青蒿琥酯通过自噬通路诱导乳腺癌细胞G2／M周期阻滞  

Artesunate induces G2/M cell cycle arrest through autophagy induction in breast cancer cells

作　　者：寿柳梅 陈凯[2] 林芳[4] 吴梦瑶[1] 沈梦[2] 龚斐然[3] 陶敏[2] 李伟[2] 

机构地区：[1]浙江中医药大学附属第一医院肿瘤二区,杭州310006 [2]苏州大学附属第一医院肿瘤科,215006 [3]苏州大学附属第一医院血液科,215006 [4]苏州大学医学部药理系,215123

出　　处：《中华实验外科杂志》2017年第4期555-558,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金（81101867、81472296、81272542、81200369、81501970）;江苏省中医药局科技项目（L213236）

摘　　要：目的探讨青蒿琥酯（ART）对乳腺癌细胞周期的影响及分子机制。方法通过流式细胞仪检测细胞周期；通过实时荧光定量聚合酶链反应（FQ-PCR）和Western blot检测p21、Beclin1基因表达；通过细胞免疫荧光检测自噬体形成；通过自噬抑制剂3-甲基腺嘌呤（3-MA）阻断自噬通路，检测ART对乳腺癌细胞细胞周期的影响是否通过自噬依赖性机制。结果对照组G2/M期细胞比例为（9.64±0.74）%，ART组为（16.67±3.29）%，两组比较差异有统计学意义（t＝3.614，P＝0.022）；ART组p21 mRNA表达水平增加（14.28±0.10）倍（t＝23.094，P＝0.000），蛋白表达增加（1.58±0.80）倍（t＝7.330，P＝0.018）；提示ART上调细胞周期蛋白依赖性激酶抑制因子（CKI）家族成员p21的表达，并使乳腺癌细胞MCF-7细胞周期阻滞于G2/M期。与对照组比较，ART组Beclin1 mRNA表达水平增加（1.71±0.14）倍（t＝8.583，P＝0.006），蛋白表达增加（1.61±0.91）倍（t＝8.553，P＝0.009）；胞质内微管相关蛋白1轻链3（LC3）荧光斑点增加（2.79±0.42）倍（t＝7.342，P＝0.018），提示ART能够诱导MCF-7细胞发生自噬。3-MA联合ART组与3-MA组比较，p21蛋白表达量、G2/M期细胞比例差异无统计学意义。提示自噬抑制剂3-MA阻断自噬通路，可削弱ART诱导的p21表达上调和G2/M期阻滞。结论ART通过诱导乳腺癌细胞自噬，导致G2/M期阻滞。Objective Autophagy is termed as type II programmed cell death （PCD）. Artesu- Mate （ART） can inhibit the proliferation of various cancer cell lines. In the present study, we investigated the regulation on cell cycle by artesunate. Methods Cell cycle was tested by flow cytometry. Gene expression level was determined by fluorescent quantitative polymerase chain reaction （ FQ - PCR） and West- ern blotting. Formation of autophagosome was determined by immunofluorescence. For cell signaling trans- duction investigation, autophagy - dependent pathway was blocked by 3 - methyladenine （3 - MA）, a classical autophagy inhibitor. Results The G2/M cell population rate of artesunate group （ 16. 67 ± 3.29） % was higher than the control group （9.64 ± 0. 74） % （ t = 3. 614, P = 0. 022）. The expression of p21 mRNA and protein of artesunate group were （ 14. 28 ± 0. 10, t = 23. 094,P = 0. 000）, （ 1.58 ± 0. 80, t =7. 330, P =0. 018） higher than that of the control group. We showed that artesunate arrested the cell cycle in the G2/M phase, which was accompanied by upregulation of p21, a member of cyclin dependent kinases inhibitor （CKI）. The expression of Beclinl mRNA, protein of artesunate group were （ 1.71± 0. 14）, （ 1.61±0. 91 ） higher than that of the control group. The microtubule - associated protein 1 light chain 3 （LC3） punctate of artesunate group was （2. 79 ± 0.42, t = 7. 342, P = 0. 018 ） higher than that of the control group. We found that artesunate could induce autophagy. Pretreated with 3 - MA, artesunate could no longer increase p21 protein expression and G2/M cell population. Conclusion Artesunate arrested cell cycle in G2/M phase through autophagy pathway dependent manner.

关 键 词：青蒿琥酯 自噬 乳腺癌 细胞周期 

分 类 号：R737.9[医药卫生—肿瘤]