Cancer Chemotherapy and Pharmacology

, Volume 63, Issue 4, pp 691–701 | Cite as

Cannabinoid receptor-independent cytotoxic effects of cannabinoids in human colorectal carcinoma cells: synergism with 5-fluorouracil

  • Sofia B. GustafssonEmail author
  • Theres Lindgren
  • Maria Jonsson
  • Stig O. P. Jacobsson
Original Article


Cannabinoids (CBs) have been found to exert antiproliferative effects upon a variety of cancer cells, including colorectal carcinoma cells. However, little is known about the signalling mechanisms behind the antitumoural effect in these cells, whether the effects are shared by endogenous lipids related to endocannabinoids, or whether such effects are synergistic with treatment paradigms currently used in the clinic. The aim of this preclinical study was to investigate the effect of synthetic and endogenous CBs and their related fatty acids on the viability of human colorectal carcinoma Caco-2 cells, and to determine whether CB effects are synergistic with those seen with the pyrimidine antagonist 5-fluorouracil (5-FU). The synthetic CB HU 210, the endogenous CB anandamide, the endogenous structural analogue of anandamide, N-arachidonoyl glycine (NAGly), as well as the related polyunsaturated fatty acids arachidonic acid and eicosapentaenoic acid showed antiproliferative and cytotoxic effects in the Caco-2 cells, as measured by using [3H]-thymidine incorporation assay, the CyQUANT proliferation assay and calcein-AM fluorescence. HU 210 was the most potent compound examined, followed by anandamide, whereas NAGly showed equal potency and efficacy as the polyunsaturated fatty acids. Furthermore, HU 210 and 5-FU produced synergistic effects in the Caco-2 cells, but not in the human colorectal carcinoma cell lines HCT116 or HT29. The compounds examined produced cytotoxic, rather than antiproliferative effects, by a mechanism not involving CB receptors, since the CB receptor antagonists AM251 and AM630 did not attenuate the effects, nor did pertussis toxin. However, α-tocopherol and the nitric oxide synthase inhibitor L-NAME attenuated the CB toxicity, suggesting involvement of oxidative stress. It is concluded that the CB system may provide new targets for the development of drugs to treat colorectal cancer.


Cannabinoids Polyunsaturated fatty acids 5-Fluorouracil Colorectal cancer Cytotoxicity 

Supplementary material

280_2008_788_MOESM1_ESM.tif (2.4 mb)
Fig. S1 Representative photomicrographs of Caco-2 cells exposed to the test compounds for three days. Shown are a control, b 100  µM 5-fluorouracil, c 100 µM arachidonic acid, d 100 µM eicosapentaenoic acid, eµM HU 210, f 30 µM AEA, and g 30 µM N-arachidonoyl glycine. (TIFF 2.35 Mb)
280_2008_788_MOESM2_ESM.tif (96 kb)
Fig. S2 Time-dependent effects of 100 ng/ml pertussis toxin (PTX) upon HU 210-induced inhibition of forskolin-stimulated cyclic AMP in Caco-2 cells. Data are means ± SEM of four different days of experiments. Statistically (one-way ANOVA with post-hoc multiple comparison test) significant differences from forskolin-induced cyclic AMP levels are indicated as *P < 0.05, ***P < 0.01 (vs. basal or 4 µM HU 210) or †P  < 0.05 (4 µM HU 210 vs. PTX). (TIFF 97 kb)


  1. 1.
    Ambs S, Merriam WG, Bennett WP, Felley-Bosco E, Ogunfusika MO, Oser SM, Klein S, Shields PG, Billiar TR, Harris CC (1998) Frequent nitric oxide synthase-2 expression in human colon adenomas: implication for tumor angiogenesis and colon cancer progression. Cancer Res 58:334–341PubMedGoogle Scholar
  2. 2.
    Athanasiou A, Clarke AB, Turner AE, Kumaran NM, Vakilpour S, Smith PA, Bagiokou D, Bradshaw TD, Westwell AD, Fang L, Lobo DN, Constantinescu CS, Calabrese V, Loesch A, Alexander SP, Clothier RH, Kendall DA, Bates TE (2007) Cannabinoid receptor agonists are mitochondrial inhibitors: a unified hypothesis of how cannabinoids modulate mitochondrial function and induce cell death. Biochem Biophys Res Commun 364:131–137PubMedCrossRefGoogle Scholar
  3. 3.
    Bifulco M, Laezza C, Pisanti S, Gazzerro P (2006) Cannabinoids and cancer: pros and cons of an antitumour strategy. Br J Pharmacol 148:123–135PubMedCrossRefGoogle Scholar
  4. 4.
    Brown AJ (2007) Novel cannabinoid receptors. Br J Pharmacol 152:567–575PubMedCrossRefGoogle Scholar
  5. 5.
    Burkey TH, Quock RM, Consroe P, Ehlert FJ, Hosohata Y, Roeske WR, Yamamura HI (1997) Relative efficacies of cannabinoid CB1 receptor agonists in the mouse brain. Eur J Pharmacol 336:295–298PubMedCrossRefGoogle Scholar
  6. 6.
    Carracedo A, Gironella M, Lorente M, Garcia S, Guzman M, Velasco G, Iovanna JL (2006) Cannabinoids induce apoptosis of pancreatic tumor cells via endoplasmic reticulum stress-related genes. Cancer Res 66:6748–6755PubMedCrossRefGoogle Scholar
  7. 7.
    Cianchi F, Cortesini C, Fantappie O, Messerini L, Schiavone N, Vannacci A, Nistri S, Sardi I, Baroni G, Marzocca C, Perna F, Mazzanti R, Bechi P, Masini E (2003) Inducible nitric oxide synthase expression in human colorectal cancer: correlation with tumor angiogenesis. Am J Pathol 162:793–801PubMedGoogle Scholar
  8. 8.
    De Petrocellis L, Bisogno T, Maccarrone M, Davis JB, Finazzi-Agro A, Di Marzo V (2001) The activity of anandamide at vanilloid VR1 receptors requires facilitated transport across the cell membrane and is limited by intracellular metabolism. J Biol Chem 276:12856–12863PubMedCrossRefGoogle Scholar
  9. 9.
    Dinh TP, Carpenter D, Leslie FM, Freund TF, Katona I, Sensi SL, Kathuria S, Piomelli D (2002) Brain monoglyceride lipase participating in endocannabinoid inactivation. Proc Natl Acad Sci USA 99:10819–10824PubMedCrossRefGoogle Scholar
  10. 10.
    Edgemond WS, Hillard CJ, Falck JR, Kearn CS, Campbell WB (1998) Human platelets and polymorphonuclear leukocytes synthesize oxygenated derivatives of arachidonylethanolamide (anandamide): their affinities for cannabinoid receptors and pathways of inactivation. Mol Pharmacol 54:180–188PubMedGoogle Scholar
  11. 11.
    Fogli S, Nieri P, Chicca A, Adinolfi B, Mariotti V, Iacopetti P, Breschi MC, Pellegrini S (2006) Cannabinoid derivatives induce cell death in pancreatic MIA PaCa–2 cells via a receptor-independent mechanism. FEBS Lett 580:1733–1739PubMedCrossRefGoogle Scholar
  12. 12.
    Galve-Roperh I, Sanchez C, Cortes ML, del Pulgar TG, Izquierdo M, Guzman M (2000) Anti-tumoral action of cannabinoids: involvement of sustained ceramide accumulation and extracellular signal-regulated kinase activation. Nat Med 6:313–319PubMedCrossRefGoogle Scholar
  13. 13.
    Giang DK, Cravatt BF (1997) Molecular characterization of human and mouse fatty acid amide hydrolases. Proc Natl Acad Sci USA 94:2238–2242PubMedCrossRefGoogle Scholar
  14. 14.
    Greenhough A, Patsos HA, Williams AC, Paraskeva C (2007) The cannabinoid delta(9)-tetrahydrocannabinol inhibits RAS-MAPK and PI3K-AKT survival signalling and induces BAD-mediated apoptosis in colorectal cancer cells. Int J Cancer 121:2172–2180PubMedCrossRefGoogle Scholar
  15. 15.
    Gunthorpe MJ, Rami HK, Jerman JC, Smart D, Gill CH, Soffin EM, Luis Hannan S, Lappin SC, Egerton J, Smith GD, Worby A, Howett L, Owen D, Nasir S, Davies CH, Thompson M, Wyman PA, Randall AD, Davis JB (2004) Identification and characterisation of SB-366791, a potent and selective vanilloid receptor (VR1/TRPV1) antagonist. Neuropharmacology 46:133–149PubMedCrossRefGoogle Scholar
  16. 16.
    Guzman M (2003) Cannabinoids: potential anticancer agents. Nat Rev Cancer 3:745–755PubMedCrossRefGoogle Scholar
  17. 17.
    Hampson AJ, Hill WA, Zan-Phillips M, Makriyannis A, Leung E, Eglen RM, Bornheim LM (1995) Anandamide hydroxylation by brain lipoxygenase:metabolite structures and potencies at the cannabinoid receptor. Biochim Biophys Acta 1259:173–179PubMedGoogle Scholar
  18. 18.
    Howlett AC, Fleming RM (1984) Cannabinoid inhibition of adenylate cyclase. Pharmacology of the response in neuroblastoma cell membranes. Mol Pharmacol 26:532–538PubMedGoogle Scholar
  19. 19.
    Howlett AC, Qualy JM, Khachatrian LL (1986) Involvement of Gi in the inhibition of adenylate cyclase by cannabimimetic drugs. Mol Pharmacol 29:307–313PubMedGoogle Scholar
  20. 20.
    Howlett AC, Champion-Dorow TM, McMahon LL, Westlake TM (1991) The cannabinoid receptor: biochemical and cellular properties in neuroblastoma cells. Pharmacol Biochem Behav 40:565–569PubMedCrossRefGoogle Scholar
  21. 21.
    Ishii I, Chun J (2002) Anandamide-induced neuroblastoma cell rounding via the CB1 cannabinoid receptors. Neuroreport 13:593–596PubMedCrossRefGoogle Scholar
  22. 22.
    Jacobsson SOP, Rongard E, Stridh M, Tiger G, Fowler CJ (2000) Serum-dependent effects of tamoxifen and cannabinoids upon C6 glioma cell viability. Biochem Pharmacol 60:1807–1813PubMedCrossRefGoogle Scholar
  23. 23.
    Jacobsson SOP, Wallin T, Fowler CJ (2001) Inhibition of rat C6 glioma cell proliferation by endogenous and synthetic cannabinoids. Relative involvement of cannabinoid and vanilloid receptors. J Pharmacol Exp Ther 299:951–959PubMedGoogle Scholar
  24. 24.
    Kaminski NE (1998) Inhibition of the cAMP signaling cascade via cannabinoid receptors: a putative mechanism of immune modulation by cannabinoid compounds. Toxicol Lett 102–103:59–63PubMedCrossRefGoogle Scholar
  25. 25.
    Katayama K, Ueda N, Kurahashi Y, Suzuki H, Yamamoto S, Kato I (1997) Distribution of anandamide amidohydrolase in rat tissues with special reference to small intestine. Biochim Biophys Acta 1347:212–218PubMedGoogle Scholar
  26. 26.
    Kozak KR, Gupta RA, Moody JS, Ji C, Boeglin WE, DuBois RN, Brash AR, Marnett LJ (2002) 15-Lipoxygenase metabolism of 2-arachidonylglycerol. Generation of a peroxisome proliferator-activated receptor alpha agonist. J Biol Chem 277:23278–23286PubMedCrossRefGoogle Scholar
  27. 27.
    Lala PK, Chakraborty C (2001) Role of nitric oxide in carcinogenesis and tumour progression. Lancet Oncol 2:149–156PubMedCrossRefGoogle Scholar
  28. 28.
    Ligresti A, Bisogno T, Matias I, De Petrocellis L, Cascio MG, Cosenza V, D’Argenio G, Scaglione G, Bifulco M, Sorrentini I, Di Marzo V (2003) Possible endocannabinoid control of colorectal cancer growth. Gastroenterology 125:677–687PubMedCrossRefGoogle Scholar
  29. 29.
    Liu J, Li H, Burstein SH, Zurier RB, Chen JD (2003) Activation and binding of peroxisome proliferator-activated receptor gamma by synthetic cannabinoid ajulemic acid. Mol Pharmacol 63:983–992PubMedCrossRefGoogle Scholar
  30. 30.
    Maccarrone M, Lorenzon T, Bari M, Melino G, Finazzi-Agro A (2000) Anandamide induces apoptosis in human cells via vanilloid receptors. Evidence for a protective role of cannabinoid receptors. J Biol Chem 275:31938–31945PubMedCrossRefGoogle Scholar
  31. 31.
    Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner TI (1990) Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 346:561–564PubMedCrossRefGoogle Scholar
  32. 32.
    McKallip RJ, Lombard C, Fisher M, Martin BR, Ryu S, Grant S, Nagarkatti PS, Nagarkatti M (2002) Targeting CB2 cannabinoid receptors as a novel therapy to treat malignant lymphoblastic disease. Blood 100:627–634PubMedCrossRefGoogle Scholar
  33. 33.
    Mimeault M, Pommery N, Wattez N, Bailly C, Henichart JP (2003) Anti-proliferative and apoptotic effects of anandamide in human prostatic cancer cell lines: implication of epidermal growth factor receptor down-regulation and ceramide production. Prostate 56:1–12PubMedCrossRefGoogle Scholar
  34. 34.
    Munro S, Thomas KL, Abu-Shaar M (1993) Molecular characterization of a peripheral receptor for cannabinoids. Nature 365:61–65PubMedCrossRefGoogle Scholar
  35. 35.
    Munson AE, Harris LS, Friedman MA, Dewey WL, Carchman RA (1975) Antineoplastic activity of cannabinoids. J Natl Cancer Inst 55:597–602PubMedGoogle Scholar
  36. 36.
    Nathan C, Xie QW (1994) Nitric oxide synthases: roles, tolls, and controls. Cell 78:915–918PubMedCrossRefGoogle Scholar
  37. 37.
    O’Sullivan SE (2007) Cannabinoids go nuclear: evidence for activation of peroxisome proliferator-activated receptors. Br J Pharmacol 152:576–582PubMedCrossRefGoogle Scholar
  38. 38.
    Pacher P, Batkai S, Kunos G (2006) The endocannabinoid system as an emerging target of pharmacotherapy. Pharmacol Rev 58:389–462PubMedCrossRefGoogle Scholar
  39. 39.
    Patsos HA, Hicks DJ, Greenhough A, Williams AC, Paraskeva C (2005) Cannabinoids and cancer: potential for colorectal cancer therapy. Biochem Soc Trans 33:712–714PubMedCrossRefGoogle Scholar
  40. 40.
    Pertwee RG (1997) Pharmacology of cannabinoid CB1 and CB2 receptors. Pharmacol Ther 74:129–180PubMedCrossRefGoogle Scholar
  41. 41.
    Poblete IM, Orliac ML, Briones R, Adler-Graschinsky E, Huidobro-Toro JP (2005) Anandamide elicits an acute release of nitric oxide through endothelial TRPV1 receptor activation in the rat arterial mesenteric bed. J Physiol 568:539–551PubMedCrossRefGoogle Scholar
  42. 42.
    Ramer R, Weinzierl U, Schwind B, Brune K, Hinz B (2003) Ceramide is involved in r(+)-methanandamide-induced cyclooxygenase-2 expression in human neuroglioma cells. Mol Pharmacol 64:1189–1198PubMedCrossRefGoogle Scholar
  43. 43.
    Rao CV (2004) Nitric oxide signaling in colon cancer chemoprevention. Mutat Res 555:107–119PubMedGoogle Scholar
  44. 44.
    Rueda D, Galve-Roperh I, Haro A, Guzman M (2000) The CB(1) cannabinoid receptor is coupled to the activation of c-Jun N-terminal kinase. Mol Pharmacol 58:814–820PubMedGoogle Scholar
  45. 45.
    Sanchez C, Rueda D, Segui B, Galve-Roperh I, Levade T, Guzman M (2001) The CB(1) cannabinoid receptor of astrocytes is coupled to sphingomyelin hydrolysis through the adaptor protein fan. Mol Pharmacol 59:955–959PubMedGoogle Scholar
  46. 46.
    Smart D, Gunthorpe MJ, Jerman JC, Nasir S, Gray J, Muir AI, Chambers JK, Randall AD, Davis JB (2000) The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1). Br J Pharmacol 129:227–230PubMedCrossRefGoogle Scholar
  47. 47.
    Snider NT, Kornilov AM, Kent UM, Hollenberg PF (2007) Anandamide metabolism by human liver and kidney microsomal cytochrome p450 enzymes to form hydroxyeicosatetraenoic and epoxyeicosatrienoic acid ethanolamides. J Pharmacol Exp Ther 321:590–597PubMedCrossRefGoogle Scholar
  48. 48.
    Tramer MR, Carroll D, Campbell FA, Reynolds DJ, Moore RA, McQuay HJ (2001) Cannabinoids for control of chemotherapy induced nausea and vomiting: quantitative systematic review. BMJ 323:16–21PubMedCrossRefGoogle Scholar
  49. 49.
    Ueda N, Yamamoto K, Kurahashi Y, Yamamoto S, Ogawa M, Matsuki N, Kudo I, Shinkai H, Shirakawa E, Tokunaga T (1995) Oxygenation of arachidonylethanolamide (anandamide) by lipoxygenases. Adv Prostaglandin Thromboxane Leukot Res 23:163–165PubMedGoogle Scholar
  50. 50.
    Velasco G, Galve-Roperh I, Sanchez C, Blazquez C, Haro A, Guzman M (2005) Cannabinoids and ceramide: two lipids acting hand-by-hand. Life Sci 77:1723–1731PubMedCrossRefGoogle Scholar
  51. 51.
    Yu M, Ives D, Ramesha CS (1997) Synthesis of prostaglandin E2 ethanolamide from anandamide by cyclooxygenase-2. J Biol Chem 272:21181–21186PubMedCrossRefGoogle Scholar
  52. 52.
    Zygmunt PM, Petersson J, Andersson DA, Chuang H, Sorgard M, Di Marzo V, Julius D, Hogestatt ED (1999) Vanilloid receptors on sensory nerves mediate the vasodilator action of anandamide. Nature 400:452–457PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Sofia B. Gustafsson
    • 1
    Email author
  • Theres Lindgren
    • 1
  • Maria Jonsson
    • 1
  • Stig O. P. Jacobsson
    • 1
  1. 1.Department of Pharmacology and Clinical NeuroscienceUmeå UniversityUmeaSweden

Personalised recommendations