摘要
目的西黄丸有消坚化结、解毒散痈的功效,是中医抗癌名方,对实体肿瘤具有一定疗效。本研究观察西黄丸含药血清对人肝癌细胞株BEL-7404细胞增殖、凋亡与自噬的影响,旨在探讨其抗肿瘤作用的机制。方法制备高、中和低剂量组西黄丸含药血清,分别处理BEL-7404细胞。MTT法检测细胞增殖;AnnexinⅤ-FITC/PI双染色流式细胞术检测细胞凋亡;蛋白质印迹法检测细胞自噬相关蛋白(LC3-Ⅰ/LC3-Ⅱ)的表达水平;单丹磺酰尸胺(monodansylcadaverine,MDC)染色后激光共聚焦显微镜下观察细胞自噬体的情况。自噬抑制剂3-甲基腺嘌呤(3-Methyladenine,3-MA)联合西黄丸含药血清处理BEL-7404细胞,检测细胞增殖、凋亡和自噬相关蛋白表达水平。结果高、中和低剂量组西黄丸含药血清处理24h后,BEL-7404细胞的增殖抑制率分别为(32.7±3.4)%、(16.8±2.2)%和(8.8±1.9)%,明显高于对照组的(3.0±0.5)%,H=21.61,P<0.001;细胞凋亡率分别为(37.8±2.1)%、(26.6±1.2)%和(15.4±0.9)%,明显高于对照组的(4.8±0.4)%,H=21.64,P<0.001。西黄丸含药血清处理24h后,BEL-7404细胞LC3-Ⅱ蛋白的表达水平升高且呈剂量依赖关系;同时,荧光染色激光共聚焦显微镜观察到细胞自噬体明显多于对照组。自噬抑制剂3-MA联合高、中和低剂量组西黄丸含药血清处理24h后,BEL-7404细胞的增殖抑制率分别为(42.3±4.5)%、(25.1±3.5)%和(18.2±2.3)%,明显高于单用同剂量组含药血清的(34.2±3.6)%、(17.2±1.8)%和(9.8±1.2)%,U高=4,U中=0,U低=0,均P<0.05;相应的,3-MA联合高、中、低剂量组西黄丸含药血清处理24h后,BEL-7404细胞的的凋亡率分别为(54.5±3.5)%、(43.2±2.9)%和(32.8±3.7)%,明显高于单用同剂量组含药血清的(38.5±2.5)%、(23.8±1.7)%和(18.8±1.3)%,U高=0,U中=0,U低=0,P<0.05。结论西黄丸含药血清能抑制BEL-7404细胞增殖,诱导其发生凋亡与自噬;抑制自噬可以增强西黄丸含药血清对BEL-7404细胞的增殖抑制和凋亡诱导效应。
OBJECTIVE Xihuang pill, a famous anti-cancer prescription of traditional Chinese medicine, has the effects of eliminating hard knot and detoxification carbuncle. Modern study confirmed that it behaved as an effective treat- ment of some solid tumors, but the concrete mechanisms are not clear. This study is to investigate the effects and mechanisms of Xihuang pill containing serum on the proliferation, apoptosis and autophagy of human hepatocellular carcinoma EEL 7404 cells. METHODS The Xihuang pills containing serum of high, middle and low-dose group and control group serum were set up. BEL-7404 cells were treated with the serum of different groups. The proliferation and apoptosis of cells were analyzed by MTT assay and flow cytometry （Annexin Ⅴ-FITC/PI double staining assay）. The expressions of autophagy-associated proteins （LC3- Ⅰ/LC3- Ⅱ ） were determined by Western blot assay. Autophgosomes in BEL-7404 cells were observed by Monodansylcadaverin （MDC） staining under laser scanning confocal microscope （LSCM）. BEL-7404 cells were treated with the combination of the autophagy inhibitor, 3-methyladenine （3-MA）,and Xihuang pill containing serum, then the proliferation and apoptosis of cells and the expressions of autophagy-assoeiated proteins were analyzed. RESULTS After the treatment of the Xihuang pill containing serum of high, middle and low-dose groups for 24 h, the proliferation inhibition rates of BEL-7404 ceils were （32.7±3.4）%, （16.8±2.2） % and （8.8±1.9）% respec tively, significantly higher than （3.0±0.5）% of the control group （H=21.61,P〈0.05）. Accordingly, the apoptosis rates of BEL-7404 cells were （37.8±2.1）%, （26.6±1.2）% and （15.4±0.9）% respectively, significantly higher than （4.8±0.4） % of the control group （H=21.64,P〈0. 001）. The Xihuang pill containing serum treatment could dose-dependently increased LC3-Ⅱ expression, and the formation of autophagosomes in BEL-7404 cells after the treatment could be observed under LSCM. After the treatment of the combination of Xihuang pill containing serum of high, middle and low-dose groups and the autophagy inhibitor 3-MA for 24 h, the growth inhibition rates of BEL-7404 cells were （42.3±4.5）%, （25.1±3.5）% and （18.2±2.3）%, respectively, significantly higher than （34.2±3.6）%, （17.2±1.8）% and （9.8 ± 1.2） % of the different drug-containing serum groups （UH = 4,UM = 0,UL = 0, P〈0.05）. Accordingly, the apoptosis rates of BEL-7404 cells were （54.5±3.5）%, （43.2±2.9）% and （32.8±3.7）% respectively, significantly higher than （38.5±2.5） %, （23. 8±1.7） % and （18.8±1.3） % of the different drug-containing serum groups （UH =0,UM=0, UL =0,P〈0.05） .CONCLUSIONS The Xihuang pill containing serum inhibits proliferation and induces apoptosis and autophagy in BEL-7404 cells. Autophagy inhibitor, 3-MA could potentiate the proliferation inhibiting and apoptosis inducing effects of Xihuang pill containing serum.
出处
《中华肿瘤防治杂志》
CAS
北大核心
2015年第22期1735-1740,共6页
Chinese Journal of Cancer Prevention and Treatment
基金
国家自然科学基金(81402040)
安徽高校省级自然科学研究重点项目(KJ2012A163)
关键词
西黄丸
肝癌
自噬
凋亡
增殖
Xihuang pill
hepatocellular carcinoma
autophagy
apoptosis
proliferation
