Characterization of Bizzy Nut extracts in estrogen-responsive MCF-7 breast cancer cells

Toxicology and Applied Pharmacology 220 (2007) 25 – 32 www.elsevier.com/locate/ytaap

Characterization of Bizzy Nut extracts in estrogen-responsive MCF-7 breast cancer cells Krystal Fontenot a,1 , Srivatcha Naragoni b,1 , Michelle Claville a , Wesley Gray a,b,⁎ a

b

Department of Chemistry, Southern University Baton Rouge, LA 70813, USA Department of Environmental Toxicology, Southern University Baton Rouge, LA 70813, USA Received 29 September 2006; revised 1 December 2006; accepted 6 December 2006 Available online 21 December 2006

Abstract Kola acuminate, also known as Bizzy Nut or Kola Nut, is a natural product that contains bioactive chemicals that possess hormonal properties. The purpose of this study was to characterize the putative phytoestrogenic compounds present in Bizzy Nut for estrogenic-like activity. As an initial step, five extracts (E1 – hexane, E2 – ether, E3 – acetone, E4 – methanol and E5 – water) were sequentially generated using solid–liquid phase extraction and their bioactivity was examined in MCF-7, MDA-MB-468 and LNCaP cancer cell models. MTT cell viability, dye exclusion, caspase activity and microscopic assessment of apoptotic cells demonstrated that extracts of Bizzy were cytotoxic to MCF-7, MDA-MB 468 and LNCaP cells. In MCF-7 cells, the acetone extract (E3) at 100 ppm elicited a potent cytotoxic response with a growth-inhibitory concentration (GI50) of 67 ppm. In contrast, E3 stimulated growth in LNCaP cells. The ether extract (E2) showed a dose-dependent cytotoxic response with a GI50 of 13 ppm in the LNCaP cell line. Examination of the apoptotic response induced by E2 and E3 paralleled the level of cell cytotoxicity observed in both cell lines. The methanol extract (E4) was the only extract that showed a time-, dose-, and estrogen-receptor-dependent stimulation of pS2 gene expression. On the other hand, the acetone extract (E3), which showed the highest degree of cytotoxicity, showed no transcription stimulation of pS2 in MCF-7 cells. Altogether, these data indicate that Bizzy contains unique active hormonal compounds that have specific biological properties that are cell line-dependent. © 2007 Published by Elsevier Inc. Keywords: Bizzy Nut; Natural products; Estrogen; Phytoestrogen; Cytotoxicity; Gene expression

Introduction There has been a growing movement in the development of nonsteroidal estrogens that modulate estrogen receptor function. These nonsteroidal estrogen receptor ligands are referred to as selective estrogen receptor modulators (SERMs). The major forces driving the development of new SERMs as therapeutic agents in estrogen receptor biology are as follows: first, SERMs have better receptor selectivity than steroidal ligands, such as estradiol; second, they produce a higher tissue-selectivity than ⁎ Corresponding author. Department of Environmental, Toxicology, 116 Lee Hall, P.O. Box 9716 Southern University-Baton Rouge, Baton Rouge, LA 70813, USA. Fax: +1 225 771 3992. E-mail addresses: [email protected] (K. Fontenot), [email protected] (S. Naragoni), [email protected] (M. Claville), [email protected], [email protected] (W. Gray). 1 Both authors contributed equally on the manuscript. 0041-008X/$ - see front matter © 2007 Published by Elsevier Inc. doi:10.1016/j.taap.2006.12.012

steroidal ligands; and third, they are more adaptable to structural modification that may result in increased potency and better pharmacokinetic and pharmacologic properties. The search for new therapeutic SERMs usually starts with natural product extracts that show some medicinal property (Wu et al., 2003; Kamatenesi-Mugisha and Oryem-Origa, 2005). Natural product extracts are normally used directly as herbal therapies to treat illnesses. The chemical agents found in natural product extracts may be used as starting material for synthesizing more potent drugs, particularly new SERMs (Halbreich and Kahn, 2000; Bush et al., 2001; Basly and Lavier, 2005). Natural teas are an important part of the human diet in a majority of cultures and are consumed in large portions on a daily basis. Kola acuminate, also known as Obi or Bizzy Nut to the ETTU people of Jamaica, is the “cure-all” bush/herbal medicine. Its appeal is not for its taste but for its reported health benefits. This herbal medicine reportedly affects many biological processes, many of which are directly, or indirectly,

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modulated by hormones. Biologically active, non-steroidal, estrogenic agents are usually found primarily in soy products, legumes and whole grains. Other non-traditional sources include medicinal teas and roots. The ethnobotanical information available on K. acuminate suggests that this natural product may contain bioactive chemicals that possess estrogenic and androgenic properties (Osadebe et al., 2004; KamatenesiMugisha and Oryem-Origa, 2005). Anecdotal reports suggest that Bizzy Nut may be useful for a number of medical purposes, such as removal of poisons from the body, birth control, control of diabetes, weight loss and relief of menstrual cramps (Robertson, 1988; Jamaican-Recipes, 2006). Given the hormonal dependency of some of these biological activities, it is possible that nonsteroidal estrogens present in Bizzy extracts are responsible for the medicinal value attributed to this natural product. Plant-derived estrogenic chemicals that influence endocrine activity in animals and play a role in the prevention of certain hormone-dependent cancers (such as breast and prostate cancers) are referred to as phytoestrogens (PEs). Several studies have demonstrated that ingestion of large quantities of PE extracts is associated with protection against breast and prostate cancers. The chemical constituents of PE extracts contain nonsteroidal compounds that are able to bind to the estrogen receptor and modulate its function (Peterson and Barnes, 1993; Arai et al., 2000; Delclos et al., 2001). For example, the PEs genistein and coumestrol are able to modulate the growth of MCF-7 cells estrogen-responsive human breast cancer cells (Stahl et al., 1998; Casanova et al., 1999). These PEs also induce several classical estrogen responses, such as induction of the progesterone receptor gene expression and increased uterine growth, when injected in immature female rats. PE extracts contain chemical agents with a number of biological activities, suggesting that these extracts are potential sources of chemical compounds that may function as SERMs (Barnes et al., 1995; Lee et al., 2004). Several SERMs have been developed which contain structural features of known PEs. For example, raloxifene, a SERM developed for the treatment of breast cancer, was synthesized from a coumarin backbone, and tamoxifen was synthesized from a stilbene nucleus (Grese et al., 1997). The usefulness of these SERMs stems from the fact that they produce differential estrogen pharmacology depending on the target tissue (Halbreich and Kahn, 2000; Krishnan et al., 2000; Bush et al., 2001; Lee et al., 2004; Ariazi and Jordan, 2006). Our laboratory is interested in natural products such as Bizzy that contain phytoestrogenic compounds which are antiestrogenic in nature. To begin to assess the health implications of Bizzy, knowledge of the quantity and type of phytoestrogen in Bizzy extracts is needed. This article describes the first report of a systematic analysis of Bizzy extract for putative estrogenic properties. A series of in vitro bioassays that reflect estrogen activity was used to generate an estrogenic profile of different Bizzy extracts. Herein, we describe the ability of two extracts of Bizzy to (1) induce cellular cytotoxicity in tumor cells, (2) stimulate proliferation of MCF-7 cells in vitro, and (3) stimulate pS2 gene expression in an estrogen-receptordependent manner.

Materials and methods Cell culture. MCF-7, LNCaP and MDA-MB 468 cells were obtained from ATCC (Rockville, MD). MCF-7 cells were maintained in phenol red-free DMEM media supplemented with 10% fetal bovine serum (FBS), insulin (1.0 ng/ml), 0.2 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin (Life Technologies, Inc.). LNCaP cells were maintained in RPMI 1640 medium supplemented with 10% FBS, 0.2 mM glutamine, 100 U/ml of penicillin, and 100 mg/ml streptomycin. MDA-MB 468 cells were maintained in Leibovitz L-15 media supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cells were maintained in 5% CO2 in a water-jacketed incubator and were passaged when they reached about 80–90% confluence using a trypsin/EDTA solution (Sigma-Aldrich, Inc). The CellTiter 96® was purchased from Promega Corp. (Madison, WI). All other reagents and solvents were of analytical grade and were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO). Solid–liquid extraction and TLC analysis. A 10.0-g sample of finely ground Bizzy root was sequentially extracted in a Soxhlet apparatus using 500 ml of 100% hexane, ether, acetone, methane or water–solvents with increasing polarity. Each extraction mixture was allowed to reflux for 24 h at temperatures corresponding to the boiling point of the respective solvent and the extraction monitored by TLC chromatography. Following extraction, particulate matter was removed by filtering the samples through a 0.45-μm glass fritted filter, and the extract evaporated to dryness using a combination of simple distillation and rotary evaporation. The residue of each extract was resuspended in DMSO to a concentration of 10,000 ppm. Aliquots (100 ppm) of each extract were run on thin-layer chromatography plates (Silica Gel Hl, Analtech; ca. 10 cm wide × 20 cm high) using a solvent of 40% v/v ether–60% v/v hexane. Analytes were visualized under ultraviolet light and the image captured digitally using a FluorChem HD Alpha Innotech system. Cell viability analysis. For experiments involving cell growth and gene induction studies, cells were grown in test media (phenol red-free DMEM media for MCF-7 cells, RPMI 1640 for LNCaP cells, and Leibovitz L-15 media for MDA-MB 468 containing 5% FBS that was stripped three times with dextran-coated charcoal) for 7 days. Cells were plated at a density of 1 × 105 cells/well, in 96-well plates, and allowed to attach overnight. The different Bizzy extracts in 0.1% DMSO were added in a series of concentrations (0–1000 ppm) to 8 of the 96 wells. As control and reference, 10− 7 M of genistein (GE) or 10− 8 M estradiol was added to separate wells of the plate. Each treatment and time point had eight replicates, and the final concentration of vehicle solvent in all treatments did not exceed 0.1% v/v in the media. After 24 h of exposure to the test compounds, the effect on cell viability and gene expression was determined. Cytotoxicity was determined by the CellTiter 96® Aqueous One non-radioactive cell proliferation assay (Promega, Madison, WI) according to manufacturer's instructions. After incubation with methyl thiazolyl tetrazolium (MTT), absorbance at 490 nm was measured using a ELX800UV universal microplate reader (Bio-Tech, Inc.). The rate of cell death was calculated as [A490 (control) − A490 (treatment) / A490 (control)] × 100%. For Tryphan blue staining, cells were plated at approximately 10,000 cells/well in 12-well plates, then induced with 500 ppm E2, E3, and 10 nM Estradiol. 24 h after induction, cells were harvested and stained with Tryphan blue. Seven random microscopic fields of cells on the hemocytometer were photographed and the number of cells in each field counted. RNA extraction and Northern blot analysis. Total RNA was obtained from cells treated with 0–500 ppm Bizzy extract, 10 nM estradiol or 100 nM GE in the presence or absence of 50 fold ICI-182,780 for 24 h by lysing with 1.0 ml of TRI reagent (Invitrogen). The RNA pellet was re-suspended in water, treated with DNase I, reprecipitated and quantified by reading the absorbance at 260 and 280 nm on a Bio Rad Spec3000. Total RNA (10–20 μg/lane) was fractionated on a 1.2% agarose–formaldehyde denaturing gel and the fractionated RNA transferred to a Hybond-N+ membrane (Amersham Pharmacia, Piscataway, NJ) by upward capillary blotting and then UV cross-linked using Bio-Rad GS Gene linker (Hercules, CA). The DNA probe (25 ng) used in Northern analysis (200 bp PCR fragment of pS2) was generated by PCR and 32P-labeled by random priming using a Promega Prime-a-Gene kit (Madison, WI) (Ramanathan

K. Fontenot et al. / Toxicology and Applied Pharmacology 220 (2007) 25–32 and Gray, 2003). Membranes were hybridized with 1.0–3.0 × 106 cpm/ml with ExpressHyb hybridization solution (Clontech, Palo Alto, CA) overnight at 50 °C in a rotisserie-style hybridization oven (Hybraid, Labnet, Woodbridge, NJ). Membranes were washed with 1× hybridization buffer (Molecular Research Center Inc., Cincinnati, OH) at 50 °C for 1 h, followed by 0.2× SSC/0.2% SDS at 50 °C for 30 min. To verify equal loading of RNA, the membrane was stripped (boiling solution of 0.5% SDS in 40 mM Tris–HCl pH 7.5, 10% glycerol and 2.0 mM EDTA) and re-hybridized with a 350-bp fragment of the glyceraldehyde-3-phosphate dehydrogenase gene as probe. Hybridized fragments were quantified on a Packard Cyclone PhosphoImager (Packard, Instrument) using OptiQuant Image analysis software. All Northern analyses were repeated multiple times (n ≥ 3) with separate blot/RNA preparation. Values from each blot were normalized against the glyceraldehyde-3-phosphate dehydrogenase (GPDH) gene. Fluorescence microscopy. MCF-7 cells (5 × 105) were grown on microscope slides and induced with 0, 100 and 500 ppm of E3 for 24 h. Cells were prepared for microscopy by adding 5 μl of a 0.1 μg/μl solution of acrydine orange and ethidium bromide stain to the slide and the cells were stained for 5 min. Two fluorescence parameters, green emission from acrydine orange (525 nm) and red emission from ethidium bromide (620 nm) were examined under a fluorescent light microscope (Nikon Optiphot) for the typical nuclear changes associated with apoptosis. Index of apoptosis was determined by the ratio of the number of cells per microscopic field with early and late apoptosis characteristics in treated samples relative to the total number of cells per microscopic field: [Apoptosis Index = (early + late apoptotic cells in sample) / total # of cells in sample].

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evaporation. We screened each extract for bioactivity using several in vitro assays. The cytotoxicity of each extract was examined in several cancer cells using the MTT assay. MCF-7 cells seeded at a density of 1 × 105 cells in a 96-well plate were grown in 5% DCC-serum for 7 days, then exposed to 100 ppm of each extract in 0.1% DMSO for 24 h. Cell death induced by each extract was calculated as [A490 (control) − A490 (extract) / A490 (control)] × 100%. Fig. 1 indicates that E3 was the only extract capable of inducing greater than 50% cell death of MCF-7 cells at 100 ppm. In contrast, E1, E2, E4 and E5 resulted in 25% stimulation in cell growth as compared to control. Of the four extracts that stimulated MCF-7 cell growth, E2 was the most potent and showed a similar increase

Analysis of Caspase 3/7 activation. Caspase 3/7 activity was determined using an (Apo-One Homogeneous Caspase-3/7 Assay kit (Promega, Madison, WI) according to the manufacturer's instructions. Briefly, cells grown in test media were plated at a density of 1 × 105 cells/well, in 96-well plates, allowed to attach, then induced with 100 ppm or 500 ppm E3 for 12 or 24 h. Following induction, samples were made 1% v/v in Triton X-100, harvested and centrifuged at 1000×g for 15 min. Caspase 3/7-like activity in the resulting supernatant was determined based on proteolytic cleavage of rhodamine 110, bis-(N-CBZ-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide) (Z-DEVDR110). The free rhodamine 110 was quantified on a SpectraMax Gemini Fluorescence Spectrophotometer with an excitation at 499 nm and emission at 521 nm using the SoftMax Pro program in static mode. The Caspase 3/7-like activity in each sample was determined from the maximum fluoresce after 90 min.

Results Bizzy Nut extract inhibits MCF-7 cells Phytoestrogens (PEs) are nonsteroidal compounds present in several commonly consumed natural teas. Bizzy tea, derived from the Kola Nut, has been reported to alleviate a vast array of ailments, many of which are hormone-dependent (Robertson, 1988; Jamaican-Recipes, 2006). As an initial step to identify and characterize medicinally relevant PEs present in Bizzy Nut, we performed solid–liquid extraction using solvents of increasing polarity. Since Bizzy Nut contains a myriad of active and non-active compounds located in different parts of the plant cell, we used solvents of different polarity to solvate the compound and generate a fingerprint of bioactive analytes present in Bizzy. Samples of finely ground Bizzy Nut (10–25 g) were sequentially extracted by Soxhlet extraction using hexane (E1), ether (E2), acetone (E3), methanol (E4) or water (E5) for 24 h. The dry weights of the extractable materials were determined gravimetrically following a combination of simple distillation and roto-vac

Fig. 1. The effect of different Bizzy Nut extracts on the viability of MCF-7 cells. MCF-7 cells were grown in the presence of 100 ppm or 500 ppm Bizzy extracts for 24 h and cell viability was determined using the (A) MTT cell viability assay or (B) Tryphan blue dye staining as described in Materials and methods. Growth-inhibitory activity of the extracts was determined as [A490 (control) − A490 (extract) / A490 (control)] × 100%. Each value represents mean ± SEM for three experiments performed in eight wells of a 96-well plate. C, control, E1, hexane extract; E2, ether extract; E3, acetone; E4, methanol extract; and E5, water extract.

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in cell growth observed with estradiol or genistein (Fig. 1, data not shown). To corroborate the MTT assay, we evaluated the effect of E3, E2 and estradiol on cell number using Tryphan blue dye exclusion. MCF-7 cells were grown in the presence and absence of 500 ppm of E2 or E3 for 24 h and the total number of viable cells determined by Tryphan blue dye staining. As a reference, cells were induced with 10 nM estradiol. Fig. 1B indicated that estradiol was stimulatory to MCF-7 cells (2.3-fold) as determined by the increase in the number of viable cells. E3 and E2 resulted in a 98% ± 3% and 60 ± 5.4% decrease in cell viability, respectively. Dose–response inhibition of MCF-7 cell growth by E3 Preliminary screening of our Bizzy extracts suggests that E3 is a growth inhibitor to the estrogen-responsive breast cancer cells MCF-7. Therefore, we determined the potency of E3 in inhibiting MCF-7 cells by performing an initial dose– response using 0, 10, 50, 100, 500 and 1000 ppm of extract. A plot of cell viability as a function of concentration is shown in Fig. 2. We determined the GI50 of E3 in MCF-7 cells by plotting the data to a downhill dose–response curve with a variable slope. Maximal inhibition of MCF-7 cell growth by E3 was achieved at 500 ppm with a GI50 of 67 ± 1.6 ppm (Fig. 2). All five extracts used in this study gave a 20–30% stimulation of MCF-7 cells at concentrations less than 50 ppm (Figs. 1 and 2, data not shown). E3 showed a 20% increase in the number of viable cells at concentrations less than 50 ppm, and a 50% inhibition in cell viability at concentrations greater than 500 ppm.

Fig. 2. Acetone extract (E3) of Bizzy induced a dose-dependent cytotoxicity in MCF-7 cells. Cells (10,000) were grown in the presence of 0–1000 ppm E3 for 24 h. Cell viability was then measured by methyl thiazolyl tetrazolium (MTT) assay. The absorbance at 490 nm was determined and expressed as percent of control. GI50 of E3 in MCF-7 cells was determined by fitting the data to a downhill dose–response curve with a variable slope according to the following equation: Y = Bottom + (Top − Bottom) / (1 + 10((logEC50−X)*Hill Slope)).

Effect of E3 in different cancer cell lines Next we determined the specificity of E3-induced cytotoxicity and ascertained whether the estrogen receptor status was a requirement for E3-dependent induction of cell cytotoxicity. The MDA-MB 468 (an estrogen-receptor-negative breast cancer cell line) and LNCaP cells (an estrogen receptor-βpositive prostate cancer cell line) were exposed to increasing concentrations of E3 from 0 to 1000 ppm and GI50 in each cell line determined (Fig. 2 and Table 1). These results indicated that the estrogen-receptor-positive MCF-7 cell is four to five times more sensitive to E3 than the estrogen-receptor-negative MDAMB 468 (GI50 = 67 ppm in MCF-7 verses GI50 = 326 ppm in MDA-MB 468). E3 demonstrated no measurable cytotoxicity in LNCaP cells at the concentration tested. Examination of the cytotoxicity of the other extracts in LNCaP cells revealed that E2, the ether extract, was 100 times more potent in inducing cytotoxicity that the other extracts tested (Table 1). E2 produced a GI50 of 13 ppm in LNCaP cells and a GI50 of 338 ppm in MCF-7 cells. Bizzy-induced apoptosis and cell cycle arrest in MCF-7 cells The observed cell cytotoxicity seen with E3 may be the result of E3 rupturing plasma membranes and the release of cell contents, indicative of a necrotic process rather than a programmed cell death. Therefore, we investigated whether the initial cellular cytotoxicity of the Bizzy extracts was related to the extent of apoptosis. On each microscope slide, 5 × 105 cells were grown, induced with 100 or 500 ppm of E3 and cells, then prepared for microscopic examination by the addition of 5 μl of a 0.1 mg/ml solution of acrydine orange and ethidium bromide stain. Slides were examined under a fluorescent light microscope for the typical nuclear changes seen with apoptosis. These changes include nuclear condensation, a crescent-like pattern of chromatin adherence to the nuclear envelope, and the presence of apoptotic bodies. Phase-contract microscopic examination of cells treated with E3 showed a dramatic decrease in cell number and cellular shrinkage (Fig. 3B). Fluorescence staining with acrydine orange and ethidium bromide revealed signs of nuclear condensation, nuclei fragmentation and membrane budding, all hallmark features of apoptosis (Fig. 3B, panels 2 and 3). Light microscope analysis of the cells indicated that at 100 ppm and 500 ppm of E3, 50%–90% of cell death occurred (Fig. 3A). The apoptotic index was determined by performing a partial dose–response of E3induced apoptosis. The number of apoptotic cells per field (70–60 cells) was divided by the total number of cells per field (120–140 cells). Fig. 3C revealed that apoptosis correlated with cytotoxicity. The 100 ppm concentration induced between 50% and 60% apoptosis as compared to untreated cells. Microscopic examination of apoptotic cells revealed that there was a dose-dependent increase in apoptosis following 24 h of exposure to E3. To corroborate the microscopic data, we measured the extent of apoptosis by ascertaining the level of Caspase 3/7 activity following

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Table 1 Growth-inhibitory effect of Bizzy extracts in different cancer cell lines Cell lines

E2 GI50 (ppm) (n = 3)

E3 GI50 (ppm) (n = 3)

E5 GI50 (ppm) (n = 3)

GE GI50 (ppm) (n = 3)

MCF-7 LNCaP MDA-MB 468

338 ± 3.17 13 ± 1.08 –

67 ± 1.17 1236 ± 1.10 326 ± 1.1

<2000*

<2000* <2000* <2000*

<2000

Cells were treated with various concentrations of Bizzy Nut extract for 24 h and the cell proliferation determined by the MTT assay as described in Materials and methods. The growth-inhibitory concentration was determined using a top down to bottom, variable slope equation [Y = Bottom + (Top − Bottom)/(1 + 10((logEC50−X)*Hill Slope))]. All three cell lines were induced with different concentrations of the extracts, only to produce the answer to the question that the extracts produce a more cytotoxic response in MCF-7 cells than in LNCaP and MDA-MB 468 cell lines. A difference in cell line will produce a difference in the response with the extracts. ⁎ represents p ≥ 0.05. n represents three independent assays performed in octaplets.

E3 exposure (Fig. 4). There was a significant increase in Caspase 3/7 activity in MCF-7 after 24 h of exposure to E3 at 100 ppm or 500 ppm. E3 elicited a 15-fold increased in Caspase 3/7 activity at 100 ppm which increased to 23-fold at 500 ppm E3. The 100 ppm concentration induced between 50% and 60% apoptosis as compared to untreated

Fig. 4. Acetone extract (E3) of Bizzy increase caspase activity. MCF-7 cells (1 × 105) were grown in a 96-well plate induced with 100 or 500 ppm E3 for 12 h and the resulting Caspase 3/7 activity determined using the Apo-One Homogeneous Caspase-3/7 assay. The Caspase 3/7 activity is expressed as fold increase relative to untreated control (n = 8, mean ± SEM).

Fig. 3. Cellular and nuclear morphology indicative of apoptosis induced by Bizzy Nut extract. MCF-7 cells were grown on microscope slides then induced with 100 or 500 ppm E3 for 24 h. Cells were stained with a solution of acrydine orange and ethidium bromide for 5 min then examined by light microscopy. (A) Bright field microcopy at 50×. (B) Confocal laser fluorescent microscopy using a 550 nm filter. (C) Apoptotic index determined by the equation: (total # of cells per microscopy field − total # of apoptotic cells per microscopy field) / total # of cells per microscopy field.

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cells. As the concentration of E3 increased, the total number of cells, the number of apoptotic cells and levels of caspase all decreased. Activation of estrogen-responsive gene by Bizzy extract The estrogenic potential of natural compounds is manifested in a variety of biological responses including induction, cell growth, and estrogen-receptor-dependent gene expression. Compounds present in Bizzy extract that are estrogenic in nature would manifest their activity through estrogen-dependent gene activation. Therefore, we examined the estrogenic activity of the different Bizzy extracts in an estrogen-responsive pS2 gene expression bioassay (Berry et al., 1998). The pS2 bioassay measured the amount of gene expression as a result of compound binding to the ER. Exposure of MCF-7 cells to 100 ppm of selective extract induced an increase in pS2 expression after 24 h (Fig. 5). Surprisingly, all the extracts, except E5, showed some level of pS2 activation at low concentration. E3, the most cytotoxic extract in MCF-7 cells, showed marginal stimulation which was unaffected by 100 nM of the anti-estrogen compound ICI. On the other hand, E4 showed a dose-dependent and estrogen-receptor-dependent stimulation of pS2 expression (Fig. 5, data not shown). At concentrations greater than 100 ppm E4 produced a 1.5- to 2.0fold increase in expression as compared to control (Fig. 5). The

activation of pS2 by E4 was dependent on the estrogen receptor, since blocking the receptor using the compound ICI-182,780 induced a repression of the pS2 gene (Fig. 5). We substantiated the observation that E4 induced expression of pS2 by determining if E4 is able to bind to the estrogen receptor. A radioligand competition assay was performed using the recombinant expressed estrogen receptor or immature rat uterus cytosol as a source of estrogen receptor. Incubation of ER sample with 10 nM 3H-estradiol in the presence of 100 ppm of E4 resulted in greater than 40% of the 3H-estradiol being displaced from the receptor suggesting that E4 has some affinity for ER. The extractability of unique phytochemicals from Bizzy Nut The extraction of usable analytes from Bizzy Nut is influenced by the analyte's chemical structure, degree of polymerization, conjugation with sugars, and cellular and subcellular location in the plant tissue. Thus, yield and type of extractable analyte vary significantly and are dependent on the polarity of the solvent and method used. Analyses of the different Bizzy Nut extracts in our bioassay suggest that we have a mixture of compounds with different biological activities. The concentration of the extracted material from each solvent expressed as μg/kg dry weight is represented in Fig. 6A. Solid–liquid extraction of Bizzy Nut resulted in a significant difference in the total amount of extract present in each of the different non-polar and polar solvents. The water extract (E5) and the methanol extract (E4) contained between 5% and 6% of the total extractable material, whereas greater than 17% of the extractable material was present in the hexane (E1) and acetone (E3) extracts (Fig. 6A). Extraction of Bizzy using ether resulted in an oil/lipid material that could not be dried under temperatures where the solvent should evaporate. As an initial step in characterizing the type of compounds present in E3, we ascertained the chemical profile of this extract using TLC and HPLC. Fig. 6B indicated the minimum number of compounds present in E3 that may be responsible for the observed bioactivities. Initial characterization of all the extracts by TLC (data not shown) indicated that each solvent extracted a unique set of compounds. E3 contained eight distinct compounds as judged by TLC; however, it contained the lowest amount of extractable compounds (Fig. 6A). The ether extract, E2, contained the least number of unique compounds, but had the highest concentration (22 mg/kg) of extractable material (Fig. 6A, data not shown). Discussion

Fig. 5. pS2 mRNA expression is modulated by Bizzy Nut extract. MCF-7 cells were grown in the absence (NT, control) or presence of 100 ppm of Bizzy extracts (E1 to E5) and 10 nM estradiol (E2) with and without 100 nM ICI for 24 h. Total RNA (10 μg) was isolated, size fractionated, transferred to membrane and probed with 32P-labeled pS2-specific probe. The resulting pS2 signal was detected by exposing the membrane to a PhosphorImager and analyzed using a Cyclone scanner (top panel). Quantification of pS2 expression was performed using OptiQuant Imagining analysis software and the DLU (digital light units) normalized against no treatment control (bottom panel).

The present study was designed to determine if Bizzy Nut contains biologically active compounds that influence estrogendependent pathways in MCF-7 cells. In this study we demonstrated that a potent cancer cell inhibitor present in Bizzy Nut is extractable by acetone. The analytes in the acetone extract were able to induce apoptosis in MCF-7 cells and regulate estrogen-dependent gene expression without significant binding to the estrogen receptor.

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Fig. 6. The extractability of bioactive compounds from Bizzy Nut using different polar solvents. Ten grams of finely ground Bizzy Nut were sequentially extracted with the indicated solvents using a Soxhlet apparatus. The extracts were clarified of particulate matter and concentrated to dryness. (A) Summary of extractable analytes from Bizzy Nut. (B) TLC analysis of 1–2 μl of E3 using 30:70% v/v of hexane/water visualized using UV-shadowing.

The World Health Organization estimated that 75–80% of the world population uses plant-based medicines. Bizzy Nut tea is consumed in large quantities in the Caribbean. At the same time estrogen-based illness are less prevalent in this region of the world. Increased circulation levels of compounds found in Bizzy Nut extract is likely to be responsible for the reports which indicate that Bizzy Nut relives menstrual cramps and may be used as a contraceptive. In establishing a putative mechanism of action for the estrogenic compounds in Bizzy Nut, we determined the effects of physiological levels of Bizzy Nut in estrogen-responsive breast cancer cells. The beneficial properties of most herbs and plants such as Bizzy Nut are generally considered to be associated with the polar-extractable components present in methanol or water extracts. However, we observed that the acetone extract (E3) inhibits the proliferation of MCF-7 cells as compared to the other four extracts. This antiproliferative effect may be mediated through compounds in the acetone extract which act as anti-estrogenic agents that may or may not require the estrogen receptors. Estrogen is known to regulate cell growth by binding to its receptor and stimulating the expression of genes (such as cyclin D1) which drive cells through the cell cycle. The E3 extract appears to produce a biphasic response in MCF-7 cells. At low concentrations, it produces an estrogenic type response and evokes an antiestrogenic response at higher doses. This behavior is not an uncommon characteristic of natural estrogenic compounds. Genistein, a well-studied phytoestrogen, has been demonstrated to have both estrogenic and anti-estrogenic properties that are concentration-dependent (Wang et al., 1996; Anderson et al.,

1998; Limer et al., 2006). In addition, several studies have demonstrated that anti-estrogens such as tamoxifen and ICI182,780 bind to estrogen receptors and induce apoptosis and growth arrest by ER-mediated or ER-independent mechanisms (Prall et al., 1998; Carroll et al., 2000). This study provided evidence that the mechanism of action of E3 extracts is mediated by anti-proliferative effects in estrogen-responsive cells. This notion was supported by the observation that there was a concentration-dependent induction of early and late apoptosis and increase in Caspase 3/7 activity in MCF-7 cells treated with E3. These results suggested that E3 is not a general cytotoxic agent. We observed no increase in necrotic cells, indicative of induction of oxidative stress or pre-inflammatory processes resulting from indiscriminate release of cytoplasm contents. It should be pointed out that we observed both early and late apoptotic cells at all concentrations of E3, suggesting that there is a time-dependent component to this process. We are currently examining the mechanism of apoptosis associated with extract E3 in MCF-7 cells. Methodologies for ascertaining the estrogenic activities of suspected estrogenic substances involve proliferation of MCF-7 cells (E-SCREEN), uterine wet weight, gene expression and enzymatic activities. As an initial step in establishing the estrogenic potential of our Bizzy Nut extracts, the extractable compounds were evaluated in three of these bioassays. We investigated the estrogenic potency of putative estrogenic substances present in E3 and E4 using pS2 gene expression as a measurable end point of estrogenicity. pS2 is an estrogenreceptor-dependent gene that requires a ligand–receptor

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complex for expression (Berry et al., 1998). We observed that E4 elicits a dose-dependent inhibition of pS2 expression within 24 h. The E4 extract-induced expression of pS2 does appear to follow the classic estrogen-receptor-dependent mechanism. On the other hand E3, represses pS2 expression in a manner that appears to be independent of the estrogen receptor, since the compound ICI was unable to de-repress pS2 in the presence of E3. The E3 extract produced an estrogenic effect at low concentration in our proliferation assays. Such biphasic response was observed for both estrogenic bioassays used in evaluation of this extract. This consistent biphasic response is not surprising because the E3 extract used is still a relatively crude extract that contains several different unidentified compounds. Nevertheless, this study provides the first systemic evaluation of the putative biologically active compounds present in Bizzy Nut. Thus, the role and mechanism of action of these biological active Bizzy Nut extracts in estrogen receptor biology require further investigation. Acknowledgments We would like to thank Dr. Ella Kelley for the critical editing of the manuscript. This study was supported partially by a National Institutes of Health MBRS-SCORE (1S06GM076530-01) and NIH INBRE (P20RR16456-01-R138954) grant (to WG). KF was the recipient of a NSF-HBCU-UP undergraduate fellowship. References Anderson, J.J., Ambrose, W.W., Garner, S.C., 1998. Biphasic effects of genistein on bone tissue in the ovariectomized, lactating rat model. Proc. Soc. Exp. Biol. Med. 217, 345–350. Arai, Y., Uehara, M., Sato, Y., Kimira, M., Eboshida, A., Adlercreutz, H., Watanabe, S., 2000. Comparison of isoflavones among dietary intake, plasma concentration and urinary excretion for accurate estimation of phytoestrogen intake. J. Epidemiol. 10, 127–135. Ariazi, E.A., Jordan, V.C., 2006. Estrogen-related receptors as emerging targets in cancer and metabolic disorders. Curr. Top Med. Chem. 6, 203–215. Barnes, S., Peterson, T.G., Coward, L., 1995. Rationale for the use of genisteincontaining soy matrices in chemoprevention trials for breast and prostate cancer. J. Cell. Biochem. Suppl. 22, 181–187. Basly, J.P., Lavier, M.C., 2005. Dietary phytoestrogens: potential selective estrogen enzyme modulators. Planta Med. 71, 287–294. Berry, M., Nunez, A.M., Chambon, P., 1998. Estrogen-responsive element of the human pS2 gene is an imperfectly palindromic sequence. Proc. Natl. Acad. Sci. U.S.A. 86, 1218–1222. Bush, T.L., Blumenthal, R., Lobo, R., Clarkson, T.B., 2001. SERMs and cardiovascular disease in women. How do these agents affect risk? Postgrad. Med. Spec. 17–24.

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