组蛋白去乙酰化酶抑制剂-FK228对人非小细胞肺癌A549细胞纺锤体检查点的影响

Histone Deacetylase Inhibitor-FK228 Inhibits the Spindle Checkpoint Function of Human Non-small-cell Lung Cancer A549 Cells

HDACi-FK228是一种新型抗肿瘤药物,但其作用机制研究的尚未十分明确,为进一步阐明FK228杀伤肿瘤细胞的机制,该文应用流式细胞术检测FK228对非小细胞肺癌A549细胞周期的影响;应用免疫荧光染色和免疫印迹检测FK228对检查点蛋白Bub1及BubR1定位和表达的影响;采用纺锤体检查点功能实验检测FK228对细胞检查点功能的影响。结果提示,FK228处理24 h后,G2/M期细胞比例由6.35%增至19.91%;FK228能够抑制检查点蛋白Bub1及BubR1着丝粒定位并上调两种蛋白表达;纺锤体检查点功能实验提示对照组经Nocodazole或Taxol处理后G2/M期细胞比例最高分别达35.74%及29.24%,而FK228处理组经上述两种药物处理后各时间点G2/M期细胞所占比例皆明显减少(最高所占比例分别仅为7.13%及6.03%),失去了随时相点的动态变化,提示FK228抑制了A549细胞纺锤体检查点在监测张力和黏附上的功能。该研究证实FK228能够通过影响纺锤体检查点蛋白着丝粒定位以及抑制纺锤体检查点的功能,进而干涉肿瘤细胞有丝分裂过程,有助于阐明HDACi杀伤肿瘤细胞的新机制。

HDACi-FK228 is a novel and promising anticancer drug. However, the underlying molecular mechanism has not been well clarified. In this study, we investigated the effect of FK228 on the spindle check- point function of human non-small-cell lung cancer A549 cells. Using the flow cytometry to detect the influence of FK228 on the A549 cell cycle; Using immunofluorescence staining to detect the centromeric localization of check- point protein Bubl and BubR1 and using Western blot to examine the checkpoint protein expression; The spindle checkpoint function of A549 cells after the FK228 treatment was determined by the spindle checkpoint function experiment. The results revealed that FK228 increased the GR/M ratio of cells from 6.35% to 19.91% after 24 h treatment; In addition, FK228 inhibited the centromeric localization of Bubl and BubR1 protein. However, western blot analysis indicated that FK228 treatment induced the protein expression of the Bub 1 and BubR1. Spindle check- point experiment indicated that the G2/M ratio in control group were increased highest to 35.74% by Nocodazole or29.24% by Taxol treatment, while the GJM ratio of FK228 group by Nocodazole or Taxol treatment in all the time point were decreased, for the highest is 7.13% by Nocodazole and 6.03% by Taxol treatment, which indicates that FK228 treatment inhibited the spindle checkpoint function of the A549 cells. Our data implicate that FK228 can inhibit the spindle checkpoint proteins Bubl and BubRl centromeric localization and inhibit the spindle checkpoint function of the A549 cells, which might be the possible reason of FK228 induced abnormal mitosis. This study con- tributes to elucidate the possible mechanism of FK228 to kill tumor cells.