Cellular and Molecular Neurobiology

, Volume 37, Issue 1, pp 101–109 | Cite as

A Single Intrathecal or Intraperitoneal Injection of CB2 Receptor Agonist Attenuates Bone Cancer Pain and Induces a Time-Dependent Modification of GRK2

  • Cui’e Lu
  • Linyu Shi
  • Bei Sun
  • Yu Zhang
  • Bailing Hou
  • Yu’e Sun
  • Zhengliang Ma
  • Xiaoping Gu
Original Research


The objective of this study was to explore the potential role of G-protein-coupled receptor kinase 2 (GRK2) in the progression of cannabinoid 2 receptor (CB2) agonist-induced analgesic effects of bone cancer pain. Female Sprague–Dawley rats, weighing 160–180 g, were utilized to establish a model of bone cancer pain induced by intra-tibia inoculation of Walker 256 mammary gland carcinoma cells. JWH-015, a selective CB2 agonist, was injected intrathecally or intraperitoneally on postoperative day 10. Bone cancer-induced pain behaviors—mechanical allodynia and ambulatory pain—were assessed on postoperative days −1 (baseline), 4, 7, and 10 and at post-treatment hours 2, 6, 24, 48, and 72. The expressions of spinal CB2 and GRK2 protein were detected by Western Blotting on postoperative days −1 (baseline), 4, 7, and 10 and at post-treatment hours 6, 24, and 72. The procedure produced prolonged mechanical allodynia, ambulatory pain, and different changes in spinal CB2 and GRK2 expression levels. Intrathecal or intraperitoneal administration of JWH-015 alleviated the induced mechanical allodynia and ambulatory pain, and inhibited the downregulation of spinal GRK2 expression. These effects were in a time-dependent manner and reversed by pretreatment of CB2 selective antagonist AM630. The results affirmed CB2 receptor agonists might serve as new treatment targets for bone cancer pain. Moreover, spinal GRK2 was an important regulator of CB2 receptor agonist-analgesia pathway.


Spinal cord CB2 JWH-015 GRK2 Bone cancer pain 



This research was supported by the National Natural Science Foundation of China (81371207, 81070892, 81171048 and 81171047) and a grant from the Department of Health of Jiangsu Province of China (XK201140, RC2011006).

Authors contributions

All of the authors read and approved the final manuscript. CEL made substantial contributions to the experiments. BLH and LYS were mainly involved in the pain behavioral tests. BS and YZ performed the surgical procedure, administration of drugs, and Western blots studies; YES, CEL, and BLH were responsible for statistical analyses. All of these individuals participated in drafting the manuscript. XPG and ZLM conceived the idea, designed the study, and helped revise the manuscript.

Compliance with Ethical Standards

Conflicts of Interest Statement

All of the authors declare no conflicts of interest.


  1. Atwood BK, Mackie K (2010) CB2: a cannabinoid receptor with an identity crisis. Br J Pharmacol 160:467–479CrossRefPubMedPubMedCentralGoogle Scholar
  2. Bab I, Zimmer A, Melamed E (2009) Cannabinoids and the skeleton: from marijuana to reversal of bone loss. Ann Med 41:560–567CrossRefPubMedGoogle Scholar
  3. Benovic JL, Strasser RH, Caron MG, Lefkowitz RJ (1986) Beta-adrenergic receptor kinase: identification of a novel protein kinase that phosphorylates the agonist-occupied form of the receptor. Proc Natl Acad Sci USA 83:2797–2801CrossRefPubMedPubMedCentralGoogle Scholar
  4. Cabral GA, Griffin-Thomas L (2009) Emerging role of the cannabinoid receptor CB2 in immune regulation: therapeutic prospects for neuroinflammation. Expert Rev Mol Med 11:e3CrossRefPubMedPubMedCentralGoogle Scholar
  5. Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL (1994) Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods 53:55–63CrossRefPubMedGoogle Scholar
  6. Clark AK, Gentry C, Bradbury EJ, McMahon SB, Malcangio M (2007) Role of spinal microglia in rat models of peripheral nerve injury and inflammation. Eur J Pain 11:223–230CrossRefPubMedGoogle Scholar
  7. DeLeo JA, Yezierski RP (2001) The role of neuroinflammation and neuroimmune activation in persistent pain. Pain 90:1–6CrossRefPubMedGoogle Scholar
  8. DeNardo DG, Johansson M, Coussens LM (2008) Immune cells as mediators of solid tumor metastasis. Cancer Metastasis Rev 27:11–18CrossRefPubMedGoogle Scholar
  9. Di Marzo V (2011) Endocannabinoid signaling in the brain: biosynthetic mechanisms in the limelight. Nat Neurosci 14:9–15CrossRefPubMedGoogle Scholar
  10. Eijkelkamp N, Heijnen CJ, Willemen HL, Deumens R, Joosten EA, Kleibeuker W et al (2010) GRK2: a novel cell-specific regulator of severity and duration of inflammatory pain. J Neurosci 30:2138–2149CrossRefPubMedPubMedCentralGoogle Scholar
  11. Facci L, Dal Toso R, Romanello S, Buriani A, Skaper SD, Leon A (1995) Mast cells express a peripheral cannabinoid receptor with differential sensitivity to anandamide and palmitoylethanolamide. Proc Natl Acad Sci USA 92:3376–3380CrossRefPubMedPubMedCentralGoogle Scholar
  12. Fernandez-Ruiz J (2009) The endocannabinoid system as a target for the treatment of motor dysfunction. Br J Pharmacol 156:1029–1040CrossRefPubMedPubMedCentralGoogle Scholar
  13. Fields RD, Stevens-Graham B (2002) New insights into neuron-glia communication. Science 298:556–562CrossRefPubMedPubMedCentralGoogle Scholar
  14. Gao YJ, Ji RR (2009) c-Fos and pERK, which is a better marker for neuronal activation and central sensitization after noxious stimulation and tissue injury? Open Pain J 2:11–17CrossRefPubMedPubMedCentralGoogle Scholar
  15. Garcia-Gutierrez MS, Ortega-Alvaro A, Busquets-Garcia A, Perez-Ortiz JM, Caltana L, Ricatti MJ et al (2013) Synaptic plasticity alterations associated with memory impairment induced by deletion of CB2 cannabinoid receptors. Neuropharmacology 73:388–396CrossRefPubMedGoogle Scholar
  16. Gu X, Zhang J, Ma Z, Wang J, Zhou X, Jin Y et al (2010) The role of N-methyl-D-aspartate receptor subunit NR2B in spinal cord in cancer pain. Eur J Pain 14:496–502CrossRefPubMedGoogle Scholar
  17. Gu X, Mei F, Liu Y, Zhang R, Zhang J, Ma Z (2011) Intrathecal administration of the cannabinoid 2 receptor agonist JWH015 can attenuate cancer pain and decrease mRNA expression of the 2B subunit of N-methyl-D-aspartic acid. Anesth Analg 113:405–411CrossRefPubMedGoogle Scholar
  18. Guo W, Wang H, Watanabe M, Shimizu K, Zou S, LaGraize SC et al (2007) Glial-cytokine-neuronal interactions underlying the mechanisms of persistent pain. J Neurosci 27:6006–6018CrossRefPubMedPubMedCentralGoogle Scholar
  19. Hausdorff WP, Caron MG, Lefkowitz RJ (1990) Turning off the signal: desensitization of beta-adrenergic receptor function. FASEB J 4:2881–2889PubMedGoogle Scholar
  20. Homan KT, Glukhova A, Tesmer JJ (2013) Regulation of G protein-coupled receptor kinases by phospholipids. Curr Med Chem 20:39–46CrossRefPubMedGoogle Scholar
  21. Howlett AC (1995) Pharmacology of cannabinoid receptors. Annu Rev Pharmacol Toxicol 35:607–634CrossRefPubMedGoogle Scholar
  22. Howlett AC, Barth F, Bonner TI, Cabral G, Casellas P, Devane WA et al (2002) International Union of Pharmacology. XXVII. Classification of cannabinoid receptors. Pharmacol Rev 54:161–202CrossRefPubMedGoogle Scholar
  23. Hylden JL, Wilcox GL (1980) Intrathecal morphine in mice: a new technique. Eur J Pharmacol 67:313–316CrossRefPubMedGoogle Scholar
  24. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D (2011) Global cancer statistics. CA Cancer J Clin 61:69–90CrossRefPubMedGoogle Scholar
  25. Jimenez-Sainz MC, Murga C, Kavelaars A, Jurado-Pueyo M, Krakstad BF, Heijnen CJ et al (2006) G protein-coupled receptor kinase 2 negatively regulates chemokine signaling at a level downstream from G protein subunits. Mol Biol Cell 17:25–31CrossRefPubMedPubMedCentralGoogle Scholar
  26. Kawasaki Y, Xu ZZ, Wang X, Park JY, Zhuang ZY, Tan PH et al (2008) Distinct roles of matrix metalloproteases in the early- and late-phase development of neuropathic pain. Nat Med 14:331–336CrossRefPubMedPubMedCentralGoogle Scholar
  27. Khan N, Afaq F, Mukhtar H (2010) Lifestyle as risk factor for cancer: evidence from human studies. Cancer Lett 293:133–143CrossRefPubMedPubMedCentralGoogle Scholar
  28. Kleibeuker W, Ledeboer A, Eijkelkamp N, Watkins LR, Maier SF, Zijlstra J et al (2007) A role for G protein-coupled receptor kinase 2 in mechanical allodynia. Eur J Neurosci 25:1696–1704CrossRefPubMedGoogle Scholar
  29. Kleibeuker W, Gabay E, Kavelaars A, Zijlstra J, Wolf G, Ziv N et al (2008) IL-1 beta signaling is required for mechanical allodynia induced by nerve injury and for the ensuing reduction in spinal cord neuronal GRK2. Brain Behav Immun 22:200–208CrossRefPubMedGoogle Scholar
  30. Lee S, Zhao YQ, Ribeiro-da-Silva A, Zhang J (2010) Distinctive response of CNS glial cells in oro-facial pain associated with injury, infection and inflammation. Mol Pain 6:79CrossRefPubMedPubMedCentralGoogle Scholar
  31. Lohse MJ, Benovic JL, Codina J, Caron MG, Lefkowitz RJ (1990) beta-Arrestin: a protein that regulates beta-adrenergic receptor function. Science 248:1547–1550CrossRefPubMedGoogle Scholar
  32. Lombardi MS, Kavelaars A, Heijnen CJ (2002) Role and modulation of G protein-coupled receptor signaling in inflammatory processes. Crit Rev Immunol 22:141–163CrossRefPubMedGoogle Scholar
  33. Lozano-Ondoua AN, Hanlon KE, Symons-Liguori AM, Largent-Milnes TM, Havelin JJ, Ferland HL et al (2013) Disease modification of breast cancer-induced bone remodeling by cannabinoid 2 receptor agonists. J Bone Miner Res 28:92–107CrossRefPubMedPubMedCentralGoogle Scholar
  34. Lu C, Liu Y, Sun B, Sun Y, Hou B, Zhang Y et al (2015) Intrathecal injection of JWH-015 attenuates bone cancer pain via time-dependent modification of pro-inflammatory cytokines expression and astrocytes activity in spinal cord. Inflammation 38:1880–1890CrossRefPubMedGoogle Scholar
  35. Mao-Ying QL, Zhao J, Dong ZQ, Wang J, Yu J, Yan MF et al (2006) A rat model of bone cancer pain induced by intra-tibia inoculation of Walker 256 mammary gland carcinoma cells. Biochem Biophys Res Commun 345:1292–1298CrossRefPubMedGoogle Scholar
  36. Marrs WR, Blankman JL, Horne EA, Thomazeau A, Lin YH, Coy J et al (2010) The serine hydrolase ABHD6 controls the accumulation and efficacy of 2-AG at cannabinoid receptors. Nat Neurosci 13:951–957CrossRefPubMedPubMedCentralGoogle Scholar
  37. Medhurst SJ, Walker K, Bowes M, Kidd BL, Glatt M, Muller M et al (2002) A rat model of bone cancer pain. Pain 96:129–140CrossRefPubMedGoogle Scholar
  38. Milligan ED, Watkins LR (2009) Pathological and protective roles of glia in chronic pain. Nat Rev Neurosci 10:23–36CrossRefPubMedPubMedCentralGoogle Scholar
  39. Peregrin S, Jurado-Pueyo M, Campos PM, Sanz-Moreno V, Ruiz-Gomez A, Crespo P et al (2006) Phosphorylation of p38 by GRK2 at the docking groove unveils a novel mechanism for inactivating p38MAPK. Curr Biol 16:2042–2047CrossRefPubMedGoogle Scholar
  40. Pertwee RG (2012) Targeting the endocannabinoid system with cannabinoid receptor agonists: pharmacological strategies and therapeutic possibilities. Philos Trans R Soc Lond B Biol Sci 367:3353–3363CrossRefPubMedPubMedCentralGoogle Scholar
  41. Porcile C, Bajetto A, Barbero S, Pirani P, Schettini G (2004) CXCR4 activation induces epidermal growth factor receptor transactivation in an ovarian cancer cell line. Ann N Y Acad Sci 1030:162–169CrossRefPubMedGoogle Scholar
  42. Reiter E, Lefkowitz RJ (2006) GRKs and beta-arrestins: roles in receptor silencing, trafficking and signaling. Trends Endocrinol Metab 17:159–165CrossRefPubMedGoogle Scholar
  43. Ren BX, Gu XP, Zheng YG, Liu CL, Wang D, Sun YE et al (2012) Intrathecal injection of metabotropic glutamate receptor subtype 3 and 5 agonist/antagonist attenuates bone cancer pain by inhibition of spinal astrocyte activation in a mouse model. Anesthesiology 116:122–132CrossRefPubMedGoogle Scholar
  44. Ribas C, Penela P, Murga C, Salcedo A, Garcia-Hoz C, Jurado-Pueyo M et al (2007) The G protein-coupled receptor kinase (GRK) interactome: role of GRKs in GPCR regulation and signaling. Biochim Biophys Acta 1768:913–922CrossRefPubMedGoogle Scholar
  45. Romero-Sandoval A, Eisenach JC (2007) Spinal cannabinoid receptor type 2 activation reduces hypersensitivity and spinal cord glial activation after paw incision. Anesthesiology 106:787–794CrossRefPubMedGoogle Scholar
  46. Romero-Sandoval A, Nutile-McMenemy N, DeLeo JA (2008) Spinal microglial and perivascular cell cannabinoid receptor type 2 activation reduces behavioral hypersensitivity without tolerance after peripheral nerve injury. Anesthesiology 108:722–734CrossRefPubMedPubMedCentralGoogle Scholar
  47. Sophocleous A, Landao-Bassonga E, Van’t Hof RJ, Idris AI, Ralston SH (2011) The type 2 cannabinoid receptor regulates bone mass and ovariectomy-induced bone loss by affecting osteoblast differentiation and bone formation. Endocrinology 152:2141–2149CrossRefPubMedGoogle Scholar
  48. Sun Y, Zhang W, Liu Y, Liu X, Ma Z, Gu X (2014) Intrathecal injection of JWH015 attenuates remifentanil-induced postoperative hyperalgesia by inhibiting activation of spinal glia in a rat model. Anesth Analg 118:841–853CrossRefPubMedGoogle Scholar
  49. van den Beuken-van Everdingen MH, de Rijke JM, Kessels AG, Schouten HC, van Kleef M, Patijn J (2007) Prevalence of pain in patients with cancer: a systematic review of the past 40 years. Ann Oncol 18:1437–1449CrossRefPubMedGoogle Scholar
  50. Vroon A, Heijnen CJ, Lombardi MS, Cobelens PM, Mayor F Jr, Caron MG et al (2004) Reduced GRK2 level in T cells potentiates chemotaxis and signaling in response to CCL4. J Leukoc Biol 75:901–909CrossRefPubMedGoogle Scholar
  51. Vroon A, Heijnen CJ, Kavelaars A (2006) GRKs and arrestins: regulators of migration and inflammation. J Leukoc Biol 80:1214–1221CrossRefPubMedGoogle Scholar
  52. Wang H, Heijnen CJ, van Velthoven CT, Willemen HL, Ishikawa Y, Zhang X et al (2013) Balancing GRK2 and EPAC1 levels prevents and relieves chronic pain. J Clin Investig 123:5023–5034CrossRefPubMedPubMedCentralGoogle Scholar
  53. Won KA, Kim MJ, Yang KY, Park JS, Lee MK, Park MK et al (2014) The glial-neuronal GRK2 pathway participates in the development of trigeminal neuropathic pain in rats. J Pain 15:250–261CrossRefPubMedGoogle Scholar
  54. Woolf CJ, Ma Q (2007) Nociceptors–noxious stimulus detectors. Neuron 55:353–364CrossRefPubMedGoogle Scholar
  55. Yao BB, Hsieh GC, Frost JM, Fan Y, Garrison TR, Daza AV et al (2008) In vitro and in vivo characterization of A-796260: a selective cannabinoid CB2 receptor agonist exhibiting analgesic activity in rodent pain models. Br J Pharmacol 153:390–401CrossRefPubMedGoogle Scholar
  56. Zhang R, Lao L (2012) A new rat model of bone cancer pain. Methods Mol Biol 851:261–273CrossRefPubMedGoogle Scholar
  57. Zhu GQ, Liu S, He DD, Liu YP, Song XJ (2014) Activation of the cAMP-PKA signaling pathway in rat dorsal root ganglion and spinal cord contributes toward induction and maintenance of bone cancer pain. Behav Pharmacol 25:267–276CrossRefPubMedGoogle Scholar
  58. Zimmermann M (1983) Ethical guidelines for investigations of experimental pain in conscious animals. Pain 16:109–110CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of AnesthesiologyAffiliated Drum-Tower Hospital of Medical College of Nanjing UniversityNanjingChina

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