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5-Azacytidine treatment induces demethylation of DAPK1 and MGMT genes and inhibits growth in canine mammary gland tumor cells

Authors Ren XL, Li HT, Song XY, Wu YH, Liu Y

Received 12 January 2018

Accepted for publication 14 March 2018

Published 15 May 2018 Volume 2018:11 Pages 2805—2813

DOI https://doi.org/10.2147/OTT.S162381

Checked for plagiarism Yes

Review by Single-blind

Peer reviewers approved by Dr Cristina Weinberg

Peer reviewer comments 2

Editor who approved publication: Dr Carlos E Vigil


Xiaoli Ren,1 Huatao Li,2 Xianyi Song,1 Yuhong Wu,1 Yun Liu1

1Heilongjiang Key Laboratory for Laboratory Animals and Comparative Medicine, Department of Veterinary Surgery, College of Veterinary Medicine, Northeast Agricultural University, Harbin, China; 2Department of Veterinary Obstetrics, College of Veterinary Medicine, Qingdao Agricultural University, Qingdao, China

Background: Canine mammary gland tumors (CMGTs) are the most common, spontaneous types of neoplasias in female dogs. Aberrant DAPK1 and MGMT methylation associated with tumor formation and development in various cancers. 5-Azacytidine is a known specific demethylation drug that covalently binds to DNA methyltransferase. However, the methylation of the DAPK1 and MGMT is unknown with respect to CMGTs. Therefore, we sought to demonstrate the effects of 5-azacytidine on the proliferation of CMGTs cell, and elucidate the potential molecular mechanisms of action in these cancerous cells.
Materials and methods: The effects of 5-azacytidine on CHMm and CHMp cell proliferation were evaluated by MTT assay. The DAPK1 and MGMT gene methylation patterns in CHMm and CHMp cells and CMGTs blood/tissue samples were analyzed by MSP assay. Effect of 5-azacytidine on the methylation of DAPK1 and MGMT gene, and DAPK1 and MGMT mRNA expression in CHMm and CHMp cells were analyzed by MSP assay and qRT-PCR assay, respectively.
Results: 5-Azacytidine may suppress the proliferation of CHMm and CHMp cells. Furthermore, the DAPK1 and MGMT genes were hypermethylated in CHMm/CHMp cells and clinical malignant tumor samples, but not in normal female dogs’ blood and tissue. However, the DAPK1 and MGMT genes were re-inducible in CHMm and CHMp cells treated with 5 μM 5-azacytidine. Meanwhile, 5-azacytidine increased the expression of DAPK1 and MGMT mRNA.
Conclusion: These results suggest that DAPK1 and MGMT methylation can serve as sensitive diagnostic biomarkers and therapeutic targets for CMGTs. 5-Azacytidine also could be a potential therapeutic candidate for CMGTs.

Keywords: canine mammary gland tumor, CHMm, CHMp, 5-azacytidine, DAPK1, MGMT, DNA methylation

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