Department of Medicine A, Hematology and Oncology, University of Münster, Münster, Germany.
Department of Medicine IV, Hematology and Oncology, University of Halle, Halle, Germany.
Int J Oncol. 2015 Mar;46(3):1192-204. doi: 10.3892/ijo.2014.2792. Epub 2014 Dec 10.
The DNA methyltransferase (DNMT) inhibitory drugs such as 5-azacytidine induce DNA hypomethylation by inhibiting DNA methyltransferases. While clinically effective, DNMT inhibitors are not curative. A combination with cytotoxic drugs might be beneficial, but this is largely unexplored. In the present study, we analyzed potential synergisms between cytotoxic drugs and 5-azacytidine in acute myeloid leukemia (AML) and non-small cell lung cancer (NSCLC) cells. Lung cancer and leukemia cell lines were exposed to low doses of 5-azacytidine with varying doses of cytarabine or etoposide for AML cells (U937 and HL60) as well as cisplatin or gemcitabine for NSCLC cells (A549 and HTB56) for 48 h. Drug interaction and potential synergism was analyzed according to the Chou-Talalay algorithm. Further analyses were based on soft agar colony formation assays, active caspase-3 staining and BrdU incorporation flow cytometry. To identify effects on DNA methylation patterns, we performed genome wide DNA methylation analysis using 450K bead arrays. Azacytidine at low doses was synergistic with cytotoxic drugs in NSCLC and in AML cell lines. Simultaneous exposure to 5-azacytidine with cytotoxic drugs showed strong synergistic activity. In colony formation assays these synergisms were repeatedly verified for 5-azacytidine (25 nM) with low doses of anticancer agents. 5-azacytidine neither affected the cell cycle nor increased apoptosis. 450K methylation bead arrays revealed 1,046 CpG sites in AML and 1,778 CpG sites in NSCLC cells with significant DNA hypomethylation (24-h exposure) to 5-azacytidine combined with the cytotoxic drugs. These CpG-sites were observed in the candidate tumor-suppressor genes MGMT and THRB. Additional incubation time after 24-h treatment led to a 4.1-fold increase of significant hypomethylated CpG-sites in NSCLC cells. These results suggest that the addition of DNA demethylating agents to cytotoxic anticancer drugs exhibits synergistic activity in AML and NSCLC. Dysregulation of an equilibrium of DNA methylation in cancer cells might increase the susceptibility for cytotoxic drugs.
DNA 甲基转移酶（DNMT）抑制剂，如 5-氮杂胞苷，通过抑制 DNA 甲基转移酶诱导 DNA 低甲基化。虽然在临床上有效，但 DNMT 抑制剂并不是治愈性的。与细胞毒性药物联合使用可能是有益的，但这在很大程度上尚未得到探索。在本研究中，我们分析了细胞毒性药物与 5-氮杂胞苷在急性髓细胞白血病（AML）和非小细胞肺癌（NSCLC）细胞中的潜在协同作用。将肺癌和白血病细胞系暴露于低剂量的 5-氮杂胞苷，并用不同剂量的阿糖胞苷或依托泊苷处理 AML 细胞（U937 和 HL60），以及顺铂或吉西他滨处理 NSCLC 细胞（A549 和 HTB56）48 小时。根据 Chou-Talalay 算法分析药物相互作用和潜在协同作用。进一步的分析基于软琼脂集落形成测定、活性 caspase-3 染色和 BrdU 掺入流式细胞术。为了鉴定对 DNA 甲基化模式的影响，我们使用 450K 珠阵列进行了全基因组 DNA 甲基化分析。低剂量的氮杂胞苷与 NSCLC 和 AML 细胞系中的细胞毒性药物具有协同作用。同时暴露于 5-氮杂胞苷和细胞毒性药物显示出强烈的协同活性。在集落形成测定中，这些协同作用在低剂量抗癌药物与 5-氮杂胞苷（25 nM）同时暴露时得到了反复验证。5-氮杂胞苷既不影响细胞周期也不增加细胞凋亡。450K 甲基化珠阵列显示 AML 中有 1046 个 CpG 位点，NSCLC 中有 1778 个 CpG 位点，在 AML 和 NSCLC 细胞中，与细胞毒性药物联合使用时，5-氮杂胞苷可导致显著的 DNA 低甲基化（24 小时暴露）。这些 CpG 位点存在于候选肿瘤抑制基因 MGMT 和 THRB 中。在 24 小时处理后，再孵育一段时间，可使 NSCLC 细胞中显著低甲基化的 CpG 位点增加 4.1 倍。这些结果表明，在 AML 和 NSCLC 中，将 DNA 去甲基化剂添加到细胞毒性抗癌药物中可表现出协同作用。癌细胞中 DNA 甲基化平衡的失调可能会增加对细胞毒性药物的敏感性。
