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Methods for Assessing Apoptosis and Anoikis in Normal Intestine/Colon and Colorectal Cancer

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Colorectal Cancer

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1765))

Abstract

Caspase-dependent apoptosis, including its distinct cell death subroutine known as anoikis, perform essential roles during organogenesis, as well as in the maintenance and repair of tissues. To this effect, the continuous renewal of the human intestinal/colon epithelium is characterized by the exfoliation by anoikis of differentiated cells, whereas immature/undifferentiated cells may occasionally undergo apoptosis in order to evacuate daughter cells that are damaged or defective. Dysregulated epithelial apoptosis is a significant component of inflammatory bowel diseases. Conversely, the acquisition of a resistance to apoptosis represents one of the hallmarks of cancer initiation and progression, including for colorectal cancer (CRC). Furthermore, the emergence of anoikis resistance constitutes a critical step in cancer progression (including CRC), as well as a limiting one that enables invasion and metastasis.

Considering the implications of apoptosis/anoikis dysregulation in gut physiopathology, it therefore becomes incumbent to understand the functional determinants that underlie such dysregulation—all the while having to monitor, assess, or evidence apoptosis and/or anoikis. In this chapter, methodologies that are typically used to assess caspase-dependent apoptosis and anoikis in intestinal/colonic normal and CRC cells, whether in vivo, ex vivo, or in cellulo, are provided.

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References

  1. Edinger AL, Thompson CB (2004) Death by design: apoptosis, necrosis and autophagy. Curr Opin Cell Biol 16:663–669

    Article  CAS  PubMed  Google Scholar 

  2. Eroglu M, Derry WB (2016) Your neighbours matter - non-autonomous control of apoptosis in development and disease. Cell Death Differ 23:1110–1118

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Galluzzi L, Bravo-San Pedro JM, Vitale I et al (2015) Essential versus accessory aspects of cell death: recommendations of the NCCD 2015. Cell Death Differ 22:58–73

    Article  CAS  PubMed  Google Scholar 

  4. Galluzzi L, Vitale I, Abrams JM et al (2012) Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012. Cell Death Differ 19:107–120

    Article  CAS  PubMed  Google Scholar 

  5. Penaloza C, Orlanski S, Ye Y et al (2008) Cell death in mammalian development. Curr Pharm Des 14:184–196

    Article  CAS  PubMed  Google Scholar 

  6. Tait SW, Ichim G, Green DR (2014) Die another way--non-apoptotic mechanisms of cell death. J Cell Sci 127:2135–2144

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Berghe TV, Linkermann A, Jouan-Lanhouet S et al (2014) Regulated necrosis: the expanding network of non-apoptotic cell death pathways. Nat Rev Mol Cell Biol 15:135–147

    Article  Google Scholar 

  8. Eisenberg-Lerner A, Bialik S, Simon HU et al (2009) Life and death partners: apoptosis, autophagy and the cross-talk between them. Cell Death Differ 16:966–975

    Article  CAS  PubMed  Google Scholar 

  9. Kroemer G, Galluzzi L, Vandenabeele P et al (2009) Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009. Cell Death Differ 16:3–11

    Article  CAS  PubMed  Google Scholar 

  10. Kroemer G, Martin SJ (2005) Caspase-independent cell death. Nat Med 11:725–730

    Article  PubMed  Google Scholar 

  11. Srivastava R (ed) (2007) Apoptosis, cell signaling and human diseases (molecular mechanisms, Vol. 1). Humana Press, Totowa

    Google Scholar 

  12. Srivastava R (ed) (2007) Apoptosis, cell signaling and human diseases (molecular mechanisms, Vol. 2). Humana Press, Totowa

    Google Scholar 

  13. Bertrand K (2011) Survival of exfoliated epithelial cells: a delicate balance between anoikis and apoptosis. J Biomed Biotechnol 2011:534139

    PubMed  PubMed Central  Google Scholar 

  14. Gilmore AP (2005) Anoikis. Cell Death Differ 12:1473–1477

    Article  CAS  PubMed  Google Scholar 

  15. Gilmore AP, Owens TW, Foster FM et al (2009) How adhesion signals reach a mitochondrial conclusion - ECM regulation of apoptosis. Curr Opin Cell Biol 21:654–661

    Article  CAS  PubMed  Google Scholar 

  16. Horbinski C, Mojesky C, Kyprianou N (2010) Live free or die: tales of homeless (cells) in cancer. Am J Pathol 177:1044–1052

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Ma Z, Liu Z, Myers DP et al (2008) Mechanotransduction and anoikis: death and the homeless cell. Cell Cycle 7:2462–2465

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Taddei ML, Giannoni E, Fiaschi T et al (2012) Anoikis: an emerging hallmark in health and diseases. J Pathol 226:380–393

    Article  CAS  PubMed  Google Scholar 

  19. Vachon PH (2011) Integrin signalling, cell survival, and anoikis: distinctions, differences, and differentiation. J Signal Transduct 2011:738137

    Article  PubMed  PubMed Central  Google Scholar 

  20. Vaux DL, Korsmeyer SJ (1999) Cell death in development. Cell 96:245–254

    Article  CAS  PubMed  Google Scholar 

  21. Marastoni S, Ligresti G, Lorenzon E et al (2008) Extracellular matrix: a matter of life and death. Connect Tissue Res 49:203–206

    Article  CAS  PubMed  Google Scholar 

  22. Murray P, Edgar D (2000) Regulation of programmed cell death by basement membranes in embryonic development. J Cell Biol 150:1215–1221

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Chiarugi P, Giannoni E (2008) Anoikis: a necessary death program for anchorage-dependent cells. Biochem Pharmacol 76:1352–1364

    Article  CAS  PubMed  Google Scholar 

  24. Dagenais M, Douglas T, Saleh M (2014) Role of programmed necrosis and cell death in intestinal inflammation. Curr Opin Gastroenterol 30:566–575

    Article  CAS  PubMed  Google Scholar 

  25. Edelblum KL, Yan F, Yamaoka T, Polk PB (2006) Regulation of apoptosis during homeostasis and disease in the intestinal epithelium. Inflamm Bowel Dis 12:413–424

    Article  PubMed  Google Scholar 

  26. Günther C, Buchen B, Neurath MF et al (2014) Regulation and pathophysiological role of epithelial turnover in the gut. Semin Cell Dev Biol 35:40–50

    Article  PubMed  Google Scholar 

  27. Günther C, Neumann H, Neurath MF et al (2013) Apoptosis, necrosis and necroptosis: cell death regulation in the intestinal epithelium. Gut 62:1062–1071

    Article  PubMed  Google Scholar 

  28. Ménard D, Beaulieu JF, Boudreau F et al (2005) Gastrointestinal tract (GI tract). In: Unsicker K, Krieglstein K (eds) Cell signalling and growth factors in development - part II. Wiley-VCH, Verlag

    Google Scholar 

  29. Negroni A, Cucchiara S, Stronati L (2015) Apoptosis, necrosis, and necroptosis in the gut and intestinal homeostasis. Mediat Inflamm 2015:250762

    Article  Google Scholar 

  30. Potten CS (1997) Epithelial cell growth and differentiation. II. Intestinal apoptosis. Am J Phys 273:G253–G2577

    CAS  Google Scholar 

  31. Tarnawski AS, Szabo I (2001) Apoptosis-programmed cell death and its relevance to gastrointestinal epithelium: survival signal from the matrix. Gastroenterology 120:294–299

    Article  CAS  PubMed  Google Scholar 

  32. Vachon PH, Cardin E, Harnois C et al (2000) Early establishment of epithelial apoptosis in the developing human small intestine. Int J Dev Biol 44:891–898

    CAS  PubMed  Google Scholar 

  33. West NJ, Courtney ED, Poullis AP et al (2009) Apoptosis in the colonic crypt, colorectal adenomata, and manipulation by chemoprevention. Cancer Epidemiol Biomark Prev 18:1680–1687

    Article  CAS  Google Scholar 

  34. Shanmugathasan M, Jothy S (2000) Apoptosis, anoikis and their relevance to the pathobiology of colon cancer. Pathol Int 50:273–279

    Article  CAS  PubMed  Google Scholar 

  35. Buchheit CL, Weigel KJ, Schafer ZT (2014) Cancer cell survival during detachment from the ECM: multiple barriers to tumour progression. Nat Rev Cancer 14:632–641

    Article  CAS  PubMed  Google Scholar 

  36. Frisch SM, Schaller M, Cieply B (2013) Mechanisms that link the oncogenic epithelial-mesenchymal transition to suppression of anoikis. J Cell Sci 126:21–29

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Guadamillas MC, Cerezo C, del Pozo MA (2011) Overcoming anoikis - pathways to anchorage independent growth in cancer. J Cell Sci 124:3189–3197

    Article  CAS  PubMed  Google Scholar 

  38. Paoli P, Giannoni E, Chiarugi P (2013) Anoikis molecular pathways and its role in cancer progression. Biochim Biophys Acta 1833:3481–3498

    Article  CAS  PubMed  Google Scholar 

  39. Crotti S, Piccoli M, Rizzolio F et al (2017) Extracellular matrix and colorectal cancer: how surrounding microenvironment affects cancer cell behavior? J Cell Physiol 232:967–975

    Article  CAS  PubMed  Google Scholar 

  40. Beaulieu J-F, Ménard D (2012) Isolation, characterization, and culture of normal human intestinal crypt and villus cells. Methods Mol Biol 806:157–173

    Article  CAS  PubMed  Google Scholar 

  41. Perreault N, Beaulieu J-F (1996) A novel method for the establishment of a pure population of nontransformed human intestinal primary epithelial cell (HIPEC) lines in long term culture. Exp Cell Res 224:354–364

    Article  CAS  PubMed  Google Scholar 

  42. Perreault N, Beaulieu J-F (1998) Primary cultures of fully differentiated and pure human intestinal epithelial cells. Exp Cell Res 245:34–42

    Article  CAS  PubMed  Google Scholar 

  43. Beaulieu J-F, Quaroni A (1991) Clonal analysis of sucrase-isomaltase expression in the human colon adenocarcinoma Caco-2 cells. Biochem J 280:599–608

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Pageot LP, Perreault N, Basora N et al (2000) Human cell models to study small intestinal functions: recapitulation of the crypt-villus axis. Microsc Res Tech 49:394–406

    Article  CAS  PubMed  Google Scholar 

  45. Gavrieli Y, Sherman Y, Ben-Sasson SA (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493–501

    Article  CAS  PubMed  Google Scholar 

  46. Beauséjour M, Thibodeau S, Demers MJ et al (2013) Suppression of anoikis in human intestinal epithelial cells: differentiation state-selective roles of α2β1, α3β1, α5β1, and α6β4 integrins. BMC Cell Biol 14:53

    Article  PubMed  PubMed Central  Google Scholar 

  47. Lévy E, Delvin E, Ménard D et al (2009) Functional development of human fetal gastrointestinal tract. Methods Mol Biol 550:205–224

    Article  PubMed  Google Scholar 

  48. Gauthier R, Laprise P, Cardin E et al (2001) Differential sensitivity to apoptosis between the human small and large intestinal mucosae: linkage with segment-specific regulation of BCL-2 homologs and involvement of signaling pathways. J Cell Biochem 82:339–355

    Article  CAS  PubMed  Google Scholar 

  49. Beauséjour M, Noël D, Thibodeau S et al (2012) Integrin/Fak/Src-mediated regulation of cell survival and anoikis in human intestinal epithelial crypt cells: selective engagement and roles of PI3-K isoform complexes. Apoptosis 17:566–578

    Article  PubMed  PubMed Central  Google Scholar 

  50. Bouchard V, Demers MJ, Thibodeau S et al (2007) Fak/Src signaling in human intestinal epithelial cell survival and anoikis: differentiation state-specific uncoupling with the PI3-K/Akt-1 and MEK/Erk pathways. J Cell Physiol 212:717–728

    Article  CAS  PubMed  Google Scholar 

  51. Bouchard V, Harnois C, Demers MJ (2008) β1 integrin/Fak/Src signaling in intestinal epithelial crypt cell survival: integration of complex regulatory mechanisms. Apoptosis 13:531–542

    Article  CAS  PubMed  Google Scholar 

  52. Demers MJ, Thibodeau S, Noël D et al (2009) Intestinal epithelial cancer cell anoikis resistance: EGFR-mediated sustained activation of Src overrides Fak-dependent signaling to MEK/Erk and/or PI3-K/Akt-1. J Cell Biochem 107:639–654

    Article  CAS  PubMed  Google Scholar 

  53. Gauthier R, Harnois C, Drolet JF et al (2001) Human intestinal epithelial cell survival: differentiation state-specific control mechanisms. Am J Physiol Cell Physiol 280:C1540–C1554

    Article  CAS  PubMed  Google Scholar 

  54. Harnois C, Demers MJ, Bouchard V et al (2004) Human intestinal epithelial crypt cell survival and death: complex modulations of Bcl-2 homologs by Fak, PI3-K/Akt-1, MEK/Erk, and p38 signaling pathways. J Cell Physiol 198(2):209–222

    Article  CAS  PubMed  Google Scholar 

  55. Klöditz K, Chen YZ, Xue D et al (2017) Programmed cell clearance: from nematodes to humans. Biochem Biophys Res Commun 482:491–497

    Article  PubMed  Google Scholar 

  56. Frisch SM (2000) Anoikis. Methods Enzymol 322:472–479

    Article  CAS  PubMed  Google Scholar 

  57. Bursch W, Kleine L, Tenniswood M (1990) The biochemistry of cell death. Biochem Cell Biol 68:1071–1074

    Article  CAS  PubMed  Google Scholar 

  58. Benoit YD, Larrivée JF, Groulx JF et al (2010) Integrin alpha8beta1 confers anoikis susceptibility to human intestinal epithelial crypt cells. Biochem Biophys Res Commun 399:434–439

    Article  CAS  PubMed  Google Scholar 

  59. Julien O, Wells JA (2017) Caspases and their substrates. Cell Death Differ 24(8):1380–1389. https://doi.org/10.1038/cdd.2017.44

    Article  CAS  PubMed  Google Scholar 

  60. Chaitanya GV, Alexander JS, Babu PP (2010) PARP-1 cleavage fragments: signatures of cell-death proteases in neurodegeneration. Cell Commun Signal 8:31

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Dufour G, Demers MJ, Gagné D et al (2004) Human intestinal epithelial cell survival and anoikis. Differentiation state-distinct regulation and roles of protein kinase B/Akt isoforms. J Biol Chem 279:44113–44122

    Article  CAS  PubMed  Google Scholar 

  62. Vachon PH, Harnois C, Grenier A et al (2002) Differentiation state-selective roles of p38 isoforms in human intestinal epithelial cell anoikis. Gastroenterology 123:1980–1991

    Article  CAS  PubMed  Google Scholar 

  63. Fiandalo MV, Kyprianou N (2012) Caspase control: protagonists of cancer cell apoptosis. Exp Oncol 34:165–175

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Vachon PH, Cardin E, Harnois C et al (2001) Early acquisition of bowel segment-specific Bcl-2 expression profiles during the development of the human ileum and colon. Histol Histopathol 16:497–510

    CAS  PubMed  Google Scholar 

  65. Anderson P (1997) Kinase cascades regulating entry into apoptosis. Microbiol Mol Biol Rev 61:33–46

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Francoeur C, Escaffit F, Vachon PH et al (2004) Proinflammatory cytokines TNF-alpha and IFN-gamma alter laminin expression under an apoptosis-independent mechanism in human intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 287:G592–G598

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

The author thanks Rona K. Graham (Département de pharmacologie-physiologie, FMSS, U. de Sherbrooke), for access to the LI-Cor Odyssey Fc imaging system and the VICTOR X multilabel plate reader spectrophotometer, and Jean-François Beaulieu (Département d’anatomie et de biologie cellulaire, FMSS, U. de Sherbrooke) for the generous gift of the HIEC-6, Caco-2/15, and HT-29 cell lines. This work was supported in part by a grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada (RGPIN 227935-2013). The author is a Researcher of the Canadian Foundation for Innovation (CFI) and a member of the FRQS-funded Centre de Recherche du CHUS.

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Correspondence to Pierre H. Vachon .

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Vachon, P.H. (2018). Methods for Assessing Apoptosis and Anoikis in Normal Intestine/Colon and Colorectal Cancer. In: Beaulieu, JF. (eds) Colorectal Cancer. Methods in Molecular Biology, vol 1765. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7765-9_7

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  • DOI: https://doi.org/10.1007/978-1-4939-7765-9_7

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