MATERIALI E METOD
BE INDET LGD HGD P Localizzazione citoplasmatica 14 2 1
Localizzazione mista 5 1 3 0
Localizzazione nucleare 3 0 6 11
<0.0005
Legenda: BE= assenza displasia; INDET= indeterminato per displasia; LGD= displasia basso grado; HGD= displasia alto grado
Figura 8: rappresentazione grafica della localizzazione subcellulare della
GST-O nei differenti gradi di displasia del tessuto di Barrett
0 10 20 30 40 50 60 70 80 90 100 BE INDET LGD HGD
DISCUSSIONE
Abbiamo visto che la GSTO pur essendo priva di attività catalitica di glutatione transferasi ha molteplici funzioni nella risposta allo stress cellulare, ed in particolare, oltre al riciclo dell’acido ascorbico, la regolazione dei canali del calcio, la modulazione dell’espressione di altre proteine, la risposta allo stress indotto da stimoli fisici e chimici e l’intervento nel metabolismo dei farmaci antineoplastici a base di arsenico. Inoltre l’importanza della GST-Ω si evince anche dall’evidente associazione di polimorfismi della stessa GST-Ω con patologie umane: pare, ad esempio, che possa esistere un’associazione tra il polimorfismo del gene che codifica per la GST-Ω1 e l’età di insorgenza del morbo di Parkinson e quello di Alzheimer. [94] Ciò concorda con il fatto che queste proteine sono coinvolte nel mantenimento dell’omeostasi ossidoriduttiva della cellula oltre ad essere implicate nell’attivazione post-trascrizionale dell’IL-1-β e, dato che nell’Alzheimer aumenta l’espressione delle citochine proinfiammatorie, si è ipotizzato che le GST-Ω possono essere implicate nello sviluppo della malattia. Come già esposto, quindi, le GST- Ω possono intervenire in numerose funzioni cellulari ma, visto il loro ruolo predominante nelle risposte allo stress cellulare, si è focalizzata l’attenzione sul comportamento di tale classe di proteine in risposta ad insulto chimico e fotodinamico.
Kodym e coll. hanno dimostrato in un studio condotto su linee cellulari murine sottoposte a stress chimico-termico la traslocazione di una proteina (p28), identificata successivamente come GST-Ω, dal citoplasma al nucleo. Tale comportamento permette di qualificare la proteina come una heat
shock protein e pertanto risulta coinvolta nell’omeostasi ossidoriduttiva
Inoltre Giri e coll. dimostrano l’efficacia dell’enzima di mediare tappe metaboliche deputate alla detossificazione di alcuni mutageni ambientali (arsenico). [95]
Alcuni lavori mostrano poi che alcuni polimorfismi della GST-Ω risultano maggiormente associati ad alcune forme neoplastiche valutate come il carcinoma mammario, l’epatocarcinoma ed il colangiocarcinoma. [96]
Sulla base di queste considerazioni è stata valutata la possibilità di studiare il comportamento della GST-Ω sul tessuto metaplastico di Barrett. Proprio partendo dalla considerazione che tale lesione precancerosa si sviluppa a seguito di un insulto chimico protratto nel tempo (rigurgito acido in corso di MRGE cronica di durata superiore a cinque anni), attendevamo una differente espressione della proteina a livello citoplasmatico sulle cellule che presentavano differenti gradi di displasia o atipie cellulari. Dalla valutazione dei risultati, che hanno dimostrato una concentrazione della proteina a livello nucleare nelle cellule con espressioni elevate di atipie (campioni tissutali con HGD), l’ipotesi che questa proteina possa indurre delle modificazioni profonde sull’omeostasi cellulare risulta fortemente supportata. Oltre a questo è interessante sottolineare come nei livelli intermedi di displasia o nell’assenza di displasia la proteina presenti una localizzazione mista o esclusivamente citoplasmatica.
Sulla base dei dati raccolti in questa tesi possiamo concludere che ancora molte valutazioni sono necessarie per comprendere il reale significato biologico e le funzioni della GST-Ω. La traslocazione nucleare così evidente, soprattutto nelle cellule con importanti alterazioni citologiche, potrebbe essere associata o al semplice insulto chimico o più probabilmente alla capacità della proteina di stimolare l’azione di geni che promuovono la trascrizione o che regolano l’omeostasi cellulare, e quindi
potrebbe permettere di compiere un ulteriore passo in avanti nella conoscenza dei meccanismi che conducono alla cancerogenesi.
Ulteriore spunto per approfondimenti futuri potrà essere la valutazione di isoforme della proteina (GST-Ω1 e GST-Ω2), attualmente in fase di valutazione iniziale, per ottenere dati ancora più specifici sulle proprietà biologiche di questo enzima.
BIBLIOGRAFIA
[1] Sleisenger & Fordtrand’s Gastrointestinal and Liver Disease. Saunders USA 2002
[2] Falk Gary W. Barrett’s Esophagus. Gastroenterol 2002;122:1569-91 [3] Devesa SS, Blot WJ, Fraumeni JF. Changing patterns in the incidence
of esophageal and gastric carcinoma in the United States. Canc
1998;83:2049-53.
[4] Bollschweiler E., Wolfgarten E., Gutschow C., Holscher AH. Demographic variations in the rising incidence of esophageal adenocarcinoma in white males. Canc 2001;92:549-55
[5] Reed PI, Jonston BJ. The changing incidence of oesophageal cancer.
Endoscopy 1993;25:606-8
[6] Pera M, Cameron AJ, Trastek VF, Carpenter HA, Zinsmeister AR. Increasing incidence of adenocarcinoma of the esophagus and esophagogastric junction. Gastroenterol 1993;104:510-3
[7] Moller H. Incidence of cancer of the esophagus, cardia and stomach in Denmark. Eur J Cancer Prev 1993;1:159-64
[8] Sodhani P, Gupta S, Prakash S, Singh V. Columnar and metaplastic cell in vault smears. Cytopathol 1999;10:122-6
[9] Jankowski JA et al. Molecular evolution of the metaplasia-dysplasia- adenocarcinoma sequence in the esophagus. Am J Pathol 1999;154(4). [10] Powell J, McConkey CC. The rising trend in oesophageal
adenocarcinoma and gastric cardia. Eur J Cancer Prev 1992;1:265-9 [11] Jankowski JA, Harrison RF, Perry I, Balkwill F, Tselepsis C. Barrett’s
metaplasia. Lancet 2000;356:2079-85.
[12] Comio M, Lapertosa G, Blanchi S, Filiberti R. Barrett Esophagus: an update. Crit Rew Oncol Hematol 2003;46(2):187-206.
[13] Sampliner RE. Practice guidelines on the diagnosis surveillance and therapy of Barrett Esophagus. The Practice Parameters Committee of the American College of Gastroenterology 1998;93(7):1028-32.
[15] Ovaska J, Miettinen M, Kivilaasko E. Adenocarcinoma arising in Barrett’s esophagus. Dig Dis Scie 1989;34:1336-9.
[16] Cameron AJ, Zinsmeister AR, Ballard DJ, Carney JA. Prevalence of columnar-lined (Barrett’s Esophagus). Gastroenterol 1990;99:918-22. [17] G.O.S.P.E. Barrett’s esophagus: epidemiological and clinical results of
a multicentricc survey. Int J Canc 1991;48:364-8.
[18] Cameron AJ, Lomboy CT. Barrett’s esophagus: age, prevalence and extent of columnar epithelium. Gastroenterol 1992;103:1241-5.
[19] Cameron AJ. Epidemiology of Barrett’s esophagus. Gastroenterol
Clinic Biologic 1994;18:D3-D4.
[20] Fahmy M, King JF. Barrett Esophagus: an acquired condition with genetic predisposition. Am J Gastroenterol 1993;88:1262-5.
[21] Eng C, Spechler SJ, Ruben R, Li FP. Familial Barrett Esophagus and adenocarcinoma of the gastroesophageal junction. Canc Epidemiol Bio
Prev 1993;2:397-9.
[22] Romero Y, Cameron AJ, Locke JR, Schaid DJ, Slezak JM, Branch CD, Melton LJ. Familial aggregation of gastroesophageal reflux in patients with Barrett’s Esophagus and esophageal adenocarcinoma.
Gastroenterol 1997;113:1149-56.
[23] Cameron AJ, Lagergren J, Henricksson C, Nyren O, Locke GR, Pedersen NL. Gastroesophageal reflux disease in monozygotic and twins. Gastroenterol 2002;122(1):55-9.
[24] Ferraris R, Bonelli L, Conio M, Fracchia M, Lapertosa G, Aste H, et al. Incidence of Barrett’s adenocarcinoma in an Italian population: an endoscopy surveillance programme. Eur J Gatroenterol Hepatol
1997;9:881-5.
[25] Spechler SJ, Robbins AH, Bloonfield Rubins H, et al. Adenocarcinoma and Barrett’s esophagus. An overrated risk?
Gastroenterol 1984;87:927-33
[26] Cameron AJ, Ott BJ, Payne WS. The incidence of adenocarcinoma in columnar lined (Barrett’s) esophagus. N Engl J Med 1985;313:857-9. [27] Bonelli V. Epidemiologia dell’esofago di Barrettt. In: GOSPE, Eds.
[28] Bremner CG, Lynch VP, Ellis FH. Barrett esophagus congenital or acquired? An experimental study of esophageal mucosa regeneration in dog. Surg 1970;68:209-6.
[29] Gillen P, Keeling P, Birne PY, West AB et al. Experimental columnar metaplasia in the canine esophagus. Brit J Surg 1988;75:113-5.
[30] Mittal RK, Fisher MJ. Electrical and mechanical inibition of the crural diaphragm during transient relaxation of the lower esophageal sphincter.
Gastroenterol 1990;99:1265-8.
[31] Sloan S, Rademaker AV, Kahrilas PJ. Determinants of gastroesophageal junction incompetence: hiatus hernia, lower sphincter, or both? Ann Int Med 1992;117:977-82.
[32] Sloan S, Karilas PJ. Impairement of esophageal emptying with jatal hernia. Gastroenterol 1991;100:596-605.
[33] Stang SJ. The main etiological factor in Barrett’s esophagus metaplasia. Am J Gastroenterol 1995:94:1315-7.
[34] Stein HJ, Hoeft S, DeMeester TR. Functional foregut abnormalities in Barrett’s esophagus. J Thor Cardiovasc Surg 1993;105:107-11.
[35] Champion G, Richter JE, Vaenzi MF, Singh S, Alexander R. Duodenogastroesophageal reflux: relationship to pH and importance in Barrett’s esophagus. Gastroenterol 1994;107:747-54.
[36] Fass R, Hell RW, garewall HS, Martinez P, Pullian G, Wendel G, Sampliner RE. Correlation of esophageal acid exposure with Barrett’s oesophagus length. GUT 2001;48:310-3.
[37] Stein HJ, Hoeft S, Korn O. Gastroduodenal function in Barrett’s esophagus. Dis Esophag 1995;8:205-10.
[38] Mullholland MW, Reid BJ, Levine DS, Rubin CE. Elevated acid gastric secretion in patients with Barrett’s metaplastic epithelium. Dig
Dis Scie 1989;34:1329-35.
[39] Collen MJ, Johnson DA. Correlation between acid output and daily ranitidine dose required for therapy in Barrett’s esophagus. Dig Dis Scie
1992;37:570-6.
[40] Katzka DA, Castell DO. Successful elimination of reflux symptoms does not insure adeguate control of acid reflux in acid reflux in patients with Barrett’s esophagus. Am J Gastroenterol 1994;89:989-91.
[41] Ouatu-Lascar R, Triadafilopoulos G. Complete elimination of reflux symptoms does not guarantee normalization of intraesophageal acid reflux in patients with Barrett’s esophagus. Am J Gastroenterol
1998;93:711-6.
[42] Peghini P, Katz PO, Bracy NA. Nocturnal recovery of acid gastric secretion with twice-daily dosing of proton pump inhibitors. Am J
Gastroenterol 1998;93:763-7.
[43] Katz PO, Anderson C, Khoury. Gastroesophageal reflux associated with nocturnal gastric acid breakthough on proton pump inhibitors.
Aliment Pharmacol Ther 1998;12:1231-4
[44] Fitzgerald RC, Omary MB, Triadafilopoulos G. Dynamic effects of acid on Barrett’s esophagus:an ex vivo proliferation and differentiation model. J Clin Invest 1996;98:2120-8
[45] Practice Parameters Committee of the American College of Gastroenterology. Updated guidelines for the diagnosis, surveillance, and therapy of Barrett’s esophagus. Am J Gastroenterol 2002;97:1888-
95
[46] P. Sharma et al. Prague classification for endoscopic diagnosis of Barrett esophagus. Am J Gastroenterol 2006 (in press)
[47] Tytgat GNJ, Hameeteman W. The neoplastic potential of columnar- lined (Barrett’s) esophagus. World J Surg 1992;16:308-12
[48] Glickman JN, Chen YY, Wang HH, Antonioll DA, Odze RD. Phenotypic characteristics of a distinctive multilayered epithelium suggests that is a precursor in the development of Barrett’s esophagus.
Am J Surg Pathol 2001;25:569-78
[49] Boch JA, Shields HM, Antonioli DA, Zwas F, Sawhney RA, Trier JS. Distribution of cytokeratin markers in Barrett’s columnar epithelium.
Gastroenterol 1997;112:760-5
[50] Sampliner RE, Fass R. Extension of squamous epithelium into the proximal stomach: a newly recognised mucosal abnormality. Endoscopy
2000;32:27-32
[51] Schmidt PH, Lee JR, Joshi V et al. Identification of a metaplastic cell lineage associated with human gastric adenocarcinoma. Lab Invest
[52] Garcia SB, Park HS, Novelli M, Wright NA. Field cancerization, clonality and epithelial stem cells: the spread of mutated clones in epithelial sheets. J Pathol 1999;187:61-81.
[53] Barrett MT, Sanchez CA, Prevo LJ et al. Evolution of neoplastic cell lineages in Barrett’s esophagus. Nat Genet 1999;22:106-9
[54] Booth C, Potten CS. Gut instincts: thoughts on intestinal epithelial stem cells. J Clin Invest 2000;105;1493-9
[55] Dahms BB, Greco MA, Strandjord SE, Rothstein FC. Barrett’s esophagus in three children after antileukemia therapy. Canc
1987;60:2896-900
[56] Jankowski JA, Harrison RF, Perry I, Balkwill F, Tselepis C. Barrett's metaplasia. Lancet. 2000 16;356(9247):2079-85
[57] Ouatu-Lascar R, Fitzgerald RC, Traidafilopoulos G. Differentiation and prolifration in Barrett’s esophagus and the effects of acid suppression. Gastroenterol 1999;117:327-35
[58] Sharma P, Sampliner R. GERD, DGER, or both in Barrett’s esophagus ? Am J Gastroenterol 1997;92:903-4
[59] Garewel H, Bernstein H, Bernstein C, Sampliner R, Payne C. Reduced bile acid induced apoptosis in „normal“ colorectal mucosa: a potential biologic marker for cancer risk. Cancer Res 1996;56:1480-3 [60] Jankowski J, Hopwood D, Pringle R, Wormsley K. Increased
expression of EGFR in Barrett’s esophagus associated with alkaline reflux: a putative model for carcinogenesis. Am J Gastroenterol
1993;56:1480-3
[61] Gillen P, Keeling P, Byrne PJ, Healy M, O’Moore RR, Hennessy TP. Implication of duodenogastric reflux in the pathogenesis of Barrett’s esophagus. Br J Surg 1988;75:540-3
[62] Tselepsis C, Perry I, Boulton R, et al. Endogenous secondary bile acid levels modify epithelial biology in Barrett’s esophagus. Gut
1999;45:A238
[63] Shirvani VN, Quatu-Lascar R, Kaur BS, Omary MB, Triadafilopoulos G. Cyclooxygenase 2 expression in Barrett’s esophagus and adenocarcinoma: ex vivo induction by bile salts and acid exposure.
[64] Weston AP, Cherian R, Horvat RT, Lawrinenko V, Dixon A, Mc Gregor D. Mucosa-associated lymphoid tissue (MALT) in Barrett’s esophagus; prospective evaluation and association with gastric MALT, MALT lymphoma and Helicobacter Pylori. Am J Gastroenterol
1997;92:800-4
[65] Goldblum JR, Vicari JJ, Falk GW et al. Inflammation and intestinal metaplasia of the gastric cardia: the role of gastroesophageal reflux and H.Pylori infection. Gastroenterol 1998;114:633-39
[66] Biddlestone LR, Barham CP, Wilkinson SP, Barr H, Shepherd NA. The histopathology of treated Barrett’s esophagus: squamous reepithelization after acid suppression and laser and photodynamic therapy. Am J Surg Pathol 1998;22:239-45
[67] Naya Mj, Pereboom D, Ortego J, Alda JO, Lanas A. Superoxide anions produced by inflammatory cells play an important part in the pathogenesis of acid and pepsin induced oesophagitis in rabbits. Gut
1997;40:175-81
[68] Jones BE, Lo CR, Liu H et al. Role of caspase and NF-kappa B signalling in hydrogen peroxide and superoxide induced hepatocyte apoptosis. Am J Phisiol 2000;278:G693-99
[69] Younes m, Schwartz MR, Finnie D, Younes A. Overexpression of Fas ligand during malignant transformation in the large bowel and in Barrett’s metaplasia of the esophagus. Hum Pathol 1999;30:1309-13 [70] Richards FM, Mc Kee SA, Rajpar MH et al. Germ-line E-cadherin
gene (CDH1) mutations predispose to familial gastric and colorectal cancer. Hum Mol Genet 1999;4:607-10
[71] Bailey T, Biddleston L, Shepherd N, Barr H, Warner P, Jankowski J. Altered cadherin/catenin complexes in the dysplasia-adenocarcinoma sequence: correlation with disease progression and dedifferentiation. Am
J Pathol 1998;152:135-44
[72] Eastman Q, Grosschedl R. Regulation of LEF-1/TCF transcription factors by Wnt and other signals. Curr Opin Cell Biology 1999;11:233-
240
[73] Perry I, Tselepsis C, Hardy R et al. TNF-α induces elevated levels of nuclear TGF/β-catenin complexes in a colon carcinoma cell line. Gut
[74] Ishikawa T, Casini AF, Nishikimi M. Molecular cloning and functional expression of rat liver glutathione-dependent dehydroascorbate reductase. J Biol Chem. 1998;273(44):28708-12. [75] Paolicchi A, Pezzini A, Saviozzi M. Localization of a GSH-dependent
dehydroascorbate reductase in rat tissues and subcellular fractions. Arch
Biochem Biophys. 1996;333(2):489-95.
[76] Board PG, Coggan M, Chelvanayagam G. Identification, characterization, and crystal structure of the Omega class glutathione transferases. J Biol Chem. 2000;275(32):24798-806.
[77] Del Bello B, Maellaro E, Sugherini L, et al. Purification of NADPH- dependent dehydroascorbate reductase from rat liver and its identification with 3 alpha-hydroxysteroid dehydrogenase. Biochem J.
1994;304 ( Pt 2):385-90.
[78] Townsend AJ, Kabler SL, Doehmer J, et al. Modeling the metabolic competency of glutathione S-transferases using genetically modified cell lines. Toxicology. 2002;181-182:265-9.
[79] Fornai F, Gesi M, Saviozzi M, et al. Immunohistochemical evidence and ultrastructural compartmentalization of a new antioxidant enzyme in the rat substantia nigra. J Neurocytol. 2001;30(2):97-105.
[80] Fornai F, Piaggi S, Gesi M, et al. Subcellular localization of a glutathione-dependent dehydroascorbate reductase within specific rat brain regions. Neuroscience. 2001;104(1):15-31.
[81] Duthie SJ, Ma A, Ross MA, Collins AR. Antioxidant supplementation decreases oxidative DNA damage in human lymphocytes. Cancer Res.
1996;56(6):1291-5.
[82] Green MH, Lowe JE, Waugh AP, et al. Effect of diet and vitamin C on DNA strand breakage in freshly-isolated human white blood cells.
Mutat Res. 1994;316(2):91-102.
[83] Noroozi M, Angerson WJ, Lean ME. Effects of flavonoids and vitamin C on oxidative DNA damage to human lymphocytes.
Am J Clin Nutr. 1998;67(6):1210-8.
[84] Stewart MS, Cameron GS, Pence BC. Antioxidant nutrients protect against UVB-induced oxidative damage to DNA of mouse keratinocytes in culture. J Invest Dermatol. 1996;106(5):1086-9.
class glutathione s-transferase-like proteins. J Biol Chem. 1999;274(8):5131-7.
[86] Petrini M, Conte A, Caracciolo F, Sabbatini A, Grassi B, Ronca G. Reversing of chlorambucil resistance by ethacrynic acid in a B-CLL patient. Br J Haematol. 1993;85(2):409-10.
[87] Laliberte RE, Perregaux DG, Hoth LR, et al. Glutathione s-transferase omega 1-1 is a target of cytokine release inhibitory drugs and may be responsible for their effect on interleukin-1beta posttranslational processing.J Biol Chem. 2003;278(19):16567-78.
[88] Boutet I, Tanguy A, Moraga D. Response of the Pacific oyster Crassostrea gigas to hydrocarbon contamination under experimental conditions. Gene. 2004;329:147-57.
[89] Board PG, Coggan M, Watson S, Gage PW, Dulhunty AF. CLIC-2 modulates cardiac ryanodine receptor Ca2+ release channels. Int J
Biochem Cell Biol. 2004;36(8):1599-612.
[90] Whitbread AK, Tetlow N, Eyre HJ, Sutherland GR, Board PG. Characterization of the human Omega class glutathione transferase genes and associated polymorphisms. Pharmacogenetics 2003;13(3):131-44.
[91] Lundell LR, Dent J, Bennett JR, et al. Endoscopic assessment of oesophagitis: clinical and functional correlates and further validation of the Los Angeles classification. Gut. 1999;45(2):172-80.
[92] Malfertheiner P, Megraud F, O'Morain C, Hungin AP, Jones R, Axon A, Graham DY, Tytgat G; European Helicobacter Pylori Study Group (EHPSG). Current concepts in the management of Helicobacter pylori infection--the Maastricht 2-2000 Consensus Report. Aliment Pharmacol
Ther. 2002;16(2):167-80.
[93] Rydell RH, Goldman H, Ransohoff DF, et al. Displasia in inflammatory bowel disease: standardized classification with provisional clinical application. Hum Pathol 1983;14:931-968.
[94] Schmuck EM, Board PG, Whitbread. Characterization of the monomethylarsonate reductase and dehydroascorbate reductase activities of Omega class glutathione transferase variants: implications for arsenic metabolism and the age-at-onset of Alzheimer's and Parkinson's diseases. Pharmacogenet Genomics. 2005;15(7):493-501.
[95] Giri U, Terry NH, Kala SV, Lieberman MW, Story MD. Elimination of the differential chemoresistance between the murine B-cell lymphoma LY-ar and LY-as cell lines after arsenic (As2O3) exposure via the overexpression of gsto1 (p28). Cancer Chemother Pharmacol.
2005;55(6):511-21.
[96] Marahatta SB, Punyarit P, Bhudisawasdi V, Paupairoj A, Wongkham S, Petmitr S. Polymorphism of glutathione S-transferase omega gene and risk of cancer. Cancer Lett. 2006;236(2):276-81.