A Comparative Study on the In Vitro Effects of the DNA Methyltransferase Inhibitor 5-Azacytidine (5-AzaC) in Breast/Mammary Cancer of Different Mammalian Species

  • Rebecca M. Harman
  • Theresa M. Curtis
  • David J. Argyle
  • Scott A. Coonrod
  • Gerlinde R. Van de WalleEmail author


Murine models are indispensible for the study of human breast cancer, but they have limitations: tumors arising spontaneously in humans must be induced in mice, and long-term follow up is limited by the short life span of rodents. In contrast, dogs and cats develop mammary tumors spontaneously and are relatively long-lived. This study examines the effects of the DNA methyltransferase (DNMT) inhibitor 5-Azacytidine (5-AzaC) on normal and tumoral mammary cell lines derived from dogs, cats and humans, as proof of concept that small companion animals are useful models of human breast cancer. Our findings show that treatment with 5-AzaC reduces in vitro tumorigenicity in all three species based on growth and invasion assays, mitochondrial activity and susceptibility to apoptosis. Interestingly, we found that the effects of 5-AzaC on gene expression varied not only between the different species but also between different tumoral cell lines within the same species, and confirmed the correlation between loss of methylation in a specific gene promotor region and increased expression of the associated gene using bisulfite sequencing. In addition, treatment with a high dose of 5-AzaC was toxic to tumoral, but not healthy, mammary cell lines from all species, indicating this drug has therapeutic potential. Importantly, we confirmed these results in primary malignant cells isolated from canine and feline adenocarcinomas. The similarities observed between the three species suggest dogs and cats can be useful models for the study of human breast cancer and the pre-clinical evaluation of novel therapeutics.


5-Azacytidine (5-AzaC) Mammary cancer cell line Primary mammary tumor Dog Cat Human 



DNA methyltransferase




Histone deacetylase


Food and Drug administration


Human normal breast epithelial cell line


Human malignant breast carcinoma cell line


Dulbecco’s modified Eagle medium


Epidermal growth factor


Oestrogen receptor


Human breast adenocarcinoma cell line


Feline normal mammary epithelial cell line


Feline mammary adenocarcinoma cell line


Feline mammary carcinoma cell line


Fetal bovine serum


Canine normal mammary epithelial cell line


Canine mammary carcinoma cell line


Canine mammary carcinoma cell line


Electric Cell-substrate Impedance Sensing


Bovine microvessel lung endothelial cells


Quantitative reverse-transcription PCR


Non-syndromic hearing impairment protein 5


Secreted frizzled-related protein 1


Netrin 4


Spleen tyrosine kinase


FK506 binding protein 6


Lysyl oxidase-like 4


Paraoxonase 1


Tripartite motif-containing 50


Oxysterol-binding protein 3


Dikkopf-related protein 3


Ubiquitin carboxy-terminal hydrolase L1


Heat shock protein 1


Glyceraldehyde 3-phosphate dehydrogenase


Hypoxanthine guaine phosphoribosyl transferase




Ribosomal Protein L30


14-3-3 protein zeta




Horseradish peroxidase


Phosphate buffered saline


Bovine serum albumin


Tris buffered saline


4 % paraformaldehyde




Canine mammary adenocarcinoma-derived cells


Feline mammary adenocarcinoma-derived cells


Extracellular matrix


Murine fibroblast cell line


Endothelial cells


Inhibitor of growth family member 1


Patient-derived xenograft



This work was supported by the Morris Animal Foundation (grant #D12MS-002). We are very grateful for the excellent technical assistance of Don Miller with the PCR and bisulfite sequencing analyses and Leen Bussche for the generation of the primary tumor cell cultures. We would like to thank Jen Olson and José Morales for sample collection and Katie Kelly for grading the tumor samples.

Author’s Contributions

RH carried out all laboratory procedures, was involved in conception and design, and manuscript writing; TC provided expertise and technical assistance with the Electric Cell-substrate Impedance (ECIS) assays; DA provided canine and feline mammary cancer cell lines and SC provided the human cell lines; DA, SC and GVdW were involved in conception and design; GVdW was involved in data analyses and manuscript writing. All authors read and approved the final manuscript.

Compliance with Etihcal Standards

Conflict Interests

The authors declare they have no competing interests.

Supplementary material

10911_2016_9350_Fig8_ESM.gif (72 kb)
Supplementary Figure 1

(A). Viability of canine, feline and human tumoral mammary cells lines/primary cells treated with 5 μM 5-AzaC as determined by MTT assays. Percent viable cells, compared to non-treated cells, set at 100 %, are shown. n = 3, *: P < 0.05. (B). Expression levels of the gene PGP9.5 in the feline cell line K12–72.1 treated with 5 and 10 μM 5-AzaC as determined by qRT-PCR. Fold changes from non-treated cells is shown. n = 3, *: P < 0.05. (GIF 71 kb)

10911_2016_9350_MOESM1_ESM.tif (5.8 mb)
High Resolution (TIFF 5927 kb)


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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Rebecca M. Harman
    • 1
  • Theresa M. Curtis
    • 2
  • David J. Argyle
    • 3
  • Scott A. Coonrod
    • 1
  • Gerlinde R. Van de Walle
    • 1
    Email author
  1. 1.Baker Institute for Animal Health, College of Veterinary MedicineCornell UniversityIthacaUSA
  2. 2.Department of Biological SciencesState University of New York at CortlandCortlandUSA
  3. 3.Royal (Dick) School of Veterinary Studies and Roslin InstituteThe University of EdinburghEdinburghUK

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