Comprehensive Proteomic Study of the Antiproliferative Activity of a Polyphenol-Enriched Rosemary Extract on Colon Cancer Cells Using Nanoliquid Chromatography–Orbitrap MS/MS

In this work, a proteomics strategy based on nanoliquid chromatography–tandem mass spectrometry (nano-LC–MS/MS) using an Orbitrap high-resolution mass spectrometer together with stable isotope dimethyl labeling (DML) is applied to quantitatively examine relative changes in the protein fraction of HT-29 human colon cancer cells treated with different concentrations of a polyphenol-enriched rosemary extract over the time. The major objective of this study was to gain insights into the antiproliferative mechanisms induced by rosemary polyphenols. Using this methodology, 1909 and 698 proteins were identified and quantified in cell extracts. The polyphenol-enriched rosemary extract treatment changed the expression of several proteins in a time- and concentration-dependent manner. Most of the altered proteins are implicated in the activation of Nrf2 transcription factor and the unfolded protein response. In conclusion, rosemary polyphenols induced proteomic changes that were related to the attenuation of aggresome formation and activation of autophagy to alleviate cellular stress.本研究采用基于轨道阱高分辨质谱仪（Orbitrap high-resolution mass spectrometer）的纳升液相色谱-串联质谱（nano-LC–MS/MS）技术，结合稳定同位素二甲基标记（DML）的蛋白质组学策略，对经不同浓度富含多酚迷迭香提取物处理不同时长的HT-29人结肠癌细胞的蛋白质组分相对丰度变化进行定量分析。本研究的核心目标在于阐明迷迭香多酚诱导的抗增殖分子机制。通过该实验方法，本研究在细胞提取物中鉴定并定量得到1909种与698种蛋白质。富含多酚的迷迭香提取物处理可使多种蛋白质的表达呈现时间与浓度依赖性的变化模式。其中，多数表达发生改变的蛋白质参与核因子E2相关因子2（Nrf2）转录因子的激活过程以及未折叠蛋白反应（unfolded protein response）。综上，迷迭香多酚可诱导蛋白质组发生改变，该改变与聚集体形成（aggresome formation）的减弱及自噬（autophagy）激活以缓解细胞应激密切相关。