Chiang Chang-Hsu, Wu Chih-Ching, Lee Li-Yu, Li Yi-Chen, Liu Hao-Ping, Hsu Chia-Wei, Lu Ya-Ching, Chang Joseph T, Cheng Ann-Joy
Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University , Kwei-Shan, Tao-Yuan 333, Taiwan.
Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University , Kwei-Shan, Tao-Yuan 333, Taiwan.
J Proteome Res. 2016 Sep 2;15(9):2981-97. doi: 10.1021/acs.jproteome.6b00138. Epub 2016 Aug 4.
The areca nut is a known carcinogen that causes oral cancer in individuals in Southeast Asia, but the molecular mechanism that leads to this malignancy is still unclear. To mimic the habit of areca nut chewing, our laboratory has established four oral cancer cell sublines (SAS, OECM1, K2, C9), which have been chronically exposed to areca nut extract (ANE). To elucidate the molecular basis of areca nut-induced oral carcinogenesis, the differential proteomes between oral cancer cells and the ANE-treated sublines were determined using isobaric mass tag (iTRAQ) labeling and multidimensional liquid chromatography-mass spectrometry (LC-MS/MS). Over 1000 proteins were identified in four sublines, and 196 proteins were found to be differentially expressed in at least two ANE-treated sublines. A bioinformatic analysis revealed that these proteins participate in several pathways, and one of the most prominent pathways was the regulation of epithelial to mesenchymal transition (EMT). In all, 24 proteins including Krt17 were confirmed to be differentially expressed in the ANE-treated sublines. To reveal additional information on the mechanism of ANE-induced carcinogenesis, Krt17 was further investigated. Krt17 knockdown significantly suppressed ANE-induced cell migration and invasion and modulated the EMT process. Furthermore, in a murine model of carcinogen-induced (arecoline cocktail, an active compound of ANE) oral cancer, Krt17 was significantly up-regulated in all hyperplastic tissues and in carcinoma tissues (p < 0.001). In conclusion, we have identified a proteome of oral cancer cells that is associated with chronic areca nut exposure. Krt17 was demonstrated to contribute to areca nut-induced oral malignancy. The results of this study contribute to risk assessment, disease prevention and other clinical applications associated with areca nut-induced oral cancer.
槟榔是一种已知的致癌物，会导致东南亚地区的人群患口腔癌，但其引发这种恶性肿瘤的分子机制仍不清楚。为模拟嚼槟榔的习惯，我们实验室建立了四个口腔癌细胞亚系（SAS、OECM1、K2、C9），它们长期暴露于槟榔提取物（ANE）中。为阐明槟榔诱导口腔癌发生的分子基础，利用等压质量标签（iTRAQ）标记和多维液相色谱 - 质谱联用（LC - MS/MS）技术测定了口腔癌细胞与经ANE处理的亚系之间的差异蛋白质组。在四个亚系中鉴定出了1000多种蛋白质，发现有196种蛋白质在至少两个经ANE处理的亚系中差异表达。生物信息学分析表明，这些蛋白质参与了多种信号通路，其中最显著的信号通路之一是上皮 - 间质转化（EMT）的调控。总共24种包括角蛋白17（Krt17）在内的蛋白质被证实在经ANE处理的亚系中差异表达。为揭示关于ANE诱导致癌机制的更多信息，对角蛋白17进行了进一步研究。敲低Krt17可显著抑制ANE诱导的细胞迁移和侵袭，并调节EMT过程。此外，在致癌物诱导（槟榔碱混合物，ANE的一种活性成分）的口腔癌小鼠模型中，Krt17在所有增生组织和癌组织中均显著上调（p < 0.001）。总之，我们鉴定出了与长期槟榔暴露相关的口腔癌细胞蛋白质组。已证明Krt17促成了槟榔诱导的口腔恶性肿瘤。本研究结果有助于与槟榔诱导的口腔癌相关的风险评估、疾病预防及其他临床应用。
