Sonic hedgehog pathway as a new target in cholangiocarcinoma therapy
Sonic hedgehog pathway as a new target in cholangiocarcinoma therapy
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Sonic Hedgehog通路作为胆管癌治疗的新靶点
DOI：
10.1055/s-0038-1668970
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发表时间：
2018
期刊：
影响因子：
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通讯作者：
RR Plentz
中科院分区：
文献类型：
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作者：
V Bhuria;J Xing;T Scholta;KC Bui;MLT Nguyen;L Wilkens;NP Malek;P Bozko;RR Plentz
目的：探讨缺氧与Shh通路激活之间的相互作用及其对胆管癌（cholangiocarcinoma，CCA）进展过程中CSCs的影响。方法：将HUCCT-1（Intrahepatic）和TFK-1（Extrahepatic）细胞系在缺氧（1%氧）和常氧（21%氧）条件下培养48小时，并加入或不加入环巴胺（Cyclopamine，Smoothened的抑制剂，一种G蛋白偶联受体）。HIF-1 α、Shh、Gli-1、干细胞转录因子的蛋白和mRNA表达分别通过免疫印迹和RT-qPCR测定Gli-1、OCT-4和NANOG的核转位（OCT-4、NANOG、SOX-2）和CD 133，通过免疫荧光显微镜研究Gli-1、OCT-4和NANOG的核转位，并通过膜联蛋白V-PI测定评估细胞凋亡诱导长达72小时。低氧导致Shh基因转录激活，并诱导Gli-1核转位，呈时间依赖性。调节CSC自我更新能力的NANOG、OCT-4和SOX-2转录因子的表达也显著增加，这与CD 133标记物水平的伴随增加有关。环巴胺抑制Shh通路导致Shh和Gli-1蛋白表达的抑制和GLi-1核转位的受损，从而导致诱导凋亡的增强。此外，它降低了NANOG，OCT-4和SOX-2的蛋白水平显着，导致实质上废除CD 133 expression.Conclusion：我们建议Shh通路作为治疗CCA，这是难治性的标准化疗的新靶点。
Objective:We investigated the interplay between hypoxia and Shh pathway activation and its effect on CSCs during cholangiocarcinoma (CCA) progression.Methodology:HUCCT-1 (Intrahepatic) and TFK-1 (Extrahepatic) cell lines were cultured under hypoxic (1% oxygen) and normoxic (21% oxygen) conditions for up to 48hr in the presence or absence of Cyclopamine (an inhibitor of Smoothened, a G-protein coupled receptor). The protein and mRNA expression of HIF-1 α, Shh, Gli-1, stem cell transcription factors (OCT-4, NANOG, SOX-2) and CD133 was determined by immunoblot and RT-qPCR respectively, nuclear translocation of Gli-1, OCT-4 and NANOG was investigated by immunofluorescence microscopy and induction of apoptosis was assessed by Annexin V-PI assay for up to 72 hrs.Results:Hypoxia led to the transcriptional activation of Shh gene and induced Gli-1 nuclear translocation in a time dependent manner. Expression of NANOG, OCT-4 and SOX-2 transcription factors, which regulate the self renewal capacity of CSCs, also increased significantly, which was associated with a concomitant increase in the CD133 marker level. Inhibition of Shh pathway by Cyclopamine led to the suppression of Shh and Gli-1 protein expression and impaired GLi-1 nuclear translocation, which resulted in enhanced induction of apoptosis. Furthermore, it decreased the protein levels of NANOG, OCT-4 and SOX-2 significantly, leading to a substantially abrogated CD133 expression.Conclusion:We propose Shh pathway as a novel target for the treatment of CCA, which is refractory to standard chemotherapy.