AUTHOR COPY ONLY Estrogen receptor a (ESR1) over- expression mediated apoptosis in Hep3BcellsbybindingwithSP1proteins

Previous studies have reported that estrogen receptors (ERs) are expressed in normal human liver, chronic hepatitis, and benign hepatic tumor tissues. However, decreased expression of ERs can be observed in hepatocellular carcinoma (HCC) and the role of ERs in HCC is not fully understood. Thus, the present study aimed to investigate the molecular mechanism induced by the overexpression of ERa (ERa (ESR1)) in Hep3B cells. We first detected the induction of apoptosis in ER-negative Hep3B cells using DNA fragmentation assay and flow cytometry. We found that ERa and ERa plus 17b-estradiol treatment increased apoptosis in Hep3B cells. Additionally, western blotting showed increased expression of active caspase 3 and tumor necrosisfactora(TNFa(TNF)) inERa-transfectedcells. To further understand the importance of SP1-bindingsitesintheTNFapromoter,ERa-negativeHep3Bcellswereco-transfectedwithERa and a wild-type TNFa plasmid or TNFa with deleted SP1 regions. Deletion of both distant and primal SP1 sites abolished the activity of ERa, and similar results were observed by blocking the expression of SP1 protein using mithramycin (MA). This result indicates that SP1 protein is essential for ERa-activated TNFa promoter activity. Co-immunoprecipitation assay further confirmed the binding interaction between ERa and SP1 in a ligand-dependent manner. Ingeneral,wedemonstratethattheoverexpressionofERamediatesapoptosisinERa-negative Hep3B cells by the binding of ERa to SP1 protein. Additionally, this ERa‐SP1 complex binds to the proximal and distal sites of the TNFa gene promoter and further induces the expression of active caspase 3 in a ligand-dependent manner.

[1]  E. Kang,et al.  Pentoxifylline Attenuates Methionine- and Choline-Deficient-Diet-Induced Steatohepatitis by Suppressing TNF-α Expression and Endoplasmic Reticulum Stress , 2012, Experimental diabetes research.

[2]  S. I. Park,et al.  Transcription Factor Sp1 Is Involved in Expressional Regulation of Coxsackie and Adenovirus Receptor in Cancer Cells , 2011, Journal of biomedicine & biotechnology.

[3]  M. Dowsett,et al.  Transient over-expression of estrogen receptor-α in breast cancer cells promotes cell survival and estrogen-independent growth , 2011, Breast Cancer Research and Treatment.

[4]  Sama F. Sleiman,et al.  Mithramycin Is a Gene-Selective Sp1 Inhibitor That Identifies a Biological Intersection between Cancer and Neurodegeneration , 2011, The Journal of Neuroscience.

[5]  B. Tzang,et al.  17β-Estradiol inhibits prostaglandin E2-induced COX-2 expressions and cell migration by suppressing Akt and ERK1/2 signaling pathways in human LoVo colon cancer cells , 2010, Molecular and Cellular Biochemistry.

[6]  Andrew J. Wilson,et al.  Apoptotic sensitivity of colon cancer cells to histone deacetylase inhibitors is mediated by an Sp1/Sp3-activated transcriptional program involving immediate-early gene induction. , 2010, Cancer research.

[7]  J. Lachuer,et al.  Overexpression of Transcription Factor Sp1 Leads to Gene Expression Perturbations and Cell Cycle Inhibition , 2009, PloS one.

[8]  F. Tsai,et al.  Akt mediates 17β-estradiol and/or estrogen receptor-α inhibition of LPS-induced tumor necresis factor-α expression and myocardial cell apoptosis by suppressing the JNK1/2-NFκB pathway , 2009, Journal of cellular and molecular medicine.

[9]  D. Ramji,et al.  The tumour necrosis factor-alpha-mediated suppression of the CCAAT/enhancer binding protein-alpha gene transcription in hepatocytes involves inhibition of autoregulation. , 2009, The international journal of biochemistry & cell biology.

[10]  A. Bahnassy,et al.  Expression of pro‐ and anti‐inflammatory cytokines in relation to apoptotic genes in Egyptian liver disease patients associated with HCV‐genotype‐4 , 2009, Journal of gastroenterology and hepatology.

[11]  H. Nam,et al.  TNF alpha-induced down-regulation of estrogen receptor alpha in MCF-7 breast cancer cells. , 2008, Molecules and cells.

[12]  S. Mishkin,et al.  Evidence for the Hormone Dependency of Hepatic Hyperplastic Nodules: Inhibition of Malignant Transformation After Exogenous 17β‐Estradiol and Tamoxifen , 2007, Hepatology.

[13]  F. Stossi,et al.  Whole-Genome Cartography of Estrogen Receptor α Binding Sites , 2007, PLoS genetics.

[14]  I. Chaudry,et al.  Downregulation of migration inhibitory factor is critical for estrogen-mediated attenuation of lung tissue damage following trauma-hemorrhage. , 2007, American journal of physiology. Lung cellular and molecular physiology.

[15]  P. Ascenzi,et al.  Estrogen signaling multiple pathways to impact gene transcription. , 2006, Current genomics.

[16]  Shin-Da Lee,et al.  Apoptotic effects of over-expressed estrogen receptor-beta on LoVo colon cancer cell is mediated by p53 signalings in a ligand-dependent manner. , 2006, The Chinese journal of physiology.

[17]  Rola Barhoumi,et al.  Analysis of estrogen receptor alpha-Sp1 interactions in breast cancer cells by fluorescence resonance energy transfer. , 2005, Molecular endocrinology.

[18]  I Kimber,et al.  Anti-proliferative effect of estrogen in breast cancer cells that re-express ERalpha is mediated by aberrant regulation of cell cycle genes. , 2005, Journal of molecular endocrinology.

[19]  Maria Sjöberg,et al.  Mechanisms of estrogen receptor signaling: convergence of genomic and nongenomic actions on target genes. , 2005, Molecular endocrinology.

[20]  D. Kardassis,et al.  Inhibition of p53-mediated transcriptional responses by mithramycin A , 2004, Oncogene.

[21]  D. Lubahn,et al.  Estrogen receptor‐α deficiency promotes increased TNF‐α secretion and bacterial killing by murine macrophages in response to microbial stimuli in vitro , 2004 .

[22]  R. Urrutia,et al.  Sp1- and Krüppel-like transcription factors , 2003, Genome Biology.

[23]  H. El‐Serag.  Hepatocellular carcinoma: an epidemiologic view. , 2002, Journal of clinical gastroenterology.

[24]  F. Bresciani,et al.  Distinct nongenomic signal transduction pathways controlled by 17beta-estradiol regulate DNA synthesis and cyclin D(1) gene transcription in HepG2 cells. , 2002, Molecular biology of the cell.

[25]  B. Katzenellenbogen,et al.  Defining the "S" in SERMs , 2002, Science.

[26]  C. Röcken,et al.  Pathology and Pathogenesis of Hepatocellular Carcinoma , 2002, Digestive Diseases.

[27]  H. Offner,et al.  Estrogen Treatment Down-Regulates TNF-α Production and Reduces the Severity of Experimental Autoimmune Encephalomyelitis in Cytokine Knockout Mice1 , 2001, The Journal of Immunology.

[28]  Jingwen Liu,et al.  Requirement of Sp1 and Estrogen Receptor α Interaction in 17β-Estradiol-Mediated Transcriptional Activation of the Low Density Lipoprotein Receptor Gene Expression. , 2001, Endocrinology.

[29]  Myles Brown,et al.  Cofactor Dynamics and Sufficiency in Estrogen Receptor–Regulated Transcription , 2000, Cell.

[30]  J. Ferlay,et al.  Estimates of the worldwide mortality from 25 cancers in 1990 , 1999, International journal of cancer.

[31]  S. Safe,et al.  Transcriptional activation of E2F1 gene expression by 17beta-estradiol in MCF-7 cells is regulated by NF-Y-Sp1/estrogen receptor interactions. , 1999, Molecular endocrinology.

[32]  A. Gurney,et al.  Toll-like receptor-2 mediates lipopolysaccharide-induced cellular signalling , 1998, Nature.

[33]  Y. Mizobuchi,et al.  Suppressive effect of oestradiol on chemical hepatocarcinogenesis in rats , 1998, Gut.

[34]  K. Grandien,et al.  Printed in U.S.A. Copyright © 1997 by The Endocrine Society Comparison of the Ligand Binding Specificity and Transcript Tissue Distribution of Estrogen Receptors � and � , 2022 .

[35]  J. Gustafsson,et al.  Cloning of a novel receptor expressed in rat prostate and ovary. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[36]  K. Roby,et al.  Mouse endometrial tumor necrosis factor-alpha messenger ribonucleic acid and protein: localization and regulation by estradiol and progesterone. , 1994, Endocrinology.

[37]  S. Nedospasov,et al.  Intracellular signals and events activated by cytokines of the tumor necrosis factor superfamily: From simple paradigms to complex mechanisms. , 2006, International review of cytology.

[38]  B. Katzenellenbogen,et al.  Biomedicine. Defining the "S" in SERMs. , 2002, Science.

[39]  H. Lorenz,et al.  A rapid and simple method for the isolation of apoptotic DNA fragments. , 1994, Nucleic acids research.