Down-regulation of Sonic hedgehog signaling pathway activity is involved in 5-fluorouracil-induced apoptosis and motility inhibition in Hep3B cells
Down-regulation of Sonic hedgehog signaling pathway activity is involved in 5-fluorouracil-induced apoptosis and motility inhibition in Hep3B cells
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Sonic Hedgehog 信号通路活性下调参与 5-氟尿嘧啶诱导的 Hep3B 细胞凋亡和运动抑制
DOI：
10.1111/j.1745-7270.2008.00456.x
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发表时间：
2008-09-01
影响因子：
3.7
通讯作者：
Zhang, Hongwei
中科院分区：
文献类型：
--
作者：
Wang, Qiyu;Huang, Shuhong;Zhang, Hongwei
Sonic hedgehog（SHh）通路在正常胚胎发生和癌发生中起着关键作用，但其在用5-氟尿嘧啶（5-FU）处理的癌细胞中的功能仍然未知。我们通过逆转录-聚合酶链反应检测了5-FU处理的人肝细胞癌（HCC）细胞系Hep 3和HepG 2中SHh信号通路基因亚组（包括SHh、SMO、PTC 1、Su（Fu）和HIP）的表达。采用台盼蓝染色法、3-（4，5-二甲基噻唑-2-基）-2，5-二苯基四氮唑溴化物染色法和末端脱氧核苷酸转移酶介导的地高辛-dUTP缺口末端标记法检测pCS 2-Glil表达载体转染联合5-FU处理后Hep 3B细胞的凋亡情况。划痕闭合实验检测细胞的运动能力。Western blot分析和免疫荧光显微镜观察PTC 1蛋白在5-FU处理的Hep 3B细胞中的表达和亚细胞定位。结果表明，经5-FU处理后，Hep 3B细胞SHh通路靶分子在mRNA和蛋白水平的表达均明显下调。Gli 1的过表达恢复了细胞活力，并在一定程度上恢复了5-FU抑制的迁移能力。此外，5-FU处理影响PTC 1蛋白的亚细胞定位，PTC 1蛋白是SHh信号通路中的关键成员。我们的数据表明，SHh信号通路的活性下调参与了5-FU诱导的凋亡和抑制运动在刺猬激活的肝癌细胞系。这意味着SHh信号通路抑制剂和5-FU为基础的化疗的组合可能是一个更有前途的策略对肝癌。
The Sonic hedgehog (SHh) pathway plays a critical role in normal embryogenesis and carcinogenesis, but its function in cancer cells treated with 5-fluorouracil (5-FU) remains unknown. We examined the expression of a subset of SHh signaling pathway genes, including SHh, SMO, PTC1, Su(Fu) and HIP in human hepatocellular carcinoma (HCC) cell lines, Hep3BandHepG2, treated with 5-FU by reverse transcription-polymerase chain reaction. Using trypan blue analysis, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay and terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assay, we also detected the apoptosis of Hep3B cells resulting from the transfection of pCS2-Glil expression vectorcombined with 5-FU treatment. The motility of the cells was detected by scratch wound closure assay. The expression and subcellular location of PTC1 protein in Hep3B cells treated by 5-FU were also investigated by Western blot analysis and immunofluorescent microscopy. The results indicated that the expression of SHh pathway target molecules at both messenger RNA and protein levels are evidently down-regulated in Hep3B cells treated with 5-FU. The overexpression of Gli1 restores cell viability and, to some extent, the migration abilities inhibited by 5-FU. Furthermore, 5-FU treatment affects the subcellular localization of PTC1 protein, a key member in SHh signaling pathway. Our data showed that the down-regulation of SHh signaling pathway activity was involved in 5-FU-induced apoptosis and the inhibition of motility in hedgehog-activated HCC cell lines. This implies that the combination of SHh signaling pathway inhibitor and 5-FU-based chemotherapy might represent a more promising strategy against HCC.