西藏新种白肉灵芝乙醇提取物抑制乳腺癌生长的化学成分及其作用机制

cancer is a public health issue and a main cause of death. Our studies aim to discover novel anti-tumour compounds from natural products. Ganoderma leucocontextum is a new species of Ganoderma sp, which was discovered from Tibet Autonomous Region of China in 2014. In our previous screening, we found that the ethanol extract of G.leucocontextum inhibited the growth of breast cancer in vitro and in vivo. We plan to purify the bioactive compounds from G. leucocontextum ethanol extract and examine its inhibitory effect on tumour cell activities. Measured by anti-cancer cell activities in vitro, G. leucocontextum ethanol extract will be isolated and purified by modern chromatographic separation methods. The structures of the purified molecules will be identified by NMR and MS spectral analysis and so on. The anti-tumour effect of active compounds will be measured by experiments including cell proliferation, cell colony formation, cell migration, cell invasion and flow cytometer. The effect of compounds are validated using cancer models in mice by inoculation of marine breast carcinoma cells line 4T1.The mRNA expression of oncogene and tumor suppressor will be tested by micro-array in mice tissue treated with the bioactive compounds and control. The results of RT-PCR experiments supported the reliability of our microarray analysis. The proteins in pathway will be identified by immunohistochemistry, immunofluorescent staining and western blot and so on. We aim to find novel compounds in G. leucocontextum and uncover their anti-tumour mechanisms. We anticipate that our studies will lead to clinical application of the medicinal mushroom G.leucocontextum in cancer intervention.

2014年，西藏灵芝属新种白肉灵芝分类学地位和命名确定之后，关于其化学成分和功能的研究迅速展开。我们前期实验结果显示：白肉灵芝乙醇提取物（GLE）能抑制乳腺癌细胞增殖和小鼠乳腺癌生长。本项目将以前期结果为基础，将对GLE采用硅胶、ODS及RPLC等方法进行分离纯化，同时在分离过程中，用乳腺癌细胞进行活性评价和指导；对获得的化合物利用波谱和理化数据进行结构鉴定；通过小鼠乳腺癌模型确证化合物整体药效；用细胞实验确定化合物对肿瘤细胞的作用方式；采用芯片测定对照组和处理组肿瘤组织mRNA表达谱，分析显著差异表达的促癌和抑癌基因，寻找靶基因；用RT-PCR验证靶基因mRNA表达量；利用免疫、荧光及Western Blotting检测靶基因蛋白表达水平，寻找靶基因相关上下游蛋白，推测活性化合物抑制肿瘤生长的信号途径。我们期望通过本项目明确白肉灵芝抗肿瘤的化学成分及作用机制，提供新的先导化合物及新资源。

药用真菌中筛选抗肿瘤天然药物发现的重要资源之一。灵芝是食药兼用菌，含有三萜、多糖等活性物质，有抗肿瘤、调节免疫、降血脂等多种功能。白肉灵芝是我国学者从西藏地区发现和鉴定的灵芝属新种（2014年），其化学成分和功效迅速成为研究热点。因此，基于前期发现的白肉灵芝乙醇提取物抑制乳腺癌细胞增殖和生长的基础，本项目系统的开展白肉灵芝乙醇提取物（GLE）抗肿瘤活性组分和化合物的功能及分子机制研究。首先，经溶剂萃取获得不同极性的组分，经乳腺癌细胞增殖活性评价，发现活性成分富集在乙酸乙酯萃取物(GLEA)，进而明确GLE和GLEA抑制乳腺癌生长的功效。接下来，经ODS分离纯化获得18个馏分，细胞增殖实验筛选出5个活性馏分（GL5-9）；在此基础上，从GL7分离纯化获得主要活性化合物Ganoderiol F。功能研究发现Ganoderiol F抑制多种乳腺癌细胞增殖；同时，发现其显著地抑制癌细胞迁移和克隆形成；流式细胞术发现Ganoderiol F使肿瘤细胞停止在G1期，停止分裂。进而发现Ganoderiol F诱导控制细胞由G1进入S期的相关蛋白Cyclin D1、D3、El和CDK4、CDK2的表达下调；并发现激活Cyclin D1-CDK4/CDK6和Cyclin E-CDK2的促癌因子c-Myc的表达下调，而抑癌因子Foxo3表达上调；另外，发现与细胞凋亡相关的促癌因子Bcl-2和Bcl-w表达降低。因此，Ganoderiol F通过作用c-Myc和Foxo3的表达，抑制Cyclin D1-CDK4/CDK6和Cyclin E-CDK2，阻止癌细胞分裂增殖，诱导细胞死亡。综上所述，本项目从抗肿瘤评价和天然产物分离结合的角度，寻找白肉灵芝醇提取物的活性部位和化合物，明确活性组分和化合物抗肿瘤功能，揭示其抗肿瘤的分子机制，完善白肉灵芝生物功能的研究，从而为其开发应用提供科学理论支持。