芹菜素诱导乳腺癌MCF-7细胞株蛋白激酶B相关性自噬性死亡  

Apigenin induces protein kinase B-associated autophagic cell death of breast cancer MCF-7 cell line

作　　者：刘博文[1] 张斌[1] 张伟然[1] 赵洪猛[1] 曹旭晨[1] 

机构地区：[1]天津医科大学附属肿瘤医院乳腺一科乳腺癌防治教育部重点实验室天津市“肿瘤防治”重点实验室,300060

出　　处：《中华实验外科杂志》2013年第9期1876-1878,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（81001186）;天津市自然科学基金资助项目（10JCYBJC14100）

摘　　要：目的 探讨芹菜素对乳腺癌MCF-7细胞株增殖的影响及其机制.方法 常规培养MCF-7细胞,用噻唑蓝（MTT）法评价芹菜素（0、10、20、40、80 μmol/L）处理乳腺癌MCF-7细胞株24、48 h的增殖抑制率；DNA ladder检测芹菜素（0、10、20、40 μmol/L）处理24 h对MCF-7细胞株凋亡的影响；、丫啶橙（AO）染色观察芹菜素（0、10、20、40μmol/L）处理24 h后MCF-7细胞自噬泡的形态及数量,然后通过流式细胞术对细胞自噬率进行定量分析；Western blot法检测芹菜素（0、10、20、40 μmol/L）处理24 h后自噬相关蛋白LC3、蛋白激酶B（Akt）及其磷酸化水平的变化；Akt cDNA瞬时转染MCF-7细胞,使其过表达,流式细胞术检测细胞自噬率的变化.结果 MTT显示,芹菜素明显抑制乳腺癌MCF-7细胞的增殖,具有浓度依赖性及时间依赖性；DNA ladder并未显示出明显的阶梯状条带,提示芹菜素不能有效的诱导MCF-7细胞凋亡；AO染色显示芹菜素可诱导MCF-7细胞株自噬,且流式细胞术显示细胞自噬率具有浓度依赖性,芹菜素20、40 μmol/L组与对照组比较差异有统计学意义（P ＜0.01）；Western blot显示,芹菜素可增加LC3Ⅱ的表达,同时降低Akt的磷酸化水平；自噬抑制剂3-甲基腺苷（3-MA）可使MCF-7的增殖抑制率降低,与对照组比较,差异有统计学意义（P ＜0.05）；Akt cDNA瞬时转染使Akt的表达明显增加,AO染色流式细胞术结果示Akt的过表达使得自噬率降低,与control cDNA转染组比较,差异有统计学意义（P＜0.01）.结论 芹菜素不能有效地诱导乳腺癌MCF-7细胞株凋亡,而是通过诱导自噬性死亡的方式抑制其增殖,该过程与芹菜素抑制Akt相关通路的活性相关.Objective To investigate the effects of apigenin on the proliferation of breast cancer MCF-7 cell line and its underlying mechanism.Methods MCF-7 cells were cultured in vitro.The proliferation of cells treated with different concentrations of apigenin （0,10,20,40,and 80 μmoL/L） was assessed by using 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTF） assay.DNA ladder was performed to detect the apoptosis-inducing effect of apigenin on MCF-7 cells.Acridine Orange （AO）dyeing was used to detect the morphology and quantity of autophagy of MCF-7 cells treated with apigenin （0,10,20,and 40 μmol/L） for 24 h,and autophagic rate was examined by flow cytometry.Western blotting was performed to detect the expression levels of LC3,protein kinase B （Akt） and its phosphorylation level after treatment of apigenin （0,10,20,and 40 μmol/L） for 24 h.Transient transfection of Akt cDNA into MCF-7 cells was performed to assess the role of Akt in the process of autophagy induced by apigenin.Results Apigenin dose-and time-dependently depressed the cell proliferation in human breast cancer MCF-7 cells.Apigenin treatment failed to form the ＂DNA ladder＂ fragmentation,indicating that apigenin didn＇t have the ability of inducing apoptosis of MCF-7 cells effectively.AO dyeing revealed the existence of autophagy induced by apigenin,and the autophagic rate was dose-dependent showed by flow cytometry （P ＜0.01）.Western blotting exhibited an increase in the expression level of LC3 Ⅱ,and a reduction in the phosphorylation level of Akt.3-methyladenine （3-MA）,a kind of autophagy inhibitor,significantly decreased the proliferation inhibition induced by apigenin （P ＜ 0.05）.Transient transfection of Akt cDNA,up-regulating the Akt expression in MCF-7 cells,significantly depressed the level of autophagy,with a significant difference compared with control cDNA transfection （P ＜ 0.01）.Conclusion Apigenin has the ability of depressing the proliferation of breast cancer MCF-7 cells th

关 键 词：芹菜素 乳腺癌 自噬 

分 类 号：R737.9[医药卫生—肿瘤]