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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 antitumor drug, but its mechanism of action has not been fully understood. To further elucidate the mechanism of FK228 killing tumor cells, the effect of FK228 on the cell cycle of non-small cell lung cancer A549 cells was examined by flow cytometry The effect of FK228 on the localization and expression of Bub1 and BubR1 were examined by immunofluorescence staining and Western blotting. The function of FK228 on the function of cell checkpoint was tested by spindle function test. The results showed that the proportion of cells in G2 / M phase was increased from 6.35% to 19.91% after FK228 treatment for 24 h. FK228 could inhibit the localization of Bub1 and BubR1 centromeres and upregulate the expression of both proteins. The functional test of spindle checkpoint The proportion of cells in G2 / M phase after treatment with Nocodazole or Taxol in control group was 35.74% and 29.24% respectively, while the proportion of cells in G2 / M phase in FK228 treated group was significantly decreased Accounting for only 7.13% and 6.03%, respectively), which lost the dynamic changes at any time point, suggesting that FK228 inhibits the function of spindle checkpoint on A549 cells in monitoring tension and adhesion. This study demonstrates that FK228 can help clarify the new mechanism of HDACi killing tumor cells by affecting the centromere localization of spindle checkpoint protein and the function of spindle checkpoint to interfere with the mitotic process of tumor cells.