5-Azacytidine treatment induces demethylation of DAPK1 and MGMT genes and inhibits growth in canine mammary gland tumor cells.
5-Azacytidine treatment induces demethylation of DAPK1 and MGMT genes and inhibits growth in canine mammary gland tumor cells.
复制标题
DOI：
10.2147/ott.s162381
复制
发表时间：
2018
影响因子：
4
通讯作者：
Liu Y
中科院分区：
文献类型：
--
作者：
Ren X;Li H;Song X;Wu Y;Liu Y
犬乳腺肿瘤（CMGT）是雌性犬中最常见的自发性肿瘤。异常DAPK 1和MGMT甲基化与多种癌症的肿瘤形成和发展相关5-氮杂胞苷是已知的特异性去甲基化药物，其共价结合DNA甲基转移酶。然而，关于CMGT，DAPK 1和MGMT的甲基化是未知的。因此，我们试图证明5-氮杂胞苷对CMGT细胞增殖的影响，并阐明这些癌细胞中的潜在分子作用机制。MTT法检测5-氮胞苷对CHMm和CHMp细胞增殖的影响。MSP法检测CHMm、CHMp细胞及CMGT血液/组织中DAPK 1和MGMT基因甲基化模式。采用MSP法和qRT-PCR法检测5-氮胞苷对CHMm和CHMp细胞DAPK 1和MGMT基因甲基化及DAPK 1和MGMT mRNA表达的影响。5-氮杂胞苷可抑制CHMm和CHMp细胞的增殖。此外，DAPK 1和MGMT基因在CHMm/CHMp细胞和临床恶性肿瘤样品中高甲基化，而在正常雌性狗的血液和组织中没有。然而，在用5 μM 5-氮杂胞苷处理的CHMm和CHMp细胞中，DAPK 1和MGMT基因可重新诱导。同时5-氮胞苷可增加DAPK 1和MGMT mRNA的表达。这些结果表明，DAPK 1和MGMT甲基化可以作为CMGT的敏感诊断生物标志物和治疗靶点。5-氮杂胞苷也可能是CMGT的潜在治疗候选药物。
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. 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. 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. 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.