JAK-STAT信号转导通路阻断剂AG490对肝癌SMMC-7721细胞移植瘤生长的影响

Effect of JAK-STAT signal transduction pathway blocker AG490 on the growth of xenograft tumor of hepatocellular carcinoma SMMC-7721 cells in nude mice

目的 :探讨Janus激酶(Janus kinase,JAK)-信号转导及转录激活因子(signal transducer and activator of transcription,STAT)信号转导通路阻断剂AG490对肝癌SMMC-7721细胞及移植瘤生长和凋亡的影响,及可能的作用机制。方法 :用人肝癌SMMC-7721细胞株建立肝细胞癌裸鼠移植瘤模型,待肿瘤生长至约150 mm3时随机分为对照组、AG490单药组、顺铂(cisplatin,DDP)单药组和AG490联合DDP组,用AG490单药或联合DDP处理各组小鼠,绘制各组小鼠的肿瘤生长曲线并计算抑瘤率,FCM法检测细胞凋亡率,免疫组织化学法和蛋白质印迹法检测移植瘤中磷酸化STAT3(phospho-STAT3,p-STAT3)及caspase-3蛋白表达的情况。结果 :与对照组相比,DDP组、AG490组和AG490联合DDP组在腹腔注射药物第10和13天时,移植瘤的体积均明显较小(P值均<0.05);AG490联合DDP组的抑瘤率为60.24%,明显高于DDP组的51.73%和AG490组的34.58%(P值均<0.05)。FCM检测结果显示,AG490组、DDP组和AG490联合DDP组移植瘤组织中肿瘤细胞的凋亡率分别为(22.4±0.8)%、(40.1±1.2)%和(43.2±1.5)%,与对照组的(11.5±0.4)%相比,差异均具有统计学意义(P值均<0.05)。免疫组织化学法检测结果显示,对照组、AG490组、DDP组和AG490联合DDP组移植瘤组织中p-STAT3蛋白的阳性表达率依次为(95.4±1.8)%、(65.8±2.4)%、(37.4±2.1)%和(20.3±1.2)%,caspase-3蛋白的阳性表达率依次为(10.7±1.1)%、(26.4±1.6)%(、53.9±3.2)%和(82.5±2.8)%。蛋白质印迹法检测结果显示,与对照组相比,AG490组、DDP组和AG490联合DDP组中p-STAT3蛋白的表达水平均明显降低(P值均<0.05),而caspase-3蛋白的表达水平均明显升高(P值均<0.05)。结论 :AG490P联合DDP可进一步提高DDP对肿瘤生长的抑制作用,阻断JAK-STAT通路中p-STAT3蛋白的表达水平,而上调凋亡相关蛋白caspase-3的表达可能是其作用机制之一。

Objective： To investigate the effect of AG490, an inhibitor of Janus kinase （JAK）-signal transducer and activator of transcription （STAT） signal transduction pathway, on the growth and apoptosis of hepatocellular carcinoma SMMC-7721 cells in vivo, and to explore the possible mechanism. Methods： The xenograft tumor models of human hepatocellular carcinoma SMMC-7721 cells in nude mice were established. When the subcutaneous transplanted tumor grew to about 150 mm3, the mice were randomly divided into four groups, including saline treated control group, AG490 treated group, cisplatin （DDP） treated group, and AG490 combined with DDP treated group. Then the growth curves of xenograft tumors in different groups were drawn, the inhibitory rates of tumor growth were calculated, and the cell cycle and apoptosis were detected by FCM. The expression levels of phospho-STAT3 （p-STAT3） and caspase-3 in xenograft tumors were detected by immunohistochemistry and Western blotting. Results： Compared with the control group, the size of transplanted tumor was significantly decreased in DDP group, AG490 group and DDP combined with AG490 group after intraperitoneal injection with drugs for 10 and 13 days （all P 〈 0.05）. The inhibitory rate of tumor growth in AG490 combined with DDP group （60.24%） was significantly higher than that in DDP group （51.73%） or AG490 group （34.58%） （both P 〈 0.05）. The apoptotic rates of DDP group [（22.4±0.8）%], AG490 group [（40.1±1.2）%] and AG490 combined with DDP group [（43.2±1.5）%] were significantly higher than that in the control group [（11.5±0.4）%] （all P 〈 0.05）. The positive rates of p-STAT3 expression in the control group, AG490 group, DDP group and AG490 combined with DDP group were （95.4±1.8）%, （65.8±2.4）%, （37.4±2.1）% and （20.3±1.2）%, respectively; while the positive rates of caspase-3 expression in the above groups were （10.7±1.1）%, （26.4±1.6）%, （53.9±3.2）% and （82.5±2.8）%, respectively. The expression level of caspase-3 was significantly increased, while the expression level of p-STAT3 was decreased after treatment with AG490, DDP or their combination （all P 〈 0.05）. Conclusion： AG490 combined with DDP can further improve the synergistic inhibitory effect of DDP on tumor growth through blocking the expression of p-STAT3 protein and up- regulating the expression of apoptosis-related protein caspase-3 in JAK2/STAT3 pathway.