Lg-1诱导人胃癌细胞系BGC-823凋亡作用及机制的探讨

Inducing effect of Lg-1 on apoptosis of human gastric cancer celland its mechanism

目的:研究新鬼臼毒素衍生物4β-4-脱氧-氮取代的异亮氨酸(5-FU基)-戊醇酯-4′-去甲表鬼臼(Lg-1)对人胃癌细胞系BGC-823的诱导凋亡作用及机制。方法:噻唑蓝(MTT)、流式细胞术和Hoechs33258染色分别检测Lg-1对BGC-823细胞增殖、细胞周期及凋亡的影响。荧光定量RT-PCR(qRT-PCR)检测药物处理后对BGC-823细胞凋亡相关基因p53、Bax、Bcl-2、p21和Caspase-3表达的影响。结果:Lg-1对BGC-823细胞增殖具有明显剂量和时间依赖的抑制作用,随药物浓度增加,抑制效应增强,药物浓度为0.01、0.1、1和10μmol/L时,Lg-1对BGC-823的抑制率分别为23.5%、30.8%、32.4%和52.1%,与依托泊苷(Vp-16)处理组(3.5%、8.7%、10.9%和21.3%)比较差异有统计学意义,P均<0.01。随时间延长,抑制作用逐渐增强,12～60hLg-1处理组对BGC-823的抑制率与Vp-16处理组比较差异有统计学意义,12h抑制率分别为9.8%和8.0%,P=0.001;24h分别为11.6%和9.0%,P=0.02;36h分别为19.4%和16.2%,P=0.014;48h分别为44.1%和41.2%,P=0.048;60h分别为65.4%和50.1%,P=0.001。细胞周期检测显示,Lg-1作用12和24h后,G2/M期的细胞含量分别为39.37%和74.36%,与对照组及Vp-16组比较均明显增加,而G0/G1期细胞含量分别为20.06%和12.25%,较对照组及Vp-16组显著减少。Hoechst33258染色显示,Lg-1处理组细胞核固缩、碎裂、凋亡样改变,药物处理后Bcl-2mRNA的表达呈降低趋势,Vp-16处理组Bcl-2mRNA表达量为0.748,Lg-1处理组为0.425,而p53、Bax、p21和Caspase-3mRNA的表达呈增高趋势,Vp-16处理组mRNA表达量分别为1.074、1.232、1.818和2.9,Lg-1处理组分别为1.903、1.393、1.911和3.097,且Lg-1处理组作用优于Vp-16处理组。结论:Lg-1可抑制胃癌细胞增殖,使细胞阻滞在G2/M期,该过程可能与其上调p53、Bax、p21、Caspase-3表达和下调Bcl-2基因的表达有关。

OBJECTIVE： To study the apoptosis inducing effect and its mechanism of 4β- N-substituted- isoleucine 5-FU-pentyl ester-4＇-demethylepipodophyllotoxin （Lg-1） in human gastric cancer cell line BGC-823. METHODS： The effect of I.g 1 on BGC 823 was observed by MTT,flow cytometry and Hoechs33258 staining. The apoptosis related genes p53, Bax, Bel-2, p21 and Caspase 3 expression were detected by fluorescent quantitative RT-PCR （qRT-PCR）. RESULTS：Lg-1 was demonstrated to exert antiproliferative activity in a dose-and time-dependence. With the increase of drug concentration,depressant effect strengthen gradually,drug concentration of 0. 01, 0. 1,1 and 10 μmol/L, Lg-1 inhibitory rate of BGC-823 were 23.5 %, 30.8 %, 32.4 %//00 and 52.1 % ,and the inhibition of drug treatment group had significant di{ference to Vp-16 treatment group 3.5% ,8.7% 10.9% and 21.3%//00 （P%0.01）. In addition,the inhibition had positive correlation to time extend,from 12 to 60 hof Lg-1 inhibitory rate of BGC-823 were 9.8%,11.6%,19.4%,44.1% and 65.4%. It also had significant difference between Lg-1 treatment group （8.0 %, 9.0 G, 16.2 % and 41.2%） and Vp-16 treatment group after 12 hto60 h（12 h,P=0.001;24 h,P=0.02;36 h,P=0.014;48 h,P=0.048;60 h,P=0.001）. The proportion of cells in G0/GI phase decreased while the proportion of cells in Gz/M phase increased after 12 and 24 hours treated with Lg-1,12 and 24 h G2/M phase of the cell contents were 39.37% and 74.36%,G0/G1 phase cell contents were 20.06% and 12.25~. Nuclear chromatin condensations and nuclear fragments were also observed in Lg-1 group cells. The expression of Bcl-2 mRNA decreased,the expression of Bcl-2 mRNA in Vp-16 treatment group was 0. 748,and Lg-1 was 0. 425. while p53, Bax, p21 ,Caspase-3 mRNA expression were increased following Lg-1 treatment, the mRNA expressions in Vp-16 treated groups were 1. 074,1. 232,1. 818 and 2.9;Lg-1 groups were 1. 903,1. 393,1. 911 and 3. 097. CONCLUSION ： Lg-1 shows obvious antieaneer activity by inducing cell cycle arrest and regulating the expression of apoptosic related gene.