芹菜素通过PI3K/Akt和MAPK信号通路诱导人大肠癌CL187细胞凋亡

Apigenin Induces Apoptosis of Human Colorectal Cancer CL187 Cells via PI3K/Akt and MAPK Signaling Pathways

目的:观察芹菜素对人大肠癌CL187细胞增殖和凋亡的作用及相关机制。方法:选择人大肠癌CL187细胞,利用芹菜素(0、30、45、60 mg·L^(-1))进行干预后,采用噻唑蓝(MTT)比色法和克隆形成实验检测芹菜素对CL187细胞的增殖作用;Hoechst 33258染色法观察细胞凋亡;实时荧光定量聚合酶链式反应(Real-time PCR)检测芹菜素对CL187细胞胱天蛋白酶-3(Caspase-3)、B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)mRNA表达水平;蛋白免疫印迹法(Western blot)观察芹菜素对细胞凋亡相关蛋白Caspase-3、Bcl-2、Bax和磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)信号通路中Akt、磷酸化(p)-Akt及丝裂原活化蛋白激酶(MAPK)信号通路中细胞外调节蛋白激酶(ERK1/2)、p-ERK1/2、c-Jun氨基末端激酶(JNK)、p-JNK、p38 MAPK、p-p38 MAPK蛋白表达的影响。结果:与空白组比较,芹菜素组细胞存活率显著降低,细胞增殖抑制率显著升高(P<0.01);与空白组比较,芹菜素组细胞克隆数和克隆形成率显著降低,克隆形成抑制率显著升高(P<0.01);不同浓度芹菜素干预CL187细胞48 h,与空白组比较,在荧光显微镜下可观察到细胞核固缩,染色质凝聚,细胞荧光反应增强等典型的细胞凋亡特征;与空白组比较,芹菜素组(45、60 mg·L^(-1))抑凋亡基因Bcl-2 mRNA表达水平显著降低(P<0.01),芹菜素组促凋亡基因Bax和Caspase-3 mRNA表达明显升高(P<0.05,P<0.01)。与空白组比较,芹菜素组促凋亡蛋白Caspase-3蛋白表达水平明显上调(P<0.05,P<0.01),芹菜素组(45、60 mg·L^(-1))促凋亡蛋白Bax蛋白表达显著上调(P<0.01),芹菜素组抑凋亡蛋白Bcl-2表达水平明显下调(P<0.05,P<0.01)。与空白组比较,芹菜素组(60 mg·L^(-1))Akt蛋白表达显著下调(P<0.01),芹菜素组(45、60 mg·L^(-1))p-Akt、ERK1/2、p-ERK1/2蛋白表达显著下调(P<0.01),芹菜素组JNK、p-JNK蛋白表达明显上调(P<0.05,P<0.01),芹菜素组(60 mg·L^(-1))p38 MAPK蛋白表达明显上调(P<0.05),芹菜素组p-p38 MAPK蛋白表达显著上调(P<0.01),芹菜素组p-Akt/Akt明显降低(P<0.05,P<0.01),芹菜素组p-ERK1/2/ERK1/2显著降低(P<0.01),芹菜素组(45、60 mg·L^(-1))p-JNK/JNK明显升高(P<0.05,P<0.01),芹菜素组p-p38 MAPK/p38 MAPK显著升高(P<0.05,P<0.01)。结论:芹菜素可抑制大肠癌CL187细胞增殖并促进细胞凋亡,其作用机制可能与抑制PI3K/Akt信号通路和调控MAPK信号通路相关蛋白的表达有关。

Objective:To study the effect of apigenin on the proliferation and apoptosis of human colon cancer CL187 cells and the underlying mechanisms.Method:Human colorectal cancer CL187 cells were treated with different concentrations of apigenin(0,30,45,60 mg·L^(-1))according to the results of the preliminary experiment.The proliferation of CL187 cells was detected by methyl thiazolyl tetrazolium(MTT)and colony formation assays,and the apoptosis was observed via Hoechst 33258 staining.Real-time fluorescence quantitative PCR was conducted to determine the mRNA levels of cysteine protease-3(Caspase-3),B-cell lymphoma-2(Bcl-2),and Bcl-2-associated X protein(Bax)in the CL187 cells treated with apigenin.Western blot was employed to measure the protein levels of Caspase-3,Bcl-2,and Bax associated with apoptosis,protein kinase B(Akt)and phosphorylated Akt(p-Akt)in phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)pathway,and extracellular signal-regulated kinases 1/2(ERK1/2),p-ERK1/2,c-Jun N-terminal kinase(JNK),p-JNK,p38 mitogen-activated protein kinase(MAPK),and p-p38 MAPK protein in MAPK pathway.Result:Compared with the blank group,the apigenin groups had low cell survival rates and high inhibition rates on cell proliferation(P<0.01).Apigenin decreased the cell clone number and clone formation rate,and increased the inhibition rate on clone formation(P<0.01).After CL187 cells were treated with different concentrations of apigenin for 48 h,typical apoptosis characteristics such as nuclear pyknosis,chromatin condensation,and enhanced fluorescence reaction were observed.Compared with blank group,45,60 mg·L^(-1)apigenin treatments down-regulated the mRNA level of anti-apoptotic gene Bcl-2(P<0.01)and all the apigenin treatments up-regulated those of the pro-apoptotic genes Bax and Caspase-3(P<0.05,P<0.01).Similarly,apigenin treatments down-regulated the protein level of Bcl-2(P<0.05,P<0.01)and up-regulated those of Caspase-3(P<0.05,P<0.01)and Bax(P<0.01,45,60 mg·L^(-1)).The blank group had higher protein level of Akt than the 60 mg·L^(-1)apigenin group(P<0.01),higher protein levels of p-Akt,ERK1/2,and p-ERK1/2 than the 45,60 mg·L^(-1)apigenin groups(P<0.01),and higher protein levels of JNK and p-JNK than the apigenin groups(P<0.05,P<0.01).Compared with blank group,60 mg·L^(-1)apigenin up-regulated the protein level of p38 MAPK(P<0.05),and all the apigenin groups up-regulated that of p-p38 MAPK(P<0.01).Furthermore,apigenin lowered the p-Akt/Akt ratio(P<0.05,P<0.01)and p-ERK1/2/ERK1/2 ratio(P<0.01),while it increased the p-JNK/JNK ratio(45,60 mg·L^(-1);P<0.05,P<0.01)and p-p38 MAPK/p38 MAPK ratio(P<0.05,P<0.01).Conclusion:Apigenin can inhibit the proliferation and promote the apoptosis of CL187 cells by inhibiting the PI3K/Akt signaling pathway and regulating the expression of proteins in the MAPK signaling pathway.