基于MEK1/2为靶点的茅术醇抑制作用、定量构效关系及抗非小细胞肺癌生物活性研究

Lung cancer is the most common cancer and ranking NO. 1 in the world. More than 85% lung cancer patients are non small cell lung cancer. Research and development of the anti-lung cancer drug from plants and herbs has been become an important research field. In recent, many researches have shown that Atractylodes Lancea, Atractylodes Macrocephala and those components (including hinesol) exhibit anti-cancer acitivity. Hinesol is one ingredient of essential oil constituents of Atractylodes Lancea and Atractylodes Macrocephala, which has the advantages of simple structure, convenience for chemical subsequents synthesis and modification. In the previous research, we found that hinesol is the potential inhibitors of MEK1/2, the results of anti non small cell lung cancer activity of hinesol in vivo and vitro showed that hinesol can inhibits proliferation and induces apoptosis via ERK/RSK2/NF-κB pathway. However, Inhibition，QSAR and anti non-small lung cancer activity of hinesol on MEK1/2 are not well understood. . In this project, we will use technologies of natural products chemistry, molecular and cell biology, organic chemistry to analyze the inhibition and QSAR of hinesol on MEK1/2; the effect of hinesol on proliferation, apoptosis and ERK/RSK2/NF-κB pathway will be measured in different non small cell lung cancer cell lines. Then, its efficacy and activity in vivo will be evaluation in tumor bearing mice. This project will became the foundation for development of novel MEK1/2 inhibitors, also provide experiments basis for its further clinical application. . In conclusion, this research will be a great development in anti-tumor activity of hinesol, Atractylodes Lancea and Atractylodes Macrocephala, also provide the foundation for development of novel MEK1/2 inhibitors and experiments basis for clinical application in cancer realm.

肺癌是十大恶性肿瘤之首，约85%为非小细胞肺癌。天然产物作为抗肺癌肿瘤药物开发的重点和捷径，备受关注。茅术醇作为苍术、白术等的挥发油成分，具有结构简单，利于后续化学合成及修饰等特点。前期研究中，我们发现茅术醇是MEK1/2的潜在抑制剂；在体内外实验显示：其可通过下调ERK/RSK2/NF-κB通路实现强效抑制非小细胞肺癌增殖、诱导凋亡等活性，但抑制、构效和调控机理不明。本课题拟将天然产物、分子、细胞生物学、有机化学等相结合，分析茅术醇对MEK1/2的抑制机制及定量构效关系；以不同非小细胞肺癌细胞为研究对象，检测茅术醇对增殖、细胞周期和凋亡的影响，检测对ERK/RSK2/NF-κB通路相关因子的影响，全面分析茅术醇的抗非小细胞肺癌的生物活性，并在荷瘤小鼠中评价其体内药效的同时，验证其活性。本项目将为以茅术醇为先导物开发新型MEK1/2抑制剂打下基础，也为其进一步在临床应用方面提供实验依据。

本项目以茅术醇为研究对象，研究其定量构效关系、抗非小细胞肺癌机制。主要成果包括：利用定点突变和SPR技术与方法，研究发现茅术醇可与茅术醇可结合至MEK1蛋白的Ala95位点，MEK2蛋白的Asp212位点；使用autodock、sybyl等软件，分析茅术醇的定量构效关系，结果显示，茅术醇-C10、-OH等位点是结合MEK1/2的关键位点；MTT比色法数据表明，茅术醇可有效抑制A549、NCI-H1661细胞的增殖，且抑制效果与茅术醇加药浓度和刺激时间呈现正相关，A549荷鼠肿瘤模型结果显示相同结果；使用流式细胞术、western blot分析茅术醇对细胞周期和凋亡的影响，结果表明，茅术醇可通过调控BCL-2家族诱导凋亡，通过抑制Cyclin D1的表达阻滞细胞周期于G0/G1期。针对茅术醇抗非小细胞肺癌的机制研究，western blot数据显示，茅术醇能够通过抑制ERK1/2、IKKα、p65等蛋白的磷酸化水平，表明茅术醇下调MEK/ERK、NF-kB通路实现非小细胞肺癌增殖抑制。此外，茅术醇可降低p-RSK2水平，提示其可能是茅术醇抑制肺癌增殖过程中MEK/ERK、NF-kB通路的关键“串话”因子。为此，构建RSK2-shRNA载体，转染A549细胞，随后加入茅术醇刺激，western blot检测MEK/ERK、NF-kB通路相关因子变化。结果表明，茅术醇通过调控ERK/RSK2/IKKα/p65通路，实现非小细胞肺癌增殖抑制。上述研究成果为以茅术醇为先导物开发抗肿瘤药物提供试验依据，也为类似天然产物的研究提供参考。