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• W2000851115 abstract "Osteoporosis is a major public health problem and the most obvious preventive strategy, hormone replacement therapy, has lost favor due to recent findings of the Women's Health Initiative regarding increased risks of breast cancer and cardiovascular disease. Resveratrol, a naturally occurring compound possessing estrogenic activity, is thought to have considerable potential for therapy of osteoporosis. In the present study, resveratrol was found to exhibit bone-protective effects equivalent to those exerted by hormone replacement therapy and decrease the risk of breast cancer in the in vivo and in vitro models. Forkhead proteins were found to be essential for both effects of resveratrol. The bone-protective effect was attributable to induction of bone morphogenetic protein-2 through Src kinase-dependent estrogen receptor activation and FOXA1 is required for resveratrol-induced estrogen receptor-dependent bone morphogenetic protein-2 expression. The tumor-suppressive effects of resveratrol were the consequence of Akt inactivation-mediated FOXO3a nuclear accumulation and activation. Resveratrol is therefore anticipated to be highly effective in management of postmenopausal osteoporosis without an increased risk of breast cancer. Osteoporosis is a major public health problem and the most obvious preventive strategy, hormone replacement therapy, has lost favor due to recent findings of the Women's Health Initiative regarding increased risks of breast cancer and cardiovascular disease. Resveratrol, a naturally occurring compound possessing estrogenic activity, is thought to have considerable potential for therapy of osteoporosis. In the present study, resveratrol was found to exhibit bone-protective effects equivalent to those exerted by hormone replacement therapy and decrease the risk of breast cancer in the in vivo and in vitro models. Forkhead proteins were found to be essential for both effects of resveratrol. The bone-protective effect was attributable to induction of bone morphogenetic protein-2 through Src kinase-dependent estrogen receptor activation and FOXA1 is required for resveratrol-induced estrogen receptor-dependent bone morphogenetic protein-2 expression. The tumor-suppressive effects of resveratrol were the consequence of Akt inactivation-mediated FOXO3a nuclear accumulation and activation. Resveratrol is therefore anticipated to be highly effective in management of postmenopausal osteoporosis without an increased risk of breast cancer. Osteoporosis is a major public health concern and is especially troublesome for menopausal and postmenopausal females who suffer from estrogen deficiency. It is widely believed that osteoporosis results from imbalance between bone resorption and bone formation leading to net bone loss (1Mundy G.R. Am. J. Clin. Nutr. 2006; 83: 427S-430SCrossref PubMed Google Scholar). However, hormone replacement therapy has lost favor due to the Women's Health Initiative finding that extended hormone replacement therapy increases the risks of both breast cancer and cardiovascular disease (2Couzin J. Science. 2003; 302: 1136-1138Crossref PubMed Scopus (15) Google Scholar). The current challenge in prevention or therapy of osteoporosis, therefore, is to identify treatments that share the protective effects of estrogen but lack its side effects. Certain natural dietary substances may act to minimize bone loss in postmenopausal women. The phytoestrogens, therefore, are potentially important in the prevention of postmenopausal osteoporosis caused by estrogen deficiency. Resveratrol is a polyphenolic phytoalexin compound found in various plants and has been characterized as a phytoestrogen based on its ability to bind to and activate estrogen receptor (ER) 3The abbreviations used are: ER, estrogen receptor; BMP-2, bone morphogenetic protein-2; OVX, ovariectomized; primary OBs, primary cultured osteoblast cells; ALP, alkaline phosphatase; BMD, bone mineral density; TNFα, tumor necrosis factorα; E2, 17β-estradiol; ERE, ER response element; ChIP, chromatin immunoprecipitation; siRNA, short interfering RNA; ERα-binding probe, non-radiolabeled probe; FRE, FOXO-responsive elements; SIR, silent information regulator; IKK, IκB kinase; siRNA, short interference RNA; ELISA, enzyme-linked immunosorbent assay; RT, reverse transcription; TRITC, tetramethylrhodamine isothiocyanate. (3Gehm B.D. McAndrews J.M. Chien P.Y. Jameson J.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14138-14143Crossref PubMed Scopus (961) Google Scholar). As a potential antioxidant, resveratrol has been reported to exhibit a wide range of biological and pharmacological properties, including 1) cancer-chemopreventive potential by blocking carcinogenesis at various stages; 2) cardiovascular protection by promoting nitric oxide production, inhibiting platelet aggregation and inflammation, and reducing toxic cholesterol level; and 3) anti-aging potential demonstrated by the effect of increasing the lifespan of yeast and flies. The mechanism studies have indicated that resveratrol acts at multiple levels, such as cellular signaling, enzymatic pathways, apoptosis, and gene expression (4Jang M. Cai L. Udeani G.O. Slowing K.V. Thomas C.F. Beecher C.W. Fong H.H. Farnsworth N.R. Kinghorn A.D. Mehta R.G. Moon R.C. Pezzuto J.M. Science. 1997; 275: 218-220Crossref PubMed Scopus (4523) Google Scholar, 5Ray P.S. Maulik G. Cordis G.A. Bertelli A.A. Bertelli A. Das D.K. Free Radic. Biol. Med. 1999; 27: 160-169Crossref PubMed Scopus (348) Google Scholar, 6Boissy P. Andersen T.L. Abdallah B.M. Kassem M. Plesner T. Delaisse J.M. Cancer Res. 2005; 65: 9943-9952Crossref PubMed Scopus (169) Google Scholar, 7de la Lastra C.A. Villegas I. Mol. Nutr. Food Res. 2005; 49: 405-430Crossref PubMed Scopus (586) Google Scholar, 8Delmas D. Jannin B. Latruffe N. Mol. Nutr. Food. Res. 2005; 49: 377-395Crossref PubMed Scopus (248) Google Scholar, 9Su J.L. Lin M.T. Hong C.C. Chang C.C. Shiah S.G. Wu C.W. Chen S.T. Chau Y.P. Kuo M.L. Carcinogenesis. 2005; 26: 1-10Crossref PubMed Scopus (99) Google Scholar, 10Delmas D. Lancon A. Colin D. Jannin B. Latruffe N. Curr. Drug Targets. 2006; 7: 423-442Crossref PubMed Scopus (333) Google Scholar). Recent studies have shown that resveratrol stimulates osteoblast differentiation and has a marked effect in reducing loss of bone mass in ovariectomized (OVX) rat model (6Boissy P. Andersen T.L. Abdallah B.M. Kassem M. Plesner T. Delaisse J.M. Cancer Res. 2005; 65: 9943-9952Crossref PubMed Scopus (169) Google Scholar, 11Mizutani K. Ikeda K. Kawai Y. Yamori Y. J. Nutr. Sci. Vitaminol. (Tokyo). 2000; 46: 78-83Crossref PubMed Scopus (103) Google Scholar, 12Andreou V. D'Addario M. Zohar R. Sukhu B. Casper R.F. Ellen R.P. Tenenbaum H.C. J. Periodontol. 2004; 75: 939-948Crossref PubMed Scopus (31) Google Scholar), indicating that resveratrol could be a preventive or therapeutic agent for osteoporosis. Details regarding the mechanisms through which resveratrol exerts its bone protection and tumor suppression are lacking. Therefore, identifying the molecular mechanisms of action is essential for understanding both the beneficial and adverse effects of resveratrol. Although the mechanisms of osteoporosis are not entirely clear, they are likely relate to decreased availability or effects of bone growth factors, such as bone morphogenetic proteins (BMPs). BMPs, structurally related to the transforming growth factor-β superfamily, were originally identified by their capacity to induce ectopic bone formation in rodents (13Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3986) Google Scholar). Among BMP family members, BMP-2 has been extensively studied and demonstrated to play a crucial role in inducing osteoblast differentiation and bone formation during embryonic skeletal development and postnatal bone remodeling (14Wozney J.M. Rosen V. Celeste A.J. Mitsock L.M. Whitters M.J. Kriz R.W. Hewick R.M. Wang E.A. Science. 1988; 242: 1528-1534Crossref PubMed Scopus (3352) Google Scholar, 15Zhao M. Harris S.E. Horn D. Geng Z. Nishimura R. Mundy G.R. Chen D. J. Cell Biol. 2002; 157: 1049-1060Crossref PubMed Scopus (156) Google Scholar). Skeletal aging studies have shown that both anabolic activity and gene expression of BMP-2 are decreased in the senile animals with osteopenia, suggesting that the decay of BMP-2 function may account for one of the molecular pathogenic mechanisms of osteoporosis (16Fleet J.C. Cashman K. Cox K. Rosen V. Endocrinology. 1996; 137: 4605-4610Crossref PubMed Scopus (81) Google Scholar, 17Matsumoto A. Yamaji K. Kawanami M. Kato H. J. Periodontal. Res. 2001; 36: 175-182Crossref PubMed Scopus (38) Google Scholar). A recent study has found a linkage of osteoporosis to specific polymorphisms in the BMP-2 gene, implicating that BMP-2 is an osteoporosis-associated gene (18Styrkarsdottir U. Cazier J.B. Kong A. Rolfsson O. Larsen H. Bjarnadottir E. Johannsdottir V.D. Sigurdardottir M.S. Bagger Y. Christiansen C. Reynisdottir I. Grant S.F. Jonasson K. Frigge M.L. Gulcher J.R. Sigurdsson G. Stefansson K. PLoS. Biol. 2003; 1: E69Crossref PubMed Scopus (220) Google Scholar). Findings of the present study reveal that resveratrol not only shares the bone-protective effects of hormone replacement therapy but also decreases the risk of occurrence of breast cancer. We found the forkhead protein, FOXA1, was involved in resveratrol-induced ER-dependent BMP-2 expression. Our findings also show that Akt inactivation-mediated FOXO3a nuclear accumulation and activity are required for the tumor suppressive effect of resveratrol. Forkhead proteins play a significant role in both resveratrol-mediated bone protective function and breast cancer suppression. We provide evidence that resveratrol would be a critical therapeutic strategy for prevention of osteoporosis. Antibodies and Reagents—Anti-BMP-2 antibody, anti-β-actin antibody, BMP-2 ELISA kit, osteopontin ELISA kit, and noggin were obtained from R&D Systems (Minneapolis, MN). The osteocalcin ELISA kit was obtained from Biocompare Inc. (San Francisco, CA). Fetal bovine serum, glutamine, and penicillin/streptomycin were purchased from HyClone (Logan, UT). Anti-p-Akt antibody, anti-Akt antibody, anti-p-Src antibody, anti-Src antibody, anti-ERα antibody, anti-ERβ antibody, anti-PCNA antibody, anti-c-Jun antibody, anti-c-Fos antibody, anti-Sp1 antibody, secondary antibodies, and protein G plus-agarose were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA). For the siRNA studies, a smart pool of double-stranded siRNA against ERα or nonspecific siRNA were obtained from Dharmacon Tech (Lafayette, CO) and used according to the manufacturer's instructions. trans-Resveratrol was obtained from Sigma. Tamoxifen and PP2 were purchased from Calbiochem Novabiochem Co. Cell Culture—The human osteoblast-like cell line MG-63 and MC3T3-E1 mouse clonal osteogenic cells were purchased from American Type Culture Collection (Manassas, VA). Cells were cultured in minimal essential medium supplemented with 10% fetal bovine serum and antibiotics (100 IU/ml penicillin G and 100 μg/ml streptomycin). Cell cultures were maintained at 37 °C in a humidified 5% CO2 atmosphere. For the cell cycle analysis, proliferating cells were serum-starved for 24 h and then treated with vehicle or resveratrol for 24 h. Cells were then subjected to flow cytometric analysis as described previously (19Su J.L. Yang P.C. Shih J.Y. Yang C.Y. Wei L.H. Hsieh C.Y. Chou C.H. Jeng Y.M. Wang M.Y. Chang K.J. Hung M.C. Kuo M.L. Cancer Cell. 2006; 9: 209-223Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Establishment of Stable Transfectants—MC3T3-E1 cells were transfected with pcDNA3 containing the dominant negative mutant Src, which were kindly provide by Dr. Ruey-Hwa Chen (Dept. of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan), or with the empty vector using Lipofectamine Plus reagent (Invitrogen). After 48 h, stably transfected cells were selected by media containing 600 μg/ml G418 for 4 weeks. Pools of six clones of MC3T3-E1/DN-Src or MC3T3-E1/vector were isolated for further studies. MDA-MB-231 cells were cotransfected with specific siRNA against FOXO3a cloned into pSuper vector and pcDNA3 in 10:1 ratio, which were kindly provide by Dr. Alex Toker (Dept. of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School). After 48 h, stably transfected cells were selected by media containing 800 μg/ml G418 for 4 weeks. Pools of 10 clones of MDA-MB-231/siFOXO3a or MDA-MB-231/siControl were isolated for further studies. BMD by DEXA—Female Wistar rats were sham-operated or OVX. BMD was measured in anesthetized rats (87 mg/kg ketamine and 13 mg/kg xylazine) after treated with various concentrations of resveratrol by orally feeding them every 2 days for 10 weeks using DEXA (QDR 4500a, Hologic, Inc.). Small animal software (Hologic, Inc.) was used to obtain BMD in the femur/tibia. The femur/tibia site consisted of the proximal half of the tibia and the entire femur. All animal work was performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the College of Medicine, National Taiwan University. Animals were maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Anchorage-independent Growth Assay—Colony-forming assays in soft agarose were performed as described previously (19Su J.L. Yang P.C. Shih J.Y. Yang C.Y. Wei L.H. Hsieh C.Y. Chou C.H. Jeng Y.M. Wang M.Y. Chang K.J. Hung M.C. Kuo M.L. Cancer Cell. 2006; 9: 209-223Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Cells were seeded in 6-well culture dishes in suspensions of 0.35% Agar noble in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum on top of a bed of 0.7% Agar noble in the same complete medium. After 3 weeks, tumor cell colonies measuring at least 50 μm were counted from six replicates per treatment under a dissecting microscope. Assaying the Levels of Osteocalcin, Osteopontin, and BMP-2—Osteocalcin, osteopontin, and BMP-2 ELISA kits were used to detect osteocalcin, osteopontin, and BMP-2 levels, respectively. Briefly, cells were treated with various concentrations of resveratrol for the indicated times. The culture medium was collected and measured for osteocalcin, osteopontin, and BMP-2, respectively. These samples were placed in 96-well microtiter plates coated with monoclonal-detective antibodies and incubated for 2 h at room temperature. After removing unbound material by washing with washing buffer (50 mm Tris, 200 mm NaCl, and 0.2% Tween 20), horseradish peroxidase-conjugated streptavidin was added to bind to the antibodies. Horseradish peroxidase catalyzed the conversion of a chromogenic substrate (tetramethylbenzidine) to a colored solution, with color intensity proportional to the amount of protein present in the sample. The absorbance of each well was measured at 450 nm. Results are presented as the percentage of change of the activity compared with the untreated control. cDNA Array Analysis—The GEArray Q series of Mouse Osteogenesis Gene Arrays from SuperArray Bioscience are application-specific cDNA arrays. GEArray Q series Mouse Osteogenesis Gene Array is designed to profile gene expression in the process of osteogenic differentiation. The genes regulating this process include growth factors and their internal cell signaling molecules, as well as the early and later differentiation genes. We used this Mouse Osteogenesis Gene Array to determine simultaneously the expression profile of the genes involved in the regulation of osteogenic differentiation. Experimental procedures and analyses were performed according to the manufacturer's instructions. Immunoprecipitation and Western Blot Analysis—Protein expression was detected by Western blot analysis as described previously (19Su J.L. Yang P.C. Shih J.Y. Yang C.Y. Wei L.H. Hsieh C.Y. Chou C.H. Jeng Y.M. Wang M.Y. Chang K.J. Hung M.C. Kuo M.L. Cancer Cell. 2006; 9: 209-223Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Equal amounts of protein were incubated with specific antibody immobilized onto protein A-Sepharose for 2 h at 4 °C with gentle rotation. Beads were washed extensively with lysis buffer, boiled, and microcentrifuged. Proteins were resolved on SDS-PAGE and transferred to nitrocellulose membrane. After blocking, blots were incubated with specific primary antibodies. After washing and incubating with secondary antibodies, immunoreactive proteins were visualized by the ECL detection system (Amersham Biosciences). Where indicated, the membranes were stripped and reprobed with another antibody. RNA Isolation and Reverse Transcriptase-PCR—Total RNA was isolated using RNAzol B and reverse transcribed into single-stranded cDNA with Moloney murine leukemia virus reverse transcriptase and random hexamers (Promega) according to the manufacturer's instructions. The primer sequences for osteocalcin were: 5′-ATGAGGACCCTCTCTCTGCTC-3′ (forward) and 5′-CTAAACGGTGGTGCCATAGAT-3′ (reverse); for osteopontin were: 5′-ATGAGACTGGCAGTGGTT-3′ (forward) and 5′-GCTTTCATTGGAGTTGCT-3′ (reverse). The primer sequences for BMPs were designed as described previously (20van der Horst G. van Bezooijen R.L. Deckers M.M. Hoogendam J. Visser A. Lowik C.W. Karperien M. Bone. 2002; 31: 661-669Crossref PubMed Scopus (65) Google Scholar). The reaction mixture was first denatured at 95 °C for 10 min. The PCR condition was 95 °C for 1 min, 52 °C for 1 min, and 72 °C for 1 min for 30 cycles, followed by 72 °C for 10 min. PCR products were visualized by ethidium bromide staining after agarose gel electrophoresis. The PCR results were repeated at least twice from a particular cDNA sample, and from at least two independent cDNA preparations. BMP-2 Promoter-luciferase Reporter Construction and Reporter Assay—The luciferase expression plasmids under the control of the BMP-2 promoter (pBMP-2-Luc) were kindly provided by Mundy G. R. (University of Texas Health Science Center at San Antonio, San Antonio, TX). To construct a plasmid containing the ERE or Sp1 site mutations in pBMP-2-Luc, we used pBMP-2-Luc promoter as a template and designed a mutated ERE site (pBMP-2-LucmERE, GGCCACTCTGACC to AACCACTCTACCT) or a mutated Sp1 site (pBMP-2-Luc-mSp1, CGGCCCGCCCGCC to CTTCCCTTCCGCC) by using the QuikChange® site-directed mutagenesis kit (Stratagene). The luciferase assays were carried out using the Dual-Luciferase® reporter assay kit (Promega) according to protocols provided by the manufacturer. Chromatin Immunoprecipitation Assay—Cells were fixed with 1% formaldehyde, washed, and lysed. The nuclei were released by processing in a Dounce homogenizer followed by lysis in 100–200 μl of nucleus lysis buffer (50 mm Tris-HCl (pH 8.0), 10 mm EDTA, and 1% SDS). One microgram of antibody was added to 0.5–1.0 ml of the lysate and rotated overnight at 4 °C. The immunocomplexes were then pulled down by using protein G-conjugated magnetic Dynabeads (Dynal Biotech), and the bound protein was eluted twice with 30 μl of 0.1 m citrate buffer (pH 3.0). The reverted DNA was purified with a Miniprep spin column (Qiagen) and then eluted in 50 μl of 10 mm Tris-HCl (pH 8.0). The BMP-2 promoter region was amplified by conventional PCR with primers 5′-GGGTTGGAACTCCAGACTGT-3′ (forward) and 5′-GAAGAGTGAGTGGACCCCAG-3′ (reverse). The PCR program was 95 °C for 10 min followed by 35 cycles at 95 °C for 45 s, 52 °C for 1 min, and 72 °C for 1 min. Input DNA and DNA recovered after immunoprecipitation were quantified using PicoGreen fluorescence (Molecular Probes, Eugene, OR). Equivalent masses of immunoprecipitation and input DNA were compared by real-time PCR as described above for RT-PCR with the following modifications. Taq polymerase was from Qiagen (Hot Start), and cycling conditions were 95 °C for 15 min followed by 45 cycles of 94 °C for 20 s, 61 °C for 1 min, and 72 °C for 40 s. Data are presented as the ratio of immunoprecipitation to input Ct values. siRNA—A 21-bp siRNA was designed against the FoxA1 transcript and synthesized by Dharmacon and transfected using Lipofectamine 2000 (Invitrogen) as described previously (21Carroll J.S. Liu X.S. Brodsky A.S. Li W. Meyer C.A. Szary A.J. Eeckhoute J. Shao W. Hestermann E.V. Geistlinger T.R. Fox E.A. Silver P.A. Brown M. Cell. 2005; 122: 33-43Abstract Full Text Full Text PDF PubMed Scopus (1081) Google Scholar). FOXO3a-siRNA (5′-GAGCUCUUGGUGGAUCAUCTT-3′) duplex (Dharmacon, 4 μm/2 × 106 cells) was transfected by Lipofectamine 2000 (Invitrogen), and lysates were prepared 48 h after transfection as described previously (22Hu M.C. Lee D.F. Xia W. Golfman L.S. Ou-Yang F. Yang J.Y. Zou Y. Bao S. Hanada N. Saso H. Kobayashi R. Hung M.C. Cell. 2004; 117: 225-237Abstract Full Text Full Text PDF PubMed Scopus (777) Google Scholar). Immunofluorescence Staining—Detection of protein expression by immunofluorescence staining was described previously (19Su J.L. Yang P.C. Shih J.Y. Yang C.Y. Wei L.H. Hsieh C.Y. Chou C.H. Jeng Y.M. Wang M.Y. Chang K.J. Hung M.C. Kuo M.L. Cancer Cell. 2006; 9: 209-223Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Briefly, cells were fixed in 3% paraformaldehyde and then blocked by incubation in 2.5% bovine serum albumin in phosphate-buffered saline. Primary antibodies as indicated were applied to the slides at a dilution of 1:50 and incubated at 4 °C overnight. The samples were treated with fluorescein isothiocyanate-conjugated or TRITC-conjugated secondary antibody (Sigma). The fluorescein isothiocyanate-labeled or TRITC-labeled cells were then analyzed by fluorescence microscopy. Orthotropic Breast Tumor Growth Assay—6-week-old female SCID mice were supplied by the animal center of the College of Medicine, National Taiwan University, Taipei, Taiwan. Mice were orthotropic inoculated with tumor cells into the mammary fat pad as described previously (22Hu M.C. Lee D.F. Xia W. Golfman L.S. Ou-Yang F. Yang J.Y. Zou Y. Bao S. Hanada N. Saso H. Kobayashi R. Hung M.C. Cell. 2004; 117: 225-237Abstract Full Text Full Text PDF PubMed Scopus (777) Google Scholar). Tumor development was followed in individual animals (eight per group) by measuring tumor length (L) and width (W) with calipers every 3 days. Tumor volume was calculated with the formula, LW2/2. All animal work was performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the College of Medicine, National Taiwan University. Fractionations of Cells and Electrophoretic Mobility Shift Assay—Nuclear extracts were prepared as described previously (22Hu M.C. Lee D.F. Xia W. Golfman L.S. Ou-Yang F. Yang J.Y. Zou Y. Bao S. Hanada N. Saso H. Kobayashi R. Hung M.C. Cell. 2004; 117: 225-237Abstract Full Text Full Text PDF PubMed Scopus (777) Google Scholar). Electrophoretic mobility shift assay for DNA binding in MC3T3-E1 cells was performed using the probe purchased from Santa Cruz Biotechnology and [α-32P]dCTP end-labeled in a 20-μl reaction mixture for 20 min at room temperature. For competition experiments, a 5-fold excess of unlabeled oligonucleotide was added to the binding reactions. The reaction products were analyzed by 5% nondenaturing PAGE using Tris (12.5 mm), boric acid (12.5 mm), and EDTA (0.25 mm), pH 8.3, for a period of 4–5 h at 280–300 V and 10–12 mA. The gels were then dried and exposed, for an appropriate time period, to Amersham™ film (Amersham Biosciences) at –70 °C while using an intensifying screen. Quantification of Apoptotic Osteoblasts in Undecalcified Bone Sections—Four-month-old female Wistar rats were subjected to a sham operation or to ovariectomy. Lumbar vertebrae (L1 to L5) were removed from female rats after 10 weeks. Apoptotic osteoblasts were detected in undecalcified plastic-embedded sections by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining using the Klenow FragEL detection kit (Oncogene) according to the manufacturer's instruction. Alkaline Phosphatase Activity Assay—Triplicate aliquots of 0.1% SDS cell lysates were used for biochemical evaluation of ALP activity using the Sigma-Aldrich kit number 104, according to the manufacturer's instruction. In Vitro Quantification of Apoptosis by Flow Cytometry—Cells were harvested and washed with phosphate-buffered saline, and hypodiploid cells were analyzed by flow cytometry as described previously (Su et al. 19Su J.L. Yang P.C. Shih J.Y. Yang C.Y. Wei L.H. Hsieh C.Y. Chou C.H. Jeng Y.M. Wang M.Y. Chang K.J. Hung M.C. Kuo M.L. Cancer Cell. 2006; 9: 209-223Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Briefly, cells were washed with phosphate-buffered saline and re-suspended in 500 μlofa buffer (0.5% Triton X-100/phosphate-buffered saline/0.05% RNase A) and incubated for 30 min. Then, 0.5 ml of propidium iodide solution (50 μg/ml) was added. Cells were then left on ice for 15–30 min. Fluorescence emitted from propidium iodide·DNA complexes was quantified after laser excitation of the fluorescent dye by fluorescence-activated cell sorting flow cytometry (BD Biosciences). Finally, the extent of apoptosis was determined by measuring DNA content of the cells below the G0/G1 peak. Real-time Quantitative RT-PCR—Real-time PCR was performed using a Roche LightCycler according to the manufacturer's protocol (Roche Applied Science). After reverse transcription reaction (20 μl) using 2 μg of total RNA, real-time PCR was carried out in a 20-μl final volume using the LightCycler-FastStart DNA Master SYBR Green I kit (Roche Applied Science). The reaction mix contained 1× LightCycler-FastStart Master SYBR Green I, 0.5 μm of each primer, 4 mm MgCl2, and 2 μl of cDNA from (20 μl of) reverse transcription reaction. The conditions of the real-time PCR were as described previously (23Zhou S. Turgeman G. Harris S.E. Leitman D.C. Komm B.S. Bodine P.V. Gazit D. Mol. Endocrinol. 2003; 17: 56-66Crossref PubMed Scopus (127) Google Scholar). Fluorescence was measured at 82 °C for 5 s. Resveratrol Increases Osteogenic Responses and Prevents Ovariectomy-induced Bone Loss—To examine the effects of resveratrol on proliferation and differentiation of osteoblastic cells, MC3T3-E1, MG63, and rat primary cultured osteoblast cells (primary OBs) were treated with various concentrations of resveratrol, and the fraction of cells in S-phase was determined. Treatment with resveratrol at the concentrations indicated in Fig. 1A significantly increased the proliferation of MC3T3-E1 and MG63 cells, closely matching the increase observed in primary OBs cultured under identical conditions. Biochemical analysis of the osteoblast differentiation marker, ALP, confirmed that enzyme activity increased in confluent preparations of these cells after treatment with resveratrol for 7 days (Fig. 1B). Expression of the osteoblast differentiation markers, osteocalcin and osteopontin, was measured by RT-PCR and ELISA assay. Resveratrol increased expression of both osteocalcin and osteopontin at the mRNA and protein levels in all three cell lines (Fig. 1C). The bone protective activity of resveratrol was also examined in ovariectomized mice. Resveratrol preserved both bone mineral density (BMD) and serum ALP activity in ovariectomized, but not sham-operated mice, and preservations were dose-dependent and significant (Fig. 1D). In addition, treatment with 10 mg/kg resveratrol restored BMD and serum ALP activity in ovariectomized mice to degrees observed in sham controls and as effectively as estradiol (Fig. 1D). To explore the anti-apoptotic effects of resveratrol in osteoblasts, MC3T3-E1 cells and primary OBs were subjected to pro-apoptotic stimuli in the absence or presence of resveratrol. The phytoestrogen prevented induction of apoptosis in MC3T3-E1 cells and primary OBs in response to etoposide or TNFα (Fig. 1E). Ovariectomy increased vertebral osteoblast apoptosis by 4.2-fold, compared with sham-operated controls (Fig. 1F). The osteoblast apoptosis was prevented in ovariectomized mice receiving either resveratrol or 17β-estradiol (Fig. 1F). These in vitro and in vivo findings support the potency and efficacy of resveratrol for prevention of both bone loss and osteoblast apoptosis. Resveratrol Prevents Breast Cancer Progression—To test the possibility that resveratrol increases the risk of breast cancer, effects of the drug on breast tumor growth were investigated. Estrogen-negative and estrogen-positive breast cancer cells were treated with vehicle, E2, or resveratrol and analyzed for extent of proliferation and anchorage-independent growth by DNA flow cytometry and the soft agar colony-forming assay, respectively. Treatment with resveratrol resulted in significant reductions in proliferation and anchorage-independent colony formation in the ER-positive (Fig. 2A) and ER-negative (Fig. 2B) breast cancer cells. In the orthotropic tumor growth assay, treatment with resveratrol reduced either E2-induced MCF-7 tumor growth or MDA-MB-231 tumor growth (Fig. 2C). In addition, the induction of tumor formation in response to treatment with E2 was significantly decreased by resveratrol (100% versus 40%, respectively). Resveratrol also reduced tumor formation in MDA-MB-231-bearing mice (100% in vehicle and E2 treatment group versus 85% in resveratrol treatment group). Tumor weight decreased by 54.6% in MCF-7-bearing mice treated with resveratrol as compared with mice treated with E2 (134 ± 33 mg versus 295 ± 47 mg, respectively). Furthermore, tumor weight decreased by 43.6% in MDA-MB-231-bearing mice treated with resveratrol as compared with the vehicle-treated group (194 ± 57 mg versus 344 ± 43 mg, respectively). Findings from these two experiments are summarized in Table 1. Resveratrol is therefore concluded to inhibit the induction and growth of breast cancer cells.TABLE 1Resveratrol decreases breas" @default.
• W2000851115 created "2016-06-24" @default.
• W2000851115 creator A5024331756 @default.
• W2000851115 creator A5069868789 @default.
• W2000851115 creator A5077185494 @default.
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• W2000851115 creator A5086650300 @default.
• W2000851115 date "2007-07-01" @default.
• W2000851115 modified "2023-10-14" @default.
• W2000851115 title "Forkhead Proteins Are Critical for Bone Morphogenetic Protein-2 Regulation and Anti-tumor Activity of Resveratrol" @default.
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