Potent genistein derivatives as inhibitors of estrogen receptor alpha ...

research paper

Research paper

Cancer Biology & Therapy 11:10, 883-892; May 15, 2011; © 2011 Landes Bioscience

Potent genistein derivatives as inhibitors of estrogen receptor alpha-positive breast cancer Radharani Marik,1 Madhan Allu,2 Ravi Anchoori,2 Vered Stearns,2 Christopher B. Umbricht1,* and Saeed Khan2,3,* Department of Surgery; Johns Hopkins University School of Medicine; 2Johns Hopkins University Cancer Research Building; Baltimore, MD USA; 3 FDA/CDER/OPS/OTR/DPQR; Silver Spring, MD USA

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Key words: structurally modified genistein, estrogen receptors, estrogen receptor alpha related breast cancer chemoprevention

The estrogen receptor (ER) is a major target for the treatment of breast cancer cells. Genistein, a soy isoflavone, possesses a structure similar to estrogen and can both mimic and antagonize estrogen effects although at high concentrations it inhibits breast cancer cell proliferation. Hence, to enhance the anticancer activity of genistein at lower concentrations, we have synthesized seven structurally modified derivatives of genistein (MA-6, MA-8, MA-11, MA-19, MA-20, MA-21 and MA-22) based on the structural requirements for an optimal anticancer effect. Among those seven, three derivatives (MA6, MA-8 and MA-19) showed high antiproliferative activity with IC50 levels in the range of 1–2.5 μM, i.e., at much lower concentrations range than genistein itself, in three ER-positive breast cancer cell lines (MCF-7, 21PT and T47D) studied. In our analysis, we noticed that at IC50 concentrations, the MA-6, MA-8 and MA-19 genistein derivatives induced apoptosis, inhibited ERα messenger RNA expression and increased the ratio of ERβ to ERα levels in a manner comparable to the parent compound genistein. Of note, these three modified genistein derivatives exerted their effects at concentrations 10–15 times lower than the parent compound, decreasing the likelihood of significant ERα pathway activation, which has been a concern for genistein. Hence, these compounds might play a useful role in breast cancer chemoprevention.

©201 1L andesBi os c i enc e. Donotdi s t r i but e. Introduction

Breast cancer is the second leading cause of cancer in the western countries compared to the Asian countries, and about 60% percent of breast cancers are detected as estrogen receptor alphapositive (ERα) cancers. ERα is essential for mammary gland development and plays a central role in breast cancer development, but ERα can mediate estrogen-induced cell proliferation in an autocrine mode in ERα positive breast cancer cell lines.1 Although estrogen acts to stimulate the growth of estrogendependent tumors in vivo, reports also suggest that estrogen and estrogen agonists can act as preventive agents to inhibit growth of carcinogen induced mammary tumors in laboratory animals.2,3 The biological actions of estrogens are mediated by interacting with two specific estrogen receptors, ERα and ERβ, which belong to the nuclear receptor superfamily of ligandregulated transcription factors.4 It is reported that the proliferative actions of 17β-estradiol (E2) are mediated via estrogen receptor ERα and can be opposed by ERβ.5-7 ERβ is expressed as several isoforms, three of which (β1, β2 or βcx, and β5) are expressed in breast tissue. ERβ1 is the only isoform with receptor binding activity, while the other isoforms can heterodimerize with ERα and may thereby further downregulate ERα-specific downstream effects.8-10 A decline of ERα expression levels is seen in malignant progression of human breast neoplasia.11 Phyto-estrogens such as genistein and resveratrol, with a structure similarity to

estrogen, may behave as partial agonists (at low concentration) as well as partial antagonists (at high concentrations) of ERα and some of the phytoestrogens show a biphasic effect on growth of breast cancer cell lines.12-15 It has been reported that genistein can inhibit cancer cell growth,16 induce apoptotic cell death and cell cycle arrest at the G2-M phase, and induce angiogenesis17 in breast cancer. To date, few reports18 have focused on improving the cytotoxic activities and the structure-activity relationships of genistein derivatives. We hypothesize that since genistein acts as antagonist to cancer cells at high treatment concentrations, structural modifications may produce compounds with better cancer growth inhibitory effects at low concentrations. Considering the above, our main objective was to synthesize and find a structurally modified derivative of genistein that: (1) has a potent antiproliferative effect at lower concentrations, (2) can inhibit ERα expression or its downstream effects, and/or (3) can maintain or induce ERβ expression levels in ERα positive breast cancer cells. Here we have investigated the growth inhibitory effects of three substituted genistein derivatives in three ER-positive breast cancer cell lines (MCF-7, 21PT and T47D) and their effects on the expression of ERα and ERβ and downstream genes. Results and Discussion Design and general synthesis of structurally modified genistein compounds. Given the favorable epidemiological data on the

*Correspondence to: Christopher B. Umbricht and Saeed Khan; Email: [email protected] and [email protected] Submitted: 12/03/10; Revised: 02/03/11; Accepted: 02/16/11 DOI: 10.4161/cbt.11.10.15184 www.landesbioscience.com

Cancer Biology & Therapy 883

©201 1L andesBi os c i enc e. Donotdi s t r i but e.

Figure 1. (A) Chemical structure of genistein. (B–F) are the schematic representations of syntheses of structurally modified genistein derivatives MA-6, MA-8, MA-11, MA-19, MA-20, MA-21, MA-22 and MA-23.

one hand, and the concerns about increased estrogenic stimulation on the other, is it possible to synthesize a “perfect” genistein? We believe a judicious modification of genistein’s structure may preserve its antitumor activity, which may in part be due to ERβ activation, while eliminating effects mediated by ERα stimulation. Genistein is a promising starting point since it shows a 20-fold higher affinity for ERβ over ERα. A wide repertoire of structurally distinct compounds bind to the ER with different degrees of affinity and potency; some are agonist, others are pure antagonists. Pure antagonists possess a bulky side

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chain that cannot be contained within the ligand-binding pocket of ERα. These side chains protrude out of the opening to the binding pocket of the secondary structure of the ER peptide, thus preventing it from adopting the agonist-bound conformation. Based on docking simulations carried out by the eHiTS docking program (SymbioSys Inc., Nashua, NH) with the cocrystal structure information of genistein bound ERα, an “ideal” genistein-like antiestrogen can be designed with a bulkier and stiffer side chain that precludes the agonist bound conformation by steric hindrance. In addition, modifying genistein analogs

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Figure 2. (A–C) Antiproliferative effects of genistein and genistein derivatives: Cell viability was evaluated by the MTT assay in the all the three cells lines after treatment with concentrations ranging from 0.1–20 μM for 96 h. The values are the mean cell survival ± SD of three independent experiments. Different colors represent different compounds, as indicated in the figure legends.

by incorporating structural features with bulky and flexible lipophilic substitutions on the genistein scaffold can increase their binding affinities for ERα. These derivatives may therefore provide a potential platform for the design of clinically useful estrogen antagonists. Such an approach should provide the basis for developing cancer therapeutic agents that can be used alone or in combination with conventional chemotherapeutics. Considering the above, in order to enhance the cytotoxic properties of genistein (Fig. 1A) by structural modifications, we prepared a number of genistein derivatives, in which genistein was linked with aliphatic chains and heterocyclic 1,2,3-triazole moieties, and the C-7 or C-4' hydroxyl groups shown were substituted with various chemical moieties (see synthesis schemes 1–4). The selective modification at the 5-OH position should eliminate 5-OH intra-molecular hydrogen bonding interaction with the C-4 carbonyl group. Since 1,2,3-triazole plays a dual role as a donor and acceptor in hydrogen-bonding interactions, the fractional nature of 1,2,3-triazole would lead to flexible molecular recognition. Furthermore, triazole has similarities to the ubiquitous amide moiety found in nature, but unlike amides it is not susceptible to cleavage. Additionally, they are nearly impossible to oxidize or reduce. These characteristics of the 1,2,3-triazole prompted us to design and synthesize triazole analogs of genistein. Figure 1B–E outlines the synthetic routes used to prepare seven modified genisteins. Compounds 1–20 were synthesized based on the schemes 1–4. Phloroglucinol was treated with 4-hydroxy phenyl acetonitrile in the presence of dry HCl to yield compound 1,19 which underwent internal clyclization with PCl5 to give compound 2 (genistein). Hydroxy functionalities of compound 2 underwent alkylation using 1,2-dibromoethane in the presence of base, yielding di- and mono-alkylated compounds 3 and 4, respectively. The Bromo functionality of compound 3 was converted to azide in the presence NaN3/ DMF to give compound 5. Click chemistry approach20 was used to obtain compound MA-6. In this approach, compound 5 was coupled with phenyl acetylene in the presence of CuSO4-sodium ascorbate-DMSO-H2O to yield the 1,2,3-triazole compound MA-6. With the same approach, MA-8 was formed from the compound 4. For the synthesis of MA-11, one of the hydroxyl functionalities of compound 2 was alkylated with the corresponding bromo compound. Compound 1 was treated with the corresponding acid chloride in the presence of base-formed compound 10. Ester hydrolysis of compound 10 using NaOH/EtOH yielded compound 11, which was coupled with N-Methyl butyl amine using general amide formation methods, resulting in compound 12 (MA-19). Compound 13


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was synthesized by alkylating the hydroxyl functionality of compound 2 with 1,2-dibromoethane in the presence of base. The Bromo functionality of compound 13 was converted to the azide functionality 14, which was used for the synthesis of compound 15 (MA-20), compound 16 (MA-22) and compound 17 (MA21), using click chemistry approach.20 Genistein derivatives show antiproliferative effects in MCF7, 21PT and T47D breast cancer cells. The seven synthesized


Table 1. IC50 concentrations (µM) of genistein and genistein derivatives in MCF-7, 21PT and T47D cells after 96 h. MCF-7

T47D

21PT

IC50 in µM

IC50 in µM

IC50 in µM 2.6

MA-8

1.0

1.1

MA-6

0.8

0.9

0.9

MA-19

1.2

1.2

0.9

Genistein

14

15

16.4

10–15 fold differences in the IC50 values were found for three derivatives compared to Genistein (p < 0.01, N = 3).

genistein derivatives (MA-6, MA-8, MA-11, MA-19, MA-20, MA-21 and MA-22) were compared for their growth inhibitory effect along with the parent compound genistein, on three ER-positive breast cancer cell lines. Cells were treated with the compounds at concentrations ranging from 0.1–20 μM for 4 days. As shown in Figure 2A–C, among the seven derivatives, three (MA-8, MA-6 and MA-19) caused effective antiproliferative activities at very low concentrations, with IC50s of 0.8–1.2 μM in MCF-7, 21PT and T47D cell lines, while MA-8 showed a slightly higher IC 50 of 2.2 μM in the 21PT cell line. As summarized in the Table 1, these IC50 levels of MA-8, MA-6 and MA-19 are about 10–15 times lower than that of the parent compound genistein in all three ERα-positive breast cancer cell lines we tested here. Genistein itself inhibited the cancer cell growth at an IC50 of 14–16 μM, while the three modified genistein compounds MA-8, MA-6 and MA-19 showed similar growth inhibitory effect at around 1 μM concentrations. This indicates that these three modified genistein compounds are effective in inhibiting growth of these ERα-positive breast cancer cells at much lower concentrations than genistein. Genistein derivatives induce apoptosis at similar levels as genistein. Isoflavones such as genistein and daidzein inhibit growth and induce apoptosis in breast cancer cells.6,21 Similar growth-inhibitory effects of various isoflavones were also seen in ER-positive pancreatic cancer cells.11,22 It has been reported that genistein can inhibit cancer cell growth,16 induce apoptotic cell death and cell cycle arrest at the G2-M phase,12,17,23 depending on cell types. To investigate whether our genistein derivativeinduced cytotoxicity differs from that of the parent compound in the breast cancer cell lines under study, we analyzed the cells with the Calcein-AM and Ethidium bromide double-staining technique. We noticed that genistein derivatives MA-8, MA-6 and MA-19 inhibit breast cancer cell growth and induce apoptosis at similar levels as genistein in all of the three cell lines tested (MCF-7, 21PT and T47D). As shown in Figure 3, we found that 21PT cells underwent apoptosis (indicated by red fluorescence) when treated with IC50 concentrations of all three genistein derivatives (MA-8, MA-6 and MA-19). In addition, caspase-3 activity measurements were used to quantify the apoptotic response to genistein and its derivatives in T47D and 21PT cell lines. Caspase-7 activity was measured for MCF-7 cells, as caspase-3 is absent in MCF-7 cells (Table 2). We found that caspase activities were significantly elevated relative to the untreated control cells after treatments with IC50 concentrations of MA-8, MA-6 and

MA-19 for 96 h and the activity enhancement was comparable to the effects of genistein itself. This type of apoptosis activation was reported previously for MDA-231 cells by the parent genistein compound in breast cancer cell lines.17 Effects of genistein derivatives on ERα and ERβ mRNA expression in 21PT breast cancer cells. Figures 4A, C and E show that ERα messenger RNA (mRNA) levels are significantly reduced following 96 h exposure to compounds MA-8, MA-6, MA-19 and genistein in all the three ERa-positive breast cancer cell lines. For MA-8, MA-6 and genistein, this was also accompanied by the inhibition of the ERα targeted downstream gene pS2. Paradoxically, MA-19 showed activation of pS2, and may be activating some other signaling pathway. All compounds increased ERβ expression in concert with the ERβ-targeted downstream gene PSN (Psoriasin, S100A7) in MCF-7 and 21PT cells, although in T47D cells the downstream effect on PSN was muted or reversed. It should be noted that we measured total ERβ transcripts, and further analysis of ERβ activation will require quantitation of individual ERβ isoforms since these may have divergent effects on downstream signaling. It has been reported that there is a negative correlation between ERα and PSN expression in breast cancer in vivo. Studies in the animal models also reported that ERβ might possibly facilitate terminal differentiation in normal mammary glands.13,24 Estrogen-induced PSN mRNA expression is specifically regulated through ERβ and PSN is a potential downstream marker of ERβ activity.25 The ratio of ERβ/ERα increased after the treatment with all the three derivatives, as shown in Figure 4B, D and F. The derivative compounds induced the ERβ/ERα ratio to the almost same levels as genistein, but achieved this effect at approximately 10-fold lower doses than genistein. It has been reported that 17β-estradiol (E2)-mediated cell proliferation occurs via high ERα expression and that can be opposed by high ERβ levels.5-7 Furthermore, reduced ERβ expression is associated with malignant progression of human breast cancers.11 A series of recent studies has renewed the interest in the ERβ pathway, after poorly standardized reagents and a failure to consider the role of ERβ isoforms26 complicated initial investigations. It has been suggested that non ligand-binding ERβ isoforms may be involved in a negative feedback loop of estrogenic stimulation, particularly by compounds with low relative ERα affinity such as genistein, further reducing ERα activation by heterodimerization.8 The prognostic significance of ERβ isoforms has also recently been confirmed in several clinical studies.9,10,27


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Materials and Methods Experimental section. General. All reagents and solvents were obtained from Aldrich, St. Louis, MO. Anhydrous solvents were used as received. Reaction progress was monitored with analytical thin-layer chromatography (TLC) plates carried out on 0.25 mm Merck F-254 silica gel glass plates. Visualization was achieved using UV illumination. 1H NMR (Nuclear Magnetic Resonance) spectra were obtained at 400 MHz on a Bruker


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Figure 3. Fluorescent images of calcein-AM (green) and Ethidium bromide (red) double stained 21PT cells treated with IC50 concentrations of MA-8, MA-6 and MA-19. Tumor cells were grown for 96 h, and subsequently analyzed for apoptosis as described in the methods section.

Avance spectrometer and are reported in parts per million downfield relative to tetramethysilane (TMS). EI-MS profiles were obtained using a Bruker Esquire 3000 plus. Crude compounds were purified by semi-preparative reversed phase HPLC using a Water Delta Prep 4000 system with a Phenomenex column C18 (30 x 4 cm, 300 A, 15 μm spherical particle size column). The results of the reverse phase HPLC analyses of our purified genistein derivatives are summarized in Tables 3 and 4. General methods and experimental data. 4-Hydroxybenzyl 2,4,6-trihydroxyphenylketone (1). Phloroglucinol (1.158 g, 9.1 mmol) and 4-hydroxyphenylacetonitrile (1.34 g, 10.0 mmol) in diethyl ether (10 mL) were cooled in an ice bath and saturated with dry HCl gas for 12 h. The precipitate after the addition of diethyl ether was collected and washed further with diethyl ether. The white precipitate thus obtained was refluxed with 2% aqueous HCl (20 mL) for 3 h and cooled. The solution was extracted twice with diethyl ether (20 mL) and the organic layer was neutralized with saturated NaHCO3 solution. Diethyl ether was removed under vacuum, yielding 0.8 g (36%) of yellow solid 1. 1H NMR (DMSO-d6) δ 12.20 (br s, 2H), 10.4 (br s, 1H), 9.20 (s, 1H), 7.05 (d, 2H, J = 8.1 Hz), 6.69 (d, 2H, J = 8.1 Hz), 5.80 (s, 2H), 4.26 (s, 2H); FABMS (m/z): 261 (M+ + 1). Genistein (2). Dimethylformamide (DMF) (4 mL) was cooled to 10°C and PCl5 (0.46 g, 2.2 mmol) was added in small portions. The mixture was allowed to stand at 55°C for 20 min. The pale pink colored solution containing N, N-dimethyl (chloromethylene) ammonium chloride was then added to the compound 1 (0.385 g, 1.48 mmol) slowly. During the addition the temperature of the reaction mixture was maintained below 27°C. The reaction mixture was then stirred at room temperature for 1 h and then worked up by adding 0.16 mL of boiling HCl (0.1 N) followed by filtering the precipitated product, yielding 0.3 g (77%) of compound 2 (genistein). 1H NMR (400 MHz, DMSO-d6) δ 12.94 (s, 1H), 10.88 (s, 1H), 9.59 (s, 1H), 8.30 (s, 1H), 7.35 (d, 2H, J = 8.7 Hz), 6.80 (d, 2H, J = 8.7 Hz), 6.36 (d, 1H, J = 2.1 Hz), 6.21 (d, 1H, J = 2.4 Hz); FABMS (m/z): 271 (M+ + 1). 7- (2-Bromo-ethoxy) -5-hydroxy-3- (4-hydroxy-phenyl) chromen-4-one (3). 7-(2-Bromo-ethoxy)-3-[4-(2-bromo-ethoxy)phenyl]-5-hydroxy-chromen-4-one (4). To the genistein 2 (0.12 g, 0.7 mmol) in DMF (45 mL) was added K 2CO3 (0.376 g, 2.7 mmol) followed by 1,2-dibromoethane (3.35 mL, 38.8

Table 2. Ratio of caspase-3 or -7 activity in treated vs. untreated control cells MA-8

MA-6

MA-19

GEN

21PT

2.50 ± 0.40

2.90 ± 0.30

3.20 ± 0.20

3.90 ± 0.23

MCF-7

3.90 ± 0.24

3.59 ± 0.41

2.11 ± 0.16

3.21 ± 0.36

T47D

1.80 ± 0.14

1.79 ± 0.32

1.92 ± 0.25

2.22 ± 0.15

The caspase 3 in 21PT and T47D cells and caspase-7 activity induction (after 5 h of drug treatment) in three breast cancer cell lines. The induction level is expressed as a ratio of caspase-3 or caspase-7 activity to that of untreated control cells. Data presented here are the mean of duplicate wells on the same plate. A greater ratio indicates a higher apoptosis induction effect.



mmol) and kept stirring at 100°C for 24 h. DMF was evaporated and the reaction mixture was diluted with ethyl acetate (EtOAc) followed by K 2CO3 filtration. The collected organic layer was concentrated and purified by the column chromatography using 60–120 SiO2 mesh with EtOAc:Hexane (1:9), yielding 0.1 g (27%) of product 3 and 0.15 g (50%) of product 4. 1H NMR (400 MHz, DMSO-d6) δ 12.95 (s, 1H), 8.60 (s, 1H), 7.55 (d, 2H, J = 8.4 Hz), 6.70 (br s, 1H), 6.4 (br s, 1H), 4.49-4.46 (t, 2H, J = 6.15 Hz), 4.40-4.36 (t, 2H, J = 6.5 Hz), 3.88-3.82 (q, 4H); FABMS (m/z): 482.7, 484.7 and 486.7 (M, M+ 2, M+ 4) for compound 3, FABMS (m/z): 376.8 (M+ + 1) for compound 4. 7-(2-Azido-ethoxy)-3-[4-(2-azido-ethoxy)-phenyl]-5-hydroxychromen-4-one (5). To the dibromo compound 3 (0.1 g, 0.2 mmol) in DMF (5 mL) was added sodium azide (0.08 g, 1.2 mmol) and continuously stirred for 12 h. DMF was removed and EtOAc was added to the reaction mixture followed by washing with the NH4Cl (conc.) solution. The separated organic layer was concentrated in vacuo and purified by column chromatography using a 60–120 SiO2 mesh with EtOAc: Hexane (3:7) to yield 0.6 g (72%) of product 5. 1H NMR (400 MHz, CDCl3) δ 12.90 (s, 1H), 7.90 (s, 1H), 7.49 (d, 2H, J = 11.7 Hz), 7.29 (s, 1H), 7.1(d, 2H, J = 11.7 Hz), 6.45-6.42(m, 1H), 6.41-6.38 (m, 1H), 4.18-4.09 (m, 4H), 3.57 (t, 4H, J = 16 Hz); FABMS (m/z): 408.9 (M+ + 1). 5-Hydroxy-7-[2-(4-phenyl [1,2,3]triazole-1-yl)-ethoxy]-3-{4[2-(4-phenyl-[1,2,3]triazol-1-yl)-ethoxy]-phenyl}-chromen-4-one (6). To the diazide 5 (0.03 g, 0.007 mmol) in DMSO-H2O (1:1, V/V, [5 mL]) was added alkyne (0.015 mL, 0.147 mmol) at room temperature followed by the catalytic addition of 1 M



Figure 4. (A, C and E) Quantitation of ERα and ERβ expression along with the corresponding targeted downstream genes pS2 and PSN (S100A7), normalized by GAPDH expression. The bars represent the percent fold induction of all four genes in cells treated with the indicated compounds at their respective IC50s over untreated control cells. Means and standard deviations of triplicate experiments are shown *p < 0.05; **p < 0.01. (B, D and F) Real time RT-PCR quantitation of ERα over ERβ ratios, in untreated controls (C, CON) and cells treated with the indicated compounds at their respective IC50s. Experiments were performed in triplicate; means and standard deviations are shown. *p < 0.05; **p < 0.01.

CuSO4 and sodium ascorbate, and the reaction was stirred for 20 h. The reaction mixture was diluted with water and the separated organic layer was concentrated and the residue was recrystallized in EtOAc: Hexane (1:1) to yield 32 mg (70%) of the solid compound 6. 1H NMR (400 MHz, CDCl3 + DMSO) δ 12.95 (s, 1H), 8.59 (s, 1H), 8.49 (s, 1H), 8.25 (s, 1H), 7.95-7.90 (m, 4H), 7.55 (d, 2H, J = 9.4 Hz), 7.60-7.20 (m, 4H), 7.35-7.30

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(m, 2H), 7.05 (d, 2H, J = 9.4 Hz), 6.65 (d, 1H, J = 2.35 Hz), 6.45 (d, 1H, J = 2.35 Hz), 5.00-4.90 (m, 4H), 4.77-4.55 (m, 4H). FABMS (m/z): 612.9 (M+ + 1). 7- (2-A zido- ethoxy) -5-hydroxy-3 - (4-hydroxy-phenyl) chromen-4-one (7). Bromo compound 4 (0.4 g, 1.06 mmol) in DMF (8 mL) was added to sodium azide (0.2 g, 3.07 mmol) and continuously stirred at room temperature for 12 h. DMF


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was removed and the reaction mixture was diluted with ethyl acetate and water, and the separated organic layer was concentrated and purified by column chromatography using a 60–120 SiO2 mesh with EtOAc:Hexane (3:7) as eluent to yield 0.26 g (72%) of compound 7 in solid form. 1H NMR (400 MHz, CDCl3) δ 12.90 (s, 1H), 7.89 (s, 1H), 7.42 (d, 2H, J = 8.4 Hz), 6.93(d, 2H, J = 8.4 Hz),6.44-6.41 (m, 1H), 6.41-6.38 (m, 1H), 5.45 (br s, 1H), 4.25 (br s, 1H), 3.75-3.65 (m, 2H). FABMS (m/z): 340 (M + + 1). 5-Hydroxy-3- (4-hydroxy-phenyl) -7-[2- (4-phenyl-[1,2,3] triazol-1-yl)-ethoxy]-chromen-4-one (8). Azide 7 (0.25 g, 0.73 mmol) in DMSO-H2O (1:1, v/v, 6 mL) was added to alkyne (0.96 mL, 0.88 mmol) at room temperature followed by addition of three drops of 1 M CuSO4, and also catalytic addition of sodium ascorbate and the reaction was stirred for 20 h. The reaction mixture was diluted with water and ethyl acetate, and the separated organic layer was concentrated and purified by column chromatography using 60–120 SiO2 mesh with EtOAc:Hexane (4:6) as eluent to yield 0.2 g (62.5%) of compound 8 as solid. 1 H NMR (400 MHz, CDCl3 + 2 drops of DMSO-d6) δ 12.85 (s, 1H), 8.85 (s, 1H), 7.95(s, 1H), 7.82-7.72 (m, 3H), 7.40-7.20 (m, 5H), 6.90-6.80 (m, 2H), 6.35-6.25 (m, 2H), 4.80-4.75 (m, 2H), 4.44-4.39 (m, 2H); FABMS (m/z): 441.9 (M+ + 1). 11- (3-{4-[10-Butyl-methyl-carbamoyl)-decyloxy]-phenyl}5-hydroxy-4-oxo-4-H-chromen-7-yloxy)-undeconoic acid butylmethyl-amide (9). Genistein 2 (0.25 g, 0.92 mmol) in acetone (50 mL) was added to K 2CO3 (0.44 g, 3.1 mmol) followed by the bromo compound shown in Figure 1C (1.23 g, 3.6 mmol) and allowed to reflux for 24 h. The reaction mixture was filtered and acetone was concentrated in vacuo to obtain the crude residue.

Table 3. High performance liquid chromatography method used for the analysis of all compounds Time (min)

Flow (mL)

% Water

% Acetonitrile 10.0

1

0.01

1.00

90.0

2

1.00

6.00

90.0

10.0

3

15.00

6.00

5.0

95.0

4

25.00

6.00

95.0

5.0

5

25.01

0.00

95.0

5.0

The residue was concentrated and purified by column chromatography using a 60–120 SiO2 mesh with EtOAc:Hexane (3:7) as eluent to yield 0.39 g (50%) of compound 9 as solid. 1H NMR (400 MHz, CDCl3) δ 12.95 (s, 1H), 7.85 (s, 1H), 7.45 (d, 1H, J = 9.8 Hz), 7.0 (d, 1H, J = 9.8 Hz), 6.45-6.35 (m, 2H), 4.10-3.95 (m, 4H), 3.35 (t, 2H, J = 7.1 Hz), 3.25 (t, 2H, J = 7.1 Hz), 3.0 (s, 3H), 2.95 (s, 3H), 2.35-2.25 (m, 4H), 1.90-1.30 (m, 44H), 1.000.85 (m, 6H); FABMS (m/z): 777.3 (M+ + 1). 11-[5, 7-Dihydroxy-3-(4-hydroxy-phenyl)-4-oxo-4H-chromen2-yl]-undecanoic acid ethyl ester (10). Ethyl 11-(chlorocarbonyl) undecanoate (1.49 g, 5.3 mmol) was added drop-wise to a stirred solution of deoxybenzoin compound 1 (0.35 g, 1.3 mmol) and potassium carbonate (1.2 g, 8.6 mmol) in anhydrous acetone (25 mL) under N2. The reaction mixture was stirred at room temperature for 2 h and then heated at 50°C for 16 h. The solvent was removed and the residue was extracted into ethyl acetate and the base was neutralized with 20% citric acid. The organic layer separated was purified by column chromatography using 60–120 SiO2 mesh with EtOAc:Hexane (1:9) as eluent to yield the oily product 10 (0.46 g, 70.7%). 1H NMR (400 MHz, CDCl3) δ


Table 4. High performance liquid chromatography results Compound

Analogues

HPLC tR in min/%purity

1

1

260.0685

261

2

2

270.0528

271 482.7

MS theoretically

MS found (M + H)

3

3

481.9364

4

4

375.9946

376.8

5

5

408.1182

408.9

6

MA-6

612.2121

612.9

7

20

339.0855

340

18.413/99.0

8

MA-8

441.1325

441.9

9

MA-11

17.267/99.0

776.534

777.3

10

10

482.2305

483

11

11

454.1992

455

12

MA-19

523.2934

548 (M + Na)

13

13

390.0103

391

14

14

353.1012

354

15

MA-20

455.1481

456

16

MA-22

17.964/99.0

469.1638

469.9

17

MA-21

17.512/99.0

485.1587

486

18

18

556.2421

578.9

19

19

356.1372

357

20

MA-23

1366.6301

1366



12.85 (s, 1H), 7.28 (d, 2H, J = 7.2 Hz), 7.20 (d, 2H, J = 7.2 Hz), 6.39 (d, 1H, J = 2.3 Hz), 6.30 (d, 1H, J = 2.3 Hz), 4.20-4.10 (m, 2H), 2.62-2.52 (m, 4H), 2.40-2.30 (m, 4H), 1.50-1.20 (m, 12H), 0.98-0.88 (m, 3H); FABMS (m/z): 483 (M+ + 1). 11-[5,7-Dihydroxy-3-(4-hydroxy-phenyl)-4-oxo-4H-chromen2-yl]-undecanoic acid (11). 10% aqueous sodium hydroxide (5 mL) was added to the solution of ester 10 (0.45 g, 0.93 mmol) in EtOH (10 mL) and the mixture was stirred at room temperature for 12 h. Ethanol was removed under reduced pressure, and the residue was dissolved in water, cooled to 0°C and acidified to PH 1 with HCl(conc.):H2O(1:1). The aqueous mixture was extracted with ethyl acetate, which was separated, dried, concentrated and purified by column chromatography using a 60–120 SiO2 mesh with EtOAc:Hexane (3:1) as eluent to yield acid 11 (0.46 g, 70.7%).1H NMR (400 MHz, CDCl3) δ 12.85 (s, 1H), 7.64-7.59 (m, 1H), 7.47-7.44 (m, 1H), 6.98-6.94 (m, 2H), 6.836.79 (m, 2H), 2.60-2.30 (m, 4H), 2.20-2.10 (m, 4H), 1.40-1.05 (m, 12H); FABMS (m/z): 455 (M+ + 1). 11-[5,7-Dihydroxy-3-(4-hydroxy-phenyl)-4-oxo-4H-chromen2-yl]-undecanoic acid butyl-methyl-amide (12). To acid 11 (0.076 g, 0.167 mmol) in anhydrous THF (10 mL) were added DIPEA (0.043, 0.33 mmol) and isobutylchloroformate (0.032 mL, 0.25 mmol) sequentially at -10°C. After 5 min, the amine shown in Figure 1D was added and stirred for 3 h. The solution was then concentrated and purified by column chromatography using a 60–120 SiO2 mesh with EtOAc:Hexane (2:8) as eluent to yield compound 12 (0.029 g, 33%). 1H NMR (400 MHz, CDCl3) δ 12.85(s, 1H), 7.7-7.0 (m, 1H), 7.58-7.53 (m, 1H), 7.32-7.29 (m, 2H), 6.90-6.86 (m, 1H), 6.68-6.65 (m, 1H), 3.40-3.35 (m, 1H), 3.28-3.25 (m, 1H), 2.93 (s, 3H), 2.60-2.55 (m, 2H), 2.32-2.27 (m, 2H), 2.13-2.06 (m, 2H), 1.40-1.20 (m, 18H); FABMS (m/z): (M + Na+: 548). 7- (3-Bromo-propoxy) -5-hydroxy-3- (4-hydroxy-phenyl) chromen-4-one (13). To genistein 2 (0.3 g, 1.1 mmol) in acetone (50 mL) was added K 2CO3 (0.53 g, 3.8 mmol) and 1,3-dibromopropane (8.5 mL, 85.3 mmol) and kept stirring at reflux for 24 h. K 2CO3 was filtered and the organic layer was concentrated and purified by column chromatography using a 60–120 SiO2 mesh with EtOAc:Hexane (2:8) as eluent to yield product 13 (0.25 g, 58%). 1H NMR (400 MHz, DMSO-d6) δ 12.95 (s, 1H), 9.65 (br s, 1H), 8.40 (s, 1H), 7.40 (d, 1H, J = 10 Hz), 6.85 (d, 1H, J = 10 Hz), 6.75-6.65 (m, 1H), 6.42-6.48 (m, 1H), 4.15-4.05 (m, 2H), 3.70-3.65 (m, 2H), 2.30-2.20 (m, 2H); FABMS (m/z): 391 (M+ + 1). 7- (3-Azido-propoxy) -5-hydroxy-3- (4-hydroxy-phenyl) chromen-4-one (14). To a solution of bromo compound 13 (0.16 g, 0.42 mmol) in anhydrous DMF (5 mL) was added sodium azide (0.082 g, 1.26 mmol) at room temperature and stirred continuously for 12 h. DMF was concentrated under reduced pressure with stirring. Ethyl acetate (EtOAc) was added to the reaction and the Ethyl acetate layer was collected and washed with NH4Cl. The separated organic layer was concentrated and purified by column chromatography using a 60–120 SiO2 mesh with EtOAc: Hexane (3:7) as eluent to yield the product 14 as solid (0.130 g, 87.2%). 1H NMR (400 MHz, CDCl3) δ 12.90 (s, 1H), 7.89 (s, 1H) 7.42 (d, 2H, J = 10 Hz), 6.93

(d, 2H, J = 10 Hz), 6.44-6.41 (m, 1H), 6.41-6.38 (m, 1H), 5.45 (br s, 1H), 4.20-4.10 (m, 2H), 3.6–3.5 (m, 2H), 2.14-2.09 (m, 2H); FABMS (m/z): 354 (M+ + 1). 5-Hydroxy-3- (4-hydroxy-phenyl) -7-[3- (4-phenyl-[1,2,3] triazol-1-yl)-propoxy]-chromen-4-one (15). To azide 14 (0.13 g, 0.36 mmol) in DMF-H2O (8 mL) (1:1, v/v) was added alkyne (0.06 mL, 0.55 mmol) followed by the addition of 1 M CuSO4 (3 drops) and a catalytic amount of sodium ascorbate, and the solution was stirred for 12 h. The reaction was then diluted with water and Ethylacetate. The Ethylacetate was concentrated and the compound was recrystallized in a (2:8) EtOAc and hexane mixture to yield compound 15 as solid (0.115 g 70%). 1H NMR (400 MHz, CDCl3 + 5 drops of DMSO-d6) δ 12.9 (s, 1H), 9.20 (s, 1H), 8.30 (s, 1H), 7.9(s, 1H), 7.77-7.22 (m, 2H), 7.3-7.2 (m, 3H), 7.38-7.30 (m, 2H), 6.84-6.78 (m, 2H), 6.35 (br s, 1H), 6.27 (br s, 1H), 4.65-4.55 (m, 2H), 4.10-4.00 (m, 2H), 2.45-2.35 (m, 2H); FABMS (m/z): 456 (M+ + 1). 5-Hydroxy-3-(4-hydroxy-phenyl)-7-[3-(4-p-tolyl-[1,2,3] triazol-1-yl)-propoxy]-chromen-4-one (16). To azide 14 (0.055 g, 0.15 mmol) in DMF (5 mL) was added alkyne (0.04 mL, 0.31 mmol) followed by the addition of 1 M CuSO4 (3 drops) and catalytic amounts of sodium ascorbate, and stirred for 12 h. The reaction mixture was diluted with Ethylacetate and water, the separated organic layer was concentrated and the residue was recrystallized in Ethylacetate and hexane mixture to yield the product 16 (0.05 g 71.14%). 1H NMR (400 MHz, CDCl3 + DMSO-d6) δ 12.70 (s, 1H), 8.90 (s, 1H), 7.74 (s, 1H), 7.10 (s, 1H), 7.58-7.51 (m, 2H), 7.22-7.16 (m, 2H), 7.09-7.04 (m, 2H), 6.78-6.72 (m, 2H), 6.25-6.21 (m, 1H), 4.55-4.45 (m, 2H), 3.953.90 (m, 2H), 2,45-2.41 (m, 2H), 2.22-2.20 (m, 3H); FABMS (m/z): 469.9 (M+ + 1). 5 - Hy d r o x y -3 - (4 - h y d r o x y - ph e n y l ) -7- [ 3 - (4 - m e t h o x y phenyl-[1,2,3]triazol-1-yl)-propoxy]-chromen-4-one (17). To azide 14 (0.026 g, 0.073 mmol) in DMF (5 mL) was added the alkyne (0.019 mL, 0.143 mmol) at room temperature followed by the addition of 1 M CuSO4 (3 drops) and a catalytic amount of sodium-ascorbate. The reaction mixture was diluted with Ethylacetate and water, and the organic layer separated was concentrated and recrystallized in Ethylacetate and hexane mixture to yield product 17 (0.025 g, 71.4%). 1H NMR (400 MHz, CDCl3 + DMSO-D6) δ12.75 (s, 1H), 8.90 (s, 1H), 7.75 (s, 1H), 7.66 (s, 1H), 7.61 (s, 1H), 7.59 (s, 1H), 7.24–7.21 (m, 1H), 7.227.0 (m, 1H), 6.8-6.75 (m, 4H), 6.35-6.25 (m, 2H), 3.70 (s, 3H), 2.46-2.43 (m, 2H); FABMS (m/z): 486 (M+ + 1). (2- (4-[7- (2-tert-Butoxycarbonylamino-ethoxy)-5-hydroxy4-oxo-4H-chromen-3-yl]-phenoxy)-ethyl)-carbamic acid tert butyl ester (18). To the genistein 2 (0.2 g, 0.74 mmol) in acetone (20 mL) was added K 2CO3 (0.35 g, 2.5 mmol) followed by the addition of 0.99 g, 4.4 mmol of the bromide shown in scheme 5. The solution was stirred at room temperature for 24 h. The reaction mixture was filtered, and the filtrate was concentrated to yield the crude residue which was purified by column chromatography using a 60–120 SiO2 mesh with EtOAc:Hexane (2:8) as eluent to yield 120mg (29%) of product 18. 1H NMR (400 MHz, CDCl3) δ 12.90 (s, 1H), 7.90(s, 1H), 7.47 (d, 2H, J = 9.6 Hz), 6.98 (d, 2H, J = 9.6 Hz), 6.44-6.39 (m, 1H), 6.39-6.37 (m, 1H), 5.05


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(br s, 2H), 4.15-4.05 (m, 4H), 3.62-3.50 (m, 4H), 1.55-1.41 (br s, 18H); FABMS (m/z): (M + Na+: 578.9). 7- (2-Amino-ethoxy) -3- [4- (2-amino-ethoxy) -phenyl]-5hydroxy-chromen-4-one (19). Diamine 18 (0.12 g, mmol) in 15 mL of TFA:DCM (2:1) was stirred at room temperature for 12 h. The reaction mixture was concentrated and the crude product was recrystallized in Ethylacetate to yield product 19 (0.053 g 70%) as solid. 1H NMR (400 MHz, CD3OD) δ 8.25 (br s, 1H) 7.55 (d, 2H, J = 10.6 Hz), 7.12 (d, 2H, J = 10.6 Hz), 6.65 (br s, 1H), δ 6.55 (br s, 1H), 4.39-4.33 (m, 2H), 4.33-4.27 (m, 2H), 3.45-3.40 (m, 4H); FABMS (m/z): 357 (M+ + 1). (1R)-1-(3-(2-(2-(4-(7-(2-(2-(3-((1R)-1-(1-(3,3-dimethyl2-oxopentanoyl)piperidine-2-carbonyloxy)-3-phenylpropyl)phenoxy)acetamido)ethoxy) -5-hydroxy-4-oxo-4H-chromen-3-yl) phenoxy)ethylamino)-2-oxoethoxy)phenyl)-3-phenylpropyl1-(3,3dimethyl-2-oxopentanoyl)piperidine-2-carboxylate (20). To a solution of the TFA salt of amine 19 (0.002 g, 0.005 mmol) in DMF (5 mL) were consecutively added 0.005g, 0.01 mmol of the the corresponding acid shown in scheme 5, HOBt (0.001 g, 0.1 mmol), NMM (0.006 mL, 0.05 mmol) and EDCI (0.002 g, 0.01 mmol) and the reaction mixture was stirred for 3 h. The reaction mixture was quenched with aqueous NH4Cl solution and extracted with Ethylacetate. The extract was dried over Na 2SO4, filtered and concentrated. The residue was purified by column chromatography using a 60–120 SiO2 mesh and EtOAc/Hexane, (2:8) as eluent to yield amide 20 as a solid (3.0 mg, 43%). 1H NMR (400 MHz, CDCl3) δ 12.85 (s, 1H), 7.85 (s, 1H), 7.50-7.43 (m, 2H), 7.43-7.25 (m, 6H), 7.23-7.11 (m, 6H), 7.03-6.95 (m, 6H), 6.90-6.84 (m, 2H), 6.44-6.40 (m, 1H), 6.40-6.37 (m, 1H), 5.83-5.76 (m, 2H), 5.36-5.32 (m, 2H), 4.58-4.54 (m, 4H), 4.21-4.12 (m, 4H), 3.86-3.78 (m, 4H), 3.73 (s, 1H), 3.43-3.33 (m, 2H), 3.23-3.12 (m, 2H), 2.742.54 (m, 4H), 2.42-2.35 (m, 2H), 2.33-2.22 (m, 2H), 2.15-2.05 (m, 2H), 1.80-1.60 (m, 14H), 1.32-1.20 (m, 11H), 0.94-0.84 (m, 6H); FABMS (m/z): 1366 (M + + 1).

tissue culture flasks. After 24 h, the culture media were changed and cells treated with different concentrations of genistein (Sigma, St. Louis, MO) and our modified genistein derivatives for 96 h. MTT assay of inhibition of cellular proliferation. Cell viability was measured using the Cell Titer 96 AQ-One Solution Cell Proliferation Assay kit from Promega Corporation (Madison, WI). Formazan absorbance was read at 490 nm in a 96-well plate reader. RNA extraction. After 96 h of incubation, RNA was purified from cell cultures (treated with IC50 compound concentrations) of all the three cell lines by organic extraction using the Trizol Reagent (Invitrogen Inc., Carlsbad, CA). Total cellular RNA was quantified by UV absorption at 260 nm using a Nanodrop spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE). Quantitative real-time and RT-PCR. Reverse transcription reactions were performed as previously described in reference 30, using random hexamer primers. PCR was then performed using gene-specific primers on approximately 1/20th to 1/40th of the resulting cDNA, depending on the abundance of the transcripts. Psoriasin and pS2 transcript levels were determined using the methods of Leygue et al.31,32 Quantification of ERα and ERβ transcript levels was performed by real time PCR assays following the method of Iwao et al. in the ABI PRISM® 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA).


Materials and Methods Cell lines and treatment. The 21PT human breast cancer cell line was derived from a primary tumor and was propagated as described in reference 28. All other human breast cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and propagated as described in reference 29. For pharmacological assays, 1.0 x 106 cells were seeded in 25 cm2 References 1.

Tan H, Zhong Y, Pan Z. Autocrine regulation of cell proliferation by estrogen receptor-alpha in estrogen receptor-alpha-positive breast cancer cell lines. BMC Cancer 2009; 9:31. 2. Hsieh CY, Santell RC, Haslam SZ, Helferich WG. Estrogenic effects of genistein on the growth of estrogen receptor-positive human breast cancer (MCF-7) cells in vitro and in vivo. Cancer Res 1998; 58:3833-8. 3. Mehta RG, Pezzuto JM. Discovery of cancer preventive agents from natural products: from plants to prevention. Curr Oncol Rep 2002; 4:478-86. 4. Pettersson K, Gustafsson JA. Role of estrogen receptor beta in estrogen action. Annu Rev Physiol 2001; 63:165-92.


5.

Conclusion

The present study establishes that the modified genistein compounds MA-8, MA-6 and MA-19 decrease the proliferation of ER-positive breast cancer cells, which is associated with decreased expression of ERα and of its signaling, while enhancing the ERβ gene expression, including the truncated isoforms that may form inactive heterodimers with ERα to the levels seen with genistein. They exerted this effect at 10- to 15-fold lower drug concentration than the parent compound, further decreasing the likelihood of significant ERα pathway activation, which has been a concern for genistein. Hence these compounds might play a useful role in breast cancer chemoprevention. Acknowledgments

Financial Support: Susan G. Komen Foundation (C.B.U.), NCI P50 CA088843-06A1 (C.B.U., V.S.), Breast Cancer Research Foundation (V.S., C.B.U.), FAMRI (S.K.).

Liu MM, Albanese C, Anderson CM, Hilty K, Webb P, Uht RM, et al. Opposing action of estrogen receptors alpha and beta on cyclin D1 gene expression. J Biol Chem 2002; 277:24353-60. 6. Strom A, Hartman J, Foster JS, Kietz S, Wimalasena J, Gustafsson JA. Estrogen receptor beta inhibits 17beta-estradiol-stimulated proliferation of the breast cancer cell line T47D. Proc Natl Acad Sci USA 2004; 101:1566-71. 7. Sotoca AM, Gelpke MD, Boeren S, Strom A, Gustafsson JA, Murk AJ, et al. Quantitative proteomics and transcriptomics addressing the estrogen receptor subtype-mediated effects in T47D breast cancer cells exposed to the phytoestrogen genistein. Mol Cell Proteomics 2011; 10:110-2170.

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11. Shaaban AM, O’Neill PA, Davies MP, Sibson R, West CR, Smith PH, et al. Declining estrogen receptor-beta expression defines malignant progression of human breast neoplasia. Am J Surg Pathol 2003; 27:1502-12. 12. Shao ZM, Wu J, Shen ZZ, Barsky SH. Genistein exerts multiple suppressive effects on human breast carcinoma cells. Cancer Res 1998; 58:4851-7. 13. Seo HS, DeNardo DG, Jacquot Y, Laios I, Vidal DS, Zambrana CR, et al. Stimulatory effect of genistein and apigenin on the growth of breast cancer cells correlates with their ability to activate ERalpha. Breast Cancer Res Treat 2006; 99:121-34. 14. Lu R, Serrero G. Resveratrol, a natural product derived from grape, exhibits antiestrogenic activity and inhibits the growth of human breast cancer cells. J Cell Physiol 1999; 179:297-304. 15. Bowers JL, Tyulmenkov VV, Jernigan SC, Klinge CM. Resveratrol acts as a mixed agonist/antagonist for estrogen receptors alpha and beta. Endocrinology 2000; 141:3657-67. 16. Dean NM, Kanemitsu M, Boynton AL. Effects of the tyrosine-kinase inhibitor genistein on DNA synthesis and phospholipid-derived second messenger generation in mouse 10T1/2 fibroblasts and rat liver T51B cells. Biochem Biophys Res Commun 1989; 165:795-801. 17. Li Y, Upadhyay S, Bhuiyan M, Sarkar FH. Induction of apoptosis in breast cancer cells MDA-MB-231 by genistein. Oncogene 1999; 18:3166-72. 18. Popiolkiewicz J, Polkowski K, Skierski JS, Mazurek AP. In vitro toxicity evaluation in the development of new anticancer drugs-genistein glycosides. Cancer Lett 2005; 229:67-75.

19. Xiao ZP, Shi DH, Li HQ, Zhang LN, Xu C, Zhu HL. Polyphenols based on isoflavones as inhibitors of Helicobacter pylori urease. Bioorg Med Chem 2007; 15:3703-10. 20. Kolb HC, Sharpless KB. The growing impact of click chemistry on drug discovery. Drug Discov Today 2003; 8:1128-37. 21. Lin YJ, Hou YC, Lin CH, Hsu YA, Sheu JJ, Lai CH, et al. Puerariae radix isoflavones and their metabolites inhibit growth and induce apoptosis in breast cancer cells. Biochem Biophys Res Commun 2009; 378:683-8. 22. Guo JM, Xiao BX, Dai DJ, Liu Q, Ma HH. Effects of daidzein on estrogen-receptor-positive and negative pancreatic cancer cells in vitro. World J Gastroenterol 2004; 10:860-3. 23. Shao ZM, Alpaugh ML, Fontana JA, Barsky SH. Genistein inhibits proliferation similarly in estrogen receptor-positive and negative human breast carcinoma cell lines characterized by p21WAF1/CIP1 induction, G2/M arrest and apoptosis. J Cell Biochem 1998; 69:44-54. 24. Forster C, Makela S, Warri A, Kietz S, Becker D, Hultenby K, et al. Involvement of estrogen receptor beta in terminal differentiation of mammary gland epithelium. Proc Natl Acad Sci USA 2002; 99:15578-83. 25. Skliris GP, Lewis A, Emberley E, Peng B, Weebadda WK, Kemp A, et al. Estrogen receptor-beta regulates psoriasin (S100A7) in human breast cancer. Breast Cancer Res Treat 2007; 104:75-85. 26. Speirs V. The evolving role of oestrogen receptor beta in clinical breast cancer. Breast Cancer Res 2008; 10:111.

27. Maehle BO, Collett K, Tretli S, Akslen LA, Grotmol T. Estrogen receptor beta—an independent prognostic marker in estrogen receptor alpha and progesterone receptor-positive breast cancer? APMIS 2009; 117:644-50. 28. Band V, Zajchowski D, Swisshelm K, Trask D, Kulesa V, Cohen C, et al. Tumor progression in four mammary epithelial cell lines derived from the same patient. Cancer Res 1990; 50:7351-7. 29. Ferguson AT, Evron E, Umbricht CB, Pandita TK, Chan TA, Hermeking H, et al. High frequency of hypermethylation at the 14-3-3-sigma locus leads to gene silencing in breast cancer. Proc Natl Acad Sci USA 2000; 97:6049-54. 30. Chen H, Rubin E, Zhang H, Chung S, Jie CC, Garrett E, et al. Identification of transcriptional targets of HOXA5. J Biol Chem 2005; 280:19373-80. 31. Leygue E, Snell L, Hiller T, Dotzlaw H, Hole K, Murphy LC, et al. Differential expression of psoriasin messenger RNA between in situ and invasive human breast carcinoma. Cancer Res 1996; 56:4606-9. 32. Park S, Song J, Joe CO, Shin I. Akt stabilizes estrogen receptor alpha with the concomitant reduction in its transcriptional activity. Cell Signal 2008; 20:1368-74. 33. Iwao K, Miyoshi Y, Egawa C, Ikeda N, Tsukamoto F, Noguchi S. Quantitative analysis of estrogen receptoralpha and -beta messenger RNA expression in breast carcinoma by real-time polymerase chain reaction. Cancer 2000; 89:1732-8.


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