Flavopiridol增强TRAIL诱导SPC-A1细胞凋亡作用机制研究  

Mechanism of the apoptosis induction by Flavopiridol synergizes TRAIL in SPC-A1 cell

作　　者：何永勤 庄莉[2,3] 刘舒媛[1] 黄小琴[1] 禇嘉祐[1] 杨昭庆[1] 

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所,云南昆明650118 [2]昆明医科大学第三附属医院 [3]云南省肿瘤医院内科,云南昆明650106

出　　处：《中华肿瘤防治杂志》2013年第10期730-733,756,共5页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家高技术发展计划(863计划,2012AA02A201)

摘　　要：目的:研究夫拉平度(FP)增强肿瘤坏死因子相关凋亡诱导配体(TRAIL)诱导非小细胞肺癌细胞SPC-A1凋亡的作用及其分子机制。方法:采用TRAIL、FP单独及联合作用的方法诱导细胞凋亡,MTT法检测各处理组细胞的增殖及活性;蛋白质印迹法检测细胞凋亡相关基因蛋白水平的表达;定量PCR检测TRAIL受体及髓系细胞白血病-1(Mcl-1)、Fas相关死亡结构域样IL-1β转化酶样抑制蛋白(FLIP)、X染色体连锁的细胞凋亡抑制因子(XIAP)和Livin等凋亡基因的转录水平表达变化。结果:100nmol/L FP处理组(F)细胞存活率为(80.26±2.43)%,100ng/mL TRAIL处理组(T)为(90.49±1.60)%,FP和TRAIL联合组(F+T)为(47.48±1.41)%,联合组的细胞存活率低于单一处理组,F=406.08,P=0.000。FP和TRAIL合用时,FLIP和XIAP分别降为对照组的4%和15%,Mcl-1与Livin的表达量甚至低于可检测水平。Caspase-3和Caspase-8的活性片段约为对照组的4倍。细胞色素c由线粒体向细胞质的释放增加约7倍,而Bcl-2家族蛋白Bcl-2、Bax、Bak和Bcl-xl的表达量基本不变。FP作用后引起DR4的mRNA表达水平(2.51±0.14)上升,P<0.05。但FP和TRAIL单独作用并未引起Mcl-1、FLIP、XIAP和Livin在转录水平的表达变化。联合组加入广谱Caspase抑制剂Z-VAD-FMK后,细胞存活率由(58.26±1.75)%上升至(88.17±2.65)%,P=0.003。说明Z-VAD-FMK可明显抑制联合处理组的凋亡效应。结论:FP能显著增强TRAIL诱导SPC-A1细胞凋亡的作用,此协同促凋亡效应与抗凋亡基因Mcl-1、FLIP、XIAP和Livin的蛋白水平表达降低以及Caspase活性增强和线粒体损伤有关,同时涉及内源性(线粒体)及外源性(非线粒体)凋亡信号通路的共同作用。OBJECTIVE：To study the synergistic mechanism between Flavopiridol （FP） and tumor necrosis factor related apoptosis inducing ligand （TRAIL） induced apoptosis in non-small cell lung cancer cell line SPC-A1. METHODS： SPC-A1 cells were treated with FP,TRAIL and FP combined with TRAIL to induce apoptosis. MTT assay was used to measure cell proliferation and viability. Expression of apoptosis related proteins were detected by using Western blotting. RT-PCR was used to measure RNA transcript expression. RESULTS .. The cell surviving rates of FP and TRAIL combina- tion treatment group （47.48 ± 1.41）%was significantly lower than that of the 100 nmol/L FP group [ （80.26 ± 2.43）% ] and 100 ng/mL TRAIL group [（90.49±1.60）5（P=0. 000）]. In the combination of TRAIL plus FP treatment group, the expression of anti-apoptotie protein FLIP（4 % ）, XIAP（15 % ）, Mcl-1, and Livin were down-regulated significantly. The expression of Mcl-1 and Livin were under detectable level. Cleaved Caspase-8 and Caspase-3 were increased,and their ac- tive fragments were about four times of the control group. The cyt0chrome e released from mitochondria into cytosol was about 7 times of the control group. However,the Bcl-2 family proteins,Bcl-2,Bax,Bak,and Bcl-xl were not altered by thecombination treatment. DR4 mRNA expression was up-regulated by FP treatment and its relative expression level was 2.514-0.14（P〈0.05）. There were no significantly changes on transcription of Mcl-1, FLIP, XIAP and Livin. The cell survival rates were raised from （58.26±1.75）% up to （88. 17±2.65）% （P〈0. 003） by using Pan-Caspase inhibitor Z-VAD-FMK,which indicated that the synergistic effect of FP plus TRAIL on apoptosis induction can be inhibited by Z-VAD-FMK. CONCLUSIONS：Our study indicated that FP synergized TRAIL- induced apoptosis in SPC-A1 cell through up-regulating DR4 transcription,down-regulating Mcl-1, FLIP, XIAP, Livin proteins but not their transcripts, as well as increasing Caspase activity and mitoch

关 键 词：夫拉平度 肿瘤坏死因子相关凋亡诱导配体 SPC-A1细胞 细胞凋亡 

分 类 号：R734.2[医药卫生—肿瘤]