Clinical & Experimental Metastasis

, Volume 29, Issue 6, pp 585–601 | Cite as

Targeting monocyte chemotactic protein-1 synthesis with bindarit induces tumor regression in prostate and breast cancer animal models

  • Massimo ZolloEmail author
  • Valeria Di Dato
  • Daniela Spano
  • Daniela De Martino
  • Lucia Liguori
  • Natascia Marino
  • Viviana Vastolo
  • Luigi Navas
  • Beatrice Garrone
  • Giorgina Mangano
  • Giuseppe Biondi
  • Angelo Guglielmotti
Research Paper


Prostate and breast cancer are major causes of death worldwide, mainly due to patient relapse upon disease recurrence through formation of metastases. Chemokines are small proteins with crucial roles in the immune system, and their regulation is finely tuned in early inflammatory responses. They are key molecules during inflammatory processes, and many studies are focusing on their regulatory functions in tumor growth and angiogenesis during metastatic cell seeding and spreading. Bindarit is an anti-inflammatory indazolic derivative that can inhibit the synthesis of MCP-1/CCL2, with a potential inhibitory function in tumor progression and metastasis formation. We show here that in vitro, bindarit can modulate cancer-cell proliferation and migration, mainly through negative regulation of TGF-β and AKT signaling, and it can impair the NF-κB signaling pathway through enhancing the expression of the NF-κB inhibitor IkB-α. In vivo administration of bindarit results in impaired metastatic disease in prostate cancer xenograft mice (PC-3M-Luc2 cells injected intra-cardially) and impairment of local tumorigenesis in syngeneic Balb/c mice injected under the mammary gland with murine breast cancer cells (4T1-Luc cells). In addition, bindarit treatment significantly decreases the infiltration of tumor-associated macrophages and myeloid-derived suppressor cells in 4T1-Luc primary tumors. Overall, our data indicate that bindarit is a good candidate for new therapies against prostate and breast tumorigenesis, with an action through impairment of inflammatory cell responses during formation of the tumor–stroma niche microenvironment.


Prostate Breast Tumor-associated macrophages Myeloid-derived suppressor cells MCP-1/CCL2 Xenograft 



We thank the Core Services platforms of CEINGE Laboratories, and Prof. Luigi del Vecchio, Head of the Cell Sorter Facility, and its facility for helpful and critical discussions. This study was funded by Angelini Grants ACRAF 004FA09369, 004FA09383 (MZ) and the European GRANT-FP7-Tumic HEALTH-F2-2008-201662 (MZ). VDD was supported by Fondazione San Paolo (IM) and Tumic EU-FP7, DS was supported by the Dipartimento di Biochimica e Biotecnologie Mediche, ‘Federico II’ University of Naples, DMD was supported by Dottorato in Medicina Molecolare e Genetica, ‘Federico II’ University of Naples, and LL was supported by Scuola di Specializzazione in Genetica Medica, ‘Federico II’ University of Naples, Italy.

Supplementary material

10585_2012_9473_MOESM1_ESM.doc (50 kb)
Supplementary material 1 (DOC 51 kb)
10585_2012_9473_MOESM2_ESM.doc (66 kb)
Supplementary material 2 (DOC 66 kb)
10585_2012_9473_MOESM3_ESM.doc (50 kb)
Supplementary material 3 (DOC 51 kb)
10585_2012_9473_MOESM4_ESM.tif (16.2 mb)
Supplementary material 4 (TIFF 16589 kb)
10585_2012_9473_MOESM5_ESM.tif (16.2 mb)
Supplementary material 5 (TIFF 16587 kb)
10585_2012_9473_MOESM6_ESM.tif (16.2 mb)
Supplementary material 6 (TIFF 16587 kb)


  1. 1.
    Havens AM et al (2008) An in vivo mouse model for human prostate cancer metastasis. Neoplasia 10(4):371–380PubMedGoogle Scholar
  2. 2.
    Spano D, Zollo M (2012) Tumor microenvironment: a main actor in the metastasis process. Clin Exp Metastasis 29(4):381–395PubMedCrossRefGoogle Scholar
  3. 3.
    Pikarsky E et al (2004) NF-kappaB functions as a tumour promoter in inflammation-associated cancer. Nature 431(7007):461–466PubMedCrossRefGoogle Scholar
  4. 4.
    Mizutani K et al (2009) The chemokine CCL2 increases prostate tumor growth and bone metastasis through macrophage and osteoclast recruitment. Neoplasia 11(11):1235–1242PubMedGoogle Scholar
  5. 5.
    Aggarwal MM et al (2010) Measurement of the bottom quark contribution to nonphotonic electron production in p + p collisions at sqrt[s] = 200 GeV. Phys Rev Lett 105(20):202301PubMedCrossRefGoogle Scholar
  6. 6.
    Lu X, Kang Y (2009) Chemokine (C-C motif) ligand 2 engages CCR2+ stromal cells of monocytic origin to promote breast cancer metastasis to lung and bone. J Biol Chem 284(42):29087–29096PubMedCrossRefGoogle Scholar
  7. 7.
    Zhang J, Lu Y, Pienta KJ (2010) Multiple roles of chemokine (C-C motif) ligand 2 in promoting prostate cancer growth. J Natl Cancer Inst 102(8):522–528PubMedCrossRefGoogle Scholar
  8. 8.
    Takahashi M et al (2009) Chemokine CCL2/MCP-1 negatively regulates metastasis in a highly bone marrow-metastatic mouse breast cancer model. Clin Exp Metastasis 26(7):817–828PubMedCrossRefGoogle Scholar
  9. 9.
    Nam JS et al (2006) Chemokine (C-C motif) ligand 2 mediates the prometastatic effect of dysadherin in human breast cancer cells. Cancer Res 66(14):7176–7184PubMedCrossRefGoogle Scholar
  10. 10.
    Zhang J, Patel L, Pienta KJ (2010) CC chemokine ligand 2 (CCL2) promotes prostate cancer tumorigenesis and metastasis. Cytokine Growth Factor Rev 21(1):41–48PubMedCrossRefGoogle Scholar
  11. 11.
    Loberg RD et al (2007) CCL2 as an important mediator of prostate cancer growth in vivo through the regulation of macrophage infiltration. Neoplasia 9(7):556–562PubMedCrossRefGoogle Scholar
  12. 12.
    Molloy AP et al (2009) Mesenchymal stem cell secretion of chemokines during differentiation into osteoblasts, and their potential role in mediating interactions with breast cancer cells. Int J Cancer 124(2):326–332PubMedCrossRefGoogle Scholar
  13. 13.
    Qian BZ et al (2011) CCL2 recruits inflammatory monocytes to facilitate breast-tumour metastasis. Nature 475(7355):222–225PubMedCrossRefGoogle Scholar
  14. 14.
    Salcedo R et al (2000) Human endothelial cells express CCR2 and respond to MCP-1: direct role of MCP-1 in angiogenesis and tumor progression. Blood 96(1):34–40PubMedGoogle Scholar
  15. 15.
    Saji H et al (2001) Significant correlation of monocyte chemoattractant protein-1 expression with neovascularization and progression of breast carcinoma. Cancer 92(5):1085–1091PubMedCrossRefGoogle Scholar
  16. 16.
    Mirolo M et al (2008) Impact of the anti-inflammatory agent bindarit on the chemokinome: selective inhibition of the monocyte chemotactic proteins. Eur Cytokine Netw 19(3):119–122PubMedGoogle Scholar
  17. 17.
    Mora E et al (2012) Bindarit: an anti-inflammatory small molecule that modulates the NFkappaB pathway. Cell Cycle 11(1):159–169PubMedCrossRefGoogle Scholar
  18. 18.
    Bhatia M et al (2008) Treatment with bindarit, an inhibitor of MCP-1 synthesis, protects mice against trinitrobenzene sulfonic acid-induced colitis. Inflamm Res 57(10):464–471PubMedCrossRefGoogle Scholar
  19. 19.
    Bhatia M et al (2005) Treatment with bindarit, a blocker of MCP-1 synthesis, protects mice against acute pancreatitis. Am J Physiol Gastrointest Liver Physiol 288(6):G1259–G1265PubMedCrossRefGoogle Scholar
  20. 20.
    Ble A et al (2011) Antiproteinuric effect of chemokine C-C motif ligand 2 inhibition in subjects with acute proliferative lupus nephritis. Am J Nephrol 34(4):367–372PubMedCrossRefGoogle Scholar
  21. 21.
    Guglielmotti A et al (2002) Amelioration of rat adjuvant arthritis by therapeutic treatment with bindarit, an inhibitor of MCP-1 and TNF-alpha production. Inflamm Res 51(5):252–258PubMedCrossRefGoogle Scholar
  22. 22.
    Ialenti A et al (2011) Inhibition of in-stent stenosis by oral administration of bindarit in porcine coronary arteries. Arterioscler Thromb Vasc Biol 31(11):2448–2454PubMedCrossRefGoogle Scholar
  23. 23.
    Rulli NE et al (2009) Amelioration of alphavirus-induced arthritis and myositis in a mouse model by treatment with bindarit, an inhibitor of monocyte chemotactic proteins. Arthritis Rheum 60(8):2513–2523PubMedCrossRefGoogle Scholar
  24. 24.
    Rulli NE et al (2011) Protection from arthritis and myositis in a mouse model of acute chikungunya virus disease by bindarit, an inhibitor of monocyte chemotactic protein-1 synthesis. J Infect Dis 204(7):1026–1030PubMedCrossRefGoogle Scholar
  25. 25.
    Zhou GX et al (2010) Protective effects of MCP-1 inhibitor on a rat model of severe acute pancreatitis. Hepatobiliary Pancreat Dis Int 9(2):201–207PubMedGoogle Scholar
  26. 26.
    Gazzaniga S et al (2007) Targeting tumor-associated macrophages and inhibition of MCP-1 reduce angiogenesis and tumor growth in a human melanoma xenograft. J Invest Dermatol 127(8):2031–2041PubMedCrossRefGoogle Scholar
  27. 27.
    D’Angelo A, Garzia L, André A, Carotenuto P, Aglio V, Guardiola O, Arrigoni G, Cossu A, Palmieri G, Aravind L, Zollo M (2004) Prune cAMP phosphodiesterase binds nm23-H1 and promotes cancer metastasis. Cancer Cell 5(2):137–149PubMedCrossRefGoogle Scholar
  28. 28.
    Roca H, Varsos Z, Pienta KJ (2008) CCL2 protects prostate cancer PC3 cells from autophagic death via phosphatidylinositol 3-kinase/AKT-dependent survivin up-regulation. J Biol Chem 283(36):25057–25073PubMedCrossRefGoogle Scholar
  29. 29.
    Roca H et al (2008) CCL2, survivin and autophagy: new links with implications in human cancer. Autophagy 4(7):969–971PubMedGoogle Scholar
  30. 30.
    Yang L et al (2008) Abrogation of TGF beta signaling in mammary carcinomas recruits Gr-1+CD11b+ myeloid cells that promote metastasis. Cancer Cell 13(1):23–35PubMedCrossRefGoogle Scholar
  31. 31.
    Mantovani A (2010) Molecular pathways linking inflammation and cancer. Curr Mol Med 10(4):369–373PubMedCrossRefGoogle Scholar
  32. 32.
    Balkwill F (2004) Cancer and the chemokine network. Nat Rev Cancer 4(7):540–550PubMedCrossRefGoogle Scholar
  33. 33.
    Lazennec G, Richmond A (2010) Chemokines and chemokine receptors: new insights into cancer-related inflammation. Trends Mol Med 16(3):133–144PubMedCrossRefGoogle Scholar
  34. 34.
    Monti P et al (2003) The CC chemokine MCP-1/CCL2 in pancreatic cancer progression: regulation of expression and potential mechanisms of antimalignant activity. Cancer Res 63(21):7451–7461PubMedGoogle Scholar
  35. 35.
    Fujimoto H et al (2009) Stromal MCP-1 in mammary tumors induces tumor-associated macrophage infiltration and contributes to tumor progression. Int J Cancer 125(6):1276–1284PubMedCrossRefGoogle Scholar
  36. 36.
    Ueno T et al (2000) Significance of macrophage chemoattractant protein-1 in macrophage recruitment, angiogenesis, and survival in human breast cancer. Clin Cancer Res 6(8):3282–3289PubMedGoogle Scholar
  37. 37.
    Spano D , Heckc C, De Antonellisa P, Christofori G, Zollo M (2012). Molecular Networks that Regulate Cancer Metastasis. Semin Cancer Biol. doi: 10.1016/j.semcancer.2012.03.006. Accessed 30 March 2012PubMedGoogle Scholar
  38. 38.
    Soria G, Ben-Baruch A (2008) The inflammatory chemokines CCL2 and CCL5 in breast cancer. Cancer Lett 267(2):271–285PubMedCrossRefGoogle Scholar
  39. 39.
    Li X et al (2009) A destructive cascade mediated by CCL2 facilitates prostate cancer growth in bone. Cancer Res 69(4):1685–1692PubMedCrossRefGoogle Scholar
  40. 40.
    Lu Y et al (2009) Activation of MCP-1/CCR2 axis promotes prostate cancer growth in bone. Clin Exp Metastasis 26(2):161–169PubMedCrossRefGoogle Scholar
  41. 41.
    Soria G et al (2008) Concomitant expression of the chemokines RANTES and MCP-1 in human breast cancer: a basis for tumor-promoting interactions. Cytokine 44(1):191–200PubMedCrossRefGoogle Scholar
  42. 42.
    Lu Y et al (2006) Monocyte chemotactic protein-1 (MCP-1) acts as a paracrine and autocrine factor for prostate cancer growth and invasion. Prostate 66(12):1311–1318PubMedCrossRefGoogle Scholar
  43. 43.
    Lu Y et al (2007) CCR2 expression correlates with prostate cancer progression. J Cell Biochem 101(3):676–685PubMedCrossRefGoogle Scholar
  44. 44.
    Lu Y et al (2007) Monocyte chemotactic protein-1 mediates prostate cancer-induced bone resorption. Cancer Res 67(8):3646–3653PubMedCrossRefGoogle Scholar
  45. 45.
    Izhak L et al (2012) Dissecting the autocrine and paracrine roles of the CCR2-CCL2 axis in tumor survival and angiogenesis. PLoS One 7(1):e28305PubMedCrossRefGoogle Scholar
  46. 46.
    van Golen KL et al (2008) CCL2 induces prostate cancer transendothelial cell migration via activation of the small GTPase Rac. J Cell Biochem 104(5):1587–1597PubMedCrossRefGoogle Scholar
  47. 47.
    Lu Y et al (2007) PTHrP-induced MCP-1 production by human bone marrow endothelial cells and osteoblasts promotes osteoclast differentiation and prostate cancer cell proliferation and invasion in vitro. Int J Cancer 121(4):724–733PubMedCrossRefGoogle Scholar
  48. 48.
    Qian DZ et al (2010) CCL2 is induced by chemotherapy and protects prostate cancer cells from docetaxel-induced cytotoxicity. Prostate 70(4):433–442PubMedGoogle Scholar
  49. 49.
    Hembruff SL et al (2010) Loss of transforming growth factor-beta signaling in mammary fibroblasts enhances CCL2 secretion to promote mammary tumor progression through macrophage-dependent and -independent mechanisms. Neoplasia 12(5):425–433PubMedGoogle Scholar
  50. 50.
    Loberg RD et al (2007) Targeting CCL2 with systemic delivery of neutralizing antibodies induces prostate cancer tumor regression in vivo. Cancer Res 67(19):9417–9424PubMedCrossRefGoogle Scholar
  51. 51.
    Zoja C et al (1998) Bindarit retards renal disease and prolongs survival in murine lupus autoimmune disease. Kidney Int 53(3):726–734PubMedCrossRefGoogle Scholar
  52. 52.
    Guglielmotti A et al (1993) Chronic inflammatory response in the rat can be blocked by bindarit. Biochem Mol Biol Int 29(4):747–756PubMedGoogle Scholar
  53. 53.
    Cioli V et al (1992) A new protein antidenaturant agent, bindarit, reduces secondary phase of adjuvant arthritis in rats. J Rheumatol 19(11):1735–1742PubMedGoogle Scholar
  54. 54.
    Craig MJ, Loberg RD (2006) CCL2 (monocyte chemoattractant protein-1) in cancer bone metastases. Cancer Metastasis Rev 25(4):611–619PubMedCrossRefGoogle Scholar
  55. 55.
    Rodrigues LR et al (2007) The role of osteopontin in tumor progression and metastasis in breast cancer. Cancer Epidemiol Biomarkers Prev 16(6):1087–1097PubMedCrossRefGoogle Scholar
  56. 56.
    Rittling SR, Chambers AF (2004) Role of osteopontin in tumour progression. Br J Cancer 90(10):1877–1881PubMedCrossRefGoogle Scholar
  57. 57.
    Solinas G et al (2010) Tumor-conditioned macrophages secrete migration-stimulating factor: a new marker for M2-polarization, influencing tumor cell motility. J Immunol 185(1):642–652PubMedCrossRefGoogle Scholar
  58. 58.
    DuPre SA, Redelman D, Hunter KW Jr (2007) The mouse mammary carcinoma 4T1: characterization of the cellular landscape of primary tumours and metastatic tumour foci. Int J Exp Pathol 88(5):351–360PubMedCrossRefGoogle Scholar
  59. 59.
    Morales JK et al (2010) GM-CSF is one of the main breast tumor-derived soluble factors involved in the differentiation of CD11b-Gr1- bone marrow progenitor cells into myeloid-derived suppressor cells. Breast Cancer Res Treat 123(1):39–49PubMedCrossRefGoogle Scholar
  60. 60.
    Roland CL et al (2009) Inhibition of vascular endothelial growth factor reduces angiogenesis and modulates immune cell infiltration of orthotopic breast cancer xenografts. Mol Cancer Ther 8(7):1761–1771PubMedCrossRefGoogle Scholar
  61. 61.
    Melancon MP et al (2010) Targeted imaging of tumor-associated M2 macrophages using a macromolecular contrast agent PG-Gd-NIR813. Biomaterials 31(25):6567–6573PubMedCrossRefGoogle Scholar
  62. 62.
    Polfliet MM et al (2006) The rat macrophage scavenger receptor CD163: expression, regulation and role in inflammatory mediator production. Immunobiology 211(6–8):419–425PubMedCrossRefGoogle Scholar
  63. 63.
    van Dongen M et al (2010) Anti-inflammatory M2 type macrophages characterize metastasized and tyrosine kinase inhibitor-treated gastrointestinal stromal tumors. Int J Cancer 127(4):899–909PubMedGoogle Scholar
  64. 64.
    Sanchez-Martin L et al (2011) The chemokine CXCL12 regulates monocyte-macrophage differentiation and RUNX3 expression. Blood 117(1):88–97PubMedCrossRefGoogle Scholar
  65. 65.
    Shabo I et al (2008) Breast cancer expression of CD163, a macrophage scavenger receptor, is related to early distant recurrence and reduced patient survival. Int J Cancer 123(4):780–786PubMedCrossRefGoogle Scholar
  66. 66.
    Asano K et al (2011) CD169-positive macrophages dominate antitumor immunity by crosspresenting dead cell-associated antigens. Immunity 34(1):85–95PubMedCrossRefGoogle Scholar
  67. 67.
    Lau SK, Chu PG, Weiss LM (2004) CD163: a specific marker of macrophages in paraffin-embedded tissue samples. Am J Clin Pathol 122(5):794–801PubMedCrossRefGoogle Scholar
  68. 68.
    Barral P et al (2010) CD169(+) macrophages present lipid antigens to mediate early activation of iNKT cells in lymph nodes. Nat Immunol 11(4):303–312PubMedCrossRefGoogle Scholar
  69. 69.
    Allavena P et al (2008) The Yin-Yang of tumor-associated macrophages in neoplastic progression and immune surveillance. Immunol Rev 222:155–161PubMedCrossRefGoogle Scholar
  70. 70.
    Roy PG, Thompson AM (2006) Cyclin D1 and breast cancer. Breast 15(6):718–727PubMedCrossRefGoogle Scholar
  71. 71.
    Fu M et al (2004) Minireview: cyclin D1: normal and abnormal functions. Endocrinology 145(12):5439–5447PubMedCrossRefGoogle Scholar
  72. 72.
    Neumeister P et al (2003) Cyclin D1 governs adhesion and motility of macrophages. Mol Biol Cell 14(5):2005–2015PubMedCrossRefGoogle Scholar
  73. 73.
    Tsou CL et al (2007) Critical roles for CCR2 and MCP-3 in monocyte mobilization from bone marrow and recruitment to inflammatory sites. J Clin Invest 117(4):902–909PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Massimo Zollo
    • 1
    • 2
    Email author
  • Valeria Di Dato
    • 1
    • 2
  • Daniela Spano
    • 1
    • 2
  • Daniela De Martino
    • 1
    • 2
  • Lucia Liguori
    • 1
  • Natascia Marino
    • 1
    • 2
  • Viviana Vastolo
    • 1
    • 2
  • Luigi Navas
    • 1
    • 4
  • Beatrice Garrone
    • 3
  • Giorgina Mangano
    • 3
  • Giuseppe Biondi
    • 3
  • Angelo Guglielmotti
    • 3
  1. 1.Centro di Ingegneria Genetica, Biotecnologie Avanzate (CEINGE)NaplesItaly
  2. 2.Dipartimento di Biochimica e Biotecnologie Mediche (DBBM)“Federico II” University of NaplesNaplesItaly
  3. 3.Angelini Research CentreRomeItaly
  4. 4.Dipartimento di Scienze Cliniche Veterinarie, Sez. di Clinica Chirurgica“Federico II” University of NaplesNaplesItaly

Personalised recommendations