苦丁冬青苷D对人HCC-1806乳腺癌细胞增殖、凋亡、自噬的影响及机制研究
Effects and Mechanism Study of Kudinoside D on Proliferation,Apoptosis and Autophagy of Human HCC-1806 Breast Cancer Cells
蒋永旭 1丁明聪 2赵泽义 2肖家军2
作者信息
- 1. 皖南医学院药学院,安徽 芜湖 241002;蚌埠市中医医院,安徽蚌埠 233080
- 2. 蚌埠市中医医院,安徽蚌埠 233080
- 折叠
摘要
目的 探讨苦丁冬青苷D(KD-D)对人HCC-1806 乳腺癌细胞增殖、凋亡、自噬的影响及机制.方法 将人HCC-1806 乳腺癌细胞作为研究对象,给予不同浓度KD-D处理后,采用CCK-8 法检测细胞存活率;EdU法检测细胞增殖能力;结晶紫染色法检测细胞克隆形成能力;流式细胞术(Annexin V-FITC/PI法)检测细胞凋亡;JC-1 染色法检测细胞线粒体膜电位;免疫荧光法检测细胞Cleaved Caspase-3、LC3、P62 蛋白表达;Western Blot法检测细胞Cleaved Caspase-3、Pan-AKT、Phosp-AKT、LC3Ⅱ蛋白表达;自噬双标mRFP-EGFP-LC3 腺病毒感染实验检测细胞自噬流.结果 与对照组比较,12.5、25、50、100、200、400 μmol·L-1 KD-D处理HCC-1806 细胞 24、48 h后,随着给药浓度增大和处理时间延长,细胞活性显著降低(P＜0.001),细胞内吸光度值显著降低(P＜0.001),细胞增殖受到抑制;60、80 μmol·L-1 KD-D组的细胞克隆形成计数显著减少(P＜0.001);50、100、150 μmol·L-1 KD-D组细胞的早期及晚期凋亡比例均显著升高(P＜0.05,P＜0.001);40、60、80 μmol·L-1 KD-D组的红色荧光比例显著下降(P＜0.001),绿色荧光比例显著升高(P＜0.01,P＜0.001),HCC-1806 细胞线粒体膜电位下降;60、80、100 μmol·L-1 KD-D组细胞的 Cleaved Caspase-3 蛋白表达均显著上调(P＜0.001),60 μmol·L-1 KD-D 组细胞的 Pan-AKT、Phosp-AKT 蛋白表达均显著上调(P＜0.001);60 μmol·L-1 KD-D组细胞的LC3 蛋白荧光小点数量显著增加(P＜0.001),P62 蛋白平均荧光强度显著降低(P＜0.001),LC3Ⅱ蛋白表达显著上调(P＜0.001),自噬小体及自噬溶酶体数量显著增加(P＜0.01,P＜0.001),自噬流激活.与 60 μmol·L-1 KD-D 组比较,KD-D+AKT 抑制剂(Afuresertib)组细胞的 Cleaved Caspase-3、Pan-AKT蛋白表达显著下调(P＜0.01,P＜0.001),Phosp-AKT蛋白表达显著上调(P＜0.001).结论 KD-D能抑制人乳腺癌HCC-1806 细胞增殖,并诱导其发生凋亡和自噬,KD-D诱导HCC-1806 细胞凋亡可能与激活AKT信号影响Cleaved Caspase-3 表达有关.
Abstract
Objective To investigate the effect and mechanism of kudinoside D(KD-D)on proliferation,apoptosis and autophagy of human HCC-1806 breast cancer cells.Methods Human HCC-1806 breast cancer cells were treated with different concentrations of KD-D,and the cell viability was detected by CCK-8 method.EdU method was used to detect cell proliferation ability;the ability of cell clone formation was detected by crystal violet staining.Apoptosis was detected by flow cytometry(Annexin V-FITC/PI).Mitochondrial membrane potential was detected by JC-1 staining.The protein expressions of Cleaved Caspase-3,LC3 and P62 were detected by immunofluorescence.The protein expressions of Cleaved Caspase-3,Pan-AKT,Phosp-AKT and LC3Ⅱ were detected by Western Blot.Autophagy double-labeled mRFP-EGFP-LC3 adenovirus infection assay was used to detect cell autophagy flow.Results Compared with the control group,HCC-1806 cells were treated with 12.5,25,50,100,200,400 μmol·L-1 KD-D for 24 and 48 hours.With the increase of drug concentration and treatment time,the cell activity was significantly decreased(P＜0.001),the intracellular absorbance value was significantly decreased(P＜0.001),and the cell proliferation was inhibited.The cell clone formation counts in 60 and 80 μmol·L-1 KD-D groups were significantly decreased(P＜0.001).The proportion of early and late apoptosis in 50,100,150 μmol·L-1 KD-D groups were significantly increased(P＜0.05,P＜0.001).The proportion of red fluorescence in 40,60 and 80 μmol·L-1 KD-D groups were significantly decreased(P＜0.001),the proportion of green fluorescence was significantly increased(P＜0.01,P＜0.001),and the mitochondrial membrane potential of HCC-1806 cells decreased.The protein expression of Cleaved Caspase-3 in 60,80,100 μmol·L-1 KD-D group were significantly up-regulated(P＜0.001),and the protein expressions of Pan-AKT and Phosp-AKT in 60 μmol·L-1 KD-D group were significantly up-regulated(P＜0.001).In the 60 μmol·L-1 KD-D group,the number of LC3 protein fluorescent dots was significantly increased(P＜0.001),the average fluorescence intensity of P62 protein was significantly decreased(P＜0.001),the expression of LC3Ⅱ protein was significantly up-regulated(P＜0.001),the number of autophagosomes and autolysosomes were significantly increased(P＜0.01,P＜0.001),and the autophagic flow was activated.Compared with 60 μmol·L-1 KD-D group,the expression of Cleaved Caspase-3 and Pan-AKT protein in KD-D+AKT inhibitor(Afuresertib)group was significantly down-regulated(P＜0.01,P＜0.001),and the expression of Phosp-AKT protein was significantly up-regulated(P＜0.001).Conclusion KD-D can inhibit the proliferation of human breast cancer HCC-1806 cells and induce apoptosis and autophagy.The apoptosis of HCC-1806 cells induced by KD-D may be related to the activation of AKT signal and the expression of Cleaved Caspase-3.
关键词
苦丁冬青苷D/人HCC-1806乳腺癌细胞/增殖/凋亡/自噬/AKT信号/Cleaved/Caspase-3Key words
Kudinoside D/HCC-1806 cells/proliferation/apoptosis/autophagy/AKT signal/Cleaved Caspase-3引用本文复制引用
基金项目
国家自然科学基金(81402818)
安徽省教育厅高等学校科研项目(YJS20210556)
出版年
2024
NETL
NSTL
万方数据