Cheng Qiuying, Ling Xiang, Haller Andrew, Nakahara Takahito, Yamanaka Kentaro, Kita Aya, Koutoku Hiroshi, Takeuchi Masahiro, Brattain Michael G, Li Fengzhi
Int J Biochem Mol Biol. 2012;3(2):179-97. Epub 2012 May 18.
YM155, a novel survivin suppressant, shows potent antitumor activity against various human cancers and is currently in phase II clinical trials. In this study, we investigated whether YM155 selectively inhibits survivin transcription. We hypothesize that inhibition of survivin transcription plays a role in YM155-mediated survivin inhibition. We found that YM155 inhibited survivin promoter activity, while it showed minimal inhibitory effect on four control gene promoters in transfection and luciferase activity assay experiments, indicating its selectivity. Transfection of various survivin promoter-luciferase constructs followed by luciferase assays revealed that the survivin core promoter (269 bp) plays a major role in YM155-mediated inhibitory effects. However, flow cytometry analysis indicated that inhibition of survivin promoter activity by YM155 is cell cycle-independent without G1 cell arrests. Electrophoretic mobility shift assays (EMSA) identified that YM155 abrogates nuclear proteins binding to the region of -149 to -71, in which Sp1 is a major candidate, and that YM155 treatment induces Sp1 re-subcellular localization without inhibiting its expression. Forced expression of Sp1 neutralized YM155-mediated downregulation of survivin promoter activity. Consistently, mutation of the identified Sp1 sites in the oligonucleotide probe diminished DNA-protein interactions in EMSA experiments, and mutation of the Sp1 sites in the survivin promoter-luciferase construct diminished survivin promoter activity. These findings indicate that YM155 inhibition of survivin expression is at least in part through its inhibition of survivin transcription by disruption of Sp1 interaction with the region of -149 to -71 in the survivin core promoter.
新型存活素抑制剂YM155对多种人类癌症显示出强大的抗肿瘤活性，目前正处于II期临床试验阶段。在本研究中，我们调查了YM155是否选择性抑制存活素转录。我们假设存活素转录的抑制在YM155介导的存活素抑制中起作用。我们发现YM155抑制存活素启动子活性，而在转染和荧光素酶活性测定实验中，它对四个对照基因启动子的抑制作用最小，表明其具有选择性。转染各种存活素启动子-荧光素酶构建体后进行荧光素酶测定，结果显示存活素核心启动子（269 bp）在YM155介导的抑制作用中起主要作用。然而，流式细胞术分析表明，YM155对存活素启动子活性的抑制不依赖细胞周期，不会导致G1期细胞停滞。电泳迁移率变动分析（EMSA）确定YM155消除了与-149至-71区域结合的核蛋白，其中Sp1是主要候选蛋白，并且YM155处理诱导Sp1重新定位到亚细胞中，而不抑制其表达。Sp1的强制表达中和了YM155介导的存活素启动子活性下调。同样，在寡核苷酸探针中鉴定出的Sp1位点发生突变会减少EMSA实验中的DNA-蛋白质相互作用，而存活素启动子-荧光素酶构建体中Sp1位点的突变会降低存活素启动子活性。这些发现表明，YM155对存活素表达的抑制至少部分是通过破坏Sp1与存活素核心启动子中-149至-71区域的相互作用来抑制存活素转录实现的。
