Breast Cancer Research and Treatment

, Volume 129, Issue 1, pp 37–47 | Cite as

Pathways mediating the effects of cannabidiol on the reduction of breast cancer cell proliferation, invasion, and metastasis

  • Sean D. McAllisterEmail author
  • Ryuichi Murase
  • Rigel T. Christian
  • Darryl Lau
  • Anne J. Zielinski
  • Juanita Allison
  • Carolina Almanza
  • Arash Pakdel
  • Jasmine Lee
  • Chandani Limbad
  • Yong Liu
  • Robert J. Debs
  • Dan H. Moore
  • Pierre-Yves Desprez
Preclinical study


Invasion and metastasis of aggressive breast cancer cells are the final and fatal steps during cancer progression. Clinically, there are still limited therapeutic interventions for aggressive and metastatic breast cancers available. Therefore, effective, targeted, and non-toxic therapies are urgently required. Id-1, an inhibitor of basic helix-loop-helix transcription factors, has recently been shown to be a key regulator of the metastatic potential of breast and additional cancers. We previously reported that cannabidiol (CBD), a cannabinoid with a low toxicity profile, down-regulated Id-1 gene expression in aggressive human breast cancer cells in culture. Using cell proliferation and invasion assays, cell flow cytometry to examine cell cycle and the formation of reactive oxygen species, and Western analysis, we determined pathways leading to the down-regulation of Id-1 expression by CBD and consequently to the inhibition of the proliferative and invasive phenotype of human breast cancer cells. Then, using the mouse 4T1 mammary tumor cell line and the ranksum test, two different syngeneic models of tumor metastasis to the lungs were chosen to determine whether treatment with CBD would reduce metastasis in vivo. We show that CBD inhibits human breast cancer cell proliferation and invasion through differential modulation of the extracellular signal-regulated kinase (ERK) and reactive oxygen species (ROS) pathways, and that both pathways lead to down-regulation of Id-1 expression. Moreover, we demonstrate that CBD up-regulates the pro-differentiation factor, Id-2. Using immune competent mice, we then show that treatment with CBD significantly reduces primary tumor mass as well as the size and number of lung metastatic foci in two models of metastasis. Our data demonstrate the efficacy of CBD in pre-clinical models of breast cancer. The results have the potential to lead to the development of novel non-toxic compounds for the treatment of breast cancer metastasis, and the information gained from these experiments broaden our knowledge of both Id-1 and cannabinoid biology as it pertains to cancer progression.


Id-1 Id-2 Helix-loop-helix Cannabinoid ERK ROS Lung metastasis 







Extracellular signal-regulated kinase


Fetal bovine serum


Inhibitor of DNA binding


Reactive oxygen species





The authors wish to thank Drs. Claudia Gravekamp and Yoko Itahana for helpful scientific discussions, and Dr. Liliana Soroceanu for critical reading of the manuscript. This study was supported by the National Institutes of Health (CA102412, CA111723, DA09978, CA082548, and CA135281), and the Research Institute at California Pacific Medical Center.

Conflict of interest

The authors have declared that no competing interests exist.

Supplementary material

10549_2010_1177_MOESM1_ESM.tif (32 kb)
Figure 1 supplementary. CBD reduces the number of metastatic foci above 1 mm in the syngeneic BALB/c mouse model. Primary tumors were generated in BALB/c mice by subcutaneous injection of 1 × 105 4T1 cells and subsequently developed lung metastases. As described above, treatment with CBD was initiated upon detection of the first palpable tumor. Visible lung metastases were counted and measured by using a dissecting microscope. Lung metastatic foci measured included those (A) <1 mm, (B) 1–2 mm, and (C) > 2 mm. Significant differences were determined using the unpaired Student’s t-test.(TIFF 32 kb)


  1. 1.
    Braun S, Harbeck N (2001) Molecular markers of metastasis in breast cancer: current understanding and prospects for novel diagnosis and prevention. Expert Rev Mol Med 3:1–14PubMedCrossRefGoogle Scholar
  2. 2.
    Perk J, Iavarone A, Benezra R (2005) Id family of helix-loop-helix proteins in cancer. Nat Rev Cancer 5(8):603–614PubMedCrossRefGoogle Scholar
  3. 3.
    Fong S, Itahana Y, Sumida T, Singh J, Coppe JP, Liu Y, Richards PC, Bennington JL, Lee NM, Debs RJ et al (2003) Id-1 as a molecular target in therapy for breast cancer cell invasion and metastasis. Proc Natl Acad Sci USA 100(23):13543–13548PubMedCrossRefGoogle Scholar
  4. 4.
    Minn AJ, Gupta GP, Siegel PM, Bos PD, Shu W, Giri DD, Viale A, Olshen AB, Gerald WL, Massague J (2005) Genes that mediate breast cancer metastasis to lung. Nature 436(7050):518–524PubMedCrossRefGoogle Scholar
  5. 5.
    Coppe JP, Smith AP, Desprez PY (2003) Id proteins in epithelial cells. Exp Cell Res 285(1):131–145PubMedCrossRefGoogle Scholar
  6. 6.
    Gupta GP, Perk J, Acharyya S, de Candia P, Mittal V, Todorova-Manova K, Gerald WL, Brogi E, Benezra R, Massague J (2007) ID genes mediate tumor reinitiation during breast cancer lung metastasis. Proc Natl Acad Sci USA 104(49):19506–19511PubMedCrossRefGoogle Scholar
  7. 7.
    Swarbrick A, Roy E, Allen T, Bishop JM (2008) Id1 cooperates with oncogenic Ras to induce metastatic mammary carcinoma by subversion of the cellular senescence response. Proc Natl Acad Sci USA 105(14):5402–5407PubMedCrossRefGoogle Scholar
  8. 8.
    Pertwee RG (1997) Pharmacology of cannabinoid CB1 and CB2 receptors. Pharmacol Ther 74(November):129–180PubMedCrossRefGoogle Scholar
  9. 9.
    McPartland JM, Russo EB (2001) Cannabis and cannabis extract: greater than the sum of the parts? J Cannabis Ther 1:103–132CrossRefGoogle Scholar
  10. 10.
    McAllister SD, Glass M (2002) CB(1) and CB(2) receptor-mediated signalling: a focus on endocannabinoids. Prostaglandins Leukot Essent Fatty Acids 66(2–3):161–171PubMedCrossRefGoogle Scholar
  11. 11.
    Showalter VM, Compton DR, Martin BR, Abood ME (1996) Evaluation of binding in a transfected cell line expressing a peripheral cannabinoid receptor (CB2): identification of cannabinoid receptor subtype selective ligands. J Pharmacol Exp Ther 278:989–999PubMedGoogle Scholar
  12. 12.
    Alozie SO, Martin BR, Harris LS, Dewey WL (1980) 3H-delta 9-Tetrahydrocannabinol, 3H-cannabinol and 3H-cannabidiol: penetration and regional distribution in rat brain. Pharmacol Biochem Behav 12(2):217–218PubMedCrossRefGoogle Scholar
  13. 13.
    Walter L, Franklin A, Witting A, Wade C, Xie Y, Kunos G, Mackie K, Stella N (2003) Nonpsychotropic cannabinoid receptors regulate microglial cell migration. J Neurosci 23(4):1398–1405PubMedGoogle Scholar
  14. 14.
    Brady KT, Balster RL (1980) The effects of delta 9-tetrahydrocannabinol alone and in combination with cannabidiol on fixed-interval performance in rhesus monkeys. Psychopharmacology 72(1):21–26PubMedCrossRefGoogle Scholar
  15. 15.
    Hiltunen AJ, Jarbe TU (1986) Cannabidiol attenuates delta 9-tetrahydrocannabinol-like discriminative stimulus effects of cannabinol. Eur J Pharmacol 125(2):301–304PubMedCrossRefGoogle Scholar
  16. 16.
    Hiltunen AJ, Jarbe TU, Wangdahl K (1988) Cannabinol and cannabidiol in combination: temperature, open-field activity, and vocalization. Pharmacol Biochem Behav 30(3):675–678PubMedCrossRefGoogle Scholar
  17. 17.
    Hollister LE, Gillespie H (1975) Interactions in man of delta-9-tetrahydrocannabinol. II. cannabinol and cannabidiol. Clin Pharmacol Ther 18(1):80–83PubMedGoogle Scholar
  18. 18.
    Abood ME, Raman C, Kim K, Moore DH (2004) Evaluation of cannabidiol in the ALS mouse model. In: ALS and other motor neuron diseases, 2–4 December 2004. Talyor and Francis, Philadelphia, USA, pp 92–93 (poster 61)Google Scholar
  19. 19.
    Nurmikko TJ, Serpell MG, Hoggart B, Toomey PJ, Morlion BJ, Haines D (2007) Sativex successfully treats neuropathic pain characterised by allodynia: a randomised, double-blind, placebo-controlled clinical trial. Pain 133(1–3):210–220PubMedCrossRefGoogle Scholar
  20. 20.
    Rog DJ, Nurmikko TJ, Young CA (2007) Oromucosal delta9-tetrahydrocannabinol/cannabidiol for neuropathic pain associated with multiple sclerosis: an uncontrolled, open-label, 2-year extension trial. Clin Ther 29(9):2068–2079PubMedCrossRefGoogle Scholar
  21. 21.
    McAllister SD, Christian RT, Horowitz MP, Garcia A, Desprez PY (2007) Cannabidiol as a novel inhibitor of Id-1 gene expression in aggressive breast cancer cells. Mol Cancer Ther 6(11):2921–2927PubMedCrossRefGoogle Scholar
  22. 22.
    Ligresti A, Moriello AS, Starowicz K, Matias I, Pisanti S, De Petrocellis L, Laezza C, Portella G, Bifulco M, Di Marzo V (2006) Antitumor activity of plant cannabinoids with emphasis on the effect of cannabidiol on human breast carcinoma. J Pharmacol Exp Ther 318(3):1375–1387PubMedCrossRefGoogle Scholar
  23. 23.
    Kaplan BL, Springs AE, Kaminski NE (2008) The profile of immune modulation by cannabidiol (CBD) involves deregulation of nuclear factor of activated T cells (NFAT). Biochem Pharmacol 76(6):726–737PubMedCrossRefGoogle Scholar
  24. 24.
    Malfait AM, Gallily R, Sumariwalla PF, Malik AS, Andreakos E, Mechoulam R, Feldmann M (2000) The nonpsychoactive cannabis constituent cannabidiol is an oral anti-arthritic therapeutic in murine collagen-induced arthritis. Proc Natl Acad Sci USA 97(17):9561–9566PubMedCrossRefGoogle Scholar
  25. 25.
    McAllister SD, Chan C, Taft RJ, Luu T, Abood ME, Moore DH, Aldape K, Yount G (2005) Cannabinoids selectively inhibit proliferation and induce death of cultured human glioblastoma multiforme cells. J NeuroOncol 74(1):31–40PubMedCrossRefGoogle Scholar
  26. 26.
    Lin CQ, Singh J, Murata K, Itahana Y, Parrinello S, Liang SH, Gillett CE, Campisi J, Desprez PY (2000) A role for Id-1 in the aggressive phenotype and steroid hormone response of human breast cancer cells. Cancer Res 60(5):1332–1340PubMedGoogle Scholar
  27. 27.
    Guzman M (2003) Cannabinoids: potential anticancer agents. Nat Rev Cancer 3(10):745–755PubMedCrossRefGoogle Scholar
  28. 28.
    McKallip RJ, Jia W, Schlomer J, Warren JW, Nagarkatti PS, Nagarkatti M (2006) Cannabidiol-induced apoptosis in human leukemia cells: a novel role of cannabidiol in the regulation of p22phox and Nox4 expression. Mol Pharmacol 70(3):897–908PubMedCrossRefGoogle Scholar
  29. 29.
    Ramer R, Hinz B (2008) Inhibition of cancer cell invasion by cannabinoids via increased expression of tissue inhibitor of matrix metalloproteinases-1. J Natl Cancer Inst 100(1):59–69PubMedCrossRefGoogle Scholar
  30. 30.
    Itahana Y, Singh J, Sumida T, Coppe JP, Parrinello S, Bennington JL, Desprez PY (2003) Role of Id-2 in the maintenance of a differentiated and noninvasive phenotype in breast cancer cells. Cancer Res 63(21):7098–7105PubMedGoogle Scholar
  31. 31.
    Stighall M, Manetopoulos C, Axelson H, Landberg G (2005) High ID2 protein expression correlates with a favourable prognosis in patients with primary breast cancer and reduces cellular invasiveness of breast cancer cells. Int J Cancer 115(3):403–411PubMedCrossRefGoogle Scholar
  32. 32.
    Massi P, Vaccani A, Bianchessi S, Costa B, Macchi P, Parolaro D (2006) The non-psychoactive cannabidiol triggers caspase activation and oxidative stress in human glioma cells. Cell Mol Life Sci 63(17):2057–2066PubMedCrossRefGoogle Scholar
  33. 33.
    Andersen MH, Sorensen RB, Schrama D, Svane IM, Becker JC, Thor Straten P (2008) Cancer treatment: the combination of vaccination with other therapies. Cancer Immunol Immunother 57(11):1735–1743PubMedCrossRefGoogle Scholar
  34. 34.
    Fong S, Debs RJ, Desprez PY (2004) Id genes and proteins as promising targets in cancer therapy. Trends Mol Med 10(8):387–392PubMedCrossRefGoogle Scholar
  35. 35.
    Ling MT, Wang X, Zhang X, Wong YC (2006) The multiple roles of Id-1 in cancer progression. Differentiation 74(9–10):481–487PubMedCrossRefGoogle Scholar
  36. 36.
    Derkinderen P, Enslen H, Girault JA (1999) The ERK/MAP-kinases cascade in the nervous system. Neuroreport 10(5):R24–R34PubMedGoogle Scholar
  37. 37.
    Pumiglia KM, Decker SJ (1997) Cell cycle arrest mediated by the MEK/mitogen-activated protein kinase pathway. Proc Natl Acad Sci USA 94(2):448–452PubMedCrossRefGoogle Scholar
  38. 38.
    Mohr S, McCormick TS, Lapetina EG (1998) Macrophages resistant to endogenously generated nitric oxide-mediated apoptosis are hypersensitive to exogenously added nitric oxide donors: dichotomous apoptotic response independent of caspase 3 and reversal by the mitogen-activated protein kinase kinase (MEK) inhibitor PD 098059. Proc Natl Acad Sci USA 95(9):5045–5050PubMedCrossRefGoogle Scholar
  39. 39.
    York RD, Yao H, Dillon T, Ellig CL, Eckert SP, McCleskey EW, Stork PJ (1998) Rap1 mediates sustained MAP kinase activation induced by nerve growth factor. Nature 392(6676):622–626PubMedCrossRefGoogle Scholar
  40. 40.
    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(3):313–319PubMedCrossRefGoogle Scholar
  41. 41.
    Sarfaraz S, Afaq F, Adhami VM, Malik A, Mukhtar H (2006) Cannabinoid receptor agonist-induced apoptosis of human prostate cancer cells LNCaP proceeds through sustained activation of ERK1/2 leading to G1 cell cycle arrest. J Biol Chem 281(51):39480–39491PubMedCrossRefGoogle Scholar
  42. 42.
    Velasco G, Galve-Roperh I, Sanchez C, Blazquez C, Guzman M (2004) Hypothesis: cannabinoid therapy for the treatment of gliomas? Neuropharmacology 47(3):315–323PubMedCrossRefGoogle Scholar
  43. 43.
    Mo FM, Offertaler L, Kunos G (2004) Atypical cannabinoid stimulates endothelial cell migration via a Gi/Go-coupled receptor distinct from CB1, CB2 or EDG-1. Eur J Pharmacol 489(1–2):21–27PubMedCrossRefGoogle Scholar
  44. 44.
    Lambeth JD (2004) NOX enzymes and the biology of reactive oxygen. Nat Rev Immunol 4(3):181–189PubMedCrossRefGoogle Scholar
  45. 45.
    Mimeault M, Hauke R, Batra SK (2008) Recent advances on the molecular mechanisms involved in the drug resistance of cancer cells and novel targeting therapies. Clin Pharmacol Ther 83(5):673–691PubMedCrossRefGoogle Scholar
  46. 46.
    Cameron MD, Schmidt EE, Kerkvliet N, Nadkarni KV, Morris VL, Groom AC, Chambers AF, MacDonald IC (2000) Temporal progression of metastasis in lung: cell survival, dormancy, and location dependence of metastatic inefficiency. Cancer Res 60(9):2541–2546PubMedGoogle Scholar
  47. 47.
    Folkman J (1990) What is the evidence that tumors are angiogenesis dependent? J Natl Cancer Inst 82(1):4–6PubMedCrossRefGoogle Scholar
  48. 48.
    Dent P, Curiel DT, Fisher PB, Grant S (2009) Synergistic combinations of signaling pathway inhibitors: mechanisms for improved cancer therapy. Drug Resist Updat 12(3):65–73PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2010

Authors and Affiliations

  • Sean D. McAllister
    • 1
    Email author
  • Ryuichi Murase
    • 1
  • Rigel T. Christian
    • 1
  • Darryl Lau
    • 1
  • Anne J. Zielinski
    • 1
  • Juanita Allison
    • 1
  • Carolina Almanza
    • 1
  • Arash Pakdel
    • 1
  • Jasmine Lee
    • 1
  • Chandani Limbad
    • 1
  • Yong Liu
    • 1
  • Robert J. Debs
    • 1
  • Dan H. Moore
    • 1
  • Pierre-Yves Desprez
    • 1
  1. 1.California Pacific Medical CenterResearch InstituteSan FranciscoUSA

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