胃癌细胞抗脱落凋亡的分子机制

Molecular Mechanisms of the Gastric Cancer Cells Resisting Anoikis

目的探求胃癌细胞抗脱落凋亡的信号转导机制。方法用soft agar实验观察信号分子抑制剂对胃癌细胞非锚定生长的影响,流式细胞仪检测信号分子抑制剂对脱落培养胃癌细胞周期的影响及凋亡诱导作用,Western印迹检测胃癌细胞脱落培养后相关信号分子表达水平及活性的变化。结果信号分子抑制剂genistein、AG17和AG825完全抑制了胃癌细胞集落的形成、阻滞细胞于细胞周期的不同时期,而且诱导细胞脱落凋亡的作用比较明显;而LY、PD和SB作用细胞后,所形成的细胞集落与对照组相比略小、数目略少,亦阻滞细胞于细胞周期的不同时期,但是诱导细胞脱落凋亡的比例较低。Western印迹检测发现,胃癌细胞脱落培养24 h后,细胞的蛋白酪氨酸磷酸化水平明显增加,ERK的表达水平无显著差异,但是ERK-p水平显著升高。结论蛋白酪氨酸激酶、HER2激酶、ERK激酶和PDGFR激酶途径是胃癌细胞抗脱落凋亡的重要信号通路和信号分子。PI-3K途径和p38MAPK分子与胃癌细胞锚定非依赖性增殖密切相关,与胃癌细胞的锚定非依赖性存活无关。

Objective To explore the mechanisms of signal transduction by which gastric cancer cells resist anoikis. Methods Soft agar assay was used to observe the effect of the specific signal molecular inhibitors on the anchorage-independent growth of the gastric cancer cells. Flow cytometry ( FCM) was employed to examine the cell cycles and apoptotic rate of the gastric cancer cells cultured in suspension with the signal molecular inhibitors. Western blot analysis was performed to study the expression level and activation of some important signal molecules of the detached gastric cancer cells. Results Signal molecular inhibitors, Genistein, AG17 and AG825 completely inhibited the cell clone formation, arrested the gastric cancer cells in different cell cycles and induced cell anoikis significantly. But when cells were treated with LY, PD and SB, its clone size was smaller and the clones were less as compared with that of control. The treatment induced anoikis to a small extent, but arrested cells at various cell cycles. Western blot revealed that, 24 hours after detachment of gastric cancer cells, phosphorylation level of protein tyrosine was increased, activation of ERK-p was increased while ERK level showed no significant difference as compared with that of control. Conclusions Protein tyrosine kinase, HER2 kinase, ERK kinase and PDGFR kinase pathway are important signal molecule and signal pathway. PI-3K pathway and p38MAPK have important function in anchorage-independent growth of the gastric cancer cells, but a lesser role in anchorage-independent survival.