CAJ | 학술논문

目的研究四嗪二甲酰胺(ZGDHu-1)诱导肺癌细胞株EBC-1凋亡的作用及分子机制.方法将不同浓度的ZGDHu-1与EBC-1细胞在体外培养,采用5′-溴-2′脱氧尿苷(BrdU)-酶联免疫吸附法(ELISA)观察ZGDHu-1对EBC-1细胞的增殖抑制作用;采用Annexin V/PI双标记染色与ELISA法测定凋亡细胞核小体等技术检测ZGDHu-1诱导EBC-1细胞凋亡的作用;采用流式细胞术检测经ZGDHu-1作用后EBC-1细胞磷酸化p38MAPK和Stat3表达的变化;采用Western blot法检测Bcl-2、Bax、Fas、p53、caspase-3蛋白表达的变化.结果 ZGDHu-1能抑制EBC-1细胞增殖,并呈现作时间和剂量依赖性,作用24、48和72 h后的IC50分别为(295±25)ng/ml、(112±8)ng/ml和(23±2)ng/ml.经ZGDHu-1作用后,EBC-1细胞中的Annexin V+/PI-细胞升高,细胞内核小体含量显著增加,二者均呈现剂量依赖性.EBC-1细胞与50、200和500 ng/ml的ZGDHu-1培养48 h后,磷酸化p38MAPK的表达率分别为67.4%、88.2%和91.1%,空白对照组为10.6%;而磷酸化Stat3的表达率分别为56.5%、43.6%和34.6%,空白对照组为89.1%.Western blot检测结果显示,随药物作用浓度的升高,Bax、Fas和p53蛋白的表达显著上调,Bcl-2的表达没有变化,caspase-3蛋白的表达则明显下调.结论 ZGDHu-1能显著抑制EBC-1细胞增殖,并诱导其凋亡.Fas介导的线粒体途径可能是ZGDHu-1诱导EBC-1细胞凋亡的通路之一,p38MAPK和Stat3激活也参与了ZGDHu-1诱导EBC-1细胞凋亡的过程.
목적 연구사진이갑선알(ZGDHu-1)유도폐암세포주EBC-1조망적작용급분자궤제.방법 장불동농도적ZGDHu-1여EBC-1세포재체외배양,채용5′-추-2′탈양뇨감(BrdU)-매련면역흡부법(ELISA)관찰ZGDHu-1대EBC-1세포적증식억제작용;채용Annexin V/PI쌍표기염색여ELISA법측정조망세포핵소체등기술검측ZGDHu-1유도EBC-1세포조망적작용;채용류식세포술검측경ZGDHu-1작용후EBC-1세포린산화p38MAPK화Stat3표체적변화;채용Western blot법검측Bcl-2、Bax、Fas、p53、caspase-3단백표체적변화.결과 ZGDHu-1능억제EBC-1세포증식,병정현작시간화제량의뢰성,작용24、48화72 h후적IC50분별위(295±25)ng/ml、(112±8)ng/ml화(23±2)ng/ml.경ZGDHu-1작용후,EBC-1세포중적Annexin V+/PI-세포승고,세포내핵소체함량현저증가,이자균정현제량의뢰성.EBC-1세포여50、200화500 ng/ml적ZGDHu-1배양48 h후,린산화p38MAPK적표체솔분별위67.4%、88.2%화91.1%,공백대조조위10.6%;이린산화Stat3적표체솔분별위56.5%、43.6%화34.6%,공백대조조위89.1%.Western blot검측결과현시,수약물작용농도적승고,Bax、Fas화p53단백적표체현저상조,Bcl-2적표체몰유변화,caspase-3단백적표체칙명현하조.결론 ZGDHu-1능현저억제EBC-1세포증식,병유도기조망.Fas개도적선립체도경가능시ZGDHu-1유도EBC-1세포조망적통로지일,p38MAPK화Stat3격활야삼여료ZGDHu-1유도EBC-1세포조망적과정.
Objective To study whether N,N′-di-(m-methylphenyi)-3,6- dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide(ZGDHu-1)inhibits proliferation and induces apoptosis in human lung carcinoma cell line EBC-1 cells and its molecular mechanism. Methods Different concentrations of ZGDHu-1 and different times of culture were used to treat EBC-1 cells in vitro. Thc inhibition of proliferation was measured by BrdU-ELISA. Cell apoptosis was detected by Annexin V/PI staining and cellular DNA fragmentation ELISA. Phosphorylated p38MAPK and STAT3 were examined by flow cytometry. The protein expressions of bcl-2, bax, p53, Fas, and caspase-3 were detected by Western blot analysis. Results ZGDHu-1 inhibited EBC-1 cell proliferation within a certain range of treating times and does, with a 24 h IC50 of (295 ± 25)ng/ml, 48 h of(112 ± 8)ng/ml and 72 h of(23 ± 2)ng/ml. The EBC-1 cell apoptosis was confirmed by Annexin V/PI labeling and cellular DNA fragmentation ELISA in a dose-related manner. When EBC-1 cells were treated with 50,200, and 500 ng/ml ZGDHu-1 for 48 h, the expression rates of phosphorp38MAPK protein were 67.4%, 88.2%, 91.1%, respectively, and that of the control was 10.6%. That of STAT3 protein were 56.5%, 43.6% and 34.6%, respectively, and that of the control was 89.1%. The expression of bax, p53 and Fas protein was significantly increased, that of bcl-2 was not changed, and that of caspase-3 was significantly decreased by the ZGDHu-1 treatment. Conclusion ZGDHu-1 can inhibit proliferation and induce apoptosis in EBC-1 cells. The mitochondrial pathway mediated by Fas may be one of its mechanisms. The apoptosis of EBC-1 cells may associate with up-regulation of phosphor-p38MAPK and down-regulation of phosphor-STAT3 in the cells.