甲基化抑制剂5-氮杂2′-脱氧胞苷对三维培养A549 细胞辐射敏感性的影响
Effect of DNA Methyltransferase Inhibitor 5-aza-2′-deoxycytidine on Radiosensitivityof the Human Lung Cancer Cells in Three-dimensional Culture
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摘要: 为探讨DNA去甲基化试剂5-氮杂-2′-脱氧胞苷(5-aza-2′-deoxycytidine，5-Aza-CdR) 对三维(3D)培养模式下的肺腺癌细胞A549 辐射敏感性的作用,开展了系列实验。使用不同浓度5-Aza-CdR 处理单层(2D)A549 细胞72 h 后，MTT法检测其对A549 细胞的增殖抑制作用。选取低浓度(2，5 μmol/L)5-Aza-CdR 预处理2D 和3D 培养的A549 细胞72 h，X射线分别辐照1，2，4，6 Gy，检测微核形成率和克隆存活。实验结果显示，不同浓度的5-Aza-CdR 均能抑制2D 的A549 细胞增殖，且呈剂量依赖性。5 μmol/L 药物预处理2D 与3D 细胞并联合辐照后诱导的细胞微核形成率均显著高于相应的对照组，并且细胞存活率显著降低。不过，较低浓度5-Aza-CdR(2μmol/L) 预处理的3D 培养A549 细胞4，6 Gy 辐照后微核数目较未用药处理组显著增加，克隆存活率较未用药组显著降低(P <0:05)，而在2D 培养A549 细胞中未观测到上述现象。研究结果表明，5-Aza-CdR 能抑制A549 细胞增殖，3D 培养A549 细胞药物预处理更能增加其辐射敏感性。结果暗示，为减少对正常细胞的毒性作用，在临床放疗中，可低剂量使用5-Aza-CdR，实现肿瘤的有效靶向治疗。5-Aza-CdR is a specific inhibitor of DNMTs which could suppress tumor growth by demethylation of genomic DNA. There have only few studies thus far concerning it as radiosensitizers in three-dimensional (3D) cells. The principal aim of this study is to evaluate the effects of 5-Aza-CdR on the radiosensitivity of A549 cells in monolayer (2D) and 3D cultures in an attempt to find out a new combination treatments with radiotherapy. The cell proliferation was detected by MTT assay after pretreated with different doses of 5-Aza-CdR for 72 h. A549 cells were treated with or without 5-Aza-CdR (2, 5 μmol/L) for 72 h before be exposed to X-rays of 1, 2, 4, 6 Gy, respectively. The DNA damage was evaluated by micronucleus assay and clonogenic assays. Pretreatment with 5-Aza-CdR inhibited the A549 cell proliferation significantly. More micronucleus were observed after irradiation in 3D cells pretreated with 2 and 5 μmol/L concentration of drug than those without treatment. The survival fractions of cells pretreated by both 2 and 5 mol/L drug reduced significantly in 3D cultures after irradiation. These significances, however, were found in 2D cells pretreated by only 5 μmol/L drug. Our results suggest that 5-Aza-CdR can inhibit the A549 cells proliferation and apparently enhance the radiosensitivity of cells in 3D cultures. Using of the low dose 5-Aza-CdR in clinical radiotherapy may reduce side effects and enhance effectively the cancer target therapy.Abstract: 5-Aza-CdR is a specific inhibitor of DNMTs which could suppress tumor growth by demethylation of genomic DNA. There have only few studies thus far concerning it as radiosensitizers in three-dimensional (3D) cells. The principal aim of this study is to evaluate the effects of 5-Aza-CdR on the radiosensitivity of A549 cells in monolayer (2D) and 3D cultures in an attempt to find out a new combination treatments with radiotherapy. The cell proliferation was detected by MTT assay after pretreated with different doses of 5-Aza-CdR for 72 h. A549 cells were treated with or without 5-Aza-CdR (2, 5 μmol/L) for 72 h before be exposed to X-rays of 1, 2, 4, 6 Gy, respectively. The DNA damage was evaluated by micronucleus assay and clonogenic assays. Pretreatment with 5-Aza-CdR inhibited the A549 cell proliferation significantly. More micronucleus were observed after irradiation in 3D cells pretreated with 2 and 5 μmol/L concentration of drug than those without treatment. The survival fractions of cells pretreated by both 2 and 5 mol/L drug reduced significantly in 3D cultures after irradiation. These significances, however, were found in 2D cells pretreated by only 5 μmol/L drug. Our results suggest that 5-Aza-CdR can inhibit the A549 cells proliferation and apparently enhance the radiosensitivity of cells in 3D cultures. Using of the low dose 5-Aza-CdR in clinical radiotherapy may reduce side effects and enhance effectively the cancer target therapy.
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