Apoptosis

, Volume 18, Issue 8, pp 1008–1016 | Cite as

AICAR induces Bax/Bak-dependent apoptosis through upregulation of the BH3-only proteins Bim and Noxa in mouse embryonic fibroblasts

  • Diana M. González-Gironès
  • Cristina Moncunill-Massaguer
  • Daniel Iglesias-Serret
  • Ana M. Cosialls
  • Alba Pérez-Perarnau
  • Claudia M. Palmeri
  • Camila Rubio-Patiño
  • Andreas Villunger
  • Gabriel Pons
  • Joan Gil
Original Paper

Abstract

5-Aminoimidazole-4-carboxamide (AICA) riboside (AICAR) is a nucleoside analogue that is phosphorylated to 5-amino-4-imidazolecarboxamide ribotide (ZMP), which acts as an AMP mimetic and activates AMP-activated protein kinase (AMPK). It has been recently described that AICAR triggers apoptosis in chronic lymphocytic leukemia (CLL) cells, and its mechanism of action is independent of AMPK as well as p53. AICAR-mediated upregulation of the BH3-only proteins BIM and NOXA correlates with apoptosis induction in CLL cells. Here we propose mouse embryonic fibroblasts (MEFs) as a useful model to analyze the mechanism of AICAR-induced apoptosis. ZMP formation was required for AICAR-induced apoptosis, though direct Ampk activation with A-769662 failed to induce apoptosis in MEFs. AICAR potently induced apoptosis in Ampkα1//α2/ MEFs, demonstrating an Ampk-independent mechanism of cell death activation. In addition, AICAR acts independently of p53, as MEFs lacking p53 also underwent apoptosis normally. Notably, MEFs lacking Bax and Bak were completely resistant to AICAR-induced apoptosis, confirming the involvement of the mitochondrial pathway in its mechanism of action. Apoptosis was preceded by ZMP-dependent but Ampk-independent modulation of the mRNA levels of different Bcl-2 family members, including Noxa, Bim and Bcl-2. Bim protein levels were accumulated upon AICAR treatment of MEFs, suggesting its role in the apoptotic process. Strikingly, MEFs lacking both Bim and Noxa displayed high resistance to AICAR. These findings support the notion that MEFs are a useful system to further dissect the mechanism of AICAR-induced apoptosis.

Keywords

AICAR Ampk Apoptosis Bim Noxa MEFs 

Abbreviations

ACC

Acetyl-CoA carboxylase

AICAR

5-Aminoimidazole-4-carboxamide (AICA) riboside or acadesine

AMPK

AMP-activated protein kinase

CLL

Chronic lymphocytic leukemia

DKO

Double knockout

MEFs

Mouse embryonic fibroblasts

MOMP

Mitochondrial outer membrane permeabilization

PI

Propidium iodide

RT-MLPA

Reverse transcriptase multiplex ligation-dependent probe amplification

RT-qPCR

Real time quantitative PCR

SEM

Standard error of the mean

WT

Wild type

ZMP

AICA ribotide

Supplementary material

10495_2013_850_MOESM1_ESM.doc (951 kb)
Supplementary material 1 (DOC 952 kb)

References

  1. 1.
    Campas C, Lopez JM, Santidrian AF, Barragan M, Bellosillo B, Colomer D, Gil J (2003) Acadesine activates AMPK and induces apoptosis in B-cell chronic lymphocytic leukemia cells but not in T lymphocytes. Blood 101:3674–3680CrossRefPubMedGoogle Scholar
  2. 2.
    Santidrian AF, Gonzalez-Girones DM, Iglesias-Serret D, Coll-Mulet L, Cosialls AM, de Frias M, Campas C, Gonzalez-Barca E, Alonso E, Labi V, Viollet B, Benito A, Pons G, Villunger A, Gil J (2010) AICAR induces apoptosis independently of AMPK and p53 through up-regulation of the BH3-only proteins BIM and NOXA in chronic lymphocytic leukemia cells. Blood 116:3023–3032CrossRefPubMedGoogle Scholar
  3. 3.
    Campas C, Santidrian AF, Domingo A, Gil J (2005) Acadesine induces apoptosis in B cells from mantle cell lymphoma and splenic marginal zone lymphoma. Leukemia 19:292–294CrossRefPubMedGoogle Scholar
  4. 4.
    Baumann P, Mandl-Weber S, Emmerich B, Straka C, Schmidmaier R (2007) Activation of adenosine monophosphate activated protein kinase inhibits growth of multiple myeloma cells. Exp Cell Res 313:3592–3603CrossRefPubMedGoogle Scholar
  5. 5.
    Sengupta TK, Leclerc GM, Hsieh-Kinser TT, Leclerc GJ, Singh I, Barredo JC (2007) Cytotoxic effect of 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) on childhood acute lymphoblastic leukemia (ALL) cells: implication for targeted therapy. Mol Cancer 6:46CrossRefPubMedGoogle Scholar
  6. 6.
    Lopez JM, Santidrian AF, Campas C, Gil J (2003) 5-Aminoimidazole-4-carboxamide riboside induces apoptosis in Jurkat cells, but the AMP-activated protein kinase is not involved. Biochem J 370:1027–1032CrossRefPubMedGoogle Scholar
  7. 7.
    Martelli AM, Chiarini F, Evangelisti C, Ognibene A, Bressanin D, Billi AM, Manzoli L, Cappellini A, McCubrey JA (2012) Targeting the liver kinase B1/AMP-activated protein kinase pathway as a therapeutic strategy for hematological malignancies. Expert Opin Ther Targets 16:729–742CrossRefPubMedGoogle Scholar
  8. 8.
    Kefas BA, Cai Y, Ling Z, Heimberg H, Hue L, Pipeleers D, Van de Casteele M (2003) AMP-activated protein kinase can induce apoptosis of insulin-producing MIN6 cells through stimulation of c-Jun-N-terminal kinase. J Mol Endocrinol 30:151–161CrossRefPubMedGoogle Scholar
  9. 9.
    Meisse D, Van de Casteele M, Beauloye C, Hainault I, Kefas BA, Rider MH, Foufelle F, Hue L (2002) Sustained activation of AMP-activated protein kinase induces c-Jun N-terminal kinase activation and apoptosis in liver cells. FEBS Lett 526:38–42CrossRefPubMedGoogle Scholar
  10. 10.
    Garcia-Gil M, Pesi R, Perna S, Allegrini S, Giannecchini M, Camici M, Tozzi MG (2003) 5′-Aminoimidazole-4-carboxamide riboside induces apoptosis in human neuroblastoma cells. Neuroscience 117:811–820CrossRefPubMedGoogle Scholar
  11. 11.
    Sauer H, Engel S, Milosevic N, Sharifpanah F, Wartenberg M (2012) Activation of AMP-kinase by AICAR induces apoptosis of DU-145 prostate cancer cells through generation of reactive oxygen species and activation of c-Jun N-terminal kinase. Int J Oncol 40:501–508PubMedGoogle Scholar
  12. 12.
    Fogarty S, Hardie DG (2010) Development of protein kinase activators: AMPK as a target in metabolic disorders and cancer. Biochim Biophys Acta 1804:581–591CrossRefPubMedGoogle Scholar
  13. 13.
    Jones RG, Plas DR, Kubek S, Buzzai M, Mu J, Xu Y, Birnbaum MJ, Thompson CB (2005) AMP-activated protein kinase induces a p53-dependent metabolic checkpoint. Mol Cell 18:283–293CrossRefPubMedGoogle Scholar
  14. 14.
    Okoshi R, Ozaki T, Yamamoto H, Ando K, Koida N, Ono S, Koda T, Kamijo T, Nakagawara A, Kizaki H (2008) Activation of AMP-activated protein kinase induces p53-dependent apoptotic cell death in response to energetic stress. J Biol Chem 283:3979–3987CrossRefPubMedGoogle Scholar
  15. 15.
    Polager S, Ginsberg D (2009) p53 and E2f: partners in life and death. Nat Rev Cancer 9:738–748CrossRefPubMedGoogle Scholar
  16. 16.
    Chipuk JE, Green DR (2008) How do BCL-2 proteins induce mitochondrial outer membrane permeabilization? Trends Cell Biol 18:157–164CrossRefPubMedGoogle Scholar
  17. 17.
    Youle RJ, Strasser A (2008) The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol 9:47–59CrossRefPubMedGoogle Scholar
  18. 18.
    Tang YC, Williams BR, Siegel JJ, Amon A (2011) Identification of aneuploidy-selective antiproliferation compounds. Cell 144:499–512CrossRefPubMedGoogle Scholar
  19. 19.
    Wei MC, Zong WX, Cheng EH, Lindsten T, Panoutsakopoulou V, Ross AJ, Roth KA, MacGregor GR, Thompson CB, Korsmeyer SJ (2001) Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 292:727–730CrossRefPubMedGoogle Scholar
  20. 20.
    Naik E, Michalak EM, Villunger A, Adams JM, Strasser A (2007) Ultraviolet radiation triggers apoptosis of fibroblasts and skin keratinocytes mainly via the BH3-only protein Noxa. J Cell Biol 176:415–424CrossRefPubMedGoogle Scholar
  21. 21.
    Laderoute KR, Amin K, Calaoagan JM, Knapp M, Le T, Orduna J, Foretz M, Viollet B (2006) 5′-AMP-activated protein kinase (AMPK) is induced by low-oxygen and glucose deprivation conditions found in solid-tumor microenvironments. Mol Cell Biol 26:5336–5347CrossRefPubMedGoogle Scholar
  22. 22.
    McMasters KM, de Oca Montes, Luna R, Pena JR, Lozano G (1996) Mdm2 deletion does not alter growth characteristics of P53-deficient embryo fibroblasts. Oncogene 13:1731–1736PubMedGoogle Scholar
  23. 23.
    Henderson JF, Paterson AR, Caldwell IC, Paul B, Chan MC, Lau KF (1972) Inhibitors of nucleoside and nucleotide metabolism. Cancer Chemother Rep 2(3):71–85Google Scholar
  24. 24.
    Cool B, Zinker B, Chiou W, Kifle L, Cao N, Perham M, Dickinson R, Adler A, Gagne G, Iyengar R, Zhao G, Marsh K, Kym P, Jung P, Camp HS, Frevert E (2006) Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome. Cell Metab 3:403–416CrossRefPubMedGoogle Scholar
  25. 25.
    Eldering E, Spek CA, Aberson HL, Grummels A, Derks IA, de Vos AF, McElgunn CJ, Schouten JP (2003) Expression profiling via novel multiplex assay allows rapid assessment of gene regulation in defined signalling pathways. Nucleic Acids Res 31:e153CrossRefPubMedGoogle Scholar
  26. 26.
    Rubio-Patino C, Palmeri CM, Perez-Perarnau A, Cosialls AM, Moncunill-Massaguer C, Gonzalez-Girones DM, Pons-Hernandez L, Lopez JM, Ventura F, Gil J, Pons G, Iglesias-Serret D (2012) Glycogen synthase kinase-3beta is involved in ligand-dependent activation of transcription and cellular localization of the glucocorticoid receptor. Mol Endocrinol 26:1508–1520CrossRefPubMedGoogle Scholar
  27. 27.
    Reed JC (1996) A day in the life of the Bcl-2 protein: does the turnover rate of Bcl-2 serve as a biological clock for cellular lifespan regulation? Leuk Res 20:109–111CrossRefPubMedGoogle Scholar
  28. 28.
    Cotsiki M, Lock RL, Cheng Y, Williams GL, Zhao J, Perera D, Freire R, Entwistle A, Golemis EA, Roberts TM, Jat PS, Gjoerup OV (2004) Simian virus 40 large T antigen targets the spindle assembly checkpoint protein Bub1. Proc Natl Acad Sci USA 101:947–952CrossRefPubMedGoogle Scholar
  29. 29.
    Guo C, Wu G, Chin JL, Bauman G, Moussa M, Wang F, Greenberg NM, Taylor SS, Xuan JW (2006) Bub1 up-regulation and hyperphosphorylation promote malignant transformation in SV40 tag-induced transgenic mouse models. Mol Cancer Res 4:957–969CrossRefPubMedGoogle Scholar
  30. 30.
    Concannon CG, Tuffy LP, Weisova P, Bonner HP, Davila D, Bonner C, Devocelle MC, Strasser A, Ward MW, Prehn JH (2010) AMP kinase-mediated activation of the BH3-only protein Bim couples energy depletion to stress-induced apoptosis. J Cell Biol 189:83–94CrossRefPubMedGoogle Scholar
  31. 31.
    Davila D, Connolly NM, Bonner H, Weisova P, Dussmann H, Concannon CG, Huber HJ, Prehn JH (2012) Two-step activation of FOXO3 by AMPK generates a coherent feed-forward loop determining excitotoxic cell fate. Cell Death Differ 19:1677–1688CrossRefPubMedGoogle Scholar
  32. 32.
    Kilbride SM, Farrelly AM, Bonner C, Ward MW, Nyhan KC, Concannon CG, Wollheim CB, Byrne MM, Prehn JH (2010) AMP-activated protein kinase mediates apoptosis in response to bioenergetic stress through activation of the pro-apoptotic Bcl-2 homology domain-3-only protein BMF. J Biol Chem 285:36199–36206CrossRefPubMedGoogle Scholar
  33. 33.
    Hallstrom TC, Mori S, Nevins JR (2008) An E2F1-dependent gene expression program that determines the balance between proliferation and cell death. Cancer Cell 13:11–22CrossRefPubMedGoogle Scholar
  34. 34.
    Weisova P, Davila D, Tuffy LP, Ward MW, Concannon CG, Prehn JH (2011) Role of 5′-adenosine monophosphate-activated protein kinase in cell survival and death responses in neurons. Antioxid Redox Signal 14:1863–1876CrossRefPubMedGoogle Scholar
  35. 35.
    Wang S, Song P, Zou MH (2012) Inhibition of AMP-activated protein kinase alpha (AMPKalpha) by doxorubicin accentuates genotoxic stress and cell death in mouse embryonic fibroblasts and cardiomyocytes: role of p53 and SIRT1. J Biol Chem 287:8001–8012CrossRefPubMedGoogle Scholar
  36. 36.
    Jeon SM, Chandel NS, Hay N (2012) AMPK regulates NADPH homeostasis to promote tumour cell survival during energy stress. Nature 485:661–665CrossRefPubMedGoogle Scholar
  37. 37.
    Liu L, Ulbrich J, Muller J, Wustefeld T, Aeberhard L, Kress TR, Muthalagu N, Rycak L, Rudalska R, Moll R, Kempa S, Zender L, Eilers M, Murphy DJ (2012) Deregulated MYC expression induces dependence upon AMPK-related kinase 5. Nature 483:608–612CrossRefPubMedGoogle Scholar
  38. 38.
    Estan MC, Calvino E, de Blas E, Boyano-Adanez Mdel C, Mena ML, Gomez-Gomez M, Rial E, Aller P (2012) 2-Deoxy-d-glucose cooperates with arsenic trioxide to induce apoptosis in leukemia cells: involvement of IGF-1R-regulated Akt/mTOR, MEK/ERK and LKB-1/AMPK signaling pathways. Biochem Pharmacol 84:1604–1616CrossRefPubMedGoogle Scholar
  39. 39.
    Canto C, Gerhart-Hines Z, Feige JN, Lagouge M, Noriega L, Milne JC, Elliott PJ, Puigserver P, Auwerx J (2009) AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity. Nature 458:1056–1060CrossRefPubMedGoogle Scholar
  40. 40.
    Keller KE, Tan IS, Lee YS (2012) SAICAR stimulates pyruvate kinase isoform M2 and promotes cancer cell survival in glucose-limited conditions. Science 338:1069–1072CrossRefPubMedGoogle Scholar
  41. 41.
    Vousden KH, Lu X (2002) Live or let die: the cell’s response to p53. Nat Rev Cancer 2:594–604CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Diana M. González-Gironès
    • 1
  • Cristina Moncunill-Massaguer
    • 1
  • Daniel Iglesias-Serret
    • 1
  • Ana M. Cosialls
    • 1
  • Alba Pérez-Perarnau
    • 1
  • Claudia M. Palmeri
    • 1
  • Camila Rubio-Patiño
    • 1
  • Andreas Villunger
    • 2
  • Gabriel Pons
    • 1
  • Joan Gil
    • 1
  1. 1.Departament de Ciències Fisiològiques IIInstitut d’Investigació Biomèdica de Bellvitge (IDIBELL)–Universitat de BarcelonaBarcelonaSpain
  2. 2.Division of Developmental ImmunologyBiocenter, Innsbruck Medical UniversityInnsbruckAustria

Personalised recommendations