基于ERK/MAPK信号通路探讨青藤碱对SW480结肠癌细胞活性抑制作用的研究

Study on the Inhibitory Effects of Sinomenine on SW480 Colon Cancer Cells Based on ERK/MAPK Signaling Pathway

目的:探讨青藤碱对SW480结肠癌细胞活性抑制作用及对细胞外调节蛋白激酶(extracellu-lar regulated protein kinase,ERK)/丝裂原活化蛋白激酶(mitogen activated protein kinase,MAPK)信号通路的影响。方法:选用SW480人结肠癌细胞株,设对照组、空白组及青藤碱不同剂量组,分别采用相应药物进行处理。采用CCK-8法检测青藤碱对SW480细胞增殖的抑制作用,采用流式细胞术分析青藤碱对SW480细胞凋亡的影响,采用蛋白免疫印迹(Western Blot)法检测各组SW480细胞B淋巴细胞瘤2(B lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2 related X protein,Bax)、含半胱氨酸的天冬氨酸蛋白水解酶3(cyste-ine-containing aspartic acid proteolytic enzymes-3,Caspase-3)、细胞外调节蛋白激酶1/2(extra-cellular regulated protein kinase 1/2,ERK1/2)、磷酸化ERK1/2(phosphorylated ERK1/2,p-ERK1/2)蛋白表达水平。结果:干预24、48、72 h后,与对照组比较,青藤碱4、8、16、20 mmol/L组SW480细胞增殖抑制率较高,且青藤碱4 mmol/L组<8 mmol/L组<16 mmol/L组及20 mmol/L组,差异有统计学意义(P<0.05)。与对照组比较,青藤碱4、8、16 mmol/L组SW4800细胞凋亡率较高,Bax、Caspase-3蛋白表达水平较高,且青藤碱4 mmol/L组<8 mmol/L组<16 mmol/L组,差异有统计学意义(P<0.05)。与对照组比较,青藤碱4、8、16 mmol/L组BcL-2、p-ERK1/2蛋白表达水平较低,且青藤碱4 mmol/L组>8 mmol/L组>16 mmol/L组,差异有统计学意义(P<0.05)。结论:青藤碱能剂量依赖性抑制SW480结肠癌细胞增殖活性,促进细胞凋亡,其作用可能与抑制ERK/MAPK信号通路活性,调节BcL-2、Bax、Caspase-3等相关凋亡蛋白表达有关。

Objective:To explore the inhibitory effect of sinomenine on the activity of SW480 colon cancer cells,its influence on ERK/MAPK signaling pathway.Methods:SW480 human colon cancer cell line was chosen and divided into the control group,the blank group and different doses groups of sinomenine,and they were treated with the corresponding medicine.CCK-8 method was adopted to detect the inhibitory effects of sinomenine on the proliferation of SW480 colon cancer cells,flow cytometry was used to analyze the influence of sinomenine on the apoptosis of SW480 cells,Western Blot to detect the expressions of Bcl-2,Bax,Caspase-3,ERK1/2 and p-ERK1/2 protein of SW480 cells in different groups.Results:After intervened for 24 h,48 h and 72 h,compared with the control group,SW480 cell proliferation inhibition rates are higher in sinomenine groups of 4,8,16 and 20 mmol/L,in the order of sinomenine group of 4 mmol/L<sinomenine group of 8 mmol/L<sinomenine group of 16 mmol/L<sinomenine group of 20 mmol/L,and the difference showed statistical meaning(P<0.05).Compared with the control group,the apoptosis rates of SW480 cells are higher in sinomenine groups of 4,8 and 16 mmol/L,the expressions of Bax and Caspase-3 protein are higher,in the order of sinomenine group of 4 mmol/L<sinomenine group of 8 mmol/L<sinomenine group of 16mmol/L,and the difference was statistically significant(P<0.05).Compared with the control group,the expressions of Bcl-2 and p-ERK1/2 protein are lower in sinomenine groups of 4,8 and 16 mmol/L,in the order of sinomenine group of 4 mmol/L>sinomenine group of 8mmol/L>sinomenine group of 16 mmol/L,and the difference showed statistical meaning(P<0.05).Conclusion:Sinomenine could inhibit the activity of SW480 colon cancer cells proliferation in the dose dependent manner and promote cellular apoptosis,and its effects might be related to inhibiting the activity of ERK/MAPK signaling pathway,regulating the expressions of the apoptosis related proteins including Bcl-2,Bax,Caspase-3 and others.