aFGF和genistein对大肠癌细胞株CCL229 PKC及ERK活性的影响

Effects of aFGF and genistein on PKC and ERK activity in human colorectal cancer cell line CCL229

目的:观察aFGF及TPK抑制剂genistein对大肠癌细胞株CCL229细胞内PKC及ERK活性的影响,探讨其信号传导途径.方法:以不同浓度的aFGF(0.15 mg/L,0.30 mg/L,0.60mg/L,1.20 mg/L)和genistein(6.00mg/L,12.00mg/L,24.00mg/L,48.00mg/L)诱导CCL229细胞,利用[γ-^(32)P]ATP掺入外源性底物的方法,液体闪烁测定PKC及ERK活性.结果:随着aFGF浓度的增加,PKC及ERK活性随之升高,与aFGF浓度呈显著正相关(P<0.05).当aFGF浓度为1.20mg/L时,PKC(胞质),PKC(胞膜)和ERK活性分别为对照组的2.60,2.79,1.77倍.genistein抑制细胞内PKC及ERK活性,且与 genistein 浓度呈剂量依赖效应(P<0.05).当genistein浓度为48.00mg/L时,PKC(胞质),PKC(胞膜)和ERK活性分别为对照组的0.41,0.36,0.50倍.genistein对aFGF诱导的PKC及ERK活性抑制更显著.结论:大肠癌细胞株CCL229中aFGF受体具有TPK活性,TPK激活后促进蛋白质和酶磷酸化,导致PKC和ERK活性升高,进一步证明PKC及ERK确是TPK的下游信号分子.

AIM: To observe the effects of aFGF and TPK inhibitor genistein on intracellular PKC and ERK activity in CCL229 cell line. METHODS: The activities of PKC and ERK in cells induced by different concentrations of aFGF (0.15 mg/L, 0.30 mg/L, 0.60 mg/L, 1.20 mg/L) and genistein (6.00 mg/L, 12.00 mg/L, 24.00 mg/L, 48.00 mg/L) were detected by incorporation of [γ-^(32)P]-ATP into exogenous substrates. RESULTS: The intracellular PKC and ERK activity increased with aFGF in a dose dependent manner (P<0.05). When the concentration of aFGF was 1.20 mg/L, the activity of PKC in cytosol and PKC in membrane and ERK was 2.60, 2.79, 1.77 times higher than control group. Genistein suppressed the intracellular PKC and ERK activity also in a dose dependent manner (P<0.05). When the concentration of genistein was 48.00 mg/L, the activity of PKC in cytosol and PKC in membrane and ERK was 0.41,0.36,0.50 times higher than that in control group, The activity of PKC and ERK decreased apparently when the cells were treated with aFGF. CONCLUSION: aFGF receptor in human colorectal cancer cell line CCL229 possesses TPK activity. Tyrosine-specific protein phosphorylation may initiate a cascade of biochemical events, which may increase the intracellular PKC and ERK activity.