, Volume 17, Issue 5, pp 464–469 | Cite as

AZA-Deoxycytidine stimulates proopiomelanocortin gene expression and ACTH secretion in human pituitary ACTH-secreting tumors

  • Maria Francesca Cassarino
  • Antonella Sesta
  • Luca Pagliardini
  • Marco Losa
  • Giovanni Lasio
  • Francesco Cavagnini
  • Francesca Pecori GiraldiEmail author



It is well known that methylation plays an important role in regulating tissue expression of proopiomelanocortin (POMC) and recent studies have shown that demethylation can occur also in vitro in neuroendocrine tumors. Aim of the present study was to evaluate whether inhibition of methylation modulates POMC expression and ACTH secretion by human corticotrope tumors.


Twenty two ACTH-secreting pituitary tumors were incubated with 5-AZA-2′-deoxycytidine (AZA), an inhibitor of DNA-methyltransferases, with or without 10 nM corticotropin-releasing hormone (CRH). Both dose response (100 nM–10 μM AZA) and time course (4–96 h) experiments were carried out for measurement of ACTH secretion and POMC gene expression.


Incubation with AZA increased constitutive POMC expression and ACTH secretion by human corticotrope adenomas. The effect appeared most notable at 24 and 48 h with 1 μM AZA. Incubation with AZA did not exert an additional stimulatory effect on CRH-stimulated POMC and ACTH.


The present study shows that AZA increases POMC gene expression and ACTH secretion in human pituitary ACTH-secreting tumors. This can be taken to indicate that mechanisms set into motion by AZA play a role in the regulation of ACTH secretion/POMC expression in tumoral corticotropes and paves the way to further studies in Cushing’s disease.


Corticotrope adenoma 5-AZA-2′-deoxycytidine ACTH Methylation POMC Cushing’s disease 


Conflict of interest

The authors state that they have no conflict of interest.


  1. 1.
    Pecori Giraldi F, Cassarino F, Pagliardini L, Asnaghi V, Cavagnini F (2011) The human POMC promoter: where do we stand? J Endocrinol Invest 34:454–460PubMedCrossRefGoogle Scholar
  2. 2.
    Newell-Price J, King P, Clark AJL (2001) The CpG island promoter of the human proopiomelanocortin gene is methylated in nonexpressing normal tissue and tumors and represses expression. Mol Endocrinol 15:338–348PubMedCrossRefGoogle Scholar
  3. 3.
    Lavender PM, Clark AJL, Besser GM, Rees LH (1991) Variable methylation of the 5′-flanking DNA of the human pro-opiomelanocortin gene. J Mol Endocrinol 6:53–61PubMedCrossRefGoogle Scholar
  4. 4.
    Gardiner-Garden M, Frommer M (1994) Transcripts and CpG islands associated with the pro-opiomelanocortin gene and other neurally expressed genes. J Mol Endocrinol 12:365–382PubMedCrossRefGoogle Scholar
  5. 5.
    Ye L, Li X, Kong X, Wang W, Bi Y, Hu L, Cui B, Li X, Ning G (2005) Hypomethylation in the promoter region of POMC gene correlates with ectopic overexpression in thymic carcinoids. J Endocrinol 185:337–343PubMedCrossRefGoogle Scholar
  6. 6.
    Mizoguchi Y, Kajiume T, Miyagawa S, Okada S, Nishi Y, Kobayashi M (2007) Steroid-dependent ACTH-produced thymic carcinoid: regulation of POMC gene expression by cortisol via methylation of its promoter region. Horm Res 67:257–262PubMedCrossRefGoogle Scholar
  7. 7.
    Invitti C, Pecori Giraldi F, De Martin M, Cavagnini F, the Study Group of the Italian Society of Endocrinology on the Pathophysiology of the Hypothalamic-Pituitary-Adrenal Axis (1999) Diagnosis and management of Cushing’s syndrome: results of an Italian multicentre study. J Clin Endocrinol Metab 84:440–448PubMedGoogle Scholar
  8. 8.
    Pecori Giraldi F, Marini E, Torchiana E, Mortini P, Dubini A, Cavagnini F (2003) Corticotrophin-releasing activity of desmopressin in Cushing’s disease. Lack of correlation between in vivo and in vitro responsiveness. J Endocrinol 177:373–379PubMedCrossRefGoogle Scholar
  9. 9.
    Pecori Giraldi F, Pesce S, Maroni P, Pagliardini L, Lasio G, Losa M, Cavagnini F (2010) Inhibitory effect of preproTRH(178-199) on ACTH secretion by human corticotrope tumours. J Neuroendocrinol 22:294–300PubMedCrossRefGoogle Scholar
  10. 10.
    Pecori Giraldi F, Pagliardini L, Cassarino MF, Losa M, Lasio G, Cavagnini F (2011) Responses to CRH and dexamethasone in a large series of human ACTH-secreting pituitary adenomas in vitro reveal manifold corticotroph tumoural phenotypes. J Neuroendocrinol 23:1214–1221PubMedCrossRefGoogle Scholar
  11. 11.
    Avgerinos PC, Chrousos GP, Nieman LK, Oldfield EH, Loriaux DL, Cutler GB Jr (1987) The corticotropin-releasing hormone test in the post-operative evaluation of patients with Cushing’s syndrome. J Clin Endocrinol Metab 65:906–913PubMedCrossRefGoogle Scholar
  12. 12.
    Garcia-Carpizo V, Ruiz-Llorente L, Fraga M, Aranda A (2011) The growing role of gene methylation on endocrine function. J Mol Endocrinol 47:R75–R89PubMedCrossRefGoogle Scholar
  13. 13.
    Zhang X, Ho SM (2011) Epigenetics meets endocrinology. J Mol Endocrinol 46:R11–R32PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Baylin SB, Jones PA (2011) A decade of exploring the cancer epigenome—biological and translational implications. Nature Rev Cancer 11:726–734CrossRefGoogle Scholar
  15. 15.
    Smith LT, Otterson GA, Plass C (2007) Unraveling the epigenetic code of cancer for therapy. Trends Genet 23:449–456PubMedCrossRefGoogle Scholar
  16. 16.
    Ehrlich S, Weiss D, Burghardt R, Infante-Duarte C, Brockhaus S, Muschler MAN, Bleich S, Lehmkuhl U, Frieling H (2010) Promoter specific DNA methylation and gene expression of POMC in acutely underweight and recovered patients with anorexia nervosa. J Psychiat Res 44:827–833CrossRefGoogle Scholar
  17. 17.
    Plagermann A, Harder T, Brunn M, Harder A, Roepke K, Wittrock-Staar M, Ziska T, Chellong K, Rodekamp E, Melchior K, Dudenhausen JW (2009) Hypothalamic proopiomelanocortin promoter methylation becomes altered by early overfeeding: an epigenetic model of obesity and the metabolic syndrome. J Physiol 20:4963–4976CrossRefGoogle Scholar
  18. 18.
    Muschler MAN, Hillemacher T, Kraus C, Kornhuber J, Bleich S, Frieling H (2010) DNA methylation of the POMC gene promoter is associated with craving in alcohol dependence. J Neural Transm 117:513–519PubMedCrossRefGoogle Scholar
  19. 19.
    Begum G, Stevens A, Bolton Smith E, Connor K, Challis JRG, Bloomfield F, White A (2012) Epigenetic changes in fetal hypothalamic energy regulating pathways are associated with maternal undernutrition and twinning. FASEB J 26:1694–1703PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Bilodeau S, Vallette-Kasic S, Gauthier Y, Figarella-Branger D, Brue T, Berthelet F, Lacroix A, Batista D, Stratakis CA, Hanson JM, Meij BP, Drouin J (2006) Role of Brg1 and HDAC2 in GR trans-repression of the pituitary POMC gene and misexpression in Cushing disease. Genes Dev 20:2871–2886PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Hofland LJ, van de Hoek J, Feelders RA, van Aken MO, van Koetsveld PM, Waaijers M, Sprij-Mooij D, Bruns C, Weckbecker G, De Herder WW, Beckers A, Lamberts SWJ (2005) The multi-ligand somatostatin analogue SOM230 inhibits ACTH secretion by cultured human corticotroph adenomas via somatostatin receptor type 5. Eur J Endocrinol 152:645–654PubMedCrossRefGoogle Scholar
  22. 22.
    Pivonello R, Ferone D, De Herder WW, Kros JM, DelBasso de Caro ML, Arvigo M, Annunziato L, Lombardi G, Colao A, Hofland L, Lamberts SWJ (2004) Dopamine receptor expression and function in corticotroph pituitary tumors. J Clin Endocrinol Metab 89:2452–2462PubMedCrossRefGoogle Scholar
  23. 23.
    Cihák A (1974) Biological effects of 5-azacytidine in eukaryotes. Oncology 60:405–422Google Scholar
  24. 24.
    Patra SK, Bettuzzi S (2009) Epigenetic DNA-(cytosine-5-carbon) modifications: 5-Aza-2′-deoxycytidine and DNA demethylation. Biochemistry 74:613–619PubMedGoogle Scholar
  25. 25.
    Otterson GA, Khleif SN, Chen W, Coxon AB, Kaye FJ (1995) CDKN2 gene silencing in lung cancer by DNA hypermethylation and kinetics of p161NK4 protein induction by 5-Aza 2′deoxycytidine. Oncogene 11:1211–1216PubMedGoogle Scholar
  26. 26.
    Bruniquel D, Schwartz RH (2003) Selective, stable demethylation of the interleukin-2 gene enhances transcription by an active process. Nature Immunol 4:235–240CrossRefGoogle Scholar
  27. 27.
    Razin A, Cedar H (1991) DNA methylation and gene expression. Microbiol Rev 55:451–458PubMedCentralPubMedGoogle Scholar
  28. 28.
    Nudi M, Ouimette JF, Drouin J (2005) Bone morphogenic protein (Smad)-mediated repression of proopiomelanocortin transcription by interference with Pitx/Tpit activity. Mol Endocrinol 19:1329–1342PubMedCrossRefGoogle Scholar
  29. 29.
    Newell-Price J (2003) Proopiomelanocortin (POMC) expression and DNA methylation—implications for Cushing’s syndrome and beyond. J Endocrinol 177:365–372PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Maria Francesca Cassarino
    • 1
  • Antonella Sesta
    • 1
  • Luca Pagliardini
    • 1
  • Marco Losa
    • 2
  • Giovanni Lasio
    • 3
  • Francesco Cavagnini
    • 1
  • Francesca Pecori Giraldi
    • 1
    • 4
    Email author
  1. 1.Neuroendocrinology Research LaboratoryIstituto Auxologico Italiano IRCCSMilanItaly
  2. 2.Department of NeurosurgeryOspedale San Raffaele IRCCSMilanItaly
  3. 3.Department of NeurosurgeryIstituto Clinico HumanitasRozzanoItaly
  4. 4.Department of Clinical Sciences and Community HealthUniversity of MilanMilanItaly

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