THE MECHANISM OF PPARγ-MEDIATED DOWNREGULATION OF SODIUM HYDROGEN EXCHANGER (NHE1) GENE EXPRESSION AND ITS INHIBITION BY ESTROGEN RECEPTOR α Zhou Ting (BSc. (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2012 i ii ACKNOWLEDGEMENTS I wish to express my heartfelt gratitude for my supervisor Professor Shazib Pervaiz. I want to thank him for taking me as an honours student during undergraduate years, and allowing me to continue my graduate study in this lab. I am grateful for all the guidance and encouragement that he has offered during all these years. To Dr. Alan Prem Kumar, thank you for giving me the initial guidance when I first entered the field of research, and throughout the course of my study. Your advice and help will always be remembered. I would also like to thank my TAC members, Prof Marie-Veronique Clement and Prof Edison Liu for there invaluable input into this project. To Marie, thank for your constructive comments during all the meeting sessions. I am also grateful for the opportunity Prof Liu offered to let me some of the crucial experiments in his lab and for all the resources he provided. My warmest thanks to all the colleagues I have worked with and learned from throughout all these years. Special thanks to Ms Kong Say Li for teaching me the ChIP techniques, and Ms Quak Ai Li for helping me establish the initial direction of the project. To NUMI girls and Team Xtream, thank you for making my PhD years enjoyable and for being great friends. Finally, I would express my deepest gratitude for my parents. Though you are not physically with me during all these years in Singapore, your encouragement and support has always been the source of my strength at every step of my endeavors. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS II SUMMARY X LIST OF FIGURES . XII LIST OF TABLES . XV LIST OF ABBREVIATIONS . XVI LIST OF PUBLICATIONS XXII 1. INTRODUCTION . 1.1 PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS (PPARS) 1.1.1 Identification of PPARs . 1.1.2 Structural domains of PPARs . 1.1.3 Mechanism of action of PPARs 1.1.4 Subtypes of PPARs 1.1.5 Ligands and physiological functions of PPARα and PPARδ . 1.1.6 Ligands of PPARγ . 1.1.7 PPARγ and adipogenesis 10 1.1.8 PPARγ and insulin sensitization . 12 1.1.9 PPARs and cancer 13 1.1.10 PPARγ and breast cancer . 15 1.2 ESTROGEN RECEPTORS (ERS) 18 1.2.1 Identification and structures of ERs 18 1.2.2 Mechanism of action of ERs 20 1.2.3 Estrogen and ERs in human breast cancer 23 1.2.4 Ligands of ERs 26 1.2.5 ERs cross talk with each other and with other signaling pathways… 29 1.3 SODIUM-HYDROGEN EXCHANGER (NHE1) . 32 iv 1.3.1 Intracellular pH regulation . 32 1.3.2 Mammalian Na+/H+ exchanger . 32 1.3.3 NHE1 and cell volume . 34 1.3.4 NHE1 and cell proliferation and differentiation 34 1.3.5 NHE1 and cell migration 35 1.3.6 NHE1 and heart disease . 35 1.3.7 NHE1 and cancer . 36 1.3.8 Regulation of NHE1 activity 39 1.3.9 Regulation of NHE1 transcription 41 1.4 REACTIVE OXYGEN/NITROGEN SPECIES (ROS/RNS) 43 1.4.1 ROS/RNS species 43 1.4.2 Intracellular sites of ROS production . 43 1.4.3 NO production and its derivatives 45 1.4.4 The antioxidant system 47 1.4.5 ROS/RNS-mediated cell death . 48 1.5 AIM OF STUDY . 49 2. MATERIALS AND METHODS 51 2.1 MATERIALS 51 2.1.1 Chemicals and reagents 51 2.1.2 Cell lines and cell culture . 52 2.1.3 Antibodies . 53 2.1.4 Plasmids and siRNAs . 54 2.1.5 Primers and Oligonucleotides . 55 2.2 METHODS 56 2.2.1 Crystal violet cell viability assay 56 2.2.2 Colony forming assays . 57 2.2.3 Immunofluorescence 57 2.2.4 Western Blot analysis . 58 v 2.2.5 Nuclear-cytoplasmic fractionation 59 2.2.6 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) . 60 2.2.7 Transient Transfection . 61 2.2.8 Luciferase gene reporter assay . 61 2.2.9 Chloramphenicol Acetyl Transferase (CAT) assays 62 2.2.10 Measurement of Reactive Oxygen Species (ROS) 62 2.2.11 Noshift Transcription Factor Assay 63 2.2.12 Chromatin immunoprecipitation assay . 63 2.2.13 Coimmunoprecipitation 65 2.2.14 Morphology studies . 66 2.2.15 Protein determination . 66 2.2.16 Statistical analysis 66 3. RESULTS 68 3A PPARγ-MEDIATED REGULATION OF NHE1 68 3A.1 PPARγ AND THE EXPRESSION OF NHE1 68 3A1.1 Identification of putative PPRE on NHE1 promoter . 68 3A1.2 Down-regulation of NHE1 by PPARγ ligands. . 73 3A1.3 Down-regulation of NHE1 by PPARγ ligands is PPARγ- dependent… 80 3A1.4 Silencing PPARγ abrogates the effect of PPARγ ligand on NHE1……. . 82 3A1.5 Pharmacologcial PPARγ antagonist abrogates the effect of PPARγ ligand on NHE1 gene expression. 84 3A.2 THE MECHANISM OF PPARγ-MEDIATED DOWNREGULATION OF NHE1 88 3A2.1 Transcription-defective PPARγ abrogates the effect of PPARγ ligand on NHE1 gene expression. 88 3A2.2 Activated PPARγ binds to the identified PPRE on NHE1 promoter…. . 92 vi 3A.3 THE MECHANISM OF ROS/RNS-MEDIATED DOWNREGULATION OF NHE1 95 3A3.1 Production of ROS/RNS by PPARγ ligands in breast cancer cells………. 96 3A3.2 ONOO– is the main RNS species produced by PPARγ ligands in breast cancer cells. . 97 3A3.3 ONOO– is partially responsible for PPARγ ligand-mediated down-regulation of NHE1 expression 102 3A.4 THE ANTI-TUMOR EFFECTS OF PPARγ LIGANDS . 109 3A4.1 PPARγ ligands induce loss of cell viability in breast cancer cells……… . 109 3A4.2 PPARγ ligands inhibit colony formation by breast cancer cells. . 111 3A4.3 Anti-tumor effect of 15d-PGJ2 is PPARγ-dependent . 114 3A4.4 Anti-tumor effects of 15d-PGJ2 is partially ROS/RNS- dependent 119 3A4.5 Reduced NHE1 expression is responsible for PPARγ-mediated anti-tumor effects . 119 3B EFFECT OF ERα ON PPARγ-MEDIATED TRANSCRIPTIONAL REGULATION . 121 3B.1 ESTROGEN BLOCKS THE EFFECT OF PPARγ ON NHE1 121 3B1.1 Regular serum versus dextran stripped serum condition. 121 3B1.2 Estrogen blocks PPARγ-mediated down-regulation of NHE1 in CS serum condition. 126 3B.2 ACTIVE ERα BLOCKS EFFECT OF PPARγ ON NHE1 129 3B2.1 Re-expression of ERα in ER negative MDA-MB-231 cells restores its response to E2 on inhibiting PPARγ-mediated down-regulation of NHE1…. . 129 3B2.2 Transient silencing of ERα in ER positive MCF-7 cells abrogates the inhibitory effect of E2 on PPARγ-mediated down-regulation of NHE1…. . 133 vii 3B2.3 Depletion of active ERα in ER positive MCF-7 cells enhances PPARγ-mediated down-regulation of NHE1. . 136 3B2.4 Re-expression of ERα in ER negative MDA-MB-231 cells blocks PPARγ-mediated down-regulationof NHE1. . 141 3B.3 TRANSCRIPTIONALLY ACTIVE ERα BLOCKS THE EFFECT OF PPARγ ON NHE1 EXPRESSION . 145 3B3.1 ERα antagonist enhances the PPARγ-mediated NHE1 repression… 145 3B3.2 ERα defective in DNA binding enhances the effect of PPARγ ligand on NHE1 down-regulation. . 149 3B.4 THE MECHANISM BY WHICH ERα BLOCKS THE EFFECT OF PPARγ ON NHE1 EXPRESSION . 153 3B4.1 ERα does not bind to the putative ERE on NHE1 promoter. . 153 3B4.2 ERα suppresses binding of PPARγ to NHE1 promoter. 155 3B4.3 ERα inhibits transactivation of PPARγ . 159 3B4.4 PPARγ inhibits binding of activated ERα to ERE . 162 3B4.5 PPARγ physically interacts with ERα . 163 3B4.6 Growth inhibitory effect by PPARγ ligand combined with ERα antagonists . 167 4. DISCUSSION . 169 4.1 4.1.1 ESTABLISHING THE RELATIONSHIP BETWEEN PPARγ ACTIVATION AND NHE1 EXPRESSION 169 Identification of NHE1 gene as a transcriptional target of PPARγ……. 169 4.1.2 The mechanism of PPARγ-mediated repression of NHE1 gene. . 171 4.1.3 Production of ROS/RNS by PPARγ ligands in breast cancer cells………… . 174 4.1.4 The mechanism of ROS/RNS-mediated repression of NHE1 gene………. 177 4.2 4.2.1 ANTI-CANCER EFFECTS OF PPARγ LIGANDS 179 PPARγ-dependent anti-cancer effects of PPARγ agonists. 179 viii 4.2.2 PPARγ-independent anti-cancer effects of PPARγ agonists 182 4.2.3 Repression of NHE1 is involved in anti-tumor effect of PPARγ ligand…… . 186 4.3 4.3.1 MECHANISM OF HOW ERα INHIBITS PPARγ-MEDIATED DOWN-REGULATION OF NHE1 189 ERα negatively interferes with PPARγ-mediated down- regulation of NHE1 gene expression. . 189 4.3.2 Unravelling the mechanism of how ERα inhibits PPARγ- mediated down-regulation of NHE1 gene expression. 192 4.3.3 Signal cross talk between ERα and PPARγ in breast cancer cells………. 195 4.4 CLINICAL SIGNIFICANCE OF PPARγ-MEDIATED BREAST CANCER THERAPY AND ITS POSSIBLE MODULATION BY ER SIGNALLING PATHWAY . 199 4.5 CONCLUSION . 201 REFERENCES 205 ix SUMMARY In addition to its role in lipid and glucose metabolism, peroxisome proliferatoractivated receptor gamma (PPAR) has been associated with the process of carcinogenesis, which therefore presents a promising target for cancer treatment. Having identified a Peroxisome Proliferator Response Element (PPRE) in the promoter region of the pH regulator, Na+/H+ exchanger (NHE1), we recently showed that exposure of ER-negative breast cancer cells to PPAR ligands repressed NHE1 expression, which could be inhibited by the PPAR antagonist, GW9662. Moreover, inhibition of NHE1 expression either by direct silencing or preincubation with PPAR ligands in breast cancer cells increased their sensitivity to doxorubicin and paclitaxel. However, recent evidence of cross talks between nuclear receptors including PPAR and the estrogen receptor (ER) pathways has been demonstrated. Here we investigated the effect of PPAR activation on NHE1 gene repression in the presence of 17-estradiol (E2) using ER-positive MCF-7 breast cancer cells. Results show that E2 prevented the strong inhibition of NHE1 expression by PPAR ligands (natural or synthetic). On the contrary, E2 was unable to prevent the inhibition of NHE1 expression by PPAR ligands in the ER -negative breast cancer cell line, MDA-MB-231 or in MCF-7 cells where ER was silenced by specific ER siRNA. This result suggested that a functional activated ER is necessary to prevent PPAR-dependent down-regulation of NHE1 by E2. Indeed, a putative ER binding site (ERE) in close proximity and upstream of the PPRE was identified; however, ER did not bind to the putative ERE. ER was found to physically associate with PPAR at the PPRE and functionally interfered with PPAR binding efficiency to NHE1 promoter. Disruption of ER-PPAR complex by antiestrogens led to increased efficacy of the anti-tumor activity of PPAR and its repressive effect on NHE1 gene expression in vivo, which could be a potential x Rockford, IL, USA. Dual-Luciferase reporter assay system was from Promega. WI, USA. 10x PBS, 10 x SDS, Tris HCl buffer (pH 7.4) were purchased from NUMI Media Preperation Facility (NUS, Singapore). 5-(and-6)-chloromethyl2’,7’-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA), 4-amino5-methylamino-2’,7’-difluorofluorescein (DAF-FM), DAPI were supplied by Molecular Probes, OR, USA. 2.1.2 Cell lines and cell culture Breast cancer cell lines MCF-7, MDA-MB231 and T47D were purchased from American Type Culture Collection (ATCC; Rockville MD). All three cell lines were maintained in RPMI supplemented with 10% FBS, 2mM L-glutamine (Hyclone) and 0.05mg/ml gentamycin sulfate. MDA-MB-231 stably transfected with pReceiver-M11 vector encoding a functional ERα gene, a kind gift from Prof Edison Liu (Genome Institute of Singapore, Singapore), (MDA-MB-231 ERα+) were cultured in phenol free RPMI medium (GIBCO, MD, USA) supplemented with 2mM L-glutamine, 5% CS-FBS and 50µg/ml G418 Geneticin (Invitrogen, CA, USA). All cells were incubated at 370C and 5% CO2 and were split when 80% confluency was reached. Fresh medium was changed regularly every days. At the time of drug treatment, medium was removed and cells were counted using haemocytometer prior to seeding into different plates. For experiments involving E2, cells were left in medium supplemented with 10% CS-FBS, 2mM Lglutamine and 0.05mg/ml gentamycin sulfate for 48 hours before replaced with fresh phenol free RPMI medium containing no serum, but supplemented with 52 2mM L-glutamine and 0.05mg/ml gentamycin. For other experiments, cells were plated in regular and complete medium in which they were usually maintained, and left for 48 hours before treatment. Cells were treated for 24h unless otherwise specified. 2.1.3 Antibodies Mouse monoclonal NHE-1(Chemicon International Inc. CA, USA) Rabbit monoclonal Progesterone Receptor A/B (Cell Signaling Technology Inc. MA, USA) Rabbit monoclonal c-Myc (Cell Signaling Technology Inc. MA, USA) Mouse monoclonal PARP (BD Pharmingen, San Jose, CA, USA) Rabbit polyclonal PPARγ (H-100) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) Mousemonoclonal PPARγ (E-8) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) Mousemonoclonal ERα (D-12) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) Rabbit polyclonal ERα (HC-20) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) Rabbit polyclonal MnSOD (BD Pharmingen, San Diego, CA, USA) Rabbit polyclonal Cu/Zn SOD (Santa Cruz Biotechnology, Santa Cruz, CA, USA) Mouse Monoclonal Anti-β-Actin (Sigma Aldrich, St Louis, MO, USA) Goat Anti-mouse IgG HRP conjugated secondary antibody (Pierce Chemical Co. Rockford, IL, USA) Goat Anti-Rabbit IgG HRP conjugated secondary antibody (Pierce Chemical Co. Rockford, IL, USA) Alexa Fluor 610-R-phycoerythrin Goat Anti-mouse IgG (Invitrogen, Eugene, Oregon, USA) 53 Alexa Fluor-488 Rabbit Anti-mouse IgG (Invitrogen, Eugene, Oregon, USA) Donkey Anti-goat IgG-Rhodamine (Santa Cruz Biotechnology, Santa Cruz, CA, USA) 2.1.4 Plasmids and siRNAs Plasmid pCMV-HA-NHE1 encoding N-terminal hemagglutinin (HA) epitope tagged-NHE1 was a kind gift from Dr. Jeffrey R. Schelling, MetroHealth Medical Centre, Cleveland, Ohio, USA. Plasmid pcmX-mPPARγ was kindly provided by Dr. Ronald M. Evans, The Salk Institute for Biological Studies, San Diego, CA, USA.Chloramphenicol acetyltransferase (CAT) pUCSS-CAT reporter plasmid constructs: -1374/+16, -850/+16, and empty vector pUCSS-CAT were kindly provided by Dr. Alexey Kolyada, Tufts University School of Medicine, Boston, USA The Renilla control vector (pRL-TK) was purchased from Promege. pIRES-Hygromycin-resistance vector, 3XPPRE-TK-Luc construct and 3XERETATA-Luc reporter construct were bought commercially from Clontech Laboratories, CA, USA. Dominant negative mPPARγ was provided by Dr. Christopher K. Glass (University of California, San Diego, CA, USA). 21-nucleotide RNAs were chemically synthesized (Qiagen). 50-GAU AGG UUU CCA UGU GAU C sequence was used to silence NHE1 gene transcription (siNHE1) and 50-AGC UUC AUA AGG CGC AUG CTT (luciferase gene sequence inverted) sequence was used as a control (control si). siRNA for ERα 54 (sc-29305) was bought from Santa Cruz Biotechnology, CA, USA. siRNA PPARγ purchased SMARTpool was from Dharmcacon Technologies (ThermoFisher Scientific, Lafeyette, CO). 2.1.5 Primers and Oligonucleotides HuNHE1-PPRE-sense Biotin-3’ 5’-CACCTGAGGTCAGGAGTTCGAGACCA- HuNHE1-PPRE-antisense 5’- TGGTCTCGAACTCCTGACCTCAGGTG-3’ PTEN-PPRE-sense 5’-GGGACCAGGACAAAGGTCACGTT-Biotin- 3’ PTEN-PPRE-antisense 5’-GGGAACGTGACCTTTGTCCTGGTC-3’ HuNHE1-ERE-sense 5’-CACCTGAGGTCAGGAGTTCGAGACCA- Biotin-3’ HuNHE1-ERE-antisense 5’- TGGTCTCGAACTCCTGACCTCAGGTG-3’ Classical-ERE-sense 5’-GGATCTAGGTCACTGTGACCCCGGATC- Biotin-3’ Classical-ERE-antisense 5’- GATCCGGGGTCACAGTGACCTAGATCC-3’ NHEnew-forward 5’-GTTGTGGCTTACCCCTGTAATCCC-3’ NHEnew-reverse 5’-GTTTCACCATGTTGGTCAGGCTG-3’ PTEN-forward 5’-CTGGCATAACGCCTACCTGGTAC-3’ PTEN-reverse 5’-CAAGTGATATCATATGTGATGCTG-3’ 55 NHE-34-forward 5’-GCCCAGGATTCTGCCCAATC-3’ NHE-34-reverse 5’-TGCCTGCTGTGGAGCCCATT-3’ NHE-77-forward 5’-CCTGAGCCTCCCTCATCTTC-3’ NHE77-reverse 5’-GTGTGTGCAAAGGTGCTGGC-3’ ERA-forward 5’-CTGGCATAACGCCTACCTGGTAC-3’ ERA-reverse 5’-GAACTTGGGTGCTGTGCTTT-3’ ERB-forward 5’-CAACCTTGTAACCCTCAGCT-3’ ERB-reverse 5’-TCTATCCTTTGGGAGTGGGC-3’ ERC-forward 5’-ATGTTTATCGGCATCTAAAG -3’ ERC-reverse 5’-TATTCCTGAGTCATCCATCG -3’ 2.2 METHODS 2.2.1 Crystal violet cell viability assay Cells were plated in 12 well plates and treated with respective drugs for desired duration. At the end of treatment, medium was removed from the wells and cells were washed once with 1xPBS. Cells were incubated with 0.5ml of crystal violet solution (0.75% crystal violet, 50% ethanol, 1.75% formaldehyde, 0.25% NaCl) for 20 minutes, before the excess crystal violet solution was washed away using distilled water. The remaining crystal violet uptaken by viable cells was dissolved in a 1% SDS in 1X PBS solution and 50µl of cell lysate from each well was transferred into 96-well microtitre plate and quantified by reading absorbance at wavelength 595nm using a spectrophotometer (TECAN, GmbH, Grödig, Austria). 56 2.2.2 Colony forming assays Cells were treated with various concentrations of respective drugs or transfected with different plasmids and siRNAs for 24 hours. The cells were pelleted by centrifugation at 1,200 rpm at the end of the treatments.15,000 of treated cells were replated onto 100mm dish in RPMI supplemented with 10% FBS, 2mM Lglutamine and 0.05mg/ml gentamycin. The cells were then allowed to grow for 10 to 21 days and replenished with complete medium from time to time. Colonies formed were then quantified by staining cells with crystal violet. The numbers of colonies were scored manually in a 2cm x 2cm grid to determine colony forming abilities of cells. Only populations containing more than 50 cells were counted as colonies. 2.2.3 Immunofluorescence Each coverslip of a 12-well plate was seeded with approximately 0.06 x 106 cells/well. Drugs were added at ~30% confluency, before the coverslips with cells were washed with 1x cold PBS at the end of designated duration of treatment. Cells were then fixed with 4% paraformaldehyde for 30 mins at room temperature. Excess paraformaldehyde was washed (rock/shake) with x PBS twice, mins each before washed (rock/shake) with x cold PBS/100mM NH4Cl for twice and rinsed once with 1x PBS. TX-100 (0.2%) was used to permeablize the cells for 10 mins at room temperature before cells were washed with 1x PBS to remove excess TX-100. The cells were incubated with primary antibodies at 1:100 dilutions, for hour (up to hours) at room temperature. 57 Excess primary antibody was then washed away with x cold PBS for mins times before addition of secondary antibodies (1: 200) conjugated to fluorophore. The cells were incubated in secondary antibodies for 45mins to hour before washed and mounted on FluorSave (Calbiochem, USA). The cells were examined and the fluorescence signals were detected using fluorescence or confocal microscopy (Olympus Fluoview FV1000). The images were analyzed using Olympus Fluoview 1.7 viewer. 2.2.4 Western Blot analysis At the end of the treatments, medium was aspirated from the wells and the cells were washed once with 1x PBS. The cells were collected by scraping on ice before pelleted down by centrifugation at 12, 000 rpm. 1X RIPA lysis buffer (50mM Tris at pH 7.5, 150mM NaCl, 1% v/v NP-40, 1% v/v deoxycholic acid, 0.1% v/v SDS and 1mM EDTA) containing 1mM PMSF, 1mM sodium vanadate, 5μg/ml leupeptin, 5μg/ml pepstatin A and 1μg/ml aprotinin was added to the cell pellets to yield total cell lysates. Equal amount of proteins from the lysates were mixed with 5X Laemelli loading dye and incubated at 37oC for mins. The samples were then resolved in different concentrations (8% or 10%) of SDSPAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) depending on the molecular weight of the target proteins using Bio-Rad Mini-PROTEAN Cell (CA, USA). Kaleidoscope prestained standards (Bio-Rad, CA, USA) and biotinyated protein markers (Cell Signaling Technology, MA, USA) were also loaded next to samples to facilitate the estimation of the molecular sizes of the target proteins. The resolved proteins were then transferred onto a nitrocellulose 58 transfer membrane by the wet transfer method at 380mA for 1h using the Bio-Rad Mini Trans-Blot Electrophoretic Transfer Cell (CA, USA) in an ice-bath. The membrane was subjected to blocking with 5% (w/v) fat-free milk in Tris-buffered saline containing 0.05% (v/v) Tween 20 (TBST) for 1h before being washed times with TBST to remove excess milk. The membrane was incubated (shake/rock) with relevant primary antibodies (refer to antibodies list above) diluted in 5% BSA in TBST at 4ºC overnight. After the membranes were washed thrice with TBST, they were probed again using appropriate HRP-conjugated secondary antibody and HRP-conjugated anti-biotin antibody (for biotinylated protein makers) in TBST containing 5% (w/v) fat-free milk at room temperature for 1hr. The membranes were washed with TBST to remove any excess unbound secondary antibody before the proteins of interest were detected with Kodak Biomax MR X-ray film (Eastman Kodak Co., Rochestor NY) by enhanced chemiluminescence using the SuperSignal Chemiluminescent Substrate. The band intensity was analyzed by ImageJ. 2.2.5 Nuclear-cytoplasmic fractionation At the end of the treatment, cells were scraped on ice and pelleted by centrifugation at 12,000 rpm, at 4oC for minutes. The cells were then resuspended and washed with 500μl ice-cold PBS. The cells were then pelleted again by centrifugation at 500 x g for minutes. Different reagents in NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology Rockford, IL, USA) were added and the duration of lysis was performed according to manufacturer’s instructions. 59 2.2.6 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) At the end of drug incubation, media was aspirated from 6-well cell culture plates before the wells were washed with PBS. Following that, 1ml TRIZOL (Invitrogen) was added into each well. The cells were left in TRIZOL for 10 minutes before being transferred to 1.7ml eppendorf tubes. To the homogenates, 0.2ml chloroform was added. The mixtures were then vortexed and centrifuged at 13000rpm for 15 mins. After centrifugation, the aqueous phase containing RNAs was transferred into fresh tubes. Equal volume of isopropanol was added to the samples and mixed thoroughly. The mixtures were then centrifuged at 13000rpm at 4ºC for 15 mins to precipitate the RNA. The RNA pellet was washed with 80% ethanol after removal of supernatant. The samples were then centrifuged at 13000rpm for another 15 mins. Supernatant was then removed and RNA pellet was left to air dry. The RNA was then dissolved in 15ul of 0.1% DEPC water before it was quantified at 260/280nm using Nanodrop. Reverse transcription (RT) was then carried out in PCR thermal cycler (Mastercycler gradient, eppendorf) at 250C for 10 minutes, followed by 370C for 60 minutes and a terminating step at 950C for 5minutes. Each RT reaction constitutes 200ng of total RNA, 1XRT buffer, 5mM MgCl2, 425uM each of dNTPs, 2uM random hexamers, 0.35U/ul RNase inhibitor, 1.1U/ul MultiScribeTM reverse transcriptase and made up to 10ul with sterile water. At the end of the reaction, cDNA content was quantified by using realtime quantitative PCR in ABI PRISM 7500 (Applied Biosystems). Fluorescence was measured with the Sequence Detection Systems 2.0 software. PCR was performed in multiplex (both 60 target and endogenous control co-amplified in the same reaction) with distinct fluorescent dyes. The target signal was normalized to endogenous control. Primers and probe for human 18S RNA, human RhoB, human PTEN and human NHE1 were purchasedas kits from Applied Biosystems (Assays on Demand). 2.2.7 Transient Transfection Calcium Phosphate Mammalian transfection kit (Clonetech Laboratories Inc., Palo Alto, CA) was used in all transient transfections. In brief, cells were plated in 6-well plates and cells were left to grow for 48h in usual maintenance medium. 2h before transfection, medium was changed to DMEM supplemented with 10% FBS, 2mM L-glutamine and 0.05mg/ml gentamycin sulfate. Each transfection mixture consists of desired amount of plasmid DNA or siRNAs in sterile water and 12.4μl of 2.5M CaCl2 in a total amount of 100μl/well. The DNA (siRNA)CaCl2 solution was then added dropwise to 100μl of DNA precipitation buffer (2X HEPES-buffered saline) while being gently vortexing. The mixture was left at room temperature for 20 minutes before 200μl of the mixture was added to the medium of each well. After 16h, the transfection medium was removed and cells were washed with 1x PBS. The cells were then left to recover in normal culture medium for hours before deprivation of serum in phenol free medium and subjected to subsequent treatments as described previously. 2.2.8 Luciferase gene reporter assay Experiments for transfection were set up as described previously. The cells were then transfected with 3μg of 3XPPRE-TK/Luc or 3XERE-TATA/Luc construct 61 together with 0.3μg Renilla plasmid using calcium phosphate transfection kit. The promoter activity was measured with a dual-luciferase assay kit (Promega) according to the manufacturer’s instruction. Briefly, medium was removed from the wells, washed once with 1x PBS, and lysed with ice-cold 100l of reporter lysis buffer. 10l of cell lysate was then mixed with 50l of luciferase substrate solution, following which 50l of stop & glow buffer was added for Renilla reading. Sirius luminometer (Berthold) was used to quantify the bioluminescence generated. The readings obtained were normalized to the protein concentrations of the corresponding samples. 2.2.9 Chloramphenicol Acetyl Transferase (CAT) assays Cells were seeded and prepared for transfection as described above. Full length (1374/+16, contains PPRE region) human NHE1 promoter and a 5’ deletion construct lacking PPRE region (-850/+16) of the human NHE1 promoter were transfected into cells respectively. Cells were treated with varying doses of drugs for 24h before the promoter activity was analyzed by CAT ELISA assay (Roche) according to manufacturer’s protocol. The levels of CAT protein were assessed using a CAT antigen capture enzyme-linked immunosorbent assay (ELISA) (Roche Molecular Biochemicals). The readings were normalized to the protein concentration of the cell lysate. 2.2.10 Measurement of Reactive Oxygen Species (ROS) Intracellular levels of ROS were measured using the oxidation-sensitive fluorescent probe dichlorodihydrofluorescein diacetate (DCFH-DA) at 5µM. 62 Similar to ROS measurement, intracellular nitric oxide (NO) was assessed using DAF-FM dye of 5µM. Briefly, medium was removed from the wells and the cells were washed once with x PBS. Trypsin was added into each well followed by serum free media. The cells collected were pelleted at 12000rpm for minutes at 220C and the pellet was resuspended in 200l of serum free media containing 5M DCFH-DA or DAF-FM in DMSO. The cells were incubated for 15 minutes at 370C. After incubation, the cells were again pelleted at 2000rpm for minutes at 40C. The pellet was then washed with ice cold x PBS and pelleted again. Cell pellet was resuspended with 500l of room temperature x PBS. The cell suspension was then filtered via 60m filter paper and the fluorescence was read at excitation  of 495nm and emission  of 525nm on a FACScan flow cytometry. 2.2.11 Noshift Transcription Factor Assay Noshift Transcription Factor kit (Novagen, EMD Chemicals Inc, USA) was used to assess in vitro binding of PPARγ and ERα to NHE1 promoter sequence. Nuclear extracts were prepared using NE-PER® Nuclear and Cytoplasmic Extraction Reagents (De Mendonca et al.) according to the manufacturer’s instructions. 2µg PPARγ antibody (E-8 and H-100, Santa Cruz) and ERα antibody (D-12) were used to assess binding of PPARγ and ERα to respective nucleotides. The experiment was set up according to manufacturer’s protocol and the final ELISA reading was recorded at absorbance of 450nm using a Spectrofluoro Plus spectrophotometer (TECAN, GmbH, Grödig, Austria). 2.2.12 Chromatin immunoprecipitation assay 63 MCF-7 cells were grown and treated in 150mm dishes before they were crosslinked with 1% formaldehyde for 10min at room temperature. Next, fixation was stopped by adding glycine stop-fix solution and the cells were washed twice with PBS at 40C for 10 minutes each. The cells were then trypsinized and scrapped on ice to be collected and pelleted at 3000 rpm for minutes. The pellet was washed and resuspended in 1x PBS before the cells were pelleted again. Next, cells were dounced on ice with 10 strokes using a homogenizer and collected by centrifugation at 40C for 10 mins at 5000rpm to pellet the nuclei. The nuclei pellet was then resuspended in 1ml digestion buffer prior to addition of 50μl of enzymatic shearing cocktail (200 U/ml) and incubate at 370C for 15 mins. The supernatant was then collected by centrifugation at 13000rpm at 40C for 10 mins. The chromatin was pre-cleared by addition of protein G beads, ChIP buffer and protease inhibitor cocktails (PIC) and rotated at 40C for 1h. The pre-cleared chromatin was immunoprecipitated with anti-PPARγ, negative control IgG and incubated overnight on a rotator at 40C. Next, protein G beads were added and precipitation was further continued for 1.5h at 40C. After pelleting, precipitates were washed sequentially for 1-3 mins with various wash buffers. The immunocomplexes were then eluted with ChIP elution buffer (1% SDS, 0.1M NaHCO3). The eluates were reverse cross-linked by heating at 650C overnight and digested with proteinase K at 420C for 2h. The purified DNA from the immunoprecipitated complexes of antibody-protein-DNA were then detected by PCR (30 cycles) using the specific primer pair spanning the NHE1 promoter 64 region. The PCR products were resolved by electrophoresis in a 2% agarose gel and visualized after ethidium bromide staining. Cross-linked cells were resuspended and washed with TritonX-100 lysis buffer before the cell pellet was suspended in SDS lysis buffer. The nuclear lysates was sonicated for times, at 20% amplitude for 15s each at 4°C. The cell debris was pelleted and the supernatant was pre-cleared using washed and pre-blocked sepharose A beads. After 2h, the beads were pelleted by centrifugation and the supernatant was incubated with respective antibodies and beads overnight on rotation at 4°C. The beads were then pelleted and washed with TSE I buffer twice, Buffer III once and TE buffer once, before the DNA was eluted at 65°C with 800 rpm shaking for 30min. The CHIP samples were de-crosslinked at 65°C overnight, extracted and purified using Phenol/Chloroform extraction method. Purified DNA was then subjected to real-time PCR and the calculated enrichment was relative to the input and normalized to human actin gene. 2.2.13 Coimmunoprecipitation MCF-7 cells were seeded at 1.78x106cells/plate in 100mm cell culture dishes. At the end of drug treatment, cells were scraped off from plates on ice and cells from four 100mm dishes were pooled together into one 50ml falcon tube. The cells were pelleted at 12,000rpm by centrifugation. Cell pellets were washed with 1x PBS before they were subjected to nuclear cytoplasmic fractionation using NEPER Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology Rockford, IL, USA) as described above. The nuclear extracts were assessed for 65 protein content, and equal amount of protein was incubated with washed protein A/G beads in 0.5ml of immunoprecipitation (IP) buffer (25mM HEPES NaOH, pH7.4, 1% NP-40, 75mM NaCl, 1mM DTT, 1mM EDTA and protease inhibitors ) for at 40C for hour. The pre-cleared samples were then rotated with 20µg of PPARγ (E-8 and H-100) antibodies at 40C overnight. Following that, protein A/G beads were added to the samples and the mixture was rotated at 40C for hour. The beads were pelleted at 12,000 rpm for minutes and washed with IP buffer times, before they were heated in 20µl of 2x loading dye at 950C for minutes. The heated samples were briefly centrifuged and the supernatant containing antibodies-captured protein was subjected to western blot analysis as described above. 2.2.14 Morphology studies The morphology of the cells was observed and recorded by Olympus camedia digital camera (C-4040ZOOM, 4.1 megapixels) attached to the light microscope (Olympus CK2) at the magnification of x 200. 2.2.15 Protein determination Protein content of cell lysates was quatified using the Coomassie Dye (PIERCE, IL, USA) in a 96-well format. 1μl of cell lysate was diluted in 200µl of Coomassie Dye and its absorbance at 595nm was recorded using a Spectrofluoro Plus spectrophotometer (TECAN, GmbH, Grödig, Austria). Protein standards were prepared using bovine serum albumin (PIERCE, IL, USA). 2.2.16 Statistical analysis 66 All experiments were performed at least times for statistical significance. Numerical data were expressed as mean + Standard Deviation. Student’s t-test was used for statistical analysis and P values < 0.05 were considered significant. 67 [...]... charcoal/dextran-treated serum Figure 18 : Estrogen blocks PPARγ -mediated down- regulation of NHE1 expression Figure 29: Re-expression of ERα blocks PPARγ -mediated downregulation of NHE1 expression in MDA-MB-2 31 cells Figure 20: Silencing ERα attenuates the inhibitory effect of E2 on PPAR mediated down- regulation of NHE1 Figure 21: Reduced ERα level enhances PPARγ -mediated downregulation of NHE1 in regular serum condition... ligand on NHE1 gene expression Figure 8: PPARγ binds to NHE1 promoter upon 15 d-PGJ2 treatment Figure 9: PPARγ ligands produce ROS/RNS in breast cancer cells Figure 10 : PPARγ ligands produce ONOO- in breast cancer cells Figure 11 : ROS/RNS contributes to down- regulation of NHE1 Figure 12 : ROS/RNS is partially responsible for 15 d-PGJ2 -mediated down- regulation of NHE1 xii Figure 13 : Effects of PPARγ ligands... ligands down- regulate NHE1 mRNA levels in human breast cancer cells Figure 4: Overexpression of PPARγ enhances the inhibition of 15 d-PGJ2 on NHE1 expressions Figure 5: Silencing PPARγ attenuates the inhibition of 15 d-PGJ2 on NHE1 expression Figure 6: PPARγ inhibitor abrogates the effects of 15 d-PGJ2 on PPARγ activity and on NHE1 expression Figure 7: Transcription-defective PPARγ abrogates the effect of. .. xiii Figure 31: The mechanism of PPARγ -mediated down- regulation of NHE1 and its inhibiton by ERα xiv LIST OF TABLES Tables RESULTS Table 1: PPRE sequences from literature xv LIST OF ABBREVIATIONS 15 d-PGJ2 15 -Deoxy-Delta -12 , 14 -protaglandin J2 AA Arachidonic acid ACO Acyl-CoA oxidase gene AF Activation function AluRRE Alu Receptor Response Element AP -1 Activatior protein 1 aP2 Adipocyte protein 2 ATP... majority of PPREs than PPARα and PPARδ, making it less dependent on the 5’ flanking sequence (Desvergne and Wahli, 19 99) 4 As a typical nuclear transcription factor, the transcriptional activity of PPAR is greatly modulated by the coactivators and corepressors it recruits to the promoters of target genes The interactions between cofactors and nuclear receptors are largely ligand-dependent In the absence of. .. Re-expression of ERα blocks PPARγ -mediated downregulation of NHE1 in MDA-MB-2 31 cells kept in regular serum condition Figure 23: ERα antagonists enhance PPARγ -mediated down- regulation of NHE1 in regular serum condition Figure 24: Transfection of DNA-binding defective ERα enhances PPAR mediated down- regulation of NHE1 in regular serum condition Figure 25: Identification of putative ERE on NHE1 promoter... al., 19 95) PPARγ expression was reported to be upregualted by co-administration of corticosteroids 6 and insulin in human adipocytes (Vidal-Puig et al., 19 97), and down- regulated by tumor necrosis factor-α (TNFα) (Xing et al., 19 97) 1. 1.5 Ligands and physiological functions of PPARα and PPARδ PPARα is activated by synthetic hypolipidemic fibrates and natural ligands such as unsaturated fatty acids and. .. controversy Other than the synthetic ligands, PPARγ is also activated by endogenous ligands, such as polyunsaturated fatty acids, arachidonic acid and eicosapentaenoic acid (Berger and Moller, 2002) Metabolic intermediates of linoleic acid, 9-HODE and 13 -HODE are able to function as PPARγ ligands (Nagy et al., 19 98) 15 -deoxy 12 , 14 -PGJ2 (15 d-PGJ2), a metabolite of the eicosanoid prostaglandin J2, is... Binding of PPARγ to PPRE is blocked in the presence of estrogen Figure 27: Transcriptional activity of PPARγ is blocked in the presence of estrogen Figure 28: Binding of ERα to ERE is blocked in the presence of PPARγ ligand Figure 29: Physical interaction between PPARγ and ERα Figure 30: ERα antagonists enhanced the effect of PPARγ ligand on colony-forming ability in MCF-7 cells xiii Figure 31: The mechanism. .. absence of ligand, PPAR recruits corepressor complex consisting of NCoR and SMRT, which repress transcription by deacetylating histones at the target gene (Chen and Evans, 19 95; Xu et al., 19 99) When PPARs are activated by ligands, they undergo conformational change to form a “charge clamp” between LBD and AF-2 domain, enabling the releasing of corepressors and recruitment of coactivators via the conserved . PPARγ 8 1. 1.7 PPARγ and adipogenesis 10 1. 1.8 PPARγ and insulin sensitization 12 1. 1.9 PPARs and cancer 13 1. 1 .10 PPARγ and breast cancer 15 1. 2 ESTROGEN RECEPTORS (ERS) 18 1. 2 .1 Identification. PPARs 1 1. 1.2 Structural domains of PPARs 2 1. 1.3 Mechanism of action of PPARs 3 1. 1.4 Subtypes of PPARs 5 1. 1.5 Ligands and physiological functions of PPARα and PPARδ 7 1. 1.6 Ligands of PPARγ. PPARγ -MEDIATED REGULATION OF NHE1 68 3A .1 PPARγ AND THE EXPRESSION OF NHE1 68 3A1 .1 Identification of putative PPRE on NHE1 promoter 68 3A1.2 Down- regulation of NHE1 by PPARγ ligands. 73 3A1.3