Autophagic Flux Assessment in Colorectal Cancer Cells

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

Abstract

Autophagy protects colorectal cancer cells against therapeutic intervention. Autophagy is a continuous process, and autophagic flux requires both autophagosome synthesis and their subsequent degradation at lysosomes. Hence, cells with elevated autophagic flux display both rapid autophagosome generation and degradation. Here, we describe an immunoblot protocol coupled to pharmaceutical inhibition of autophagosome clearance to monitor autophagic flux levels between colorectal cancer cell lines.

Key words

Autophagy Colorectal cancer cells Immunoblots LC3 Bafilomycin A1 

Notes

Acknowledgments

This work was supported by grants from the Canadian Institutes of Health Research (CIHR) and the Cancer Research Society (CRS) to S.J. and by junior faculty salary awards from the CIHR and Fond de Recherche du Québec—Santé (FRQS) to S.J.. Steve Jean is a member of the FRSQ-Funded Centre de Recherche du CHUS.

References

  1. 1.
    Mizushima N, Komatsu M (2011) Autophagy: renovation of cells and tissues. Cell 147(4):728–741.  https://doi.org/10.1016/j.cell.2011.10.026CrossRefPubMedGoogle Scholar
  2. 2.
    Levine B, Kroemer G (2008) Autophagy in the pathogenesis of disease. Cell 132(1):27–42.  https://doi.org/10.1016/j.cell.2007.12.018CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Galluzzi L, Pietrocola F, Bravo-San Pedro JM, Amaravadi RK, Baehrecke EH, Cecconi F, Codogno P, Debnath J, Gewirtz DA, Karantza V, Kimmelman A, Kumar S, Levine B, Maiuri MC, Martin SJ, Penninger J, Piacentini M, Rubinsztein DC, Simon HU, Simonsen A, Thorburn AM, Velasco G, Ryan KM, Kroemer G (2015) Autophagy in malignant transformation and cancer progression. EMBO J 34(7):856–880.  https://doi.org/10.15252/embj.201490784CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Kimmelman AC (2011) The dynamic nature of autophagy in cancer. Genes Dev 25(19):1999–2010.  https://doi.org/10.1101/gad.17558811CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Burada F, Nicoli ER, Ciurea ME, Uscatu DC, Ioana M, Gheonea DI (2015) Autophagy in colorectal cancer: an important switch from physiology to pathology. World J Gastrointest Oncol 7(11):271–284.  https://doi.org/10.4251/wjgo.v7.i11.271CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Groulx JF, Khalfaoui T, Benoit YD, Bernatchez G, Carrier JC, Basora N, Beaulieu JF (2012) Autophagy is active in normal colon mucosa. Autophagy 8(6):893–902.  https://doi.org/10.4161/auto.19738CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Sato K, Tsuchihara K, Fujii S, Sugiyama M, Goya T, Atomi Y, Ueno T, Ochiai A, Esumi H (2007) Autophagy is activated in colorectal cancer cells and contributes to the tolerance to nutrient deprivation. Cancer Res 67(20):9677–9684.  https://doi.org/10.1158/0008-5472.CAN-07-1462CrossRefPubMedGoogle Scholar
  8. 8.
    Zheng HY, Zhang XY, Wang XF, Sun BC (2012) Autophagy enhances the aggressiveness of human colorectal cancer cells and their ability to adapt to apoptotic stimulus. Cancer Biol Med 9(2):105–110.  https://doi.org/10.3969/j.issn.2095-3941.2012.02.004PubMedPubMedCentralGoogle Scholar
  9. 9.
    Klionsky DJ et al (2016) Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12(1):1–222.  https://doi.org/10.1080/15548627.2015.1100356CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Mizushima N, Yoshimori T (2007) How to interpret LC3 immunoblotting. Autophagy 3(6):542–545CrossRefPubMedGoogle Scholar
  11. 11.
    Nakatogawa H, Ichimura Y, Ohsumi Y (2007) Atg8, a ubiquitin-like protein required for autophagosome formation, mediates membrane tethering and hemifusion. Cell 130(1):165–178.  https://doi.org/10.1016/j.cell.2007.05.021CrossRefPubMedGoogle Scholar
  12. 12.
    Watanabe Y, Tanaka M (2011) p62/SQSTM1 in autophagic clearance of a non-ubiquitylated substrate. J Cell Sci 124(Pt 16):2692–2701.  https://doi.org/10.1242/jcs.081232CrossRefPubMedGoogle Scholar
  13. 13.
    Fujita N, Hayashi-Nishino M, Fukumoto H, Omori H, Yamamoto A, Noda T, Yoshimori T (2008) An Atg4B mutant hampers the lipidation of LC3 paralogues and causes defects in autophagosome closure. Mol Biol Cell 19(11):4651–4659.  https://doi.org/10.1091/mbc.E08-03-0312CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Chen Y, Klionsky DJ (2011) The regulation of autophagy—unanswered questions. J Cell Sci 124(Pt 2):161–170.  https://doi.org/10.1242/jcs.064576CrossRefPubMedGoogle Scholar
  15. 15.
    Kimura S, Fujita N, Noda T, Yoshimori T (2009) Monitoring autophagy in mammalian cultured cells through the dynamics of LC3. Methods Enzymol 452:1–12.  https://doi.org/10.1016/S0076-6879(08)03601-XCrossRefPubMedGoogle Scholar
  16. 16.
    Mauvezin C, Nagy P, Juhasz G, Neufeld TP (2015) Autophagosome-lysosome fusion is independent of V-ATPase-mediated acidification. Nat Commun 6:7007.  https://doi.org/10.1038/ncomms8007CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Anatomy and Cell Biology, Faculty of Medicine and Health SciencesUniversité de SherbrookeSherbrookeCanada

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