基于磷酸化蛋白质组学调控网络的片仔癀逆转大肠癌耐药机制研究

The existence of multidrug Resisitance (MDR) of tumor restraines the effect of chemotherapy on colorectal cancer (CRC) ,and attracting more and more attention. It has been shown that abnormal modification of many proteins, especially phosphorylation, plays a key role in the development of MDR. Pien Tze Huang (PZH), a well-known traditional Chinese formula, has been demonstrated to be clinically effective in the treatment of various malignancies including colorectal cancer (CRC). Our previous studies showed that PZH displays multi-target, multi-pathway characteristics in the treatment of CRC. In addition, PZH can significantly inhibit the drug resistance, migration, invasion of the human colorectal cell. However, the mechanism of anti-drug resistance of PZH remains largely unclear. In this study we will screen the differential proteins between 5-Fu resistance cells and normal CRC cells using the phosphoproteomics and MALDI-TOF/TOF, build the signaling pathway network among the phosphorylated protein and the target protein using bioinformatics, find the key target protein. We will also detect the effect of PZH on the multiple targets in the protein phophorylation in vitro and in vivo using western blot technique, clarify the mechanisms of its reverse action of 5-Fu resistance. This study can provide a theoretical basis for the treatment of colorectal cancer and provide new ideas for the study of Traditional Chinese Medicine.

肿瘤多药耐药（Multidrug Resistance, MDR）的存在极大地限制了大肠癌化疗的效果，是目前大肠癌研究的热点。研究表明多种蛋白质的异常磷酸化修饰在MDR中发挥着关键作用。片仔癀是传统名贵中成药，对大肠癌疗效明确。课题组前期研究表明片仔癀治疗大肠癌具有多靶点、多通路的特点，能显著抑制大肠癌的耐药、迁移和侵袭，但其作用机制尚未阐明。本课题采用磷酸化蛋白质组学技术分析俩株5-Fu耐药细胞和非耐药细胞磷酸化蛋白的差异表达，由MALDI-TOF/MS质谱鉴定蛋白，找出共同的差异表达磷酸化蛋白，再用生物信息学构建与大肠癌耐药相关的磷酸化蛋白-靶点网络，找出关键节点蛋白；通过体内外实验，研究片仔癀对大肠癌耐药的逆转作用，用western blot法检测片仔癀对磷酸化蛋白网络中关键节点蛋白表达的影响，系统地阐明片仔癀逆转大肠癌耐药的作用机制，以期为临床片仔癀治疗大肠癌提供进一步的理依据。

片仔癀是传统名贵中成药，具有清热解毒、消肿散结、活血化瘀、扶正祛邪的功效，治疗大肠癌等恶性肿瘤疗效明确，其作用机制表现在可通过调控STAT3、Hedgehog、PI3K/Akt等多条信号通路来调节Bcl-2、Bax、cyclin D1、CDK4、VEGF等具有调控细胞凋亡、增殖、肿瘤血管新生等靶基因的表达，最终发挥抗大肠癌的作用。然而，关于片仔癀对于逆转大肠癌耐药作用的研究尚未见报道。因此，本项目（1）采用磷酸化组学技术筛选片仔癀逆转大肠癌耐药的可能作用靶点，进一步通过体内外实验研究片仔癀对上述差异表达的基因及其通路的影响；进而深入地研究目的基因与耐药的关系及其作用靶点，较全面系统地阐明片仔癀抗耐药的作用机制。研究结果发现1.5倍差异的磷酸化蛋白位点有214个，其中上调46个，下调168个；KEGG通路富集发现主要相关的耐药通路有蛋白转运通路、剪接体信号通路、HIF信号通路、凋亡信号通路等；（2）片仔癀均具有抑制大肠癌耐药细胞的活力、可促进HCT-8/5-FU细胞凋亡、抑制耐药细胞增殖及外排，同时体内实验也发现片仔癀可抑制HCT-8/5-FU移植瘤细胞增殖及细胞外排。而通过调控包括多个基因、蛋白及磷酸化蛋白的表达可能是片仔癀发挥逆转大肠癌作用的重要机制。在本基金项目的资助下，课题组已发表SCI源论文4篇，核心期刊1篇。

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