Artigo Acesso aberto Revisado por pares

Role of Sirtuin Histone Deacetylase SIRT1 in Prostate Cancer

2008; Elsevier BV; Volume: 284; Issue: 6 Linguagem: Inglês

10.1074/jbc.m807869200

ISSN

1083-351X

Autores

Brittney Jung‐Hynes, Minakshi Nihal, Weixiong Zhong, Nihal Ahmad,

Tópico(s)

Autophagy in Disease and Therapy

Resumo

Prostate cancer (PCa) is a major age-related malignancy, and according to estimates from the American Cancer Society, a man's chance of developing this cancer significantly increases with increasing age, from 1 in 10,149 by age 39 to 1 in 38 by age 59 to 1 in 7 by age 70. Therefore, it is important to identify the causal connection between mechanisms of aging and PCa. Employing in vitro and in vivo approaches, in this study, we tested the hypothesis that SIRT1, which belongs to the Sir2 (silent information regulator 2) family of sirtuin class III histone deacetylases, is overexpressed in PCa, and its inhibition will have antiproliferative effects in human PCa cells. Our data demonstrated that SIRT1 was significantly overexpressed in human PCa cells (DU145, LNCaP, 22Rν1, and PC3) compared with normal prostate epithelial cells (PrEC) at protein, mRNA, and enzymatic activity levels. SIRT1 was also found to be overexpressed in human PCa tissues compared with adjacent normal prostate tissue. Interestingly, our data demonstrated that SIRT1 inhibition via nicotinamide and sirtinol (at the activity level) as well as via short hairpin RNA-mediated RNA interference (at the genetic level) resulted in a significant inhibition in the growth and viability of human PCa cells while having no effect on normal prostate epithelial cells. Further, we found that inhibition of SIRT1 caused an increase in FOXO1 acetylation and transcriptional activation in PCa cells. Our data suggested that SIRT1, via inhibiting FOXO1 activation, could contribute to the development of PCa. We suggest that SIRT1 could serve as a target toward developing novel strategies for PCa management. Prostate cancer (PCa) is a major age-related malignancy, and according to estimates from the American Cancer Society, a man's chance of developing this cancer significantly increases with increasing age, from 1 in 10,149 by age 39 to 1 in 38 by age 59 to 1 in 7 by age 70. Therefore, it is important to identify the causal connection between mechanisms of aging and PCa. Employing in vitro and in vivo approaches, in this study, we tested the hypothesis that SIRT1, which belongs to the Sir2 (silent information regulator 2) family of sirtuin class III histone deacetylases, is overexpressed in PCa, and its inhibition will have antiproliferative effects in human PCa cells. Our data demonstrated that SIRT1 was significantly overexpressed in human PCa cells (DU145, LNCaP, 22Rν1, and PC3) compared with normal prostate epithelial cells (PrEC) at protein, mRNA, and enzymatic activity levels. SIRT1 was also found to be overexpressed in human PCa tissues compared with adjacent normal prostate tissue. Interestingly, our data demonstrated that SIRT1 inhibition via nicotinamide and sirtinol (at the activity level) as well as via short hairpin RNA-mediated RNA interference (at the genetic level) resulted in a significant inhibition in the growth and viability of human PCa cells while having no effect on normal prostate epithelial cells. Further, we found that inhibition of SIRT1 caused an increase in FOXO1 acetylation and transcriptional activation in PCa cells. Our data suggested that SIRT1, via inhibiting FOXO1 activation, could contribute to the development of PCa. We suggest that SIRT1 could serve as a target toward developing novel strategies for PCa management. Prostate cancer (PCa) 2The abbreviations used are: PCa, prostate cancer; PBS, phosphate-buffered saline; shRNA, short hairpin RNA. 2The abbreviations used are: PCa, prostate cancer; PBS, phosphate-buffered saline; shRNA, short hairpin RNA. is a major age-related malignancy and is rarely seen in men younger than 40 years; the incidence rises rapidly with each decade thereafter. Because the present life expectancy has significantly improved and Americans are living longer, it is believed that more cases of PCa will be diagnosed in the future. According to one prediction, by year 2010, the number of annual PCa cases will skyrocket to 330,000. Thus, it will be immensely useful to better understand the molecular mechanism and connection between aging and PCa. Unraveling determinants such as genes and gene-products involved in aging and that have a connection with PCa could be exploited in designing novel targets and approaches for the management of this age-related neoplasm. We hypothesized that sirtuins (Sirt proteins), which are nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases, could serve as a connection between aging and cancer. Originally discovered in yeast, sirtuins are a unique class of type III histone deacetylases that utilize NAD+ as a cofactor for their functions (1Longo V.D. Kennedy B.K. Cell.. 2006; 126: 257-268Google Scholar, 2Marmorstein R. Biochem. Soc. Trans.. 2004; 32: 904-909Google Scholar, 3Michan S. Sinclair D. Biochem. J.. 2007; 404: 1-13Google Scholar, 4Sauve A.A. Wolberger C. Schramm V.L. Boeke J.D. Annu. Rev. Biochem.. 2006; 75: 435-465Google Scholar). Seven homologs of yeast Sir2 have been identified in the human genome. Called SIRT1 to -7, they all contain a highly conserved catalytic domain, and despite their enzymatic activity on histone substrates in vitro, Sirt proteins predominantly target nonhistone proteins for deacetylation, in both the nucleus and the cytoplasm (1Longo V.D. Kennedy B.K. Cell.. 2006; 126: 257-268Google Scholar, 2Marmorstein R. Biochem. Soc. Trans.. 2004; 32: 904-909Google Scholar, 3Michan S. Sinclair D. Biochem. J.. 2007; 404: 1-13Google Scholar, 4Sauve A.A. Wolberger C. Schramm V.L. Boeke J.D. Annu. Rev. Biochem.. 2006; 75: 435-465Google Scholar). Each sirtuin is characterized by a conserved 275-amino acid catalytic core domain and unique N-terminal and/or C-terminal sequences of variable length. The catalytic core domain may act preferentially as a mono-ADP-ribosyltransferase or NAD+-dependent deacetylase (3Michan S. Sinclair D. Biochem. J.. 2007; 404: 1-13Google Scholar). Their functions and locations differ greatly, and SIRT1 is the best characterized member among the mammalian sirtuins (1Longo V.D. Kennedy B.K. Cell.. 2006; 126: 257-268Google Scholar, 2Marmorstein R. Biochem. Soc. Trans.. 2004; 32: 904-909Google Scholar, 3Michan S. Sinclair D. Biochem. J.. 2007; 404: 1-13Google Scholar, 4Sauve A.A. Wolberger C. Schramm V.L. Boeke J.D. Annu. Rev. Biochem.. 2006; 75: 435-465Google Scholar). SIRT1 has been reported to be a nuclear as well as cytoplasmic protein and demonstrated to be involved in a number of cellular processes, including gene silencing at telomere and mating loci, DNA repair, recombination, and aging (1Longo V.D. Kennedy B.K. Cell.. 2006; 126: 257-268Google Scholar, 2Marmorstein R. Biochem. Soc. Trans.. 2004; 32: 904-909Google Scholar, 3Michan S. Sinclair D. Biochem. J.. 2007; 404: 1-13Google Scholar, 4Sauve A.A. Wolberger C. Schramm V.L. Boeke J.D. Annu. Rev. Biochem.. 2006; 75: 435-465Google Scholar, 5Haigis M.C. Guarente L.P. Genes Dev.. 2006; 20: 2913-2921Google Scholar, 6Ying W. Front. Biosci.. 2006; 11: 3129-3148Google Scholar). Recent studies have demonstrated that SIRT1 plays an important role in the regulation of cell death/survival and stress response in mammals. SIRT1 promotes cell survival by inhibiting apoptosis or cellular senescence induced by stresses, including DNA damage and oxidative stress (1Longo V.D. Kennedy B.K. Cell.. 2006; 126: 257-268Google Scholar, 2Marmorstein R. Biochem. Soc. Trans.. 2004; 32: 904-909Google Scholar, 3Michan S. Sinclair D. Biochem. J.. 2007; 404: 1-13Google Scholar, 4Sauve A.A. Wolberger C. Schramm V.L. Boeke J.D. Annu. Rev. Biochem.. 2006; 75: 435-465Google Scholar, 5Haigis M.C. Guarente L.P. Genes Dev.. 2006; 20: 2913-2921Google Scholar, 6Ying W. Front. Biosci.. 2006; 11: 3129-3148Google Scholar). An increasing number of proteins have been identified as substrates of SIRT1, including p53 (7Cheng H.L. Mostoslavsky R. Saito S. Manis J.P. Gu Y. Patel P. Bronson R. Appella E. Alt F.W. Chua K.F. Proc. Natl. Acad. Sci. U. S. A.. 2003; 100: 10794-10799Google Scholar, 8Langley E. Pearson M. Faretta M. Bauer U.M. Frye R.A. Minucci S. Pelicci P.G. Kouzarides T. EMBO J.. 2002; 21: 2383-2396Google Scholar, 9Luo J. 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Farzati B. Giovane A. Napoli C. Biochim. Biophys. Acta.. 2008; 1784: 936-945Google Scholar), repair protein Ku70 (11Brunet A. Sweeney L.B. Sturgill J.F. Chua K.F. Greer P.L. Lin Y. Tran H. Ross S.E. Mostoslavsky R. Cohen H.Y. Hu L.S. Cheng H.L. Jedrychowski M.P. Gygi S.P. Sinclair D.A. Alt F.W. Greenberg M.E. Science.. 2004; 303: 2011-2015Google Scholar, 17Cohen H.Y. Lavu S. Bitterman K.J. Hekking B. Imahiyerobo T.A. Miller C. Frye R. Ploegh H. Kessler B.M. Sinclair D.A. Mol. Cell.. 2004; 13: 627-638Google Scholar, 18Jeong J. Juhn K. Lee H. Kim S.H. Min B.H. Lee K.M. Cho M.H. Park G.H. Lee K.H. Exp. Mol. Med.. 2007; 39: 8-13Google Scholar), p300 (19Bouras T. Fu M. Sauve A.A. Wang F. Quong A.A. Perkins N.D. Hay R.T. Gu W. Pestell R.G. J. Biol. Chem.. 2005; 280: 10264-10276Google Scholar), Rb (19Bouras T. Fu M. Sauve A.A. Wang F. Quong A.A. Perkins N.D. Hay R.T. Gu W. Pestell R.G. J. Biol. Chem.. 2005; 280: 10264-10276Google Scholar, 20Wong S. Weber J.D. Biochem. J.. 2007; 407: 451-460Google Scholar), and p73 (19Bouras T. Fu M. Sauve A.A. Wang F. Quong A.A. Perkins N.D. Hay R.T. Gu W. Pestell R.G. J. Biol. Chem.. 2005; 280: 10264-10276Google Scholar, 21Dai J.M. Wang Z.Y. Sun D.C. Lin R.X. Wang S.Q. J. Cell Physiol.. 2007; 210: 161-166Google Scholar) just to name a few. Interestingly, SIRT1 has been shown to negatively regulate proliferative signaling via regulating (i) p53 function (7Cheng H.L. Mostoslavsky R. Saito S. Manis J.P. Gu Y. Patel P. Bronson R. Appella E. Alt F.W. Chua K.F. Proc. Natl. Acad. Sci. U. S. A.. 2003; 100: 10794-10799Google Scholar, 8Langley E. Pearson M. Faretta M. Bauer U.M. Frye R.A. Minucci S. Pelicci P.G. Kouzarides T. EMBO J.. 2002; 21: 2383-2396Google Scholar, 9Luo J. Nikolaev A.Y. Imai S. Chen D. Su F. Shiloh A. Guarente L. Gu W. Cell.. 2001; 19: 137-148Google Scholar, 10Vaziri H. Dessain S.K. Ng E.E. Imai S.I. Frye R.A. Pandita T.K. Guarente L. Weinberg R.A. Cell.. 2001; 19: 149-159Google Scholar, 19Bouras T. 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Hikasa M. Iijima K. Eto M. Kozaki K. Akishita M. Ouchi Y. Kaneki M. Oncogene.. 2006; 25: 176-185Google Scholar). Improper regulation of sirtuin proteins has been reported in a number of diseases, including Bowen's disease (25Hida Y. Kubo Y. Murao K. Arase S. Arch. Dermatol. Res.. 2007; 299: 103-106Google Scholar), type I diabetic nephropathy (26Tikoo K. Tripathi D.N. Kabra D.G. Sharma V. Gaikwad A.B. FEBS Lett.. 2007; 581: 1071-1078Google Scholar), Alzheimer disease and amyotrophic lateral sclerosis (27Kim D. Nguyen M.D. Dobbin M.M. Fischer A. Sananbenesi F. Rodgers J.T. Delalle I. Baur J.A. Sui G. Armour S.M. Puigserver P. Sinclair D.A. Tsai L.H. EMBO J.. 2007; 26: 3169-3179Google Scholar), and nonalchoholic fatty liver disease (28Deng X.Q. Chen L.L. Li N.X. Liver Int.. 2007; 27: 708-715Google Scholar). There are four reported human forkhead family members, FOXO1 (FKHR) (29Galili N. Davis R.J. Fredericks W.J. Mukhopadhyay S. Rauscher III, F.J. Emanuel B.S. Rovera G. Barr F.G. Nat. Genet.. 1993; 5: 230-235Google Scholar), FOXO3a (FKHRL1) (30Anderson M.J. Viars C.S. Czekay S. Cavenee W.K. Arden K.C. Genomics.. 1998; 47: 187-199Google Scholar), FOXO4 (ARX) (31Parry P. Wei Y. Evans G. Genes Chromosomes Cancer.. 1994; 11: 79-84Google Scholar), and FOXO6 (32Jacobs F.M. van der Heide L.P. Wijchers P.J. Burbach J.P. Hoekman M.F. Smidt M.P. J. Biol. Chem.. 2003; 278: 35959-35967Google Scholar), which have been shown to regulate a variety of cellular processes, including cell differentiation, transformation, and metabolism (11Brunet A. Sweeney L.B. Sturgill J.F. Chua K.F. Greer P.L. Lin Y. Tran H. Ross S.E. Mostoslavsky R. Cohen H.Y. Hu L.S. Cheng H.L. Jedrychowski M.P. Gygi S.P. Sinclair D.A. Alt F.W. Greenberg M.E. Science.. 2004; 303: 2011-2015Google Scholar, 12Frescas D. Valenti L. Accili D. J. Biol. Chem.. 2005; 280: 20589-20595Google Scholar, 13Kobayashi Y. Furukawa-Hibi Y. Chen C. Horio Y. Isobe K. Ikeda K. Motoyama N. Int. J. Mol. 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Cell.. 2004; 116: 551-563Google Scholar); however, the connection between SIRT1 and the FOXO factors in PCa is not well understood. In this study, we have demonstrated that SIRT1 is significantly overexpressed in human PCa cell lines compared with normal human prostate epithelial cells. Further, we found that SIRT1 is significantly overexpressed in prostate cancer tissue samples versus adjacent normal prostate epithelium in patients with PCa. Furthermore, our data demonstrated that SIRT1 inhibition by nicotinamide, sirtinol, or shRNA-mediated RNA interference causes an inhibition in growth and cell viability of human PCa cells while having no effect on normal prostate epithelial cells. We have also found that inhibition caused an increase in acetylated FOXO1 protein with a concomitant increase in FOXO1 transcriptional activity. Our data suggest that SIRT1 may be promoting PCa cell growth via inhibiting FOXO1. Cell Culture—The human prostate carcinoma cell lines (viz. LNCaP, 22Rν1, DU145, and PC3 (obtained from ATCC) were maintained in RPMI 1640, minimum Eagle's, and F12K media (ATCC) supplemented with fetal bovine serum and antibiotics (penicillin/streptomycin). Normal human prostate epithelial cells PrEC (Cambrex) and NPEC (Celprogen) were maintained at standard cell culture conditions in PrEBM medium or human prostate culture complete growth medium, respectively, with growth factors and supplements as recommended by the vendors (Cambrex and Celprogen). Normal human keratinocytes, NHEK (Invitrogen), were maintained in keratinocyte-SFM medium (Invitrogen). N/Tert-1 keratinocytes were obtained from the Brigham and Women's Hospital Cell Culture Core Facility (Boston, MA) and maintained in keratinocyte-SFM medium. Human mammary epithelial cells (Cambrex) were maintained in mammary epithelial cell growth medium with supplements (Cambrex). Normal human bronchial epithelial cells (Cambrex) were maintained in bronchial epithelial cell basal medium supplemented with BEGM Single Quots (Cambrex). All cells were maintained at standard cell culture conditions (37 °C, 5% CO2 in a humidified incubator) as recommended by the vendors. Primary Cell Culture—Prostate tissue was obtained under an approved Institutional Review Board protocol from men (ages 44-66) undergoing cystoprostatectomy for bladder cancer at the University of Wisconsin Hospital and Clinics. Histology confirmed the absence of cancer in the tissue. Prostate epithelial cultures were established, as described by others (37Jarrard D.F. Sarkar S. Shi Y. Yeager T.R. Magrane G. Kinoshita H. Nassif N. Meisner L. Newton M.A. Waldman F.M. Reznikoff C.A. Cancer Res.. 1999; 59: 2957-2964Google Scholar, 38Reznikoff C.A. Loretz L.J. Pesciotta D.M. Oberley T.D. Ignjatovic M.M. J. Cell Physiol.. 1987; 131: 285-301Google Scholar). Briefly, prostate tissues were minced with a scalpel and digested in a solution containing collagenase (500 units/ml; Sigma) and plated on collagen-coated plates. Cells were maintained in Ham's F-12 medium (Invitrogen) supplemented with 0.25 units/ml regular insulin, 1 μg/ml hydrocortisone, 5 μg/ml human transferrin, 2.7 mg/ml dextrose, 0.1 mm nonessential amino acids, 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mm l-glutamine, 10 ng/ml cholera toxin, 25 μg/ml bovine pituitary extract, and 1% fetal bovine serum. Cells were passaged using trypsin-EDTA. Preparation of Whole Cell Protein Lysates and Western Blot Analysis—PCa cells were washed with ice-cold PBS, trypsinized, and collected by centrifugation. Cell lysates were prepared using 1× radioimmune precipitation buffer, with freshly added phenylmethylsulfonyl fluoride and protease inhibitor mixture (Cell Signaling), and protein concentration was determined with a BCA protein assay (Pierce). Primary culture protein (designated P326 and P218) was extracted by freeze thawing three times in ECB buffer, and protein concentration was determined with a BCA protein assay. For immunoblot analysis, 30-40 μg of protein was subjected to SDS-PAGE and transferred onto a nitrocellulose membrane. Immunoblot analysis was performed using a variety of primary antibodies (anti-SIRT1, anti-TATA-binding protein TBP (Abcam), anti-FOXO1, anti-FOXO3a, anti-FOXO4 (Cell Signaling), anti-actin, and anti-Ac-FKHR (Santa Cruz Biotechnology, Inc.) and a variety of secondary antibodies (goat anti-rabbit and goat anti-mouse horseradish peroxidase-conjugated antibodies (Upstate) and donkey anti-goat horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology) followed by chemiluminescent detection. The quantification of protein was performed by a digital analyses of protein bands (TIFF images) using UN-SCAN-IT software. Preparation of Nuclear and Cytosolic Protein Lysates—Following treatments, the medium was aspirated, and the cells were washed twice with ice-cold 1× PBS. Cytoplasmic lysis buffer (10 mmol/liter HEPES (pH 7.9), 10 mmol/liter KCl, 0.1 mmol/liter EDTA, 0.1 mmol/liter dithiothreitol, 1 mmol/liter phenylmethylsulfonyl fluoride, 10 μg/ml protease inhibitor mixture) was added, and cells were scraped off. The lysate was then incubated on ice for 15 min. 10% Nonidet P-40 was added to the suspension, which was then centrifuged at 14,000 × g at 4 °C for 2 min. Supernatant was collected for cytosolic protein lysate. The remaining cell pellet was resuspended in nuclear extraction buffer (20 mmol/liter HEPES (pH 7.9), 0.4 mol/liter NaCl, 1 mmol/liter EDTA, 1 mmol/liter EGTA, 1 mmol/liter dithiothreitol, 2 mmol/liter phenylmethylsulfonyl fluoride, and 10 μg/ml protease inhibitor mixture). Suspension was incubated on ice for 30 min and then centrifuged at 14,000 × g for 10 min at 4 °C. Supernatant was collected for nuclear protein lysate. Nuclear and cytosolic protein concentrations were determined with a BCA protein assay (Pierce). Both nuclear and cytosolic lysates were used for subsequent Western blotting experiments (described above). Immunofluorescence—For detection of SIRT1 by immunofluorescence, the cells were plated and grown on BD Falcon CultureSlides (BD Biosciences) until a confluence of 80% was reached. The cells were fixed and then blocked for 1 h at room temperature in 10% normal goat serum (Caltag Laboratories) in PBS. Following blocking, rabbit anti-SIRT1 antibody (Santa Cruz Biotechnology) was added and allowed to incubate for 2 h at room temperature. Primary antibody was removed, and Alexa Fluor 555 goat anti-rabbit secondary antibody (Molecular Probes, Inc.) was then added and incubated for 1 h at room temperature in the dark. PBS-diluted 4′,6-diamidino-2-phenylindole, dihydrochloride (Pierce) counterstain was used for nuclear staining. Cells were mounted with the ProLong anti-fade kit as per the vendor's protocol (Molecular Probes) and examined under a Bio-Rad Radiance 2100 MP Rainbow confocal/multiphoton system. Immunoprecipitation and SIRT1 Enzyme Activity Assay—For immunoprecipitation of SIRT1 protein, the lysates containing 500 μg of total protein were incubated with rabbit anti-SIRT1 antibody (Abcam) overnight at 4 °C with constant rotation. The specific antibody-antigen complex was collected by precipitation with Protein A-agarose beads (Pierce) for 2.5 h at 4 °C with constant rotation. SIRT1 activity was determined in immunoprecipitates from cells using the SIRT1 fluorimetric drug discovery kit (AK-555; Biomol) as per the vendor's protocol. Quantitative Real Time Reverse Transcription-PCR—RNA was isolated using TRIzol reagent (Invitrogen) according to the vendor's protocol. RNA was treated with DNase (Invitrogen), and first strand cDNA was transcribed with 300 ng of random primers, 10 mm dNTPs, and 200 units of Moloney murine leukemia virus reverse transcriptase (Invitrogen). Quantitative reverse transcription-PCR was performed in triplicate with Platinum SYBR Green quantitative PCR SuperMix-UDG (Invitrogen) with 50 ng of first strand cDNA and a 0.2 μm concentration each of forward and reverse primers for SIRT1 (forward (5′-TGCTGGCCTAATAGAGTGGCA-3′) and reverse (5′-CTCAGCGCCATGGAAAATGT-3′) with a product size of 102 bp) or glyceraldehyde-3-phosphate dehydrogenase (forward (5′-GAAGGTGAAGGTCGGAGTC-3′) and reverse (5′-GAAGATGGTGATGGGATTTC-3′) with a product size of 236 bp). The samples were cycled once for 50 °C for 2 min for UDG incubation followed by 94 °C for 2 min and then 40 cycles of 94 °C for 15 s and 55 °C for 30 s each. Relative SIRT1 mRNA was calculated using the ΔΔCt comparative method using glyceraldehyde-3-phosphate dehydrogenase as an endogenous control. Purity of product was checked by dissociation curve analysis as well as running the samples on 3% agarose gel. Human Tissues and Immunohistochemistry—Paraffin-embedded tissue slides containing human prostate cancer tissue with adjacent normal prostate tissue and a custom tissue microarray containing cancer and normal prostatic tissue from 41 patients (3-9 samples per patient depending on heterogenicity) with varying grades of PCa were obtained from the Department of Pathology and Laboratory Medicine, University of Wisconsin (Madison, WI). The slides were deparaffinized and blocked for endogenous peroxidases with a 3% H2O2 in a double-distilled H2O incubation. 1 mm EDTA, pH 8.0, was heated to a boil, and tissue slides were then boiled for 3 min for antigen retrieval. Slides were then blocked in 1.5% normal goat serum (Caltag Laboratories) in PBS for 1 h at room temperature in a humidified chamber followed by incubation with a rabbit anti-SIRT1 antibody (Santa Cruz Biotechnology) overnight at 4 °C in a humidified chamber. The slides were then incubated with goat anti-rabbit secondary antibody (Upstate) for 1 h at room temperature in a humidified chamber, followed by cross-reaction with freshly prepared liquid 3,3′-diaminobenzidine substrate chromogen system, 20 μl of 3,3′-diaminobenzidine chromogen/1 ml of substrate buffer (DakoCytomation). Hematoxylin (Vector Laboratories) was diluted 1:5 in double-distilled H2O and used as a nuclear counterstain. Finally, slides were dehydrated and mounted with coverslips followed by microscopic analysis with digital image capture. SIRT1 staining was semiquantitatively graded as negative (-), weak (+), moderate (++), or strong (+++) staining in 50% of cells examined. Statistical analysis of the data were performed using Fisher's exact test. Treatment of Cells with Nicotinamide—Cells were grown to 60% confluence and then treated with 150 μm, 300 μm, 5 mm, or 20 mm nicotinamide (Acros Organics) dissolved in the growth medium. Cells were incubated with the nicotinamide treatment for 24 h, after which they were used for subsequent experiments. Treatment of Cells with Sirtinol—Cells were grown to 60% confluence and then treated with 30 or 120 μm sirtinol (Sigma; dissolved in DMSO). Cells were incubated with sirtinol for 24 or 48 h, after which they were used for subsequent experiments. Transfection with SIRT1 Short Hairpin RNA (shRNA)—Short hairpin SIRT1 clone V2HS_20109 (sequenced with U6 5′-TGT GGA AAG GAC GAA ACA CC sequencing primers) cloned into a pSHAG-MAGIC2 vector was purchased from Open Biosystems (Huntsville, AL). The transfections were done according to the protocol supplied with the RNAintro shRNA transfection kit (Open Biosystems). Plasmid DNA from cultures was prepared according to the protocol given in Qiagen Plasmid Maxi Kit (Qiagen, Valencia, CA). Appropriate restriction digests were performed to confirm correct shRNA plasmid DNA. Plasmid DNA was then diluted in serum-free medium and mixed with Arrest-In diluted in serum-free medium and incubated for 10 min at room temperature. The DNA·Arrest-In complex mixture was added to 60-80% confluent cells and incubated for 6 h at 37 °C in 5% CO2 in a humidified incubator. After 6 h, transfection medium was removed, and medium with serum was added and incubated in a humidified chamber at 37 °C in 5% CO2 for 48 h. The cells were then harvested, and further studies were performed. Trypan Blue Exclusion Assay—Following treatments, cells were trypsinized and collected in a 1.5-ml Eppendorf tube. The cells were pelleted by centrifugation and resuspended in PBS (120 μl). Trypan blue (0.4% in PBS; 10 μl) was added to a smaller aliquot (10 μl) of cell suspension, and the number of cells (viable unstained and nonviable blue) were counted. Luciferase Reporter Assay—PCa cells were transfected with phRL-TK (Promega) and 3×IRSLuc-FOXO1 (15Yang Y. Hou H. Haller E.M. Nicosia S.V. Bai W. EMBO J.. 2005; 24: 1021-1032Google Scholar) or FGHRE-Luc (33Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell.. 1999; 96: 857-868Google Scholar) (Addgene plasmid 1789) by Lipofectamine 2000 (Invitrogen) as per the vendor's protocol. Transfected cells were then collected and replated into 12-well plates 48 h post-transfection so that they were 75% confluent the following day. 24 h postreplating, cells were treated in triplicate with medium, DMSO (vehicle), or sirtinol (30 or 120 μm for 24 h). Then cell lysates were prepared, and luciferase activity was determined using the dual luciferase assay system (Promega, WI). Luciferase activity was normalized to Renilla luciferase activity. Statistical Analysis—Statistical analyses were performed with Student's t test for independent samples, and the data are expressed as means ± S.E. unless specified otherwise. Statistically significant p values are provided for each individual experiment. Aging, an inevitable process in living organisms, has been linked to several unwanted disease conditions, including several types of cancers. Studies suggest that certain genetic and epigenetic alterations are accumulated during aging and appear to possess a direct role in cell transformation. These events show a clear evolution during aging and are reversed in cancer. An interesting example of this is that telomere length (controlled by genetic and epigenetic modifications) decreases with ag

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