AIM： To explore the effect of gastrin 17 （G17） on β-catenin/T cell factor-4 （Tcf-4） signaling in colonic cancer cell line Colo320WT. METHODS： The pCR3.1/GR plasmid, which expresses gastrin receptor, cholecystok...AIM： To explore the effect of gastrin 17 （G17） on β-catenin/T cell factor-4 （Tcf-4） signaling in colonic cancer cell line Colo320WT. METHODS： The pCR3.1/GR plasmid, which expresses gastrin receptor, cholecystokinin-2 receptor （CCK-2R）, was transfected into a colonic cancer cell line Colo320 by Lipofectamine ^TM 2000 and the stably expressing CCK-2R clones were screened by G418. The expression levels of gastrin receptor in the Colo320 and the transfected Colo320WT cell line were assayed by RTPCR. Colo320WT cells were treated with G17 in a time-dependent manner （0, 1, 6, 12, 24 and 48 h）, then with L365,260 （Gastrin17 receptor blocker） for 30 rain, and with G17 again for 12 h or L365,260 for 12 h. Expression levels of β-catenin in a TX-100 soluble fraction and TX-100 insoluble fraction of Colo320WT cells treated with G17 were detected by co-immuniprecipation and Western blot. Immunocytochemistry was used to examine the distribution of β-catenin in CoLoWT320 cells. Expression levels of c-myc and cyclin D1 in Colo320WT cells treated with G17 were assayed by Western blot. RESULTS： Expression levels of β-catenin in the TX-100 solution fraction decreased apparently in a time- dependent fashion and reached the highest level after G17 treatment for 12 h, while expression levels of β-catenin in the TX-100 insoluble fraction were just on the contrary. Immunocytochemistry showed that β-catenin was translocated from the cell membranes into the cytoplasm and nucleus under G17 treatment. Expression levels of c-myc and cyclin D1 in the G17- treated Colo320WT cells were markedly higher compared to the untreated Colo320WT cells. In addition, the aforementioned G17-stimulated responses were blocked by L365,260.CONCLUSION： Gastrin17 activates β-catenin/Tcf-4 signaling in Colo320WT cells, thereby leading to over- expression of c-myc and cyclin D1.展开更多
目的探讨头框转录因子O家族4(class O of forkhead box transcriptionfactor 4,FOXO4)对喉癌细胞增殖凋亡能力的影响。方法应用Western blot法检测喉癌组织及对应癌旁组织中FOXO4的表达水平。细胞转染FOXO4过表达载体(p-EGFP-C1/FOXO4组...目的探讨头框转录因子O家族4(class O of forkhead box transcriptionfactor 4,FOXO4)对喉癌细胞增殖凋亡能力的影响。方法应用Western blot法检测喉癌组织及对应癌旁组织中FOXO4的表达水平。细胞转染FOXO4过表达载体(p-EGFP-C1/FOXO4组)和空载体(p-EGFP-C1组),同时设置未转染组,未转染组中只加入转染试剂。Western blot法检测转染后细胞中FOXO4蛋白水平。四甲基偶氮唑蓝(MTT)检测细胞增殖,流式细胞术检测细胞凋亡,Western blot检测细胞中活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved Caspase-3)、Caspase-3、活化的含半胱氨酸的天冬氨酸蛋白水解酶9(Cleaved Caspase-9)、Caspase-9、β-连环蛋白(β-catenin)、Wnt1表达水平。用Wnt/β-catenin信号通路激活剂作用于转染p-EGFP-C1/FOXO4后的喉癌细胞(激活剂组),检测细胞增殖、凋亡情况。结果 FOXO4在喉癌组织中表达水平明显低于癌旁组织(P=0.000)。p-EGFP-C1/FOXO4组细胞中FOXO4表达水平明显高于未转染组(P=0.000)。p-EGFP-C1/FOXO4组细胞存活率及β-catenin、Wnt1水平明显低于未转染组(P=0.002,P=0.004,P=0.006),细胞凋亡率及Cleaved Caspase-3、Caspase-3、Cleaved Caspase-9、C a s p a s e-9表达水平均明显高于未转染组(P=0.0 0 2,P=0.001,h P<0.05,P=0.004,j P<0.05)。Wnt/β-catenin信号通路激活剂可以部分逆转FOXO4抑增殖和促凋亡作用。结论 FOXO4能够促进人喉癌细胞凋亡,抑制喉癌细胞增殖,作用机制可能与Wnt/β-catenin信号通路有关。展开更多
基金Supported by the National Natural Science Foundation of China, No. 30470782
文摘AIM： To explore the effect of gastrin 17 （G17） on β-catenin/T cell factor-4 （Tcf-4） signaling in colonic cancer cell line Colo320WT. METHODS： The pCR3.1/GR plasmid, which expresses gastrin receptor, cholecystokinin-2 receptor （CCK-2R）, was transfected into a colonic cancer cell line Colo320 by Lipofectamine ^TM 2000 and the stably expressing CCK-2R clones were screened by G418. The expression levels of gastrin receptor in the Colo320 and the transfected Colo320WT cell line were assayed by RTPCR. Colo320WT cells were treated with G17 in a time-dependent manner （0, 1, 6, 12, 24 and 48 h）, then with L365,260 （Gastrin17 receptor blocker） for 30 rain, and with G17 again for 12 h or L365,260 for 12 h. Expression levels of β-catenin in a TX-100 soluble fraction and TX-100 insoluble fraction of Colo320WT cells treated with G17 were detected by co-immuniprecipation and Western blot. Immunocytochemistry was used to examine the distribution of β-catenin in CoLoWT320 cells. Expression levels of c-myc and cyclin D1 in Colo320WT cells treated with G17 were assayed by Western blot. RESULTS： Expression levels of β-catenin in the TX-100 solution fraction decreased apparently in a time- dependent fashion and reached the highest level after G17 treatment for 12 h, while expression levels of β-catenin in the TX-100 insoluble fraction were just on the contrary. Immunocytochemistry showed that β-catenin was translocated from the cell membranes into the cytoplasm and nucleus under G17 treatment. Expression levels of c-myc and cyclin D1 in the G17- treated Colo320WT cells were markedly higher compared to the untreated Colo320WT cells. In addition, the aforementioned G17-stimulated responses were blocked by L365,260.CONCLUSION： Gastrin17 activates β-catenin/Tcf-4 signaling in Colo320WT cells, thereby leading to over- expression of c-myc and cyclin D1.
文摘目的探讨头框转录因子O家族4(class O of forkhead box transcriptionfactor 4,FOXO4)对喉癌细胞增殖凋亡能力的影响。方法应用Western blot法检测喉癌组织及对应癌旁组织中FOXO4的表达水平。细胞转染FOXO4过表达载体(p-EGFP-C1/FOXO4组)和空载体(p-EGFP-C1组),同时设置未转染组,未转染组中只加入转染试剂。Western blot法检测转染后细胞中FOXO4蛋白水平。四甲基偶氮唑蓝(MTT)检测细胞增殖,流式细胞术检测细胞凋亡,Western blot检测细胞中活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved Caspase-3)、Caspase-3、活化的含半胱氨酸的天冬氨酸蛋白水解酶9(Cleaved Caspase-9)、Caspase-9、β-连环蛋白(β-catenin)、Wnt1表达水平。用Wnt/β-catenin信号通路激活剂作用于转染p-EGFP-C1/FOXO4后的喉癌细胞(激活剂组),检测细胞增殖、凋亡情况。结果 FOXO4在喉癌组织中表达水平明显低于癌旁组织(P=0.000)。p-EGFP-C1/FOXO4组细胞中FOXO4表达水平明显高于未转染组(P=0.000)。p-EGFP-C1/FOXO4组细胞存活率及β-catenin、Wnt1水平明显低于未转染组(P=0.002,P=0.004,P=0.006),细胞凋亡率及Cleaved Caspase-3、Caspase-3、Cleaved Caspase-9、C a s p a s e-9表达水平均明显高于未转染组(P=0.0 0 2,P=0.001,h P<0.05,P=0.004,j P<0.05)。Wnt/β-catenin信号通路激活剂可以部分逆转FOXO4抑增殖和促凋亡作用。结论 FOXO4能够促进人喉癌细胞凋亡,抑制喉癌细胞增殖,作用机制可能与Wnt/β-catenin信号通路有关。
