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 ZolloEmail author
Research Paper


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.


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



α1 Acid glycoprotein


Human breast cancer resistance protein


Bioluminescence imaging


Cell index


Dulbecco’s modified Eagle’s medium




Granulocyte colony-stimulating factor


Granulocyte-macrophage colony-stimulating factor








Monocyte chemotactic protein 1


Myeloid-derived suppressor cells


Macrophage inflammatory protein 1a


Matrix metalloproteinase 9




Phosphate-buffered saline


Polyethylene glycol




Real-time cell analysis


Real-time cell electronic sensor


Stem cell factor


Standard error


Standard error of the mean


Tumor-associated macrophages



We would like to thank: Prof. Eugene Lukanidin for sharing the S100A4 antibodies used in this study, Prof. Luigi del Vecchio and Dr. Maddalena Raia for technical advice with the FACS analyses, Dr. Donatella Montanaro for technical advice with histological analyses, and Prof. Francesco Salvatore for supporting the project with the instrumentation required for in vivo imaging in mice. We also thank Viviana Vastolo for technical assistance in in vivo experiments. Associazione Italiana per la ricerca sul Cancro AIRC (MZ), Associazione Italiana per la lotta al Neuroblastoma (MZ). This study was also supported in part by the Intramural Research Program of the National Cancer Institute (PSS). DS was supported by the Dipartimento di Biochimica e Biotecnologie Mediche, ‘Federico II’ University of Naples; VDD was supported by the Fondazione San Paolo (IM) and Tumic; DMD was supported by a Dottorato in Medicina Molecolare, ‘Federico II’ University of Naples; GDV was supported by a Dottorato in Medicina Molecolare, ‘Federico II’ University of Naples; and CM was supported by a Dottorato in Produzione e Sanità degli Alimenti di Origine Animale, ‘Federico II’ University of Naples.

Conflict of interest

The authors declare that they have no competing interests as defined by Clinical & Experimental Metastasis, or other interests that might be perceived as influencing the results and discussion reported in this manuscript.

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)


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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
    Email author
  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

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