Response Profiling Using Shotgun Proteomics Enables Establishing Global Metallodrug Mechanisms of Action - cytoplasmic proteins of arsenic trioxide-treated SW480 epithelial cells

Response profiling using shotgun proteomics for establishing global metallodrug mechanisms of action in two colon carcinoma cell lines, HCT116 and SW480, was applied and evaluated with the clinically approved arsenic trioxide. Surprisingly, the complete established mechanism of action of arsenic trioxide was observed by protein regulations in SW480, but not in HCT116 cells. Comparing the basal protein expression in the two cell lines revealed an 80% convergence of protein identifications, but with significant expression differences, which in turn seem affect the extent of protein regulation. For example, while a clear-cut redox response was observed in SW480 cells upon arsenic treatment, such an effect was lacking in HCT116 cells and the latter express drastically higher levels of the involved redox proteins. Response profiling was then used to investigate four anticancer metallodrugs (KP46, KP772, KP1339 and KP1537) by means of functional groups of proteins and mapped their effects according to DNA repair, endocytosis, protection from oxidative stress, protection from endoplasmatic reticulum (ER) stress, cell adhesion and mitochondrial function. We were able to characterize the global effects of these metallodrugs on the proteome and generate hypotheses on hitherto unrecognized mechanisms. Significant differences in the two colon cell lines strongly suggest that knowledge of mechanistic hallmarks of anticancer metallodrug action are imperative for the design of clinical studies and that outcome may be enhanced by means of patient stratification strategies according to these hallmarks.

本研究采用鸟枪法蛋白质组学（shotgun proteomics）响应谱分析技术，针对两种结肠癌细胞系HCT116与SW480，开展金属药物全局作用机制的解析，并以临床获批药物三氧化二砷（arsenic trioxide）进行方法验证。令人意外的是，在SW480细胞中可通过蛋白质表达调控现象，完整复现三氧化二砷已知的作用机制，但该现象并未在HCT116细胞中观测到。对两种细胞系的基础蛋白质表达水平进行比较后发现，二者的蛋白质鉴定重合度达80%，但表达水平存在显著差异，而该差异似乎会影响蛋白质调控的幅度。例如，三氧化二砷处理后，SW480细胞中可观测到明确的氧化应激响应，但HCT116细胞中并无该效应；且后者所涉及的氧化应激相关蛋白质的表达水平显著更高。随后，本研究利用蛋白质功能类群分析，通过响应谱分析技术对四种抗肿瘤金属药物（KP46、KP772、KP1339及KP1537）开展研究，并依据DNA修复、胞吞作用、氧化应激防护、内质网（ER）应激防护、细胞黏附及线粒体功能等通路，绘制了各药物的效应图谱。本研究成功表征了这些金属药物对蛋白质组的全局调控效应，并针对此前未被阐明的作用机制提出了全新假说。两种结肠癌细胞系间存在的显著差异强烈表明，阐明抗肿瘤金属药物的作用机制特征，对临床研究的设计至关重要；且通过基于这些特征的患者分层策略，有望提升临床试验的结局。