AG490通过JAK2/STAT3信号通路对肝癌细胞裸鼠移植瘤的抑制作用及机制

Inhibitory effects of AG490 on transplanted tumor of nude mice with live cancer through JAK2/STAT3 signal pathway and its mechanism

目的:探讨JAK2/STAT3信号通路在AG490干预下对人肝细胞癌裸鼠移植瘤生长、凋亡的影响以及与相关基因表达之间的关系。方法:建立人肝癌细胞裸鼠移植瘤模型,将BALB/C-nu裸鼠随机平均分成3组,按照实验设计分为对照组(生理盐水)、低浓度AG490组(浓度4 mg/kg的AG490)、高浓度AG490组(浓度为8 mg/kg的AG490),而后按照实验设计分别给予生理盐水或AG490处理裸鼠。观察期间记录各组裸鼠体质量、瘤重、肿瘤体积、计算肿瘤生长抑制率;通过流式细胞法检测细胞周期变化和凋亡情况;蛋白质印迹法检测细胞中p-JAK2、JAK2、p-STAT3、STAT3、MMP-2、Bcl-2、Bcl-xl及Bax蛋白表达的变化;免疫组织化学检测移植瘤组织中p-JAK2和p-STAT3蛋白表达。结果:实验组裸鼠瘤重、肿瘤体积均小于对照组,抑瘤率高于对照组,差异均具有统计学意义(P<0.05)。流式细胞分析结果显示低浓度AG490组(73.98±0.90)%、高浓度AG490组(81.39±1.66)%的细胞周期G0/G1期较对照组(59.30±1.13)%明显增加(P<0.05),同样低浓度AG490组(33.32±1.16)%和高浓度AG490组(52.22±1.06)%的细胞凋亡率较对照组(1.50±0.12)%明显增高(P<0.05)。Western blot结果显示低浓度AG490组(1.073±0.028)、(0.955±0.025)和高浓度AG490组(0.763±0.023)、(0.751±0.037)的p-JAK2,p-STAT3蛋白表达均较对照组(1.265±0.012)、(1.477±0.020)明显降低(P<0.05)。同时下调通路下游蛋白MMP-2,Bcl-2,Bcl-xl和上调Bax表达水平。免疫组化结果显示AG490组移植瘤肿瘤组织p-JAK2和p-STAT3蛋白表达较对照组明显降低(P<0.05)。结论:AG490能够通过抑制JAK2/STAT3信号通路促进人肝癌细胞的凋亡、抑制增殖和侵袭作用达到抑制裸鼠移植瘤的生长,提示JAK2/STAT3通路在肝细胞癌恶性演进过程中发挥了重要作用。

Objective：To explore the effect of JAK/STAT signal pathway inhibitor AG490 on growth and apoptosis of transplanted tumor of nude mice with human liver cancer and its relationship with the expression of related genes. Methods：The models of transplanted tumor of nude mice with human liver cancer were established. The model mice were randomly divided into 3 groups ：control group（normal saline）,low concentration AG490 group（4 mg/kg AG490）,high concentration AG490 group（8 mg/kg AG490）,then saline or AG490 was used to treat the nude mice with the requirements. The growth situations of transplanted tumor in different groups were observed during the treatment,including the body weight,the tumor weight and volume and the inhibiting rates of growth of transplanted tumor. Cell cycle distribution and cell apoptosis were detected by flow cytometry. Western blot was used to detect the expression levels of p-JAK2,JAK2,p-STAT3,STAT3,MMP-2,Bcl-2,Bcl-xl and Bax. The expressions of p-JAK2 and p-STAT3 were observed by immunohistochemistry. Results：Significant differences in tumor weight,volumes,and tumor growth inhibition rates were found between the treatment groups and the control group（P〈0.05）. Flow cytometric analysis demonstrated the G0/G1 phase in cell cycle was increased in low concentration AG490 group（73.98±0.90）% and high concentration AG490 group（81.39±1.66）% than in control group（59.30±1.13）%,so did cell apoptosis rate of low concentration AG490 group（33.32 ±1.16）% and high concentration AG490 group（52.22 ±1.06）% between control group（1.50 ±0.12）%. Western blot demonstrated both low concentration AG490group（1.073±0.028）（0.955±0.025）and high concentration AG490 group（0.763±0.023）（0.751±0.037）had lower level of pJAK2 and p-STAT3 than control group（1.265±0.012）（1.477±0.020）. It shown that the expression of MMP-2,Bcl-2 and Bcl-xl were down-regulated while the expression of Bax was up-regulated after being treated with AG490. Compared with those of control group,the expressions of p-JAK2 and p-STAT3 in AG490 group were decreased by immunohistochemistry（P〈0.05）. Conclusion：AG490 could promote apoptosis and inhibit the cell growth and invasion by blocking JAK/STAT signal pathway,thereby inhibiting the growth of transplanted tumor of nude mice. The results suggest that the expression of JAK2/STAT3 signal pathway may play an important role in the evolution of liver cancer.