Archives of Toxicology

, Volume 78, Issue 2, pp 74–85 | Cite as

Cytoskeletal disruption is the key factor that triggers apoptosis in okadaic acid-treated neuroblastoma cells

  • A. G. Cabado
  • F. Leira
  • M. R. Vieytes
  • J. M. Vieites
  • L. M. Botana
Molecular Toxicology
First Online:
Received:
Accepted:

Abstract

Okadaic acid (OA) is a tumour promoter that induces apoptosis in several cell models. Following previous findings, the objective of this work was to elucidate the pathways involved in OA-triggered apoptosis in BE(2)-M17 cells by using a combination of pharmacological agents and apoptosis-related assays. OA-induced apoptosis involves disruption of F-actin cytoskeleton, activation of caspase-3, collapse of mitochondrial membrane potential, DNA fragmentation and decreased levels of monomeric Bcl-2 and Bax proteins. All the agents tested were unable to obliterate changes in F-actin levels, caspase-3 activation or DNA fragmentation, but all of them prevented OA-induced decrease of mitochondrial potential and changes in Bax/Bcl-2 levels. Taken together, these results demonstrate that collapse of mitochondrial membrane potential is accessory in the execution of apoptosis, which is directly dependent on cytoskeletal changes. Mitochondrial changes are mediated by complex associations among the Bcl-2 proteins. Cytochrome c release from mitochondria is a late event, occurring 24 h after OA exposure. Moreover, okadaic acid triggers activation of upstream caspases resembling the extrinsic pathway of apoptosis.

Keywords

Apoptosis Okadaic acid Cytoskeleton Mitochondria Neuroblastoma cells 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgements

The authors thank CACTI (Centre for Scientific and Technical Support to Research) of the University of Vigo, Spain, for technical assistance with the confocal microscopy. This work was funded with grants REN2001-2959-C04-03/MAR from Ministerio de Ciencia y Tecnología (MCYT), Xunta Galicia (PGIDT99INN26101 and PGIDT00MAR26101PR), and MCYT(DGI) BMC2000-0441. The experiments complied with the current laws of Spain.

References

  1. Aplin AE, Juliano RL (1999) Integrin and cytoeskeletal regulation of growth factor signaling to the MAP kinase pathway. J Cell Sci 112:695–706PubMedGoogle Scholar
  2. Aplin A, Howe A, Alahari SK, Juliano RL (1998) Signal transduction and signal modulation by cell adhesion receptors. Pharmacol Rev 50:197–264PubMedGoogle Scholar
  3. Aplin AE, Short SM, Juliano RL (1999) Anchorage-dependent regulation of the mitogen-activated protein kinase cascade by growth factors is supported by a variety of integrin α chains. J Biol Chem 274:31223–31228CrossRefPubMedGoogle Scholar
  4. Ayllon V, Cayla X (2002) The anti-apoptotic molecules Bcl-xL and Bcl-w target protein phosphatase 1α to Bad. Eur J Immunol 32:1847–55CrossRefPubMedGoogle Scholar
  5. Baldacini O, Lutun P, Girardot R, Monteil H (1993) Effect of okadaic acid on the cytotoxic activity of Clostridium difficile toxin B and Clostridium sordellii toxin L. Nat Toxins 1:361–368PubMedGoogle Scholar
  6. Benito A, Lerga A, Silva M, Leon J et al (1997) Apoptosis of human myeloid leukemia cells induced by an inhibitor of protein phosphatases (okadaic acid) is prevented by Bcl-2 and Bcl-XL. Leukemia 11:940–944CrossRefPubMedGoogle Scholar
  7. Bialojan C, Takai A (1988) Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics. Biochem J 256:283–290PubMedGoogle Scholar
  8. Bissell MJ, Nelson WJ (1999) Cell-to-cell contact and extracellular matrix Integration of form and function: the central role of adhesion molecules. Curr Opin Cell Biol 11:537–539CrossRefGoogle Scholar
  9. Blankson H, Holen I, Seglen PO (1995) Disruption of the cytokeratin cytoskeleton and inhibition of hepatocytic autophagy by okadaic acid. Exp Cell Res 218:522–530CrossRefPubMedGoogle Scholar
  10. Bredesen DE (2000) Apoptosis: overview and signal transduction pathways. J Neurotrauma 17:801–810PubMedGoogle Scholar
  11. Burridge K, Chrzanowska-Wodnicka M (1996) Focal adhesions, contractility and signaling. Annu Rev Cell Dev Biol 12:463–518Google Scholar
  12. Cabado AG, Yu FH, Kapus A, Lukacs G et al (1996) Distinct structural domains confer cAMP sensitivity and ATP dependence to the Na+/H+ exchanger NHE3 isoform. J Biol Chem 271:3590–3599CrossRefPubMedGoogle Scholar
  13. Cabado AG, Leira F, Vieites JM, Vieytes MR, Botana LM (2003) Caspase-8 activation initiates okadaic acid-induced apoptosis in neuroblastoma. In: Villalba A, Reguera B, Romalde JL, Bieras R (eds) Molluscan shellfish safety. Consellería de Pesca e Asuntos Maritimos da Xunta de Galicia and Interngoverment Oceanographic Commission of UNESCO, Santiago de Compostela, pp 107–117Google Scholar
  14. Carbott DE, Duan L (2002) Phosphoinositol 3 kinase inhibitor, LY294002 increases bcl-2 protein and inhibits okadaic acid-induced apoptosis in Bcl-2 expressing renal epithelial cells. Apoptosis 7:69–76CrossRefPubMedGoogle Scholar
  15. Davis MA, Chang SH, Trump BF (1996) Differential selectivity of normal and H-ras oncogene transformed rat kidney epithelial cells to okadaic acid-induced apoptosis. Toxicol Appl Pharmacol 141:93–101CrossRefPubMedGoogle Scholar
  16. Desagher S, Martinou JC (2000) Mitochondria as the central control point of apoptosis. Trends Cell Biol 10:369–377PubMedGoogle Scholar
  17. Fiorentini C, Matarrese P, Fattorossi A, Donelli G (1996) Okadaic acid induces changes in the organization of F-actin in intestinal cells. Toxicon 34:937–945CrossRefPubMedGoogle Scholar
  18. Fladmark KE, Brustugun OT, Hoveland R, Boe R et al (1999) Ultrarapid caspase-3 dependent apoptosis induction by serine/threonine phosphatase inhibitors. Cell Death Differ 6:1099–1108CrossRefPubMedGoogle Scholar
  19. Frey T (1997) Correlated flow cytometric analysis of terminal events in apoptosis reveals the absence of some changes in some model systems. Cytometry 28:253–263CrossRefPubMedGoogle Scholar
  20. Frisch SM, Ruoslahti E (1997) Integrins and anoikis. Curr Opin Cell Biol 9:701–706Google Scholar
  21. Frisch SM, Vuori K, Ruoslahti E, Chan-Hui PY (1996) Control of adhesion-dependent cell survival by focal adhesion kinase. J Cell Biol 134:793–799PubMedGoogle Scholar
  22. Gao G, Dou P (2000) N-terminal cleavage of Bax by calpain generates a potent proapoptotic 18-kDa fragment that promotes Bcl-2 independent cytochorme c release and apoptotic cell death. J Cell Biochem 80:53–72CrossRefPubMedGoogle Scholar
  23. Giancotti FG, Ruoslahti E (1999) Integrin signaling. Science 285:1028–1032CrossRefPubMedGoogle Scholar
  24. Gottlieb RA (2000) Mitochondria: execution central. FEBS Letters 482:6–12CrossRefPubMedGoogle Scholar
  25. Hengartner MO (2000) The biochemistry of apoptosis. Nature 407:770–776CrossRefPubMedGoogle Scholar
  26. Jarvis WD, Turner AJ, Povirk LF et al (1994) Induction of apoptosis DNA fragmentation and cell death in HL-60 human promyelocytic leukemia cells by pharmacological inhibitors of protein kinase C. Cancer Res 54:1707–1714PubMedGoogle Scholar
  27. Ke N, Godzik A, Reed JC (2001) Bcl-B: a novel Bcl-2 family member that differentially binds and regulates Bax and Bak. J Biol Chem 376:229–236Google Scholar
  28. Khwaja, A, Downward J (1997) Lack of correlation between activation of Jun-NH2-terminal kinase and induction of apoptosis after detachment of epithelial cells. J Cell Biol 139:1017–1023CrossRefPubMedGoogle Scholar
  29. Kolb TM, Chang SH, Davis MA (2002) Biochemical and morphological events during okadaic acid-induced apoptosis of Tsc2-null ERC-18 cell line. Toxicol Pathol 30:235–246CrossRefPubMedGoogle Scholar
  30. Kreienbühl P, Keller H, Niggli V (1992) Protein phosphatase inhibitors okadaic acid and calyculin A alter cell shape and F-actin distribution and inhibit stimulus-dependent increases in cytoskeletal actin of human neutrophils. Blood 80:2911–2919PubMedGoogle Scholar
  31. Leira F, Alvarez C et al. (2000) Study of cytoskeletal changes induced by okadaic acid in BE(2)-M17 cells by means of a quantitative fluorimetric microplate assay. INVITOX2000, Pueblo Acantilado, AlicanteGoogle Scholar
  32. Leira F, Vieites JM, Vieytes MR, Botana LM (2001) Apoptotic events induced by the phosphatase inhibitor okadaic acid in normal human lung fibroblasts. Toxicol In Vitro 15:199–208CrossRefPubMedGoogle Scholar
  33. Leira F, Alvarez C, Vieites JM, Vieytes MR, Botana LM (2002a) Characterization of distinct apoptotic changes induced by okadaic acid and yessotoxin in the BE(2)-M17 cell line. Toxicol In Vitro 16:23–31CrossRefPubMedGoogle Scholar
  34. Leira F, Cabado AG, Vieytes MR, Roman Y et al (2002b) Characterization of F-actin depolymerization as a major toxin event induced by pectenotoxin-6 in neuroblastoma cells. Biochem Pharmacol 63:1979–1988CrossRefPubMedGoogle Scholar
  35. Leira F, Alvarez C, Cabado AG, Vieites JM et al (2003) Development of a F-actin-based live-cell fluorimetric microplate assay for diarrhetic shellfish toxins. Anal Biochem 317:129–135CrossRefPubMedGoogle Scholar
  36. Loeffler M, Kroemer G (2000) The mitochondrion in cell death control: certainties and incognita. Exp Cell Res 256:19–26CrossRefPubMedGoogle Scholar
  37. Macías-Silva M, García-Sáinz JA (1994) Inhibition of hormone-stimulated inositol phosphate production and disruption of cytoskeletal structure. Effects of okadaic acid, microcystin, chlorpromazine, W7 and nystatin. Toxicon 32:105–112CrossRefPubMedGoogle Scholar
  38. Marushige Y, Marushige K (1998) Alterations in focal adhesion and cytoeskeletal proteins during apoptosis. Anticancer Res 18:31–38Google Scholar
  39. Maruyama W, Irie S, Sato TA (2000) Morphological changes in the nucleus and actin cytoskeleton in the process of Fas-induced apoptosis in Jurkat T cells. Histochem J 32:495–503CrossRefPubMedGoogle Scholar
  40. Mashima T, Naito M, Noguchi K, Miller DK et al (1997) Actin cleavage by CPP-32/apopain during the development of apoptosis. Oncogene 14:1007–1012CrossRefPubMedGoogle Scholar
  41. Millward TA, Zolnierowicz S, Hemmings BA (1999) Regulation of protein kinase cascades by protein phosphatase 2A. Trends Biochem Sci 24:186–191PubMedGoogle Scholar
  42. Moreau V, Way M (1999) In vitro approaches to study actin and microtubule dependent cell processes. Curr Opin Cell Biol 11:152–158CrossRefPubMedGoogle Scholar
  43. Morimoto Y, Ohba T, Kobayashi S, Haneji T (1997) The protein phosphatase inhibitor okadaic acid and calyculin A induces apoptosis in human osteoblastic cells. Exp Cell Res 230:181–186CrossRefPubMedGoogle Scholar
  44. Niggli V, Djafarzadeh S, Keller H (1999) Stimulus-induced selective association of actin-associated proteins (α-actinin) and protein kinase C isoforms with the cytoskeleton of human neutrophils. Exp Cell Res 250:558–568CrossRefPubMedGoogle Scholar
  45. Nuydens R, de Jong M, Van Den Kieboom G, Heers C et al (1998) Okadaic acid-induced apoptosis in neuronal cells: evidence for an abortive mitotic attempt. J Neurochem 70:1124–1133PubMedGoogle Scholar
  46. Parlato S, Giammarioli AM, Logozzi M, Matarrese P et al (2000) CD95 (APO-1/Fas) linkage to the actin cytoeskeleton through ezrin in human T lymphocytes: a novel regulatory mechanism of the CD95 apoptotic pathway. EMBO J 19:5123–5134CrossRefPubMedGoogle Scholar
  47. Pedersen PL (1999) Mitochondrial events in the life and death of animal cells: a brief overview. J Bioenerg and Biomembr 31:291–304CrossRefGoogle Scholar
  48. Riordan FA, Foroni L, Hoffbrand AV, Mehta AB et al (1998) Okadaic acid-induced apoptosis of HL60 leukemia cells is preceded by destabilization of bcl-2 mRNA and downregulation of bcl-2 protein. FEBS Lett 435:195–198PubMedGoogle Scholar
  49. Rossini GP, Sgarbi N, Malaguti C (2001) The toxic responses induced by okadaic acid involve processing of multiple caspase isoforms. Toxicon 39:763–770CrossRefPubMedGoogle Scholar
  50. Ruvolo PP, Clark W (2002) A functional role for the B56 α-subunit of protein phosphatase 2A in ceramide-mediated regulation of Bcl-2 phosphorylation status and function. J Biol Chem 277:22847–22852CrossRefPubMedGoogle Scholar
  51. Santini MT, Rainaldi G, Indovina PL (2000) Apoptosis, cell adhesion and the extracellular matrix in the three-dimensional growth of multicellular tumor spheroids. Crit Rev Oncol Hematol 36:75–87CrossRefPubMedGoogle Scholar
  52. Savill J, Fadok V (2000) Corpse clearance defines the meaning of cell death. Nature 407:784–788CrossRefPubMedGoogle Scholar
  53. Schoenenberger C, Steinmetz MO, Stoffler D, Mandinova A et al (1999) Structure, assembly and dynamics of actin filaments in situ and in vitro. Microsc Res Tech 47:38–50CrossRefPubMedGoogle Scholar
  54. Séité P, Ruchaud S, Hillion J, Gendron MC et al (2000) Ectopic expression of Bcl-2 switches over nuclear signalling for cAMP-induced apoptosis to granulocytic differentiation. Cell Death Differ 7:1081–1089CrossRefPubMedGoogle Scholar
  55. Slee EA, Adrain C, MartinSJ (1999) Serial killers: ordering caspase events in apoptosis. Cell Death Differ 6:1067–1074PubMedGoogle Scholar
  56. Small JV, Rottner K, Kaverina I (1999) Functional design in the actin cytoskeleton. Curr Opin Cell Biol 11:54–60Google Scholar
  57. Tergau F, Weichert J, Quentin I, Optiz R (1997) Inhibitors of ser/thr phosphatases 1 and 2A induce apoptosis in pituitary GH3 cells. Naunyn Schmiedebergs Arch Pharmacol 356:8–16PubMedGoogle Scholar
  58. Thornberry NA, Lazebnik Y (1998) Caspases: enemies within. Science 281:1312–1316CrossRefPubMedGoogle Scholar
  59. Tsujimoto Y, Shimizu S. (2000) VDAC regulation by the Bcl-2 family of proteins. Cell Death Differ 7:1174–1181PubMedGoogle Scholar
  60. van de Water B, Tijdens IB, Verbrugge A, Huigsloot M et al (2000) Cleavage of the actin-capping protein a-adducin at Asp-Asp-Ser-Asp633-Ala by caspase-3 is preceded by its phosphorylation on serine726 in cisplatin-induced apoptosis of renal epithelial cells. J Biol Chem 275:25805–25813CrossRefPubMedGoogle Scholar
  61. von Zezschwitz C, Vorwerk H, Tergau F, Steinfelder HJ et al (1997) Apooptosis induction by inhibitors of Ser/Thr phosphatases 1 and 2A is associated with transglutaminase activation in two different human epithelial tumour lines. FEBS Lett 413:147–151CrossRefPubMedGoogle Scholar
  62. Yan Y Shay JW, Wright WE, Mumby MC (1997) Inhibition of protein phosphatase activity induces p53-dependent apoptosis in the absence of p53 transactivation. J Biol Chem 272:15220–15226CrossRefPubMedGoogle Scholar
  63. Yano Y, Sakon M, Kambayashi J, Kawasaki T et al (1995) Cytoskeletal reorganization of human platelets induced by the protein phosphatase 1/2A inhibitors okadaic acid and calyculin A. Biochem J 307:439–449PubMedGoogle Scholar
  64. Yasumoto T, Oshima Y et al (1978) Occurrence of a new type of shellfish poisoning in the Tohoku district. Bull Jpn Soc Sci Fish 44:1249–1255Google Scholar
  65. Yoon HS, Moon SC, Kim ND, Park BS et al (2000) Genistein produces reduction in growth and induces apoptosis of rat RPE-J cells. Curr Eye Res 20:215–224CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • A. G. Cabado
    • 1
  • F. Leira
    • 1
  • M. R. Vieytes
    • 2
  • J. M. Vieites
    • 1
  • L. M. Botana
    • 3
  1. 1.ANFACO-CECOPESCACampus Universitario de VigoVigo (Pontevedra)Spain
  2. 2.Dpto. FisiologíaFacultad de Veterinaria de LugoLugo (USC)Spain
  3. 3.Dpto. FarmacologíaFacultad de Veterinaria de LugoLugo (USC)Spain

Personalised recommendations