Inhibition of the Androgen Receptor by Antisense Oligonucleotides Regulates the Biological Activity of Androgens in SZ95 Sebocytes

Abstract

One novel strategy for the blockade of the androgen receptor could be the selective inhibition of androgen receptor by antisense oligonucleotides or small interfering RNA molecules. Here we describe the down regulation of the androgen receptor in cultured human SZ95 sebocytes with antisense oligonucleotides modified with phosphorothioates and 2′-O-methylribosyl residues. The ability of antisense oligonucleotides to cross the cellular membrane was enhanced by establishing a transient transfection system based on cationic lipid vesicles. Both antisense oligonucleotide types administered caused assumedly translational arrest. Dose-dependent inhibition of androgen receptor protein expression was observed after SZ95 sebocyte transfection with modified phosphorothioate oligonucleotides and modified 2′-O-methylribonucleotides which were directed against the translational start of the androgen receptor mRNA. The strongest transient inhibition of androgen receptor expression was detected after 14 hours with 1.0 μM antisense 2′-O-methylribonucleotides (88±1.3%, p<0.001). With longer recovery times than 24 hours, androgen receptor protein expression returned to the native control levels. Inhibition of the expression of androgen receptor by antisense oligonucleotides, reduced the enhanced proliferation of SZ95 sebocytes challenged by testosterone and 5α-dihydrotestosterone. This administration opens new therapeutic possibilities in androgen-associated skin diseases, since we could also show androgen inhibition with these antisense oligonucleotides in a reconstituted human epidermis model (Horm Metab Res 2007; 39:157-165).

Keywords

androgens - sebaceous glands - transient transfection - biological activity

References

• 1 Liao G, Chen LY, Zhang A, Godavarthy A, Xia F, Ghosh JC, Li H, Chen JD. Regulation of androgen receptor activity by the nuclear receptor corepressor SMRT.  J Biol Chem. 2003;  278 5052-5061
• 2 Verrijdt G, Haelens A, Claessens F. Selective DNA recognition by the androgen receptor as a mechanism for hormone-specific regulation of gene expression.  Mol Genet Metab. 2003;  78 175-185
• 3 Zouboulis CC. Human skin: an independent peripheral endocrine organ.  Horm Res. 2000;  54 230-242
• 4 Hirawat S, Budman DR, Kreis W. The androgen receptor: structure, mutations, and antiandrogens.  Cancer Invest. 2003;  21 400-417
• 5 Lee DK, Chang C. Endocrine mechanisms of disease: expression and degradation of androgen receptor: mechanism and clinical implication.  J Clin Endocrinol Metab. 2003;  88 4043-4054
• 6 Culig Z, Klocker H, Bartsch G, Steiner H, Hobisch A. Androgen receptors in prostate cancer.  J Urol. 2003;  170 1363-1369
• 7 Khalkhali-Ellis Z, Handa RJ, Price Jr RH, Adams BD, Callaghan JJ, Hendrix MJ. Androgen receptors in human synoviocytes and androgen regulation of interleukin 1beta (IL-1beta) induced IL-6 production: a link between hypoandrogenicity and rheumatoid arthritis?.  J Rheumatol. 2002;  29 1843-1846
• 8 Ibanez L, Ong KK, Mongan N, Jaaskelainen J, Marcos MV, Hughes IA, De Zegher F, Dunger DB. Androgen receptor gene CAG repeats polymorphism in the development of ovarian hyperandrogenism.  J Clin Endocrinol Metab. 2003;  88 3333-3338
• 9 Sasaki M, Sakuragi N, Dahiya R. The CAG repeats in exon 1 of the androgen receptor gene are significantly longer in endometrial cancer patients.  Biochem Biophys Res Commun. 2003;  305 1105-1108
• 10 Chen W, Thiboutot D, Zouboulis ChC. Cutaneous androgen metabolism: basic research and clinical perspectives.  J Invest Dermatol. 2002;  119 992-1007
• 11 Elias P, Feingold K. Coordinate regulation of epidermal differentiation and barrier homeostasis.  Skin Pharmacol Appl Skin Physiol. 2001;  14 28-34
• 12 Kao JS, Garg A, Mao-Qiang M, Crumrine D, Ghadially R, Feingold KR, Elias PM. Testosterone perturbs epidermal permeability barrier homeostasis.  J Invest Dermatol. 2001;  116 443-451
• 13 Ashcroft G, Mills S. Androgen receptor-mediated inhibition of cutaneous wound healing.  J Clin Invest. 2002;  110 615-624
• 14 Zouboulis ChC, Degitz K. Androgen action on human skin - from basic research to clinical significance.  Exp Dermatol. 2004;  4 5-10
• 15 Cronauer MV, Schulz WA, Burchardt T, Anastasiadis AG, De La Taille A, Ackermann R, Burchardt M. The androgen receptor in hormone-refractory prostate cancer: relevance of different mechanisms of androgen receptor signalling.  Int J Oncol. 2003;  23 10095-10102
• 16 Hoffmann J, Sommer A. Steroid hormone receptors as targets for the therapy of breast and prostate cancer-recent advances, mechanisms of resistance and new approaches.  J Steroid Biochem Mol Biol. 2005;  93 191-200
• 17 Chi KN, Gleave ME. Antisense approaches in prostate cancer.  Expert Opin Biol Ther. 2004;  6 927-936
• 18 Hamy F, Brondani V, Spoerri R, Rigo S, Stamm C, Klimkait T. Specific block of androgen receptor activity by antisense oligonucleotides.  Prostate Cancer Prostatic Disc. 2003;  6 27-33
• 19 Haag P, Bektic J, Bartsch G, Klocker H, Eder IE. Androgen receptor down regulation by small interference RNA induces cell growth inhibition in androgen sensitive as well as in androgen independent prostate cancer cells.  J Steroid Biochem Mol Biol. 2005;  96 251-258
• 20 Ko YJ, Devi GR, London CA, Kayas A, Reddy MT, Iversen PL, Bubley GJ, Balk SP. Androgen receptor down-regulation in prostate cancer with phosphorodiamidate morpholino antisense oligomers.  J Urol. 2004;  172 1140-1144
• 21 Fimmel S, Kurfurst R, Bonté F, Zouboulis CC. Responsiveness to androgens and effectiveness of antisense oligonucleotides against the androgen receptor on human epidermal keratinocytes is dependent on the age of the donor and the location of cell origin.  Horm Metab Res. 2007;  39 157-165
• 22 Zouboulis ChC, Seltmann H, Neitzel H, Orfanos CE. Establishment and characterization of an immortalized human sebaceous gland cell line (SZ95).  J Invest Dermatol. 1999;  113 1011-1020
• 23 Boudon C, Lobaccaro JM, Lumbroso S, Ogur G, Ocal G, Belon C, Sultan C. A new deletion of the 5alpha-reductase type 2 gene in a Turkish family with 5 alpha-reductase deficiency.  Clin Endocrinol. 1995;  43 183-188
• 24 Fimmel S, Saborowski A, Orfanos CE, Zouboulis ChC. Development of efficient transient transfection systems for introducing antisense oligonucleotides into human epithelial skin cells.  Horm Res. 2000;  54 306-311
• 25 Oshima A, Kyle JW, Miller RD, Hoffmann JW, Powell PP, Grubb JH, Sly WS, Tropak M, Guise KS, Gravel RA. Cloning, sequencing, and expression of cDNA for human beta- glucuronidase.  Proc Natl Acad Sci USA. 1987;  84 685-689
• 26 Wróbel A, Seltmann H, Fimmel S, Müller-Decker K, Tsukada M, Bogdanoff B, Mandt N, Blume-Peytavi U, Orfanos CE, Zouboulis ChC. Differentiation and apoptosis in human immortalized sebocytes.  J Invest Dermatol. 2003;  120 175-181
• 27 Gillies RJ, Didier N, Denton M. Determination of cell number in monolayer cultures.  Anal Biochem. 1986;  159 109-113
• 28 Nirde P, Georget V, Terouanne B, Galifer RB, Belon C, Sultan C. Quantitation of androgen receptor messenger RNA from genital skin fibroblasts by reverse transcription-competitive polymerase chain reaction.  J Steroid Biochem Mol Biol. 1998;  66 35-43
• 29 Kemppainen JA, Lane MV, Sar M, Wilson EM. Androgen receptor phosphorylation, turnover, nuclear transport, and transcriptional activation. Specificity for steroids and antihormones.  J Biol Chem. 1992;  267 968-974
• 30 Kimura K, Markowski M, Bowen C, Gelmann EP. Androgen blocks apoptosis of hormone- dependent prostate cancer cells.  Cancer Res. 2001;  61 5611-5618
• 31 Gad YZ, Berkovitz GD, Migeon CJ, Brown TR. Studies of up-regulation of androgen receptors in genital skin fibroblasts.  Mol Cell Endocrinol. 1988;  57 205-213
• 32 Chen W, Yang CC, Sheu HM, Seltmann H, Zouboulis ChC. Expression of peroxisome proliferator-activated receptor and CCAAT/enhancer binding protein transcription factors in cultured human sebocytes.  J Invest Dermatol. 2003;  121 441-447
• 33 Marcusson EG, Bhat B, Manoharan M, Bennett CF, Dean NM. Phosphorothioate oligodeoxyribonucleotides dissociate from cationic lipids before entering the nucleus.  Nucleic Acid Res. 1998;  26 2016-2023
• 34 Wang L, Prakash RK, Stein CA, Koehn RK, Ruffne DE. Progress in the delivery of therapeutic oligonucleotides: organ/cellular distribution and targeted delivery of oligonucleotides in vivo.  Antisense Nucleic Acid Drug Dev. 2003;  13 169-189
• 35 Monia BP, Sasmor H, Johnston JF, Freier SM, Lesnik EA, Muller M, Geiger T, Altmann KH, Moser H, Fabbro D. Sequence-specific antitumor activity of a phosphorothioate oligodeoxyribonucleotide targeted to human C-raf kinase supports an antisense mechanism in vivo.  Proc Natl Acad Sci USA. 1996;  93 15481-15484
• 36 Agrawal S, Temsamani J, Galbraith W, Tang J. Pharmacokinetics of antisense oligonucleotides.  Clin Pharmacokinet. 1995;  28 7-16
• 37 Crooke ST, Lemonidis KM, Neilson L, Griffey R, Lesnik EA, Monia BP. Kinetic characteristics of Escherichia coli RNase H1: cleavage of various antisense oligonucleotide- RNA duplexes.  Biochem J. 1995;  312 599-608
• 38 Kurreck J, Wyszko E, Gillen C, Erdmann VA. Design of antisense oligonucleotides stabilized by locked nucleic acids.  Nucleic Acids Res. 2002;  30 1911-1918
• 39 Baker BF, Lot SS, Condon TP, Cheng S, Flournoy F, Lesnik EA, Sasmor H, Bennett CF. 2′-O-(2-Methoxy)ethyl-modified anti-intercellular adhesion molecule 1 (ICAM-1) oligonucleotides selectively increase the ICAM-1 mRNA level and inhibit formation of the ICAM-1 translation initiation complex in human umbilical vein endothelial cells.  J Biol Chem. 1997;  272 1994-2000
• 40 Geary RS, Watanabe TA, Truong L, Freier S, Lesnik EA, Sioufi NB, Sasmor H, Manoharan M, Levin AA. Pharmacokinetic properties of 2′-O-(2-methoxy-ethyl)-modified oligonucleotide analogs in rats.  J Pharmacol Exp Ther. 2001;  296 890-897
• 41 Schmitz JC, Yu D, Agrawal S, Chu E. Effect of 2′-O-methyl antisense ORNs on expression of thymidylate synthase in human colon cancer RKO cells.  Nucleic Acids Res. 2001;  29 415-422
• 42 Spiller DG, Giles RV, Broughton CM, Grzybowski J, Ruddell CJ, Tidd DM, Clark RE. The influence of target protein half-life on the effectiveness of antisense oligonucleotide analog- mediated biologic responses.  Antisense Nucleic Acid Drug Dev. 1998;  8 281-293
• 43 Ranganathan G, Song W, Dean N, Monia B, Barger SW, Kern PA. Regulation of lipoprotein lipase by protein kinase C alpha in 3T3-F442A adipocytes.  J Biol Chem. 2002;  277 38669-38675
• 44 Reid J, Betney R, Watt K, McEwan IJ. The androgen receptor transactivation domain: the interplay between protein conformation and protein-protein interactions.  Biochem Soc Trans. 2003;  31 1042-1046
• 45 Grino PB, Griffin JE, Wilson JD. Testosterone at high concentrations interacts with the human androgen receptor similarly to dihydrotestosterone.  Endocrinology. 1990;  126 1165-1172
• 46 Fritsch M, Orfanos CE, Zouboulis ChC. Sebocytes are the key regulators of androgen homeostasis in human skin.  J Invest Dermatol. 2001;  116 793-800
• 47 Rosenfield RL, Deplewski D, Kentsis A, Ciletti N. Mechanism of androgen induction of sebocyte differentiation.  Dermatology. 1998;  196 43-46
• 48 Birrell SN, Bentel JM, Hickey TE, Ricciardelli C, Weger MA, Horsfall DJ, Tilley WD. Androgens induce divergent proliferative responses in human breast cancer cell lines.  J Steroid Biochem Mol Biol. 1995;  52 459-467
• 49 Song CS, Jung MH, Supakar PC, Chen S, Vellanoweth RL, Chatterjee B, Roy AK. Regulation of androgen action by receptor gene inhibition.  Ann N Y Acad Sci. 1995;  761 97-108
• 50 Yeap BB, Krueger RG, Leedman PJ. Differential posttranscriptional regulation of androgen receptor gene expression by androgen in prostate and breast cancer cells.  Endocrinology. 1999;  40 3282-3291
• 51 Krongrad A, Wilson CM, Wilson JD, Allman DR, McPhaul MJ. Androgen increases androgen receptor protein while decreasing receptor mRNA in LNCaP cells.  Mol Cell Endocrinol. 1991;  76 79-88
• 52 Wolf DA, Herzinger T, Hermeking H, Blaschke D, Horz W. Transcriptional and posttranscriptional regulation of human androgen receptor expression by androgen.  Mol Endocrinol. 1993;  7 924-936
• 53 van Erp PE, Wingens M. Does antisense make sense in Dermatology?.  Acta Derm Venereol. 2001;  81 385-391
• 54 Verma DD, Verma S, Blume G, Fahr A. Liposomes increase skin penetration of entrapped and non-entrapped hydrophilic substances into human skin: a skin penetration and confocal laser scanning microscopy study.  Eur J Pharm Biopharm. 2003;  55 271-277

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