ERK和SAPK/JNK通路在神经酰胺致HT-29细胞凋亡中的作用

Mechanisms of ERK and SAPK/JNK pathways of ceramide-induced apoptosis in HT-29 cells

目的:探讨ERK和SAPK/JNK信号途径在C2-神经酰胺诱导人结肠癌HT-29细胞凋亡中的作用。方法:分别用不同剂量C2-神经酰胺(0、12.5、25.0和50.0μmol/L)处理HT-29细胞24h,分别采用AO/EB染色法观察HT-29细胞凋亡的形态学变化,计算细胞凋亡率;Western Blotting法检测ERK、p-ERK蛋白表达。再分别用不同剂量C2-神经酰胺(0、12.5、25.0和50.0μmol/L)处理HT-29细胞24h,并选择50.0μmol/LC2-神经酰胺作用3、6、12、24h后,用SAPK/JNK检测试剂盒结合Western Blotting方法检测phospho-c-Jun蛋白的表达。结果:与溶剂对照组比较,随着C2-神经酰胺剂量的增加,各剂量组HT-29细胞的凋亡率增加(P<0.05),ERK总蛋白和p-ERK的表达呈现下降趋势,存在剂量-效应关系(<0.05)。不同浓度和不同时间的C2-神经酰胺作用下,HT-29细胞中phospho-c-jun的表达无明显变化(P>0.05)。结论:C2-神经酰胺能够直接诱导人结肠癌HT-29细胞发生凋亡,在此过程中SAPK/JNK活性无显著变化,抑制ERK信号转导通路可能是其诱导凋亡的机制。

OBJECTIVE：To study the mechanisms of ERK and SAPK/JNK pathways of ceramide-induced apoptosis in HT-29 cells. METHODS：HT-29 cells were treated with C2-ceramide for 24 hours at different doses （0,12.5,25.0,50.0μmol/L）. AO/EB fluorescent staining was used to identify the morphological changes of HT-29 cells and evaluate the rate of apoptosis in 200 cells. The protein expressions of ERK and phosphorylated ERK（p-ERK） were evaluated by Western Blotting. The activity of phospho-c-Jun in different doses and treatment time of C2-ceramide groups was assessed using SAPK/JNK assay and Western Blotting. RESULTS：Apoptosis rate was increased and the expressions of ERK and p-ERK were reduced in a dose-dependent manner （P0.05）. The activity of phospho-c-jun in different doses and treatment time showed no obvious changes （P0.05）. CONCLUSION：C2-ceramide could directly induce apoptosis in human colon carcinoma HT-29 cells in vitro,and C2-ceramide could inhibit the ERK pathway without activating SAPK/JNK pathway during this process.