, (11)} comprises the response of four inactive state. mammalian protein kinases to repair cell damage or induce Our gene expression studies revealed several potential apoptosis. The ISR is one of three components of the mediators of the growth inhibitory effects of the MeOH unfolded protein response (UPR), which is regulated by three extract, including increased expression of the apoptotic signaling pathways PERK/ATF4, IREI/XBP1 and ATF6 gene GDF15 and decreased expression of the cell cycle

699 ANTICANCER RESEARCH 27: 697-712 (2007) related genes HELLS and PLK4. The MeOH extract and antisense cRNA with the BioArray high yield RNA transcript actein induced the expression of HSD17B7, which labeling kit (Enzo Life Sciences; Farmingdale, NY, USA), according functions in cholesterol biosynthesis (15) and also converts to a modified Affymetrix protocol. cRNA (15 Ìg) was fragmented into the final probe and hybridized to human U133A 2.0 gene chips estrone to estradiol (16). The latter effect could contribute (Affymetrix, Inc.; Santa Clara, CA, USA), comprised of more than to the purported estrogenic activity of black cohosh. 22,000 probe sets. The Institute for Cancer Genetics Core Facility at the Columbia Genome Center performed the hybridization. Materials and Methods Western blot analysis and real-time quantitative RT-PCR. For Materials. All solvents and reagents were reagent grade; H2O was Western blot analysis, cells were treated for increasing times with distilled and deionized. Actein was obtained from ChromaDex approximately half the IC50 and the IC50 concentration, measured (Laguna Hills, CA, USA; lot number 01355-101), purity 89% by at 48 h, of the MeOH extract; Western blot analysis was HPLC, and from Planta Analytica (Danbury, CT, USA; lot number performed, as described previously (10). Antibody to DDIT3 was PA-A-037), purity was over 95% by HPLC. All gene expression obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). treatments were performed in duplicate with actein from the two For real-time quantitative RT-PCR, total RNA was isolated sources. Actein was dissolved in dimethylsulfoxide (DMSO: Sigma; using Trizol (Invitrogen), and purified with the RNeasy Kit St. Louis, MO, USA) prior to addition to the cell cultures. (Qiagen; Valencia, CA, USA). mRNA sequences were obtained from the public GeneBank database (www.ncbi.nlm.nih.gov), and Extraction and isolation procedures. Black cohosh raw material primers were designed using Primer3 software from the (Pure World Botanicals; South Hackensack, NJ, USA) was Massachusetts Institute of Technology (frodo.wi.mit.edu/cgi- collected in the United States and identified by the New York bin/primer3/primer3_www.cgi). Primer sequences used in qPCR Botanical Garden. Each lot of the raw material was compared with are listed in Table IA. All primers were synthesized by Invitrogen the authentic samples using HPLC. Black cohosh roots and Company, and quality was confirmed in pre-PCR by no primer rhizomes (lot number 9-2677; Pure World Botanicals) were dimer and only one peak in the dissociation curve, and by only one extracted with MeOH/H-20 (80%), MeOH (100%) (HPLC, Figure band in agarose gel electrophoresis. All PCR products were in the 1A, B), ethanol (70%) or isopropanol (40%) overnight [4 times 140-180 bp range. Comparative quantitative PCR (18) was (each for 12)], filtered and evaporated to dryness in vacuo. The performed in the Mx3000p (Stratagene; La Jolla, CA, USA) real- four extracts had similar chemical profiles for both the major time PCR machine, using QuantiTect SYBR Green PCR kit triterpene glycosides and phenolic constituents, but the relative (Qiagen). PCR was carried out in a total 20 Ìl volume containing a amount and ratios of some of the major triterpene glycosides in the final concentration of 1 x reaction buffer, 300 nM each forward and methanolic extract were significantly different from those in the reverse primer and 6 Ìl of 1:60 diluted cDNA (as about 10 ng input other three extracts (data not shown). RNA). The PCR was hot-started at 95ÆC for 15 min, and then 40 cycles of 94ÆC for 20 sec, 54ÆC for 30 sec, 72ÆC for 30 sec, and Cell culture, proliferation assays and cell cycle analysis. MDA-MB- then followed by disassociation curve measurement from 54 to 453 (ER–, Her2 overexpressing) and MCF7 (ER+, Her2 low) were 94ÆC. No template control (NTC) and no reverse transcription obtained from the ATCC (Manassas, VA, USA). Cell proliferation control (NRT) were run together with every assay and all samples was determined using the Coulter Counter assay. Cells were seeded were run at least in duplicate (2 technical replicates of at least 2 at 6x104 cells per well in 24-well plates (0.875 cm diameter); 24 h biological sample replicates; for MCF7 cells, DMSO control 2 later the medium was replaced with fresh medium with or without technical replicates). PCR data was saved and analyzed with the indicated test materials, in triplicate, and the number of software Mxpro (Stratagene). Comparative quantitative analysis attached viable cells was counted 96 h later, using a Coulter was performed based on the delt-deltCt method using the Counter, model ZF (Coulter Electronics Inc., Hialeah, FL, USA) housekeeping gene GAPDH as internal control. and the IC50 values calculated (17). Cell viability was calculated by comparing cell counts in treated samples relative to cell counts in Gene expression analysis. We analyzed 2 replicate cultures after the DMSO control and converted to percentage. For cell cycle treating MDA-MB-453 cells with the MeOH extract at 40 Ìg/ml or analysis the cells were plated (3x105) onto 6-cm dishes and allowed actein at 20 or 40 Ìg/mL for 6 or 24 h and added a third replicate to attach for 24 h. Then the medium was replaced with DMEM after treating with actein at 40 Ìg/mL for 24 h. We analyzed 2 containing 10% FBS and DMSO or the MeOH extract. After 24 h replicate culture after treating MCF7 cells with the MeOH extract the cells were analyzed by DNA flow cytometry, as described at 40 Ìg/ml for 24 h or actein at 20 or 40 Ìg/ml for 24 h. All previously (10). For cell growth assays, the data are expressed as analyses, from the cel file to the list of differentially expressed mean ± standard deviation. Control and treated cells were genes, were performed using the AffyLimmaGUI package in the compared using the student’s t-test (p<0.05). open-source Bioconductor suite. All samples were normalized to remove chip-dependent regularities using the GCRMA method of RNA isolation and oligonucleotide microarray analysis. Total cellular Irizarry et al. (19). The statistical significance of differential RNA was extracted using Trizol (Invitrogen; Carlsbad, CA, USA) expression was calculated using the empirical Bayesian LIMMA according to the manufacture’s protocol with minor modifications, (LI Model for MicroArrays) method of Smyth et al. (20). A cut-off and then purified twice with Qiagen’s RNeasy column. High quality B>0 was used for the statistical significance of gene expression. total RNA (8 Ìg) was reverse transcribed with T7-(dT)24 primer The genes that displayed significant changes in levels of and Super Script III reverse transcriptase (Invitrogen). After expression were assigned to Gene Ontology categories and KEGG purification, cDNA was in vitro transcribed into biotin labeled Pathways using the Onto-express and Pathway-express web-servers

700 Einbond et al: Effects of Black Cohosh on Gene Expression in Breast Cancer Cells

Table I. Primer sequences, effects of the MeOH extract on cell cycle distribution and gene expression profiles in MDA-MB-453 cells.

A. Designed primer sequence used in RT-PCR.

Symbol Forward sequence Reverse sequence

GAPD ggcctccaaggagtaagacc aggggtctacatggcaactg ATF3 tgggaggactccagaagatg gacagctctccaatggcttc CYP1A1 ccaccaagaactgcttagcc gctccttgaccatcttctgc GDF15 ctccgaagactccagattcc agagatacgcaggtgcaggt DDIT3 tggaaagcagcgcatgaagga aaaggtgggtagtgtggcccaa DDIT4 agtgccctccaagacagaga gtatgtgtccccaatgcaca CCNE2 ccgaagagcactgaaaaacc tagggcaatcaatcacagca SLC7A11 tgtgtggggtcctgtcacta gcagggcgtattatgaggag MCM10 cgtcagtgagcagcatgaat tcccgttcccatttgtagag CYP1B1 cccaccaaggctgagacagtga gccgcaagcatctgatgacga HMGCR gacctttccagagcaagcac agctgacgtacccctgacat HMGCS1 ccccagtgtggtaaaattgg tggcctggacttaacattcc INSIG1 gacagtcacctcggagaacc caccaaaggcccaaagatag GRP78 tgaaactgtgggaggtgtca tttgtcaggggtctttcacc PERK tggctgtgataatgcttcca gtggttggtcttggaggaga CEBP‚ ccaaaccaaccgcacatgcaga caacagcaacaagcccgtagga ATF4 ccaacaacagcaaggaggat gtgtcatccaacgtggtcag GADD34 ggaggctgaagacagtggag cctctagggacactggttgc ABCC3 tggttttagacgaggccacagc agtgttaagccggtgtgcgatg

B. Effects of the MeOH extract on cell cycle distribution in MDA-MB-453 cells at 24 h.

Concentration (Ìg/mL) % of cells in each cell cycle phase

subG1 G1 S G2/M

DMSO 1.63 (0.07) 62.65 (1.06) 26.35 (0.49) 9.72 (0.03) MeOH 20 1.96 (0.43) 66.20 (0.14) 24.15 (0.21) 7.82 (0.47) MeOH 40 0.94 (0.23) 69.35 (0.21) 22.30 (0.57) 7.65 (1.13)

C. Differentially expressed genes (B>0) after treating MDA-MB-453 cells with the MeOH extract at 40 Ìg/ml for 24 h.

Treatment Number of genes M>0 Up M<0 Down Ratio up/down

6 h 128 72 56 1.29:1 24 h 133 35 98 0.36:1

D. Four genes altered at both 6 and 24 h after treating MDA-MB-453 cells with the MeOH extract at 40 Ìg/ml (B>0).

Category Gene symbol Gene name Fold-change of gene expression with MeOH (40 Ìg/ml) treatment

6 h 24 h

Lipid related INSIG1 Insulin induced gene 1 3.52 0.41 Lipid related SQLE epoxidase 2.82 0.62 Phase 1 CYP1B1 Cytochrome P450, family 0.42 1.76 NA DCC1 NA –0.46 –0.43

In Table A, primers were designed using Primer3 software from the Massachusetts Institute of Technology (frodo.wi.mit.edu/cgi- bin/primer3/primer3_www.cgi). In Table B, the cells were treated with 0 or 40 Ìg/ml of the MeOH extract from black cohosh and analyzed at 24 h by DNA flow cytometry. The values indicate the % of cells in the indicated phases of the cell cycle. The control contained 0.08% DMSO (standard denation in parentheses). In Tables C and D, exponentially dividing cultures of MDA-MB-453 cells were treated with the MeOH extract at 40 Ìg/ml and then collected for RNA extraction at 6 or 24 h. cRNA (15 Ìg) was fragmented into the final probe and hybridized to human U133A 2.0 gene chips. For additional details, see Materials and Methods. Fold-change (log) is the mean of the ratio of hybridization signals in MeOH treated versus DMSO control-treated cells.

701 ANTICANCER RESEARCH 27: 697-712 (2007)

Table II. Genes altered after treating MDA-MB-453 cells with the MeOH extract at 40 Ìg/ml for 6 or 24 h, B>0, |M|>1.

A. 6 h

Category Symbol Name M-fold

Up-regulated Apoptosis GDF15 growth differentiation factor 15 1.58 Cell cycle regulation HYAL3 hyaluronoglucosaminidase 3 1.19 Cholesterol biosynthesis MVD mevalonate (diphospho) decarboxylase 4.65 HMGCS1 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (soluble) 3.50 SC4MOL sterol-C4-methyl oxidase-like 3.05 MVK (mevalonic aciduria) 3.05 SQLE squalene epoxidase 2.82 DHCR7 7-dehydrocholesterol reductase 2.48 HSD17B7 hydroxysteroid (17-beta) dehydrogenase 7 2.20 FDFT1 farnesyl-diphosphate 1 1.98 LSS synthase (2,3-oxidosqualene-lanosterol cyclase) 1.74 IDI1 isopentenyl-diphosphate delta 2.46 HMGCR 3-hydroxy-3-methylglutaryl-Coenzyme A reductase 2.20 NSDHL NAD(P) dependent dehydrogenase-like 2.05 FDPS farnesyl diphosphate synthase (farnesyl synthetase, 1.82 dimethylallyltranstransferase, ) SC5DL sterol-C5-desaturase (ERG3 delta-5-desaturase homolog, fungal)-like 1.77 EBP emopamil binding protein (sterol isomerase) 1.02 Fatty acid ELOVL6 ELOVL family member 6, elongation of long chain fatty acids 1.69 (FEN1/Elo2, SUR4/Elo3-like, yeast) LIPG lipase, endothelial 4.83 LPIN1 lipin 1 4.61 ADPN adiponutrin 3.66 ACLY ATP citrate 1.34 Sterol related INSIG1 insulin induced gene 1 3.52 NA LOC81558 NA 1.58 C14orf1 14 open reading frame 1 1.27 TBC1D17 TBC1 domain family, member 17 1.08 Phase 1 CYP51A1 cytochrome P450, family 51, subfamily A, polypeptide 1 1.57 CYP1B1 cytochrome P450, family 1, subfamily B, polypeptide 1 1.43 Signal transduction PTGER4 prostaglandin E receptor 4 (subtype EP4) 1.09 Transcription regulation MAGEA2 melanoma antigen family A, 2 1.65 BATF basic leucine zipper transcription factor, ATF-like 1.07 Transport STXBP1 syntaxin binding protein 1 1.90 Down-regulated Apoptosis TXNIP thioredoxin interacting protein –1.80 Cell cycle regulation CDK5R1 cyclin-dependent kinase 5, regulatory subunit 1 (p35) –1.27 NA C18orf1 chromosome 18 open reading frame 1 –1.01

B. 24 h

Category Symbol Name M-fold

Up-regulated Apoptosis ADORA1 adenosine A1 receptor 1.35 Ca2+ homeostasis STC1 stanniocalcin 1 1.61 CACNA1D calcium channel, voltage-dependent, L type, alpha 1D subunit 1.37 Cell cycle regulation CYFIP2 cytoplasmic FMR1 interacting protein 2 1.41 Cellular interaction NEU1 sialidase 1 (lysosomal sialidase) 1.01 Lipid related UGT1A10 UDP glycosyltransferase 1 family, polypeptide A10 1.33 TPP1 tripeptidyl peptidase I 1.15 Phase 1 CYP1A1 cytochrome P450, family 1, subfamily A, polypeptide 1 4.08 CYP1B1 cytochrome P450, family 1, subfamily B, polypeptide 1 1.76 TGF‚R assembly FMOD fibromodulin 1.14 Transport ABCC3 ATP-binding cassette, sub-family C (CFTR/MRP), member 3 1.82 Table II. continued

702 Einbond et al: Effects of Black Cohosh on Gene Expression in Breast Cancer Cells

Table II. continued

Category Symbol Name M-fold

Down-regulated Cell adhesion CNTNAP2 contactin associated protein-like 2 –1.25 Cell cycle regulation PLK4 polo-like kinase 4 (Drosophila) –1.82 HELLS helicase, lymphoid-specific –1.64 FBXO5 F-box protein 5 –1.22 KNTC2 kinetochore associated 2 –1.14 MYCBP c-myc binding protein –1.03 MCM10 MCM10 minichromosome maintenance deficient 10 (S. cerevisiae) –1.01 Cell growth regulation SOCS2 suppressor of cytokine signaling 2 –1.25 NA FLJ23311 NA –1.44 BM039 NA –1.34 RIBC2 RIB43A domain with coiled-coils 2 –1.31 FLJ13220 NA –1.19 KIAA0101 KIAA0101 –1.10 WDHD1 WD repeat and HMG-box DNA binding protein 1 –1.02 Post-translation METTL4 methyltransferase like 4 –1.25 Protein folding PPIA peptidylprolyl isomerase A (cyclophilin A) –1.03 RNA processing EXOSC9 exosome component 9 –1.04 Signal transduction ARHGAP19 Rho GTPase activating protein 19 –1.00 Stress response RPS6KA5 ribosomal protein S6 kinase, 90 kDa, polypeptide 5 –1.22

In Tables A and B assays were performed as described in Table I. Fold-change (log) is the mean of the ratio of hybridization signals in actein treated versus DMSO control treated cells. NA, designates function not known.

(21). Intersections between treatments were calculated using the findings, Burdette et al. (2003) reported that the MeOH Gene Traffic program. Clustering was performed with the Program extract, compared to the 2-propanolic (40%) or ethanolic Cluster 3.0 (22) using hierarchical clustering with average-linkage (75%) extracts, showed the highest binding activity to the clustering. Average linkage clustering is based on the idea that the 2 serotonin receptors, 5-HT and 5-HT . For our gene distance between clusters is the average of the distance of all of the 7 1A expression studies, we chose the 100% MeOH extract at edges in one cluster with all of the edges in the other cluster (23). The pictures were displayed with JavaTreeview which is an update 40 Ìg/ml. The effects on cell cycle distribution after of Eisen’s program Treeview (24). exposing MDA-MB-453 cells to 0, 20 or 40 Ìg/mL of the MeOH extract for 24 h are shown in Table IB. At 24 h the Results MeOH extract induced a dose-dependent increase in the percent of cells in G1. Growth inhibitory and cell cycle effects of the methanolic extract. Our previous experiments indicated that the effects Alterations in gene expression induced by the methanolic of the ethyl acetate fraction or actein from black cohosh extract. To identify genes and signaling pathways affected by were greater on the MDA-MB-453 ER– Her2 exposure to the MeOH extract, we used microarray analysis overexpressing cell line than on the MCF7 ER+ Her2 low and an unbiased informatics approach. We treated cell line (10). In addition, Her2 overexpressing breast exponentially dividing MDA-MB-453 cells with the MeOH cancers have a poorer clinical prognosis. Therefore, in the extract at 40 Ìg/ml and collected RNA at 6 and 24 h. We present study we focused on the latter cell line. We selected the shorter duration of treatment, 6 h, to reveal compared the effects of extracts prepared using different primary effects of the extract. We first constructed an solvents - ethanol, isopropanol, and methanol, to see which overview of the number of genes altered by treatment with extract induced the greatest inhibition of cell growth. The the MeOH extract for 6 or 24 h, i.e., B >0 (Table IC). The concentrations that induced 50% inhibition of cell number of genes significantly altered was 128 at 6 h and 133 proliferation at 96 h, the IC50, were as follows: MeOH at 24 h. More genes were induced than repressed by a factor (100%): 30 Ìg/ml; MeOH/H2O (80%): 120 Ìg/ml; ethanol of 1.3-fold at 6 h, but the inverse (0.4-fold) was true at 24 h. (70%): 50 Ìg/ml; isopropanol (40%): 200 Ìg/ml (Figure To compare the effects of treating the cells with the 1D), compared to 6 Ìg/ml for the purified triterpene MeOH extract for two durations of time, we used the glycoside actein (data not shown). In agreement with our program Gene Traffic. Intersections are defined as those

703 ANTICANCER RESEARCH 27: 697-712 (2007) genes whose expression is altered under all of the conditions cell cycle control genes PLK4 and FBOX5 and DNA repair being compared. Only four of the 128 genes altered at 6 h gene EXO1 that were repressed after treatment with the were also altered at 24 h (Table ID). Three of the four MeOH extract at 40 Ìg/mL for 24 h. genes altered at both 6 and 24 h, INSIG1, SQLE and CYP1B1, were up-regulated (Table ID); these genes The effects of the methanolic extract on expression of specific mediate cellular responses to diverse stresses. We therefore mRNAs determined by real-time RT-PCR. To confirm some of determined the effect of the MeOH extract on the the above-described MeOH extract-induced changes in gene expression of the ER stress chaperone GRP78 which expression detected by microarray analysis, we also treated mediates the unfolded protein response (UPR) (12, 13) and MDA-MB-453 cells with 40 Ìg/mL of the MeOH extract for found an 0.7-fold activation. (Figure 1C, Table IV) 6 or 24 h and then performed real-time RT-PCR analysis. We analyzed 18 genes involved in cellular responses to stress or Genes altered after treatment with the MeOH extract for 6 or cell cycle control. The RT-PCR results are displayed in 24 h. We tabulated the genes most highly altered after Figure 3 and Table III and are consistent with those obtained treatment with the MeOH extract at 6 and 24 h {B>0 and in the microarray analysis. Thus, mRNAs for the ISR genes absolute value of fold-M>1.0}. At 6 h, these included 32 up- GRP78, ATF4 and ATF3, the DNA damage gene DDIT4, regulated genes and 3 down-regulated genes and are listed and the lipid genes increased at 6 h, but did not further in Table IIA. The up-regulated genes included genes related significantly increase at 24 h (Figure 3A, B); whereas the to lipid biosynthesis (INSIG1, HMGCR and HSD17B7), apoptotic and/or induced genes DDIT3, GADD34 and apoptosis (GDF15) and phase 1 detoxification (CYP1B1 and GDF15, cytochromes CYP1A1 and CYP1B1 and transporter CYP51A1), while the down-regulated genes included anti- ABCC3 showed a progressive increase in expression of the apoptotic (TXNIP) and cell cycle related (CDK5R1) genes. related mRNAs after treatment with 40 Ìg/mL of the MeOH HSD17B7 mediates both the postsqualene biosynthesis of extract for 6 or 24 h (Figure 3C, D). The cell cycle related cholesterol and the conversion of estrone to ‚-estradiol (15). gene CCNE2 showed a progressive decrease after treatment At 24 h, there were 11 up-regulated and 19 down-regulated with 40 Ìg/mL for 6 or 24 h. genes (Table IIB). The up-regulated genes included Phase I (CYP1A1 and CYP1B1) and transport (ABCC3) genes and The effects of the MeOH extract on expression of DDIT3 the down-regulated genes included cell cycle related (PLK4, protein in MDA-MB-453 cells. As described above, treatment HELLS and FBXO5) and protein related (METTL4 and with the MeOH extract caused an increase in the expression PPIA) genes. of the ISR apoptotic gene DDIT3, as assayed by RT-PCR analysis (Figure 3C, Table III). Western blot analysis Hierarchical clustering of alterations in gene expression after indicated that the MeOH extract induced a dose-dependent treating cells with the methanolic extract. We used increase in the level of DDIT3 protein after treatment with hierarchical clustering to reveal genes that are coordinately 20 or 40 Ìg/ml for 8 h (Figure 4). regulated (Figure 2A). We restricted probesets to those that corresponded to an absolute value of M (log fold) greater Comparison of the effects of the methanolic extract and actein than 0.7-fold (the value for GRP78 at 6 h) for at least one of on gene expression profiles. In studies to be published in the treatment conditions. The threshold for color in the detail elsewhere we have examined the effect of the single hierarchical clustering map is an absolute value of M greater triterpene glycoside actein at two doses and after two than 3 log fold. The full hierarchical clustering map, is durations of treatment on human breast cancer cells. The shown in Figure 2A, which contains 971 probesets; specific methanol extract contains about 2.3% actein and greater subcategories of these probesets are shown in Figure 2a, b than 10% triterpene glycosides and 4% polyphenols (Figure and c. We selected clusters containing the highly altered 1A, B). Using hierarchical clustering, we observed similar genes CYP1A1, INSIG1 and HELLS. patterns of expression after treating MDA-MB-453 cells Figure 2a contains a cluster of genes which, like CYP1A1 with the MeOH extract at 40 Ìg/ml or actein at 20 Ìg/ml for and CYP1B1, were mainly activated after treatment with the 6 or 24 h (data not shown). In subsequent studies, we MeOH extract at 40 Ìg/mL, for either 6 or 24 h, including therefore compared the genes altered by these two genes related to apoptosis (GDF15), cell cycle regulation treatments. The respective IC50 values for the MeOH (CYFIP2), and calcium metabolism (STC1). The cluster of extract and actein were 30 or 6 Ìg/ml. Thus at the doses genes displayed in Figure 2b contains the genes INSIG1 and selected actein was about 2.5 times as potent, as assayed by SQLE which were up-regulated after treatment with 40 Ìg/mL the inhibition of cell proliferation. of the MeOH extract for 6 h. This cluster contained additional We first constructed an overview of the number of genes genes that are related to lipid biosynthesis, including HMGCR in common to the two agents, each at 6 and 24 h. There and HSD17B7. Figure 2c contains, in addition to HELLS, the were no genes in common to the two doses after the two

704 Einbond et al: Effects of Black Cohosh on Gene Expression in Breast Cancer Cells

Figure 2. Hierarchical clustering of differentially expressed genes analyzed on U133A2.0 Affymetrix chips after treating MDA-MB-453 cells with the MeOH extract at 40 Ìg/mL for 6 or 24 hours. Clustering was performed with the Program Cluster 3.0 (28). We restricted probesets to those that corresponded to an absolute value of M (log fold) >0.7 for at least one of the conditions. The threshold for color in the hierarchical clustering map is M >3 log fold. Fold change indicates relative expression in actein versus DMSO control cells. To pick the blowup region, the area containing a specific gene was expanded to include a well-defined expression pattern. A) 971 genes, (a) up-regulated gene region amplified for CYP1A1, (b) up-regulated gene region amplified for INSIG1, (c) down-regulated gene region amplified for HELLS; red, up-regulated; green, down-regulated.

durations of treatment. At 6 h, actein altered 26 of the 128 and stress response gene FJL 23311 (Table IV A, B). genes altered by the MeOH extract; the ratio of up to down- However, in contrast to the MeOH extract, at 6 h, actein did regulated genes was 12:1. Whereas at 24 h, actein altered 39 not induce expression of the apoptotic gene IER3 and the of the 133 genes altered by the MeOH extract; the ratio of phase 1 gene CYP1B1, and, at 24 h, actein did not repress up to down-regulated genes was 0.26:1. We then compared expression of the cell cycle genes PLK4 and FBXO5 or the the effects of actein and the MeOH extract on gene stress gene RPS6KA5 (Table IV C, D). expression profiles. When MDA-MB-453 cells were treated with 20 Ìg/ml of actein for 6 h, we also found increased Gene expression analysis of the effects of the MeOH extract expression of GRP78 and the lipid biosynthetic genes on MCF7 cells. To determine whether the effects were cell INSIG1, HMGCR, HMGCS1 and HSD17B7, and at 24 h type specific, we tested the effects of the methanol extract we also found decreased expression of the cell cycle genes on MCF7 cells. We treated MCF7 cells with the MeOH HELLS and MCM10, the RNA metabolism gene RIBC2, extract at 40 Ìg/ml for 24 h. As reported for doxorubicin

705 ANTICANCER RESEARCH 27: 697-712 (2007)

Figure 3. Real-time RT-PCR analysis after treating MDA-MB-453 cells with the MeOH extract at 40 Ìg/mL for 6 or 24 hours. The cells were treated with 0 or 40 Ìg/mL of the MeOH extract and, after 6 or 24 hours, extracts were prepared and analyzed by real-time RT-PCR, as described in Materials and Methods. Fold change indicates relative expression in actein versus DMSO control cells. A, B, C and D display different patterns of gene expression.

on MCF7 cells (25), the effects of the MeOH extract on glycoside actein, exerts growth inhibitory effects on human gene expression levels were less pronounced on MCF7 breast cancer cells. We treated MDA-MB-453 human breast cells. There were no statistically significant effects (B>0) cancer cells with the extract at one concentration, 40 Ìg/mL, on the MCF7 cells; we did, however, observe an increase and collected RNA at 6 and 24 h for gene expression in the expression of the stress response genes GRP78, analysis. MDA-MB-453 cells overexpress the Her2, FGF and PERK, CEBPb and phase I genes CYP1A1 and CYP1B1 AR receptors and are mutant for p53. They also express the (fold>0.5) and the ISR genes ATF3, GADD34, DDIT3, prolactin-inducible protein, suggesting an apocrine origin GDF15 and SLC7A11 (fold>1.1) and decreased (26). Using an unbiased informatics approach we identified expression of the estrogen receptor gene (ESR1; –1.4) and genes and signaling pathways whose expression was altered CCNE2 (–3-01). We confirmed the effects on 6 stress by exposure of the cells to the MeOH extract. To determine genes using real-time RT-PCR (p<0.05): (fold) GRP78 the intrasample variation, at least two replicates of each (1.1), ATF4 (1.24), ATF3 (2.39), CYP1A1 (2.86), CYP1B1 microarray were performed. We examined the effects for two (1.48), GDF15 (2.21) and SLC7A11 (3.34). durations of time to increase the robustness of the analysis. The quantitative PCR results shown in Table II confirm the Discussion accuracy of the microarray results. We identified four genes that were altered (B>0) after This study employed gene expression profiles to yield treatment for 6 or 24 h (Table ID); three of these genes, insights into the mechanisms by which the MeOH extract of INSIG1, SQLE and CYP1B1, are induced in response to black cohosh, alone and compared to the triterpene cellular stresses. The ISR component of the UPR (Figure

706 Einbond et al: Effects of Black Cohosh on Gene Expression in Breast Cancer Cells

Table III. Comparison of real-time PCR and microarray analysis after treating MDA-MB-453 cells with the MeOH extract at 40 Ìg/ml for 6 or 24 hours.

A. 6 h

Category Affymetrix number Gene Fold-change relative to DMSO (p-value)

MeOH treatment MeOH treatment (6 h, 40 Ìg/mL) (6 h, 40 Ìg/mL) Microarray RT-PCR

Stress response 202887_s_at DDIT4 1.58 1.7 211936_at GRP78 (HSPA5) 0.71 1.18 202672_s_at ATF3 0.33 0.96 218696_at PERK 0.26 0.27 (0.11) 212501_at CEBP≤ 0.39 0.17 (0.72) 37028_at GADD34 0.001 –0.18 200779_at ATF4 0.34 0.2 (0.50) Promote apoptosis 221577_x_at GDF15 1.58 1.25 209383_at DDIT3 0.0034 0.19 (0.67) Transport 209921_at SLC7A11 1.89 1.47 214979_at ABCC3 0.0038 –0.35 (0.76) Cell cycle 211814_s_at CCNE2 –0.87 –0.18 (0.14) 220651_s_at MCM10 –0.27 –0.93 (0.36) Sterol related/Phase I 205822_s_at HMGCS1 3.5 4.43 201625_s_at INSIG1 3.52 3.15 202540_s_at HMGCR 2.2 2.77 205749_at CYP1A1 4.04 2.03 202434_at CYP1B1 1.43 0.47 (0.44)

B. 24 h

Category Affymetrix number Gene Fold-change relative to DMSO (p-value)

MeOH treatment MeOH treatment (24 h, 40 Ìg/mL) (24 h, 40 Ìg/mL) Microarray RT-PCR

Stress response 202887_s_at DDIT4 0.54 0.93 211936_at GRP78 (HSPA5) 0.056 0.70 202672_s_at ATF3 0.011 0.50 (0.06) 218696_at PERK 0.096 0.61 (0.28) 212501_at CEBP≤ 0.22 0.44 (0.25) 37028_at GADD34 0.17 1.03 (0.15) 200779_at ATF4 0.066 0.17 (0.38) Promote apoptosis 221577_x_at GDF15 2.89 2.35 (0.11) 209383_at DDIT3 0.38 0.74 (0.17) Transport 209921_at SLC7A11 2.31 1.09 (0.09) 214979_at ABCC3 1.82 1.76 Cell cycle 211814_s_at CCNE2 –1.52 –0.55 220651_s_at MCM10 –1.10 –0.6 Sterol related/Phase I 205822_s_at HMGCS1 0.87 1.45 201625_s_at INSIG1 0.41 0.94 (0.06) 202540_s_at HMGCR 0.15 0.92 205749_at CYP1A1 4.08 5.75 202434_at CYP1B1 2.75 1.87 (0.17)

Microarray analysis: In Tables A and B assays were performed as described in Table I. Fold-change (log) is the mean of the ratio of hybridization signals in actein treated versus DMSO control treated cells. In Tables A and B all B values are >0. RT-PCR analysis: In Tables A and B exponentially dividing cultures of MDA-MB-453 cells were treated with actein at two concentrations, 20 or 40 Ìg/ml, and then collected for RNA extraction at 6 or 24 h. Real-time RT-PCR was performed as described in the Materials and Methods section. All p-values are <0.05, unless otherwise indicated in Tables A and B.

707 ANTICANCER RESEARCH 27: 697-712 (2007)

Figure 4. Effects of actein on the level of DDIT3 protein. Western blot analysis of extracts obtained from MDA-MB-453 cells treated with the MeOH extract. The cells were treated with 0, 20 or 40 Ìg/mL of the MeOH extract and after 1, 3, or 8 h extracts were prepared and analyzed by Western blotting with an antibody to DIT3; an antibody to ‚-actin was used as a loading control.

1B) is composed of two temporal phases: 1) rescue (GDF15) and lipid biosynthetic genes at 6 h and both molecules, such as GRP78, phospho-PERK, phospho- repressed the expression of cell cycle and DNA replication eIF2· and 2) apoptotic molecules, such as DDIT3, genes at 24 h (Table IVA, B). Unique to the MeOH extract caspase-12 and caspase-7 (27). Our microarray analysis were induction of the cytochromes CYP1A1 and CYP1B1 indicated that the MeOH extract induced expression of the and multidrug resistance gene ABCC3 (Table IVC, D) ISR rescue gene GRP78, apoptotic genes GDF15 and (30). Presumably, these differences reflect the effects of IER3, cytochromes CYP1A1 and CYP1B1 and lipid compounds, in addition to actein, present in the MeOH related genes INSIG1, HMGCR and HSD17B7 (Table II extract or to synergistic or antagonistic effects. The and III). Real-time RT-PCR confirmed that the induction differences could also be related to the specific doses and of the ISR rescue gene GRP78, the ISR transcription durations of treatment that we analyzed. Nevertheless, both factors ATF4 and ATF3, the p53-dependent DNA damage the MeOH extract and actein appear to induce two phases response gene DDIT4 (28) and the lipid related genes did of the integrated stress response, the survival phase or the not increase with increasing time, whereas the induction of apoptotic phase, depending on the duration of treatment. the ISR apoptotic genes (DDIT3 and GDF15) The exposure of cells to the MeOH extract appears to progressively increased with time (Figure 3 and Table III). result in a complex array of cellular stress responses. The Thus the extract may induce the ISR survival or apoptotic primary molecules targeted by the extract may play a role responses, depending on the duration of exposure. MEFs in cellular processes involving calcium since the MeOH lacking the ISR gene ATF3 were more resistant to the extract altered the expression of several genes involved in growth inhibitory effects of the MeOH extract at low calcium homeostasis such as GRP78, IER3, TPCN1, concentrations (unpublished results), suggesting that ATF3 SLC3A2, STC1, CACNAID, and CYB5M (Tables II, III, may mediate the effects at low concentrations and other IV). Our finding that the MeOH extract activated the pathways may function at higher concentrations. expression of PTGER4 (prostaglandin receptor 4; EP4) is a Microarray and real-time RT-PCR analysis showed that cause for concern and requires further study, since lipid biosynthesis was up-regulated at 6 h of exposure to 40 prostaglandin E2 may regulate neoplastic cell function via Ìg/ml of the MeOH extract; but not at 24 h (Tables II, III). the EP2/4 receptors (31). Since two low molecular weight inhibitors of the ISR The effects of the MeOH extract on expression of genes inhibit cholesterol biosynthesis and the CYP51 related to the UPR are not specific to the MDA-MB-453 cell (29), the MeOH extract may also inhibit CYP51, but this line since we found that treatment of the ER-positive MCF7 requires further study. The MeOH extract induced the human breast cancer cell line with the MeOH extract also expression of HSD17B7, which converts estrone to ‚- induces increased expression of GRP78, ATF4, ATF3, estradiol; the purported estrogenic effects of black cohosh GDF15, SLC7A11, CYP1A1 and CYP1B1 assayed by real- may be explained in part by this induced expression. time RT-PCR. Black cohosh extracts are a popular dietary To ascertain the nature of the components responsible supplement, taken for menopausal and other symptoms. Over for the observed effects of the MeOH extract, we compared the counter preparations of black cohosh typically contain the effects of the MeOH extract and the triterpene isopropanolic (Remifemin) or ethanolic extracts of black glycoside actein Both induced UPR (GRP78), apoptotic cohosh. Since the MeOH extract contained the same peaks

708 Einbond et al: Effects of Black Cohosh on Gene Expression in Breast Cancer Cells

Table IV. Genes altered after treating MDA-MB-453 cells with the MeOH extract at 40 Ìg/ml or actein at 20 Ìg/ml for 6 h or 24 h, B>0, |M|-fold>0.7. A, B. Genes altered by both the MeOH extract and actein; C, D. Genes altered by the MeOH extract, but not actein.

A. 6 h

Category Symbol Name M-fold

Ac 20 Ìg/mL MeOH 40 Ìg/mL

Up-regulated Cell cycle regulation MAC30 NA 0.57 1.06 Cholesterol related MVD mevalonate (diphospho) decarboxylase 2.27 4.65 HMGCS1 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (soluble) 2.75 3.50 MVK mevalonate kinase (mevalonic aciduria) 2.44 3.05 SC4MOL sterol-C4-methyl oxidase-like 2.00 3.05 SQLE squalene epoxidase 2.22 2.82 DHCR7 7-dehydrocholesterol reductase 1.84 2.48 IDI1 isopentenyl-diphosphate delta isomerase 1.95 2.46 HMGCR 3-hydroxy-3-methylglutaryl-Coenzyme A reductase 1.51 2.20 NSDHL NAD(P) dependent steroid dehydrogenase-like 1.47 2.05 FDFT1 farnesyl-diphosphate farnesyltransferase 1 1.31 1.98 FDPS farnesyl diphosphate synthase ( synthetase, dimethylallyltranstransferase, geranyltranstransferase) 1.21 1.82 SC5DL sterol-C5-desaturase (ERG3 delta-5-desaturase homolog, fungal)-like 1.18 1.77 LSS lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase) 1.22 1.74 Fatty acid metabolism SCD stearoyl-CoA desaturase (delta-9-desaturase) 1.32 1.90 ELOVL6 ELOVL family member 6, elongation of long chain fatty acids (FEN1/Elo2, SUR4/Elo3-like, yeast) 1.25 1.69 Lipid related ADPN adiponutrin 2.57 3.66 LIPG lipase, endothelial 1.39 4.83 LPIN1 lipin 1 3.96 4.61 Steroid related HSD17B7 hydroxysteroid (17-beta) dehydrogenase 7 1.54 2.20 INSIG1 insulin induced gene 1 2.65 3.52 Stress response HSPA5 heat shock 70 kDa protein 5 (glucose-regulated protein, 78 kDa) 0.65 0.71 Transcription regulation MAGEA2 melanoma antigen family A, 2 1.08 1.65

B. 24 h

Category Symbol Name M-fold

Ac 20 Ìg/mL MeOH 40 Ìg/mL

Up-regulated Cellular interaction NEU1 sialidase 1 (lysosomal sialidase) 0.85 1.01 Lipid metabolism TPP1 tripeptidyl peptidase I 1.80 1.15 Down-regulated Cell cycle regulation CHEK1 CHK1 checkpoint homolog (S. pombe) –0.80 –0.78 RFC5 replication factor C (activator 1) 5, 36.5 kDa –0.85 –0.82 ASF1B ASF1 anti-silencing function 1 homolog B (S. cerevisiae) –1.29 –0.88 POLD1 polymerase (DNA directed), delta 1, catalytic subunit 125 kDa –0.85 –0.96 MCM10 MCM10 minichromosome maintenance deficient 10 (S. cerevisiae) –1.85 –1.01 HELLS helicase, lymphoid-specific –2.27 –1.64 DNA repair/ replication LIG1 I, DNA, ATP-dependent –1.04 –0.71 EXO1 exonuclease 1 –1.01 –0.88 DUT dUTP pyrophosphatase –1.31 –0.99 RNA related RIBC2 RIB43A domain with coiled-coils 2 –1.41 –1.31 Stress response FLJ23311 NA (E2F transcription factor 8) –1.21 –1.44 NA MTB NA –1.07 –0.80 RFWD3 ring finger and WD repeat domain 3 –1.04 –0.74

Table IV. continued

709 ANTICANCER RESEARCH 27: 697-712 (2007)

Table IV. continued

C. 6 h

Category Symbol Name M-fold MeOH 40 Ìg/mL

Up-regulated Apoptosis related IER3 immediate early response 3 0.89 Cell cycle regulation HYAL3 hyaluronoglucosaminidase 3 1.19 MAC30 NA 0.97 SAT spermidine/spermine N1-acetyltransferase 0.89 Steroid related CYP1B1 cytochrome P450, family 1, subfamily B, polypeptide 1 0.85 SREBF1 sterol regulatory element binding transcription factor 1 0.71 NA C14orf1 chromosome 14 open reading frame 1 1.27 Down-regulated Cell cycle regulation CDK5R1 cyclin-dependent kinase 5, regulatory subunit 1 (p35) –1.27 RNA related ASE-1 NA –0.83 NA MDM1 Mdm4, transformed 3T3 cell double minute 1, p53 binding protein (mouse) –0.84

D. 24 h

Category Symbol Name M-fold MeOH 40 Ìg/mL

Up-regulated Calcium homeostasis CACNA1D calcium channel, voltage-dependent, L type, alpha 1D subunit 1.37 STC1 stanniocalcin 1 1.10 Cell cycle regulation CYR61 -rich, angiogenic inducer, 61 0.67 Steroid related CYP1A1 cytochrome P450, family 1, subfamily A, polypeptide 1 4.08 CYP1B1 cytochrome P450, family 1, subfamily B, polypeptide 1 1.76 UGT1A10 UDP glycosyltransferase 1 family, polypeptide A10 1.33 Transcription repressor BCL6 B-cell CLL/lymphoma 6 ( finger protein 51) 0.88 Transport ABCC3 ATP-binding cassette, sub-family C (CFTR/MRP), member 3 1.82 SLC22A18 solute carrier family 22 (organic cation transporter), member 18 0.74 NA C9orf7 chromosome 9 open reading frame 7 0.93 Down-regulated Cell cycle regulation PLK4 polo-like kinase 4 (Drosophila) –1.82 FBXO5 F-box protein 5 –1.22 SMCHD1 structural maintenance of flexible hinge domain containing 1 –0.99 ESPL1 extra spindle poles like 1 (S. cerevisiae) –0.88 RECQL RecQ protein-like (DNA helicase Q1-like) –0.70 Cellular stress response RPS6KA5 ribosomal protein S6 kinase, 90 kDa, polypeptide 5 –1.22 STMN1 stathmin 1/oncoprotein 18 –0.79 Signal transduction ARHGAP19 Rho GTPase activating protein 19 –1.00 CDC42EP4 CDC42 effector protein (Rho GTPase binding) 4 –0.93 GLRB glycine receptor, beta –0.90 SMAD5 SMAD, mothers against DPP homolog 5 (Drosophila) –0.86 Potassium channel CNTNAP2 contactin associated protein-like 2 –1.25 Post-translation METTL4 methyltransferase like 4 –1.25 MGC21416 NA (Yip1 domain family, member 6) –0.73

Differentially expressed genes after treating MDA-MB-453 cells with the MeOH extract at 40 Ìg/ml or actein at 20 Ìg/ml for 6 h or 24 h. In Tables A and B (genes in common), C and D (genes not in common) assays were performed as described in Table π. Fold-change (log) is the mean of the ratio of hybridization signals in MeOH extract treated versus DMSO control treated cells. NA, designates function not known.

as the ethanolic or isopropanolic extracts, our results are known. The exposure times of 6 or 24 h may not be sufficient relevant to these extracts. The pharmacokinetics and to demonstrate the cumulative effects of exposure over metabolites of extracts of black cohosh and of actein are not longer periods of time. It is possible that the effective

710 Einbond et al: Effects of Black Cohosh on Gene Expression in Breast Cancer Cells concentration might be lower because of these cumulative 9 Garita-Hernandez M, Calzado MA, Caballero FJ, Macho A, effects and because of possible concentration of the Munoz E, Meier B, Brattstrom A, Fiebich BL and Appel K: compound in specific tissues, as reported for EGCG (32). The growth inhibitory activity of the Cimicifuga racemosa extract Ze 450 is mediated through estrogen and progesterone The MeOH extract may be selective for malignant versus receptors-independent pathways. Planta Med 72: 317-323, nonmalignant cells, since this appears to be the case for the 2006. ethyl acetate fraction or actein, but this requires further study 10 Einbond LS, Shimizu M, Xiao D, Nuntanakorn P, Lim JT, (10). Since we have shown that relatively low concentrations Suzui M, Seter C, Pertel T, Kennelly EJ, Kronenberg F and of the ethyl acetate fraction or actein synergize with several Weinstein IB: Growth inhibitory activity of extracts and classes of chemotherapy agents in inhibiting the proliferation purified components of black cohosh on human breast cancer of human breast cancer cells, extracts of black cohosh might cells. Breast Cancer Res Treat 83: 221-231, 2004. enhance the efficacy of other agents with respect to breast 11 Harding HP, Zhang Y, Zeng H, Novoa I, Lu PD, Calfon M, Sadri N, Yun C, Popko B, Paules R, Stojdl DF, Bell JC, cancer prevention and therapy (33). Hettmann T, Leiden JM and Ron D: An integrated stress response regulates metabolism and resistance to Acknowledgements oxidative stress. Mol Cell 11: 619-633, 2003. 12 Benjamin IJ: Viewing a stressful episode of ER: is ATF6 the We thank Dr. Shannon Brightman, Alejandro Ramirez, Sung-Min triage nurse? Circ Res 98: 1120-1122, 2006. Park and Hyun Park for excellent technical assistance, and Dr. 13 Xu C, Bailly-Maitre B and Reed JC: Endoplasmic reticulum Rong Cheng for assistance in bioinformatics analysis. stress: cell life and death decisions. J Clin Invest 115: 2656- This research was supported by NIH-NCCAM Grant 3P50 2664, 2005. AT00090-02S2, NIH-NCCAM K01 AT001692-01A2 and ¡π∏- 14 Zhang K and Kaufman RJ: The unfolded protein response: a ¡CCAM R21AT002930-01A1. This research was also supported stress signaling pathway critical for health and disease. by the Susan G. Komen Breast Cancer Foundation Grant Neurology 66: S102-S109, 2006. BCTR0402502 to L. S. E; and by awards from the T. J. Martell 15 Marijanovic Z, Laubner D, Moller G, Gege C, Husen B, Foundation and the National Foundation for Cancer Research to Adamski J and Breitling R: Closing the gap: identification of I. B. W. 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25 Mallory JC, Crudden G, Oliva A, Saunders C, Stromberg A and 31 Sales KJ, Katz AA, Davis M, Hinz S, Soeters RP, Hofmeyr Craven RJ: A novel group of genes regulates susceptibility to MD, Millar RP and Jabbour HN: Cyclooxygenase-2 expression antineoplastic drugs in highly tumorigenic breast cancer cells. and prostaglandin E(2) synthesis are up-regulated in Mol Pharmacol 68: 1747-1756, 2005. carcinomas of the cervix: a possible autocrine/paracrine 26 de Longueville F, Lacroix M, Barbuto AM, Bertholet V, Gallo regulation of neoplastic cell function via EP2/EP4 receptors. J D, Larsimont D, Marcq L, Zammatteo N, Boffe S, Leclercq G Clin Endocrinol Metab 86: 2243-2249, 2001. and Remacle J: Molecular characterization of breast cancer cell 32 Shimizu M, Deguchi A, Lim JT, Moriwaki H, Kopelovich L lines by a low-density microarray. Int J Oncol 27: 881-892, 2005. and Weinstein IB: (–)-Epigallocatechin gallate and polyphenon 27 Wu Y, Zhang H, Dong Y, Park YM and Ip C: Endoplasmic E inhibit growth and activation of the epidermal growth factor reticulum stress signal mediators are targets of selenium action. receptor and human epidermal growth factor receptor-2 Cancer Res 65: 9073-9079, 2005. signaling pathways in human colon cancer cells. Clin Cancer 28 Ellisen LW, Ramsayer KD, Johannessen CM, Yang A, Beppu Res 11: 2735-2746, 2005. H, Minda K, Oliner JD, McKeon F and Haber DA: REDD1, a 33 Einbond LS, Shimizu M, Nuntanakorn P, Seter C, Cheng R, developmentally regulated transcriptional target of p63 and p53, Jiang B, Kronenberg F, Kennelly EJ and Weinstein IB: Actein links p63 to regulation of reactive oxygen species. Mol Cell 10: and a fraction of black cohosh potentiate antiproliferative 995-1005, 2002. effects of chemotherapy agents on human breast cancer cells. 29 Harding HP, Zhang Y, Khersonsky S, Marciniak S, Scheuner Planta Med 72: 1200-1206, 2006. D, Kaufman RJ, Javitt N, Chang YT and Ron D: Bioactive small molecules reveal antagonism between the integrated stress response and sterol-regulated gene expression. Cell Metab 2: 361-371, 2005. 30 Dalton TP, Puga A and Shertzer HG: Induction of cellular oxidative stress by aryl hydrocarbon receptor activation. Chem Received December 19, 2006 Biol Interact 141: 77-95, 2002. Accepted January 3, 2007

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