小陷胸汤通过下调FACT表达阻碍静止期肺癌细胞重新激活抗转移的作用和分子机制研究

Recurrence and metastasis are the main cause of mortality of patients with lung cancer. Blocking cell cycle re-entry of quiescent cancer cells is the key to prevent cancer recurrence and metastasis. But the therapeutic target and intervention method are lacking. Facilitates Chromatin Transcription (FACT) is a member of the histone chaperone family. We found that (1) FACT can promote proliferative switch of quiescent lung cancer cells; (2) Xiaoxianxiong Decoction can effectively hinder the quiescent lung cancer cells entering the active phase and concomitantly inhibit the expression of FACT. In this project, a post-surgery model on nude mice will be used to dynamically analyze the effect of Xiaoxianxiong Decoction and FACT on metastasis by Caliper IVIS Lumina II. Together with the flow cytometry, qPCR, WB, ChIP and other methods , this project will confirm the hypothesis that, by down-regulation of the expression of histone chaperone FACT, Xiaoxianxiong decoction can block cell cycle re-entry of quiescent lung cancer cells and prevent recurrence and metastasis. This project has a potential to establish new therapeutic target and method to prevent the recurrence and metastasis, and provide evidence for prevention and treatment of cancer with traditional Chinese medicine.

复发转移是导致肺癌患者死亡的根本原因。阻断处于静止期状态肿瘤细胞的激活被认为是降低复发转移的关键。但是，作用靶点及干预手段明显匮乏。促染色质转录复合物（FAcilitates Chromatin Transcription，FACT）是组蛋白分子伴侣中的一员。申请人通过前期研究发现，（1）FACT对静止期肺癌细胞的激活起到关键促进作用；（2）小陷胸汤能有效阻碍静止期肺癌细胞的激活并抑制FACT的表达。本项目将建立裸鼠肺癌术后复发转移模型，采用活体成像系统动态监测小陷胸汤及FACT表达水平对复发转移的影响；通过结合流式细胞术、qPCR、WB，ChIP和体外实验平台，从细胞、蛋白和基因水平阐明本项目的科学假说，即小陷胸汤通过下调FACT基因表达阻碍静止期肺癌细胞的激活从而达到抗复发转移作用。本项目将为防治肺癌复发转移提供新的作用靶点和干预手段，为中医药的防治恶性肿瘤提供理论依据。

肺癌复发转移是导致肺癌患者死亡的根本原因，而肺癌休眠细胞重新激活及快速增殖是导致复发和转移的关键，但其针对性的治疗手段尚属空白，是目前临床治疗的热点和难点。本项目通过接触抑制或血清清除法建立肺癌静止期细胞、DNA/RNA双染流式检测静止期细胞比例。进一步采用ChIP、RT-PCR和Western Blot检测小陷胸汤对FACT及c-MYC的表达活性、采用小陷胸汤体内外干预小鼠肺癌皮下瘤模型对抗肺癌作用进行动物实验验证。最后，对小陷胸汤进行拆方研究，并采用流式检测和小鼠肺癌原位模型对有效成分抗肺癌活性进行验证。结果表明，小陷胸汤可以通过降低FACT和c-MYC mRNA和蛋白水平阻碍肺癌静止期细胞的激活，同时可降低FACT和c-MYC启动子的H3K4三甲基化和RNA聚合酶II募集，同时动物实验也证明小陷胸汤体内外干预均可抑制小鼠肺癌皮下移植瘤的生长。最后，拆方实验结果证实黄连碱是小陷胸汤中抗肺癌休静止期细胞激活的活性成分，肺原位模型结果表明，黄连碱具有抑制小鼠体内肺癌生长和转移的作用。本项目的研究救过为预防或者延缓肺癌的复发转移提供新的靶点和治疗手段，并为肺癌“从痰论治”理论思想的科学内涵及现代应用提供有力的证据。