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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
Article

Abstract

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.

Keywords

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

Abbreviations

DNMT

DNA methyltransferase

5-AzaC

5-Azacytidine

HDAC

Histone deacetylase

FDA

Food and Drug administration

MCF10A

Human normal breast epithelial cell line

MCF10CA1a

Human malignant breast carcinoma cell line

DMEM

Dulbecco’s modified Eagle medium

EGF

Epidermal growth factor

ER

Oestrogen receptor

MCF7

Human breast adenocarcinoma cell line

FMEC

Feline normal mammary epithelial cell line

K12-72.1

Feline mammary adenocarcinoma cell line

CAT-MT

Feline mammary carcinoma cell line

FBS

Fetal bovine serum

CMEC

Canine normal mammary epithelial cell line

REM134

Canine mammary carcinoma cell line

CMT12

Canine mammary carcinoma cell line

ECIS

Electric Cell-substrate Impedance Sensing

BLMVEC

Bovine microvessel lung endothelial cells

qRT-PCR

Quantitative reverse-transcription PCR

DFNA5

Non-syndromic hearing impairment protein 5

SFRP1

Secreted frizzled-related protein 1

NTN4

Netrin 4

SYK

Spleen tyrosine kinase

FKBP6

FK506 binding protein 6

LOXL4

Lysyl oxidase-like 4

PON1

Paraoxonase 1

TRIM50

Tripartite motif-containing 50

OSPBL3

Oxysterol-binding protein 3

DKK3

Dikkopf-related protein 3

PGP9.5

Ubiquitin carboxy-terminal hydrolase L1

HSPBC

Heat shock protein 1

GAPDH

Glyceraldehyde 3-phosphate dehydrogenase

HPRT

Hypoxanthine guaine phosphoribosyl transferase

UBI

Polyubiquitin

RPL30

Ribosomal Protein L30

YWHAZ

14-3-3 protein zeta

ICC

Immunocytochemistry

HRP

Horseradish peroxidase

PBS

Phosphate buffered saline

BSA

Bovine serum albumin

TBS

Tris buffered saline

PFA

4 % paraformaldehyde

AEC

3-amino-9-ethylcarbazole

CMADC

Canine mammary adenocarcinoma-derived cells

FMADC

Feline mammary adenocarcinoma-derived cells

ECM

Extracellular matrix

NIH-3T3

Murine fibroblast cell line

EC

Endothelial cells

ING1

Inhibitor of growth family member 1

PDX

Patient-derived xenograft

Notes

Acknowledgments

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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