Clinical & Experimental Metastasis

, Volume 30, Issue 1, pp 47–68 | Cite as

Dipyridamole prevents triple-negative breast-cancer progression

  • Daniela Spano
  • Jean-Claude Marshall
  • Natascia Marino
  • Daniela De Martino
  • Alessia Romano
  • Maria Nunzia Scoppettuolo
  • Anna Maria Bello
  • Valeria Di Dato
  • Luigi Navas
  • Gennaro De Vita
  • Chiara Medaglia
  • Patricia S. Steeg
  • Massimo Zollo
Research Paper

Abstract

Dipyridamole is a widely prescribed drug in ischemic disorders, and it is here investigated for potential clinical use as a new treatment for breast cancer. Xenograft mice bearing triple-negative breast cancer 4T1-Luc or MDA-MB-231T cells were generated. In these in vivo models, dipyridamole effects were investigated for primary tumor growth, metastasis formation, cell cycle, apoptosis, signaling pathways, immune cell infiltration, and serum inflammatory cytokines levels. Dipyridamole significantly reduced primary tumor growth and metastasis formation by intraperitoneal administration. Treatment with 15 mg/kg/day dipyridamole reduced mean primary tumor size by 67.5 % (p = 0.0433), while treatment with 30 mg/kg/day dipyridamole resulted in an almost a total reduction in primary tumors (p = 0.0182). Experimental metastasis assays show dipyridamole reduces metastasis formation by 47.5 % in the MDA-MB-231T xenograft model (p = 0.0122), and by 50.26 % in the 4T1-Luc xenograft model (p = 0.0292). In vivo dipyridamole decreased activated β-catenin by 38.64 % (p < 0.0001), phospho-ERK1/2 by 25.05 % (p = 0.0129), phospho-p65 by 67.82 % (p < 0.0001) and doubled the expression of IkBα (p = 0.0019), thus revealing significant effects on Wnt, ERK1/2-MAPK and NF-kB pathways in both animal models. Moreover dipyridamole significantly decreased the infiltration of tumor-associated macrophages and myeloid-derived suppressor cells in primary tumors (p < 0.005), and the inflammatory cytokines levels in the sera of the treated mice. We suggest that when used at appropriate doses and with the correct mode of administration, dipyridamole is a promising agent for breast-cancer treatment, thus also implying its potential use in other cancers that show those highly activated pathways.

Keywords

Dipyridamole Metastasis ERK1/2-MAPK Wnt NF-kB Immune cell infiltration Tumor microenvironment 

Abbreviations

AGP

α1 Acid glycoprotein

BCRP/ABCG2

Human breast cancer resistance protein

BLI

Bioluminescence imaging

CI

Cell index

DMEM

Dulbecco’s modified Eagle’s medium

DMSO

Dimethylsulfoxide

G-CSF

Granulocyte colony-stimulating factor

GM-CSF

Granulocyte-macrophage colony-stimulating factor

IHC

Immunohistochemistry

IL-1α

Interleukin-1α

IL-1β

Interleukin-1β

MCP-1

Monocyte chemotactic protein 1

MDSCs

Myeloid-derived suppressor cells

MIP-1a

Macrophage inflammatory protein 1a

MMP9

Matrix metalloproteinase 9

MTS

(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)

PBS

Phosphate-buffered saline

PEG

Polyethylene glycol

PGK

Phosphoglucokinase

RTCA

Real-time cell analysis

RT-CES

Real-time cell electronic sensor

SCF

Stem cell factor

SE

Standard error

SEM

Standard error of the mean

TAMs

Tumor-associated macrophages

Supplementary material

10585_2012_9506_MOESM1_ESM.tif (15.8 mb)
Figure S1. Representative caspase-3 activity assay (of two experiments performed with similar results) in 4T1-Luc cells treated for 24 h with PBS-PEG or dipyridamole (as indicated). Data are means ±SE. * p = 0.03 (Students’ t-test). (TIFF 16188 kb)
10585_2012_9506_MOESM2_ESM.tif (15.8 mb)
Figure S2. Dipyridamole affects 4T1-Luc primary tumor growth in vivo. Time-course of bioluminescent signals from 4T1-Luc tumor cells implanted into the mouse mammary fat pad at day 0, followed by intraperitoneally administered treatments with PBS-PEG or 15 mg/kg/day (A) or 30 mg/kg/day (B) dipyridamole (as indicated). Data are total flux means ±SE. (A) * p = 0.0433; (B) * p = 0.0182 (ANOVA). (TIFF 16184 kb)
10585_2012_9506_MOESM3_ESM.tif (16.1 mb)
Figure S3.In vivo effects of dipyridamole. Statistical analyses on sections from 4T1-Luc primary tumors (A) and MDA-MB-231T lung metastases (B, C) stained for cleaved caspase-3 (A, C) and Ki67 (B). No significant differences were observed for all of the analyzed markers. (TIFF 16453 kb)
10585_2012_9506_MOESM4_ESM.tif (15.8 mb)
Figure S4. Dipyridamole toxicity in vivo. Time-course of mean weights of athymic nude mice injected with MDA-MB-231T cells via the tail vein and treated with vehicle or 30 mg/kg/day dipyridamole, delivery either by oral gavage or intraperitoneally (as indicated). There are no significant differences between the body weights of these three treatment groups. (TIFF 16194 kb)
10585_2012_9506_MOESM5_ESM.tif (15.8 mb)
Figure S5.In vitro effects of dipyridamole. (A) Immunoblotting for IkBα in 4T1-Luc cells treated for 4 h with PBS-PEG or dipyridamole (as indicated). β-actin used as control for equal loading. (B) Expression of PGK gene, a non-target of Wnt, ERK1/2-MAPK and NF-kB pathways, in 4T1-Luc cells treated for 24 h with PBS-PEG or dipyridamole (as indicated). Data are means ±SEM. (TIFF 16189 kb)
10585_2012_9506_MOESM6_ESM.tif (15.8 mb)
Figure S6. Dipyridamole affects in vivo the expression of known targets of Wnt pathway. Immunoblotting for cyclin D1 and c-Myc in tumors from mice implanted with 4T1-Luc cells in the mammary fat pad and treated with PBS-PEG or dipyridamole (as indicated). β-actin used as control for equal loading. (TIFF 16188 kb)

References

  1. 1.
    Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D (2011) Global cancer statistics. CA Cancer J Clin 61:69–90PubMedCrossRefGoogle Scholar
  2. 2.
    Steeg PS (2006) Tumor metastasis: mechanistic insights and clinical challenges. Nat Med 12:895–904PubMedCrossRefGoogle Scholar
  3. 3.
    Schaper W (2005) Dipyridamole, an underestimated vascular protective drug. Cardiovasc Drugs Ther 19:357–363PubMedCrossRefGoogle Scholar
  4. 4.
    Belt JA, Marina NM, Phelps DA, Crawford CR (1993) Nucleoside transport in normal and neoplastic cells. Adv Enzyme Regul 33:235–252PubMedCrossRefGoogle Scholar
  5. 5.
    Walling J (2006) From methotrexate to pemetrexed and beyond. A review of the pharmacodynamic and clinical properties of antifolates. Invest New Drugs 24:37–77PubMedCrossRefGoogle Scholar
  6. 6.
    Howell SB, Hom D, Sanga R, Vick JS, Abramson IS (1989) Comparison of the synergistic potentiation of etoposide, doxorubicin, and vinblastine cytotoxicity by dipyridamole. Cancer Res 49:3178–3183PubMedGoogle Scholar
  7. 7.
    Ramu N, Ramu A (1989) Circumvention of adriamycin resistance by dipyridamole analogues: a structure-activity relationship study. Int J Cancer 43:487–491PubMedCrossRefGoogle Scholar
  8. 8.
    Lehman NL, Danenberg PV (2000) Modulation of RTX cytotoxicity by thymidine and dipyridamole in vitro: implications for chemotherapy. Cancer Chemother Pharmacol 45:142–148PubMedCrossRefGoogle Scholar
  9. 9.
    Nelson JA, Drake S (1984) Potentiation of methotrexate toxicity by dipyridamole. Cancer Res 44:2493–2496PubMedGoogle Scholar
  10. 10.
    Desai PB, Sridhar R (1992) Potentiation of cytotoxicity of mitoxantrone toward CHO-K1 cells in vitro by dipyridamole. Pharm Res 9:178–181PubMedCrossRefGoogle Scholar
  11. 11.
    Boyer CR, Karjian PL, Wahl GM, Pegram M, Neuteboom ST (2002) Nucleoside transport inhibitors, dipyridamole and p-nitrobenzylthioinosine, selectively potentiate the antitumor activity of NB1011. Anticancer Drugs 13:29–36PubMedCrossRefGoogle Scholar
  12. 12.
    Rodrigues M, Barbosa F Jr, Perussi JR (2004) Dipyridamole increases the cytotoxicity of cisplatin in human larynx cancer cells in vitro. Braz J Med Biol Res 37:591–599PubMedCrossRefGoogle Scholar
  13. 13.
    Sato S, Kohno K, Hidaka K, Hisatsugu T, Kuwano M, Komiyama S (1993) Differentially potentiating effects by dipyridamole on cytotoxicity of 5-fluorouracil against three human maxillary cancer cell lines derived from a single tumor. Anticancer Drug Des 8:289–297PubMedGoogle Scholar
  14. 14.
    Kennedy DG, Van den Berg HW, Clarke R, Murphy RF (1986) Enhancement of methotrexate cytotoxicity towards the MDA.MB.436 human breast cancer cell line by dipyridamole. The role of methotrexate polyglutamates. Biochem Pharmacol 35:3053–3056PubMedCrossRefGoogle Scholar
  15. 15.
    Goda AE, Yoshida T, Horinaka M, Yasuda T, Shiraishi T, Wakada M, Sakai T (2008) Mechanisms of enhancement of TRAIL tumoricidal activity against human cancer cells of different origin by dipyridamole. Oncogene 27:3435–3445PubMedCrossRefGoogle Scholar
  16. 16.
    Zhang Y, Gupta A, Wang H, Zhou L, Vethanayagam RR, Unadkat JD, Mao Q (2005) BCRP transports dipyridamole and is inhibited by calcium channel blockers. Pharm Res 22:2023–2034PubMedCrossRefGoogle Scholar
  17. 17.
    Haimeur A, Conseil G, Deeley RG, Cole SP (2004) The MRP-related and BCRP/ABCG2 multidrug resistance proteins: biology, substrate specificity and regulation. Curr Drug Metab 5:21–53PubMedCrossRefGoogle Scholar
  18. 18.
    Eisert WG (2002) Dipyridamole. In: Michelson AD (ed) Platelets. Academic Press, Amsterdam, pp 803–815Google Scholar
  19. 19.
    White H, Jamieson DG (2010) Review of the ESPRIT Study: aspirin plus dipyridamole versus aspirin alone for prevention of vascular events after a noncardioembolic, mild-to-moderate ischemic stroke or transient ischemic attack. Postgrad Med 122:227–229PubMedCrossRefGoogle Scholar
  20. 20.
    Tsuruo T, Fujita N (2008) Platelet aggregation in the formation of tumor metastasis. Proc Jpn Acad Ser B Phys Biol Sci 84:189–198PubMedCrossRefGoogle Scholar
  21. 21.
    Nierodzik ML, Karpatkin S (2006) Thrombin induces tumor growth, metastasis, and angiogenesis: evidence for a thrombin-regulated dormant tumor phenotype. Cancer Cell 10:355–362PubMedCrossRefGoogle Scholar
  22. 22.
    Isacoff WH, Bendetti JK, Barstis JJ, Jazieh AR, Macdonald JS, Philip PA (2007) Phase II trial of infusional fluorouracil, leucovorin, mitomycin, and dipyridamole in locally advanced unresectable pancreatic adenocarcinoma: SWOG S9700. J Clin Oncol 25:1665–1669PubMedCrossRefGoogle Scholar
  23. 23.
    Raschko JW, Synold TW, Chow W, Coluzzi P, Hamasaki V, Leong LA, Margolin KA, Morgan RJ, Shibata SI, Somlo G, Tetef ML, Yen Y, ter Veer A, Doroshow JH (2000) A phase I study of carboplatin and etoposide administered in conjunction with dipyridamole, prochlorperazine and cyclosporine A. Cancer Chemother Pharmacol 46:403–410PubMedCrossRefGoogle Scholar
  24. 24.
    Burch PA, Ghosh C, Schroeder G, Allmer C, Woodhouse CL, Goldberg RM, Addo F, Bernath AM, Tschetter LK, Windschitl HE, Cobau CD (2000) Phase II evaluation of continuous-infusion 5-fluorouracil, leucovorin, mitomycin-C, and oral dipyridamole in advanced measurable pancreatic cancer: a North Central Cancer Treatment Group Trial. Am J Clin Oncol 23:534–537PubMedCrossRefGoogle Scholar
  25. 25.
    Wenzel J, Zeisig R, Fichtner I (2009) Inhibition of breast cancer metastasis by dual liposomes to disturb complex formation. Int J Pharm 370:121–128PubMedCrossRefGoogle Scholar
  26. 26.
    Wenzel J, Zeisig R, Haider W, Habedank S, Fichtner I (2010) Inhibition of pulmonary metastasis in a human MT3 breast cancer xenograft model by dual liposomes preventing intravasal fibrin clot formation. Breast Cancer Res Treat 121:13–22PubMedCrossRefGoogle Scholar
  27. 27.
    Foulkes WD, Smith IE, Reis-Filho JS (2010) Triple-negative breast cancer. N Engl J Med 363:1938–1948PubMedCrossRefGoogle Scholar
  28. 28.
    Xing JZ, Zhu L, Gabos S, Xie L (2006) Microelectronic cell sensor assay for detection of cytotoxicity and prediction of acute toxicity. Toxicol in Vitro 20:995–1004PubMedCrossRefGoogle Scholar
  29. 29.
    Spano D, Cimmino F, Capasso M, D’Angelo F, Zambrano N, Terracciano L, Iolascon A (2008) Changes of the hepatic proteome in hepatitis B-infected mouse model at early stages of fibrosis. J Proteome Res 7:2642–2653PubMedCrossRefGoogle Scholar
  30. 30.
    Chuang KA, Lieu CH, Tsai WJ, Wu MH, Chen YC, Liao JF, Wang CC, Kuo YC (2010) Evaluation of anti-Wnt/β-catenin signaling agents by pGL4-TOP transfected stable cells with a luciferase reporter system. Braz J Med Biol Res 43:931–941PubMedCrossRefGoogle Scholar
  31. 31.
    Vlad A, Röhrs S, Klein-Hitpass L, Müller O (2008) The first five years of the Wnt targetome. Cell Signal 20:795–802PubMedCrossRefGoogle Scholar
  32. 32.
    Weyrich AS, Denis MM, Kuhlmann-Eyre JR, Spencer ED, Dixon DA, Marathe GK, McIntyre TM, Zimmerman GA, Prescott SM (2005) Dipyridamole selectively inhibits inflammatory gene expression in platelet-monocyte aggregates. Circulation 111:633–642PubMedCrossRefGoogle Scholar
  33. 33.
    Chakrabarti S, Blair P, Wu C, Freedman JE (2007) Redox state of dipyridamole is a critical determinant for its beneficial antioxidant and antiinflammatory effects. J Cardiovasc Pharmacol 50:449–457PubMedCrossRefGoogle Scholar
  34. 34.
    Sethi G, Ahn KS, Sung B, Aggarwal BB (2008) Pinitol targets nuclear factor-kappaB activation pathway leading to inhibition of gene products associated with proliferation, apoptosis, invasion, and angiogenesis. Mol Cancer Ther 7:1604–1614PubMedCrossRefGoogle Scholar
  35. 35.
    Kawakami H, Tomita M, Matsuda T, Ohta T, Tanaka Y, Fujii M, Hatano M, Tokuhisa T, Mori N (2005) Transcriptional activation of survivin through the NF-kappaB pathway by human T-cell leukemia virus type I tax. Int J Cancer 115:967–974PubMedCrossRefGoogle Scholar
  36. 36.
    Mantovani A (2010) Molecular pathways linking inflammation and cancer. Curr Mol Med 10:369–373PubMedCrossRefGoogle Scholar
  37. 37.
    Boye K, Maelandsmo GM (2010) S100A4 and metastasis: a small actor playing many roles. Am J Pathol 176:528–535PubMedCrossRefGoogle Scholar
  38. 38.
    Stein U, Arlt F, Walther W, Smith J, Waldman T, Harris ED, Mertins SD, Heizmann CW, Allard D, Birchmeier W, Schlag PM, Shoemaker RH (2006) The metastasis-associated gene S100A4 is a novel target of beta-catenin/T-cell factor signaling in colon cancer. Gastroenterology 131:1486–1500PubMedCrossRefGoogle Scholar
  39. 39.
    Grotterød I, Maelandsmo GM, Boye K (2010) Signal transduction mechanisms involved in S100A4-induced activation of the transcription factor NF-kappaB. BMC Cancer 10:241PubMedCrossRefGoogle Scholar
  40. 40.
    Haskó G, Kuhel DG, Chen JF, Schwarzschild MA, Deitch EA, Mabley JG, Marton A, Szabó C (2000) Adenosine inhibits IL-12 and TNF-[alpha] production via adenosine A2a receptor-dependent and independent mechanisms. FASEB J 14:2065–2074PubMedCrossRefGoogle Scholar
  41. 41.
    Budd GT, Jayaraj A, Grabowski D, Adelstein D, Bauer L, Boyett J, Bukowski R, Murthy S, Weick J (1990) Phase I trial of dipyridamole with 5-fluorouracil and folinic acid. Cancer Res 50:7206–7211PubMedGoogle Scholar
  42. 42.
    Chakravarthy A, Abrams RA, Yeo CJ, Korman LT, Donehower RC, Hruban RH, Zahurek ML, Grochow LB, O’Reilly S, Hurwitz H, Jaffee EM, Lillemoe KD, Cameron JL (2000) Intensified adjuvant combined modality therapy for resected periampullary adenocarcinoma: acceptable toxicity and suggestion of improved 1-year disease-free survival. Int J Radiat Oncol Biol Phys 48:1089–1096PubMedCrossRefGoogle Scholar
  43. 43.
    Willson JK, Fischer PH, Tutsch K, Alberti D, Simon K, Hamilton RD, Bruggink J, Koeller JM, Tormey DC, Earhart RH, Ranhosky A, Trump DL (1988) Phase I clinical trial of a combination of dipyridamole and acivicin based upon inhibition of nucleoside salvage. Cancer Res 48:5585–5590PubMedGoogle Scholar
  44. 44.
    Goel R, Cleary SM, Horton C, Balis FM, Zimm S, Kirmani S, Howell SB (1989) Selective intraperitoneal biochemical modulation of methotrexate by dipyridamole. J Clin Oncol 7:262–269PubMedGoogle Scholar
  45. 45.
    Isonishi S, Kirmani S, Kim S, Plaxe SC, Braly PS, McClay EF, Howell SB (1991) Phase I and pharmacokinetic trial of intraperitoneal etoposide in combination with the multidrug-resistance-modulating agent dipyridamole. J Natl Cancer Inst 83:621–626PubMedCrossRefGoogle Scholar
  46. 46.
    Mahony C, Wolfram KM, Cocchetto DM, Bjornsson TD (1982) Dipyridamol kinetics. Clin Pharmacol Ther 31:330–338PubMedCrossRefGoogle Scholar
  47. 47.
    Curtin NJ, Bowman KJ, Turner RN, Huang B, Loughlin PJ, Calvert AH, Golding BT, Griffin RJ, Newell DR (1999) Potentiation of the cytotoxicity of thymidylate synthase (TS) inhibitors by dipyridamole analogues with reduced alpha1-acid glycoprotein binding. Br J Cancer 80:1738–1746PubMedCrossRefGoogle Scholar
  48. 48.
    Huang C, Jacobson K, Schaller MD (2004) MAP kinases and cell migration. J Cell Sci 117:4619–4628PubMedCrossRefGoogle Scholar
  49. 49.
    Kaler P, Godasi BN, Augenlicht L, Klampfer L (2009) The NF-kappaB/AKT-dependent Induction of Wnt Signaling in Colon Cancer Cells by Macrophages and IL-1beta. Cancer Microenviron 2:69–80CrossRefGoogle Scholar
  50. 50.
    Kim D, Rath O, Kolch W, Cho KH (2007) A hidden oncogenic positive feedback loop caused by crosstalk between Wnt and ERK pathways. Oncogene 26:4571–4579PubMedCrossRefGoogle Scholar
  51. 51.
    Chuderland D, Seger R (2005) Protein-protein interactions in the regulation of the extracellular signal-regulated kinase. Mol Biotechnol 29:57–74PubMedCrossRefGoogle Scholar
  52. 52.
    Solinas G, Germano G, Mantovani A, Allavena P (2009) Tumor-associated macrophages (TAM) as major players of the cancer-related inflammation. J Leukoc Biol 86:1065–1073PubMedCrossRefGoogle Scholar
  53. 53.
    Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646–674PubMedCrossRefGoogle Scholar
  54. 54.
    Karin M (2006) Nuclear factor-kappaB in cancer development and progression. Nature 441:431–436PubMedCrossRefGoogle Scholar
  55. 55.
    Pikarsky E, Porat RM, Stein I, Abramovitch R, Amit S, Kasem S, Gutkovich-Pyest E, Urieli-Shoval S, Galun E, Ben-Neriah Y (2004) NF-kappaB functions as a tumour promoter in inflammation-associated cancer. Nature 431:461–466PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Daniela Spano
    • 1
    • 2
  • Jean-Claude Marshall
    • 3
  • Natascia Marino
    • 1
    • 3
  • Daniela De Martino
    • 1
    • 2
  • Alessia Romano
    • 2
  • Maria Nunzia Scoppettuolo
    • 1
    • 2
  • Anna Maria Bello
    • 1
    • 2
  • Valeria Di Dato
    • 1
    • 2
  • Luigi Navas
    • 1
    • 4
  • Gennaro De Vita
    • 1
    • 2
  • Chiara Medaglia
    • 1
    • 2
  • Patricia S. Steeg
    • 3
  • Massimo Zollo
    • 1
    • 2
  1. 1.Centro di Ingegneria Genetica (CEINGE) Biotecnologie AvanzateNaplesItaly
  2. 2.Dipartimento di Biochimica e Biotecnologie Mediche‘Federico II’ University of NaplesNaplesItaly
  3. 3.Women’s Cancers Section, Laboratory of Molecular PharmacologyNational Cancer InstituteBethesdaUSA
  4. 4.Dipartimento di Scienze Cliniche Veterinarie, Sezione di Clinica Chirurgica‘Federico II’ University of NaplesNaplesItaly

Personalised recommendations