Physiological intestinal oxygen modulates the Caco-2 cell model and increases sensitivity to the phytocannabinoid cannabidiol

  • Tara Macpherson
  • Jane A. Armstrong
  • David N. Criddle
  • Karen L. WrightEmail author


The Caco-2 cell model is widely used as a model of colon cancer and small intestinal epithelium but, like most cell models, is cultured in atmospheric oxygen conditions (∼21%). This does not reflect the physiological oxygen range found in the colon. In this study, we investigated the effect of adapting the Caco-2 cell line to routine culturing in a physiological oxygen (5%) environment. Under these conditions, cells maintain a number of key characteristics of the Caco-2 model, such as increased formation of tight junctions and alkaline phosphatase expression over the differentiation period and maintenance of barrier function. However, these cells exhibit differential oxidative metabolism, proliferate less and become larger during differentiation. In addition, these cells were more sensitive to cannabidiol-induced antiproliferative actions through changes in cellular energetics: from a drop of oxygen consumption rate and loss of mitochondrial membrane integrity in cells treated under atmospheric conditions to an increase in reactive oxygen species in intact mitochondria in cells treated under low-oxygen conditions. Inclusion of an additional physiological parameter, sodium butyrate, into the medium revealed a cannabidiol-induced proliferative response at low doses. These effects could impact on its development as an anticancer therapeutic, but overall, the data supports the principle that culturing cells in microenvironments that more closely mimic the in vivo conditions is important for drug screening and mechanism of action studies.


Colon Cannabinoid Normoxia Oxidative metabolism Butyrate 



T. Macpherson is partly funded by a Novartis Pharmaceuticals UK Ltd (Horsham, UK) studentship with matched funding from the Biotechnology and Biological Sciences Research Council (BBSRC, Swindon, UK). K. Wright is partly funded by a Peel Trust Lectureship from The Dowager Countess Eleanor Peel Trust. We would also like to thank Dr Dave Clancy for useful discussions on data interpretation and statistics and Dr Jane Andre for assistance with the confocal imaging (both from Faculty of Health and Medicine, Lancaster University).


  1. Athanasiou A.; Clarke A.; Turner A.; Kumaran N.; Vakilpour S.; Smith P.; Bagiokou D.; Bradshaw T.; Westwell A.; Fang L.; Lobo D.; Constantinescu C.; Calabrese V.; Loesch A.; Alexander S.; Clothier R.; Kendall D.; Bates T. Cannabinoid receptor agonists are mitochondrial inhibitors: a unified hypothesis of how cannabinoids modulate mitochondrial function and induce cell death. Biochem. Biophys. Res. Commun. 364: 131–137; 2007.PubMedCrossRefGoogle Scholar
  2. Aviello G.; Romano B.; Borrelli F.; Capasso R.; Gallo L.; Piscitelli F.; Di Marzo V.; Izzo A. Chemopreventive effect of the non-psychotropic phytocannabinoid cannabidiol on experimental colon cancer. J. Mol. Med. 90: 925–934; 2012.PubMedCrossRefGoogle Scholar
  3. Bauer E.; Williams B. A.; Smidt H.; Verstegen M. W.; Mosenthin R. Influence of the gastrointestinal microbiota on development of the immune system in young animals. Curr. Issue Intest. Microbiol. 7: 35–51; 2006.Google Scholar
  4. Bugaut M. Occurrence, absorption and metabolism of short chain fatty acids in the digestive tract of mammals. Comp. Biochem. Physiol. B 86: 439–472; 1987.PubMedCrossRefGoogle Scholar
  5. Chantret I.; Barbat A.; Dussaulx E.; Brattain M. G.; Zweibaum A. Epithelial polarity, villin expression, and enterocytic differentiation of cultured human colon carcinoma cells: a survey of twenty cell lines. Cancer Res. 48: 1936–1942; 1988.PubMedGoogle Scholar
  6. Chiu P.; Karler R.; Craven C.; Olsen D. M.; Turkanis S. A. The influence of delta9-tetrahydrocannabinol, cannabinol and cannabidiol on tissue oxygen consumption. Res. Commun. Chem. Pathol. Pharmacol. 12: 267–286; 1975.PubMedGoogle Scholar
  7. Csete M.; Ourednik J.; Ourednik V.; Sakaguchi D.; NilsenHamilton M. Oxygen in the cultivation of stem cells. Stem Cell Biol. Develop Plast. 1049: 1–8; 2005.Google Scholar
  8. Donohoe D. R.; Garge N.; Zhang X.; Sun W.; O’Connell T. M.; Bunger M. K.; Bultman S. J. The microbiome and butyrate regulate energy metabolism and autophagy in the mammalian colon. Cell Metab. 13: 517–526; 2011.PubMedCentralPubMedCrossRefGoogle Scholar
  9. Dutta R. C.; Dutta A. K. Cell-interactive 3D-scaffold; advances and applications. Biotechnol. Adv. 27: 334–339; 2009.PubMedCrossRefGoogle Scholar
  10. Espey M. G. Role of oxygen gradients in shaping redox relationships between the human intestine and its microbiota. Free Radic. Biol. Med. 55: 130–140; 2013.PubMedCrossRefGoogle Scholar
  11. Hara A.; Hibi T.; Yoshioka M.; Toda K.; Watanabe N.; Hayashi A.; Iwao Y.; Saito H.; Watanabe T.; Tsuchiya M. Changes of proliferative activity and phenotypes in spontaneous differentiation of a colon cancer cell line. Jpn. J. Cancer Res. 84: 625–632; 1993.PubMedCrossRefGoogle Scholar
  12. Ivanovic Z. Hypoxia or in situ normoxia: the stem cell paradigm. J. Cell. Physiol. 219: 271–275; 2009.PubMedCrossRefGoogle Scholar
  13. Ligresti A.; Moriello A. S.; Starowicz K.; Matias I.; Pisanti S.; De Petrocellis L.; Laezza C.; Portella G.; Bifulco M.; Di Marzo V. Antitumor activity of plant cannabinoids with emphasis on the effect of cannabidiol on human breast carcinoma. J. Pharmacol. Exp. Ther. 318: 1375–1387; 2006.PubMedCrossRefGoogle Scholar
  14. Massi P.; Vaccani A.; Ceruti S.; Colombo A.; Abbracchio M. P.; Parolaro D. Antitumor effects of cannabidiol, a nonpsychoactive cannabinoid, on human glioma cell lines. J. Pharmacol. Exp. Ther. 308: 838–845; 2004.PubMedCrossRefGoogle Scholar
  15. McAllister S. D.; Murase R.; Christian R. T.; Lau D.; Zielinski A. J.; Allison J.; Almanza C.; Pakdel A.; Lee J.; Limbad C.; Liu Y.; Debs R. J.; Moore D. H.; Desprez P. Y. Pathways mediating the effects of cannabidiol on the reduction of breast cancer cell proliferation, invasion, and metastasis. Breast Cancer Res. Treat. 129: 37–47; 2011.PubMedCentralPubMedCrossRefGoogle Scholar
  16. McKallip R. J.; Jia W.; Schlomer J.; Warren J. W.; Nagarkatti P. S.; Nagarkatti M. Cannabidiol-induced apoptosis in human leukemia cells: a novel role of cannabidiol in the regulation of p22phox and Nox4 expression. Mol. Pharmacol. 70: 897–908; 2006.PubMedCrossRefGoogle Scholar
  17. Morrison S. J.; Csete M.; Groves A. K.; Melega W.; Wold B.; Anderson D. J. Culture in reduced levels of oxygen promotes clonogenic sympathoadrenal differentiation by isolated neural crest stem cells. J. Neurosci. 20: 7370–7376; 2000.PubMedGoogle Scholar
  18. Pinto M.; Robineleson S.; Appay M.; Kedinger M.; Triadou N.; Dussaulx E.; Lacroix B.; Simonassmann P.; Haffen K.; Fogh J.; Zweibaum A. Enterocyte-like differentiation and polarization of the human-colon carcinoma cell-line Caco-2 in culture. Biol. Cell. 47: 323–330; 1983.Google Scholar
  19. Prasad S.; Czepiel M.; Cetinkaya C.; Smigielska K.; Weli S. C.; Lysdahl H.; Gabrielsen A.; Petersen K.; Ehlers N.; Fink T. Continuous hypoxic culturing maintains activation of Notch and allows long-term propagation of human embryonic stem cells without spontaneous differentiation. Cell Prolif. 42: 63–74; 2009.PubMedCrossRefGoogle Scholar
  20. Rocha F. G.; Whang E. E. Intestinal tissue engineering: from regenerative medicine to model systems. J. Surg. Res. 120: 320–325; 2004.PubMedCrossRefGoogle Scholar
  21. Roeselers G.; Ponomarenko M.; Lukovac S.; Wortelboer H. M. Ex vivo systems to study host-microbiota interactions in the gastrointestinal tract. Best Pract. Res. Clin. Gastroenterol. 27: 101–113; 2013.PubMedCrossRefGoogle Scholar
  22. Ruhaak L. R.; Felth J.; Karlsson P. C.; Rafter J. J.; Verpoorte R.; Bohlin L. Evaluation of the cyclooxygenase inhibiting effects of six major cannabinoids isolated from Cannabis sativa. Biol. Pharm. Bull. 34: 774–778; 2011.PubMedCrossRefGoogle Scholar
  23. Scheppach W. Effects of short chain fatty acids on gut morphology and function. Gut 35: S35–S38; 1994.PubMedCentralPubMedCrossRefGoogle Scholar
  24. Shin E.; Forsyth N. R.; Fricker R. A. The effect of physiological oxygen levels on GABAergic neuronal differentiation from mouse embryonic stem cells. Stem Cell Stud. 2: e3; 2012.CrossRefGoogle Scholar
  25. Shipp C.; Derhovanessian E.; Pawelec G. Effect of culture at low oxygen tension on the expression of heat shock proteins in a panel of melanoma cell lines. PLoS ONE 7: e37475; 2012.PubMedCentralPubMedCrossRefGoogle Scholar
  26. Shrivastava A.; Kuzontkoski P. M.; Groopman J. E.; Prasad A. Cannabidiol induces programmed cell death in breast cancer cells by coordinating the cross-talk between apoptosis and autophagy. Mol. Cancer Ther. 10: 1161–1172; 2011.PubMedCrossRefGoogle Scholar
  27. Solinas M.; Massi P.; Cinquina V.; Valenti M.; Bolognini D.; Gariboldi M.; Monti E.; Rubino T.; Parolaro D. Cannabidiol, a non-psychoactive cannabinoid compound, inhibits proliferation and invasion in U87-MG and T98G glioma cells through a multitarget effect. PLoS ONE 8: e76918; 2013.PubMedCentralPubMedCrossRefGoogle Scholar
  28. Van De Walle J.; Hendrickx A.; Romier B.; Larondelle Y.; Schneider Y. Inflammatory parameters in Caco-2 cells: effect of stimuli nature, concentration, combination and cell differentiation. Toxicol. in Vitro 24: 1441–1449; 2010.CrossRefGoogle Scholar
  29. Webster M.; Witkin K. L.; Cohen-Fix O. Sizing up the nucleus: nuclear shape, size and nuclear-envelope assembly. J. Cell Sci. 122: 1477–1486; 2009.PubMedCentralPubMedCrossRefGoogle Scholar
  30. Young E. W.; Beebe D. J. Fundamentals of microfluidic cell culture in controlled microenvironments. Chem. Soc. Rev. 39: 1036–1048; 2010.PubMedCentralPubMedCrossRefGoogle Scholar
  31. Yu D.; Waby J. S.; Chirakkal H.; Staton C. A.; Corfe B. M. Butyrate suppresses expression of neuropilin I in colorectal cell lines through inhibition of Sp1 transactivation. Mol. Cancer 9: 276; 2010.PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2014

Authors and Affiliations

  • Tara Macpherson
    • 1
  • Jane A. Armstrong
    • 2
  • David N. Criddle
    • 2
  • Karen L. Wright
    • 1
    Email author
  1. 1.Faculty of Health and Medicine, Division of Biomedical and Life SciencesLancaster UniversityLancasterUK
  2. 2.NIHR Liverpool Pancreas Biomedical Research Unit, Department of Molecular and Clinical Cancer MedicineRoyal Liverpool University HospitalLiverpoolUK

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