Liang Ye, Xu Wenhua, Liu Shihai, Chi Jingwei, Zhang Jisheng, Sui Aihua, Wang Liping, Liang Zhijuan, Li Dan, Chen Yuanbin, Niu Haitao
Key Laboratory, Department of Urology and Andrology, Qingdao, China.
Department of Urology, Affiliated Hospital of Qingdao University, Qingdao, China.
Cell Physiol Biochem. 2018;45(5):2054-2070. doi: 10.1159/000488042. Epub 2018 Mar 7.
BACKGROUND/AIMS: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential anti-cancer agent due to its selective toxicity. However, many human non-small cell lung cancer (NSCLC) cells are partially resistant to TRAIL, thereby limiting its clinical application. Therefore, there is a need for the development of novel adjuvant therapeutic agents to be used in combination with TRAIL.
In this study, the effect of N-acetyl-glucosamine (GlcNAc), a type of monosaccharide derived from chitosan, combined with TRAIL was evaluated in vitro and in vivo. Thirty NSCLC clinical samples were used to detect the expression of death receptor (DR) 4 and 5. After GlcNAc and TRAIL co-treatment, DR expression was determined by real-time PCR and western blotting. Cycloheximide was used to detect the protein half-life to further understand the correlation between GlcNAc and the metabolic rate of DR. Non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to detect receptor clustering, and the localization of DR was visualized by immunofluorescence under a confocal microscope. Furthermore, a co-immunoprecipitation assay was performed to analyze the formation of death-inducing signaling complex (DISC). O-linked glycan expression levels were evaluated following DR5 overexpression and RNA interference mediated knockdown.
We found that the clinical samples expressed higher levels of DR5 than DR4, and GlcNAc co-treatment improved the effect of TRAIL-induced apoptosis by activating DR5 accumulation and clustering, which in turn recruited the apoptosis-initiating protease caspase-8 to form DISC, and initiated apoptosis. Furthermore, GlcNAc promoted DR5 clustering by improving its O-glycosylation.
These results uncovered the molecular mechanism by which GlcNAc sensitizes cancer cells to TRAIL-induced apoptosis, thereby highlighting a novel effective agent for TRAIL-mediated NSCLC-targeted therapy.
背景/目的：肿瘤坏死因子相关凋亡诱导配体（TRAIL）因其选择性毒性而成为一种潜在的抗癌药物。然而，许多人非小细胞肺癌（NSCLC）细胞对TRAIL存在部分抗性，从而限制了其临床应用。因此，需要开发新型辅助治疗药物与TRAIL联合使用。
在本研究中，评估了壳聚糖衍生的一种单糖N-乙酰葡糖胺（GlcNAc）与TRAIL联合使用在体外和体内的效果。使用30例NSCLC临床样本检测死亡受体（DR）4和5的表达。GlcNAc和TRAIL联合处理后，通过实时PCR和蛋白质印迹法测定DR表达。使用放线菌酮检测蛋白质半衰期，以进一步了解GlcNAc与DR代谢率之间的相关性。使用非还原十二烷基硫酸钠-聚丙烯酰胺凝胶电泳检测受体聚集，并在共聚焦显微镜下通过免疫荧光观察DR的定位。此外，进行了免疫共沉淀试验以分析死亡诱导信号复合物（DISC）的形成。在DR5过表达和RNA干扰介导的敲低后评估O-连接聚糖表达水平。
我们发现临床样本中DR5的表达水平高于DR4，GlcNAc联合处理通过激活DR5的积累和聚集提高了TRAIL诱导凋亡的效果，这反过来又招募了凋亡起始蛋白酶caspase-8形成DISC，并启动凋亡。此外，GlcNAc通过改善其O-糖基化促进DR5聚集。
这些结果揭示了GlcNAc使癌细胞对TRAIL诱导的凋亡敏感的分子机制，从而突出了一种用于TRAIL介导的NSCLC靶向治疗的新型有效药物。
