整合蛋白α5β1过表达诱导人肝癌细胞SMMC-7721“失巢凋亡”机理  

Overexpression of Integrin α5β1 Induces Anoikis in Human Hepatocellular Carcinoma Cells SMMC-7721

作　　者：雷霆雯[1] 梁玉龙[2] 查锡良[2] 

机构地区：[1]贵阳医学院生物化学教研室,贵阳550004 [2]复旦大学上海医学院生物化学与分子生物学系,上海200032

出　　处：《中国生物化学与分子生物学报》2004年第1期112-118,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金资助项目 (No .3 0 0 0 0 0 83 );上海市科委资助项目 (No .0 0JC14 0 42 )~~

摘　　要：为了进一步阐明整合蛋白α5 β1过表达诱导非粘着状态的人肝癌细胞SMMC 772 1失巢凋亡的机理 ,利用poly HEME阻断细胞贴壁 ,诱导失巢凋亡 ,在poly HEME包被的 96孔板中检测细胞生存率 .利用Western印迹法检测了与细胞信号转导密切相关的粘着斑激酶 (FAK)、蛋白激酶B(PKB)及生存蛋白survivin的表达水平 .α5 β1过表达细胞株在去粘附状态下的生存率明显低于对照细胞 .在生长 8d时 ,以整合蛋白不同亚单位转染的α5 β1 772 1、β1 772 1和α5 772 1细胞 ,活细胞数分别降至对照组的 4 0 %、4 5 %和 83% .这说明整合蛋白α5 β1过表达细胞株 ,特别是α5 β1 772 1和 β1 772 1细胞的失巢凋亡明显加快 .细胞悬浮生长 2 4h后 ,α5 β1过表达细胞株都有不同程度的凋亡发生 ;同时粘着斑激酶 (FAK)和蛋白激酶B(PKB)表达量及其磷酸化水平均低于对照细胞 ,而survivin表达水平在α5 β1 772 1中最高 ,在 β1 772 1中最低 .在给予天然细胞外基质纤粘连蛋白的条件下 ,α5 β1 772 1和 β1 772 1细胞的FAK和PKB的磷酸化水平却呈升高趋势 .结果说明 ,FAK和PKB的变化与细胞的粘附状态密切相关 ,并且参与整合蛋白α5 β1过表达细胞在去粘附状态下失巢凋亡的发生 .To further investigate the mechanisms that underly anoikis induced by overexpression of integrin α5β1 and blockade of cell attachment, the anoikis detection assay was exploited and survival rates were also analyzed in the 96-well under the same condition. Phosphorylation levels of focal adhesion kinase (FAK) and protein kinase B (PKB) were detected by Western blot analysis in the integrin α5β1 overexpressing cells cultured for 24 hours in the poly-HEME-coated Petri dishes. These results showed that the survival rates of α5β1-7721, β1-7721 and α5-7721 cells were decreased to 40%, 45% and 83%, respectively. Concomitantly, apoptosis occurred in the α5β1-7721 and β1-7721 cells to some extents. Moreover, FAK phosphorylation was declined in the α5β1-7721, β1-7721 and α5-7721 cells, while decrease of PKB phosphorylation only took place in β1-7721 and α5-7721 cells when the cells were seeded onto poly-HEME-coated Petri dishes. Meanwhile, Survivin, an inhibitor of apoptosis, was decreased in the β1-7721 cells. However, phosphorylation levels of FAK and PKB in α5β1-7721 and β1-7721 cells were elevated compared with mocked cells when they were grown on the fibronectin-coated dishes. These findings suggested that FAK and/or PKB might be involved in anoikis of α5β1-7721 and β1-7721 cells.

关 键 词：整合蛋白 失巢凋亡 粘着斑激酶 蛋白激酶B 肝癌细胞 

分 类 号：R735.7[医药卫生—肿瘤]