土木香内酯通过调节细胞周期相关蛋白的表达抑制慢性粒细胞白血病耐药细胞株K562／ADR增殖

Alantolactone inhibits the proliferation of K562/ADR cells through regulating expression of cell cycle-related proteins

目的研究土木香内酯对慢性粒细胞白血病（CML）耐药细胞株K562／ADR增殖、周期分布以及周期相关蛋白的影响。方法选用0、1．0、2．0、4．0、6．0、8．0、10．0μmol／L土木香内酯作用K562／ADR细胞12、24、48h，四甲基偶氮唑蓝（MTr）比色法检测土木香内酯对K562／ADR细胞的增殖抑制率，流式细胞术检测土木香内酯对K562／ADR细胞周期分布的影响，Westernblot检测周期相关蛋白的表达。结果土木香内酯能够显著抑制K562／ADR细胞增殖，半数抑制浓度为5．0μmol／L。流式细胞术检测结果显示，不同浓度（0、2．5、5．0、7．5μmol／L）的土木香内酯能够使K562／ADR细胞周期阻滞在G2／M期，G2／M期细胞比例由（15．8±1．7）％分别提高到（21．0±2．4）％、（26．4±2．7）％、（30．1±3．9）％（P〈0．05）。土木香内酯能够明显降低CDKl、CyclinBl的表达，上调周期抑制蛋白p21的表达。此外，土木香内酯能够有效地抑制bcr-abl融合蛋白的表达水平。结论土木香内酯可能通过调节细胞周期相关蛋白的表达介导K562／ADR细胞G2／M期阻滞，抑制细胞的增殖。

Objective To investigate the effects of alantolactone on cell proliferation, cell-cycle and cell cycle-related proteins in human chronic myelogenous leukemia drug-resistant cell line K562/ADR. Methods K562/ADR cells were treated with 0, 1.0, 2.0, 4.0, 6.0, 8.0, and 10.0 μmol/L of alantolactone for 12, 24 and 48 h, with its cell viability analyzed by MTT assay. Flow cytometry was used to examine the effect of alantolactone on the cell-cycle of K562/ADR cells. The cell cycle-related proteins were analyzed by using Western blot after treatment with alantolactone. Results The results of MTT showed that alantolactone effectively inhibited the proliferation of K562/ADR cells in dose and time-dependent way, and the IC5o value of alantolactone in K562/ADR cells was about 5 μmol/L. Flow cytometric analysis displayed that alantolactone could arrest cell cycle at G2/M phase. The percentage of accumulated cells in the G2/M phase was increased from （15.8±1.7） % in the control group to （21.0±2.4） %, （26.4±2.7） %, and （30.1±3.9） % in cells treated with 2.5, 5.0, and 7.5 μmol/L of alantolactone for 24 h, respectively （P 〈 0.05）. Alantolactone significantly decreased the expression of CDK1 and CyclinB1 and increased the expression of cyclin-dependent kinase inhibitor p21. Meanwhile, the treatment of K562/ADR with alantolactone led to a dose-dependent decrease in bcr-abl protein levels. Conclusion Alantolactone can significantly inhibit the proliferation and cell-cycle arrest in GJM phase of K562/ADR cells, in which mechanism may be associated with the regulation of cell cycle-related proteins and downregulation of bcr-abl protein.