Qiao Shu-Kai, Wang Ying, Niu Zhi-Yun, Tan Jin-Man, Wang Jun-Li
Department of Hematology, The Second Hospital of Hebei Medical University; Shijiazhuang 050000, Hebei Province, China.
Department of Hematology, The Second Hospital of Hebei Medical University; Shijiazhuang 050000, Hebei Province, China. E-mail:
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2016 Feb;24(1):131-7. doi: 10.7534/j.issn.1009-2137.2016.01.026.
To investigate the effects of artesunate (ART) on proliferation, cell cycle and apoptosis of SKM-1 cells in vitro and to explore the underlying mechanisms.
After SKM-1 cells were treated with different concentrations of ART, the cell proliferation was determined by CCK-8 method. Apoptosis and distribution of cell cycle were detected by flow cytometry. Both DCFH-DA fluorescent probe and Fluo-3-Am fluorescent probe were used to detect the changes of intracellular reactive oxygen species (ROS) and calcium ion concentration. Western blot was used to measure the protein levels of BCL-2, BAX, BAD, P-BAD, survivin and XIAP.
ART obviously inhibited the growth of SKM-1 cells in time and dose-dependent manners (r = -0.841; r = 0.-786). The antioxidant trolox-pretreatment significantly decreased the growth inhibition effect of ART on SKM-1 cells. Caspase inhibitor Ac-DEVD-CHO partially reduced the growth inhibition effect of ART on SKM-1 cells. After treatment with ART for 24 hours, the apoptosis of SKM-1 cells was found, the cell cycle of SKM-1 was arrested in G0/G1 phase, ART could elevate the levels of calciumion and reactive orygen. ART could significantly down-regulate the protein expression levels of P-BAD and survivin in SKM-1 cells, and showed a highly negative correlation with ART dose (r = -0.909; r = -0.849). On the contrary, ART had no significant effect on expression levels of BAD and XIAP in SKM-1 cells, and after ART treatment, although BCL-2 protein expression was not significantly different when compared with control group, but the BCL-2/BAX ratio significantly decreased and highly negatively correlated with ART dose (r = -0.866).
The ART significantly suppresses the cell proliferation, induces the apoptosis and promoted cell cycle arrest at G0/G1 phase in SKM-1 cells. The mechanisms of ART anti-MDS is associated with the increase of intracellular calciumion concentration and ROS levels. In addition, the pro-apoptotic activity of ART may be involved in the regulation of BCL-2 /BAX ratio and the expressions of P-bad and survivin.
探讨青蒿琥酯（ART）对SKM-1细胞体外增殖、细胞周期及凋亡的影响，并探究其潜在机制。
用不同浓度的ART处理SKM-1细胞后，采用CCK-8法检测细胞增殖情况。通过流式细胞术检测细胞凋亡及细胞周期分布。使用DCFH-DA荧光探针和Fluo-3-Am荧光探针检测细胞内活性氧（ROS）和钙离子浓度的变化。采用蛋白质印迹法检测BCL-2、BAX、BAD、P-BAD、生存素和XIAP的蛋白水平。
ART明显以时间和剂量依赖性方式抑制SKM-1细胞的生长（r = -0.841；r = -0.786）。抗氧化剂托可索仑预处理显著降低了ART对SKM-1细胞的生长抑制作用。半胱天冬酶抑制剂Ac-DEVD-CHO部分降低了ART对SKM-1细胞的生长抑制作用。用ART处理24小时后，发现SKM-1细胞发生凋亡，SKM-1细胞周期停滞在G0/G1期，ART可提高钙离子和活性氧水平。ART可显著下调SKM-1细胞中P-BAD和生存素的蛋白表达水平，且与ART剂量呈高度负相关（r = -0.909；r = -0.849）。相反，ART对SKM-1细胞中BAD和XIAP的表达水平无显著影响，ART处理后，虽然BCL-2蛋白表达与对照组相比无显著差异，但BCL-2/BAX比值显著降低且与ART剂量呈高度负相关（r = -0.866）。
ART显著抑制SKM-1细胞的增殖，诱导其凋亡并促进细胞周期停滞在G0/G1期。ART抗骨髓增生异常综合征的机制与细胞内钙离子浓度和ROS水平升高有关。此外，ART的促凋亡活性可能参与了BCL-2 /BAX比值以及P-bad和生存素表达的调节。
