阻断磷脂酶C-γ1信号通路对大肠癌细胞株LoVo细胞增殖与凋亡的影响

背景与目的:磷脂酶C-γ1(phospholipase C gamma 1,PLC-γ1)是跨膜信号转导中关键和重要的一个信号中介,是细胞增殖与细胞凋亡调控的一个重要分子,最近研究发现它在大肠癌等许多肿瘤组织中呈过表达状态,与肿瘤的发生、发展有密切关系.本研究主要探讨阻断PLC-γ1信号通路后对大肠癌LoVo细胞增殖、凋亡的影响,及其上述影响的信号机制.方法:以人大肠癌LoVo细胞作为研究模型,利用PLC-γ1特异的化学阻断剂U73122处理以阻断LoVo细胞中PLC-γ1信号通路,通过绘制细胞生长曲线、PI单染的流式细胞仪检测细胞周期而评估对其细胞增殖的影响,通过细胞形态观察及DNA片段琼脂糖凝胶电泳评估是否启动细胞凋亡,同时检测阻断PLC-γ1信号通路后HSP70、Caspase-3表达水平的变化来探讨可能的信号机制.结果:阻断PLC-γ1信号通路明显减缓大肠癌LoVo细胞的生长,其细胞增殖抑制率随药物作用时间和浓度的增加逐渐增高,10 μmol/L U73122作用24、48 h后其抑制效果可分别达到35%和45%,使LoVo细胞G1期细胞比例增加,而S期细胞比例降低,延缓细胞从G1期向S期的过渡,即抑制细胞周期的进行,阻断PLC-γ1信号通路后LoVo细胞未能出现凋亡特征性的形态学改变,DNA琼脂糖凝胶电泳未能检测到凋亡特征的梯状带的出现,不能引起Caspase-3的激活,同时PLC-γ1信号通路的阻断可上调HSP70的表达水平,HSP70的分子伴侣作用可能是其抑制大肠癌细胞周期进行的机制.结论:阻断磷脂酶C-γ1信号通路能够抑制大肠癌LoVo细胞的过度增殖、抑制其细胞周期的进行,其机制可能是通过上调热休克蛋白70的表达水平而实现,但不能启动LoVo细胞凋亡,磷脂酶C-γ1不是调控LoVo细胞凋亡的关键信号分子.

Effects of blocking phospholipase C-gamma1 signaling pathway on proliferation and apoptosis of human colorectal cancer cell line LoVo

BACKGROUND & OBJECTIVE: Phospholipase C-gamma 1 (PLC-gamma1) is a vital signal transducer in transmembrane signaling, which regulates cell proliferation and apoptosis. It is overexpressed in many cancers, such as colorectal cancer, which indicates that it is closely related to the genesis and development of tumors. This study was to explore the effects of blocking PLC-gamma1 signaling pathway on the proliferation and apoptosis of human colorectal cancer cell line LoVo, and investigate the signaling mechanisms. METHODS: LoVo cells were treated with PLC-gamma1-specific chemical blocking agent U73122. Cell proliferation was examined by cell counting, MTT assay, and flow cytometry (FCM). Cell apoptosis was observed under a microscope, and measured by agarose gel electrophoresis and FCM with PI simple staining. The expression of hot shock protein 70(HSP70) and Caspase-3 in LoVo cells were detected by Western blot. RESULTS: The proliferation of LoVo cells was inhibited after blocking PLC-gamma1 signaling pathway and the effect was enhanced along with the increasing concentration of U73122. The inhibition rate reached 35% and 45% when treated with 10 micromol/L U73122 for 24 h and 48 h respectively. After blocking PLC-gamma1 signaling pathway, the G1 phase proportion of LoVo cells was increased while the S phase proportion was decreased? no apoptosis-specific cell shrinkage was found under a light microscope, and no apoptosis-specific DNA ladder was found by agarose gel electrophoresis? no activated Caspase-3 was detected by Western blot, while increased expression of HSP70 was detected. CONCLUSIONS: Blocking PLC-gamma1 signaling pathway can inhibit the proliferation and cell cycle progress of LoVo cells, which may be due to the up-regulated expression of HSP70. PLC-gamma1 is not a vital signal molecule regulating the apoptosis of LoVo cells.