低浓度丰加霉素对人白血病K562细胞集落形成抑制作用的机制研究

Inhibitory Effects of Toyocamycin at Low Concentrations on Colony Forming of Human Leukemia Cell Line K562

目的初步探讨低浓度丰加霉素对人白血病K562细胞集落形成抑制作用的机制。方法甲基纤维素集落形成实验检测低浓度丰加霉素对人白血病K562细胞集落形成能力的影响；CCK-8法检测低浓度丰加霉素对K562细胞的生长抑制率；AnnexinV／PI双染流式细胞仪检测低浓度丰加霉素作用下的K562细胞凋亡率；PI单染流式细胞仪检测药物作用后细胞的周期分布改变；Western免疫印迹和实时定量PCR检测周期相关分子表达水平变化。结果低浓度丰加霉素对人白血病K562细胞具有较强的集落形成抑制作用；可明显抑制K562细胞的生长，呈时间一剂量依赖性；尽管短时间（48h）的药物处理仅出现轻度的细胞凋亡和周期阻滞，但10nmol/L和30nmol/L的丰加霉素长时间（7d）作用后，K562细胞G0／G1期比例分别是（62．3±1．7）％和（76．9±0．7）％，与对照组（38．9±1．1）％相比差异具有高度统计学意义（P〈0．01）；低浓度丰加霉素长时间作用后诱导K562细胞周期相关分子P16蛋白水平和转录水平的高表达。结论丰加霉素在低浓度，长时间作用于人白血病K562细胞后，具有较强的集落形成抑制和生长抑制作用，此作用可能与诱导细胞周期相关分子p16高表达，导致细胞G0／G1期阻滞有关。

Objective To clarify the inhibitory effects of toyocamycin at low concentrations on colony forming of human leukemia cell line K562 and the action mechanism. Methods Methyl cellulose colony forming assay was used to study the inhibitory effects of toyocamycin on K562 cells. CCK-8 assay was employed to detect the growth inhibition rate of K562 cells treated with toyocamycin at low concentrations. The effects of toyocamycin on apoptosis and cell cycle alteration in K562 cells were determined by flow cytometry using AnnexinV/PI double staining and PI staining respec- tively. Western blot was used to detect the expression of cell cycle-relative proteins, and real-time PCR analysis was used to detect the expression of p16 mRNA in K562 cells treated with toyocamy- cin. Results Toyocamycin at low concentration could strongly inhibit the colony forming and growth of K562 cells in a time- and dose-dependent manner. Mild apoptosis and cell cycle arrest were observed in K562 cells treated with toyocamycin for 48 h, but treatment with toyocamycin （10 and 30 nmol/L） for 7 days could obviously increase the proportion of cells in G0/G1 phase （62.3% 1.7% and 76. 9% 0. 7% vs 38.9% 1.1%, P 〈0. 01 ） . Low concentration of toyocamycin could induce the high expression of p16 mRNA and protein in K562 cells after a long-time treatment. Conclusion Toyocamycin at at low concentrations with prolonged treatment can obviously inhibit the colony formation and growth of K562 cells, which might be related to G0/G1 phase arrest as a result of in- duced over-expression of pl6 by toyocamycin