视黄醇X受体小分子配体衍生物对宫颈癌细胞周期及细胞凋亡的影响

(厦门大学药学院,福建 厦门 361102)

视黄醇X受体; 细胞周期阻滞; 细胞凋亡; 宫颈癌

Effects of Small-molecule Ligand Derivatives Targeting Retinoid X Receptor on Cell Cycle and Apoptosis of Cervical Cancer Cells
ZHANG Xiaoli,TU Xuhuang,XIE Guobin,JIANG Fuquan,ZHANG Xiaokun*

(School of Pharmaceutical Sciences,Xiamen University,Xiamen 361102,China)

retinoid X receptor; cell cycle arrest; cell apoptosis; cervical cancer

DOI: 10.6043/j.issn.0438-0479.201803021

备注

为筛选视黄醇X受体α(RXRα)配体XS-23的衍生物中作用于细胞周期的化合物,通过蛋白免疫印迹检测细胞周期G0/G1期检查点关键负调控因子p21 WAF1/CIP1(简称p21)、细胞M期周期蛋白Cyclin B1的表达水平,从中筛选到两个编号分别为05和06的化合物,可导致人宫颈癌HeLa细胞中p21表达上调和Cyclin B1表达下调.用流式细胞术和siRNA干扰技术检测化合物对细胞周期的影响及其RXRα依赖性,结果显示:化合物05和06可引起G0/G1期细胞比例显著增加,同时G2/M期细胞比例显著下降,且化合物05和06引起p21表达水平上调的作用在一定程度上依赖于RXRα.用Annexin V-FITC/PI试剂盒检测细胞凋亡率,同时检测聚腺苷二磷酸核糖聚合酶(PARP)切割蛋白水平及其RXRα依赖性,结果表明:化合物05和06均能够引起HeLa细胞凋亡率显著增加,并导致PARP切割且具有一定的RXRα依赖性.进而用计算机模拟的方法对化合物与RXRα进行分子对接,模拟化合物与RXRα可能的结合位点,结果显示化合物05和06可能与RXRα蛋白的helix12、helix3、helix4 形成的表面区域结合.综上,筛选出的化合物05和06可能通过与RXRα表面结合,阻滞HeLa细胞周期于G0/G1期,最终诱导HeLa细胞凋亡.

To screen compounds functioning on cell cycles by the derivatives of XS-23 targeting retinoid X receptor(RXRα),we determind the expression of p21 WAF1/CIP1(p21),a key negative regulator of G0/G1 phase of cell cycle,and Cyclin B1 which highly expressed in the M phase,using Western blotting,and identified two compounds 05 and 06,which resulted in up-regulation of p21 expression and down-regulation of Cyclin B1 expression in HeLa cervical cancer cells.Then,flow cytometry and siRNA interference techniques were used to evaluate the effect of the compounds on cell cycles in HeLa cells as well as RXRα dependence.The results showed that compounds 05 and 06 were able to make the proportion of cells significantly increase in G0/G1 phase,while decrease in the G2/M phase,and the effect of up-regulating p21 decreased was paritially RXRα-dependent.Apoptosis rate was detected using Annexin V-FITC/PI reagent,and poly ADP-ribose polymerase(PARP)cleavage level and its RXRα-dependence were detected with Western blotting.The results showed that both compounds 05 and 06 caused significant increase in apoptosis rate of HeLa cells,and led to PARP cleavage which was partially RXRα-dependent.Moreover,computer modeling was used to dock the possible binding sites of the compounds with RXRα.The results showed that compounds 05 and 06 could bind to the small molecule binding region formed by helix12,helix3 and helix4 on the surface of RXRα.In summary,compounds 05 and 06 might bind to the surface of RXRα,subsequently block cell cycle in G0/G1 phase and ultimately induce apoptosis of HeLa cells.