异甘草素对人宫颈癌细胞增殖的抑制作用

Inhibitory effect of isoliquiritigenin on proliferation of human cervical cancer cells in vitro and in vivo

目的鉴于异甘草素(ISL)对不同肿瘤细胞系的不同作用,研究ISL对人宫颈鳞状上皮癌细胞(CaSki)癌细胞的影响及有关机制.方法体外研究采用MTT法测定细胞增殖;流式细胞仪检测细胞周期;逆转录聚合酶链反应(RT-PCR)分析细胞周期蛋白B1(cyclin B1)mRNA表达;Western免疫印迹分析cyclin B1, P34cdc2和磷酸化P34cdc2(phospho-cdc2, 酪氨酸15)蛋白表达;体内研究通过建立人宫颈癌CaSki细胞裸鼠移植瘤模型观察抑瘤率.结果 ISL浓度依赖性(10～80 μmol·L-1)抑制CaSki细胞增殖,IC50为(19.3±3.3)μmol·L-1,且呈时间依赖性.ISL 20 μmol·L-1作用3 d对细胞抑制率为82.3%;ISL浓度依赖性(10～80 μmol·L-1)干扰CaSki细胞周期,细胞周期阻滞于S和G2/M期,S和G2/M期的百分率分别从23.1%和8.2%上升到43.2%和32.1%;同时G0/G1期细胞百分率从68.7%下降到24.7%;RT-PCR分析显示,ISL(10～80 μmol·L-1)显著抑制cyclin B1 mRNA表达,抑制率最高可达61.7%,且呈时间依赖性;Western免疫印迹分析显示,ISL作用24 h cyclin B1蛋白表达开始不同程度下降,48 h下降明显,P34cdc2变化较小,ISL作用16 h后磷酸化P34cdc2(酪氨酸15)蛋白水平明显改变;ISL(20～500 mg·kg-1·d-1)剂量依赖性抑制CaSki细胞裸鼠皮下移植瘤的生长,抑瘤率分别为36.8%,51.2%和84.6%,且对动物体重和外周血白细胞数均无明显影响.结论 ISL可以显著抑制人宫颈癌细胞体内外增殖,其机制与将细胞周期阻滞于S和G2/M期及影响细胞周期因子cyclin B1的表达和改变P34cdc2的磷酸化水平有关.

AIM In view of the different effect of isoliquiritigenin（ISL） on proliferation of different tumors, to study the effect of ISL on proliferation of human cervical cancer cells in vitro and in vivo and its related mechanisms. METHODS Inhibitory effect of cell proliferation was evaluated by MTT method; cell cycle distributions were measured by flow cytometric assay; RT-PCR was used to analyze the expression of cyclin B1 mRNA; Western blotting analysis was used to detect the protein expression of cyclin B1 and phosphorylation change of P34^cac2. The inhibitory rate in vivo was evaluated in nude mice by means of human cervical cancer CaSki implantation. RE- SULTS ISL （ 10 - 80 μmol·L^-1 ） suppressed proliferation of human cervical cancer cells in a concentration- and time-dependent manner, the IC50 of CaSki cells exposed to ISL for 48 h was （19.3 - 3.3 ） μmol·L^-1, the inhibition rate of CaSki cells exposed to ISL （ 20 μmol·L^-1 ） Was 82. 3% on d 3 of culture. DNA flowcytometric analysis indicated that ISL induced the cell cycle arrest at S and G2/M phase concentration-depen-dently at 48 h. ISL down-regulated the expression of cyclin B1 mRNA, at the concentration of 80 μmol·L^-1 inhibitory rate reached the maximum （61.7%）. Western blotting analysis showed that treatment of CaSki cells with ISL remarkably decreased the expression of cyclin B1 at 48h and enhanced the level of P34^cdc2 phosphorylation from 16 h; ISL was shown to inhibit human xenograft in nude mice with no apparent toxicity to the animals, the inhibition rate reached 84.6% at 500 mg·kg^-1·d^-1 for 10 d. CONCLUSION ISL can significantly inhibit proliferation of the human cervical cancer cells and induce cell cycle arrest at the S and G2/M phase, and this induction is associated with the influence on expression of cyclin B1 and phosphorylation of P34^cdc2 protein.