文蛤多肽靶向微管蛋白抑制肺癌生长的分子机制

Tubulin inhibitors are widely used clinically to treat solid tumors. However, these kinds of agents usually possesses severe toxicity including bone marrow suppression and neurotoxicity. In addition, drug resistance is another problem for tubulin inhibitors. Therefore, finding novel tubulin inhibitors is promising. Mere15 is a novel polypeptide isolated from Meretrix meretrix Linnaeus with molecular weight of 15 KD. Previous study in our laboratory reveals that Mere15 displays potent anticancer activity both in vitro and in vivo on solid tumor and human leukemia cells. Mere15 induces cell death via mitochondrial pathway. Further study confirmed that Mere15 causes the disassembly of microtubule cytoskeleton in both human lung cancer A549 and human leukemia K562 cancer cells. Low concentration of Mere15 does not affect the interaction between the colchicines and tubulin, while high concentration of the polypeptide increases the binding of tubulin with colchicines, suggesting that there is a novel binding site on tubulin interacted with Mere15. In the present study, we plan to: 1) identify the novel binding site on tubulin using photoaffinity labeling and light scattering approaches ; 2) dissect the binding peptide of Mere15 interacting with tubulin by site-directed mutagenesis and molecular docking experiments; 3)study the interaction of tubulin with the binding peptide; 4) study the anticancer effects of binding peptide on the resistant cells to tubulin inhibitors. The study is of great significance to understand the dynamics of tubulin assembly/disassembly. The study also provides a useful strategy for developing novel tubulin inhibitors.

靶向微管药物是临床上广泛应用的抗肿瘤药物, 但这类药物存在骨髓抑制、神经毒性等副作用，并容易产生耐药性。文蛤多肽（Mere15）是由海洋生物文蛤中获得的抗肿瘤多肽，前期研究表明，Mere15具有良好的体内外抗肿瘤活性, 对人肺癌A549细胞移植性裸鼠具有显著的抑制作用，Mere15可以通过线粒体途径诱导肿瘤细胞凋亡, 进一步的研究发现Mere15可以抑制肿瘤细胞微管聚合, 低浓度的Mere15不影响秋水仙碱与微管的相互作用，高浓度反而促进二者的相互作用, 说明Mere15具有新颖的作用靶点。本研究拟采用光亲和标记、分子对接等技术鉴定Mere15在微管蛋白分子上的结合位点；利用定点突变、光散射实验等手段确定与微管蛋白结合的多肽片段与氨基酸残基；研究结合多肽与微管蛋白的相互作用；研究结合多肽对微管耐药细胞的抗肿瘤作用，阐明Mere15抑制微管聚合的动力学机制，为发展新型的抗微管药物奠定基础。

肺癌是影响人类健康的重大疾病，目前尚缺乏安全有效的治疗措施。文蛤多肽Mere15对肺癌显示了良好的体内外抗肿瘤活性，但其作用的分子机制上不清楚。本研究揭示了Mere15抗肿瘤作用的分子机制，鉴定了Mere15分子上与微管蛋白相互作用的多肽片段，证实Mere15分子上一段45个氨基酸残基的序列可以与微管蛋白发生特异性相互作用。同时证实Mere15可以通过AMPK途径诱导肿瘤细胞自噬，自噬作用对肿瘤细胞产生显著的抑制作用。通过Transwell及划痕实验证实，Mere15还可以显著抑制肺癌细胞的迁移与管腔形成，Western blot 证实PI3K/Akt/mTOR对Mere15的抗转移作用发挥关键影响，体内实验证实，Mere15可以抑制多种肿瘤细胞裸鼠移植性肿瘤生长。研究结果对发展Mere15作为新型的抗肿瘤药物提供了依据。课题共发表SCI论文8篇，申请国家发明专利一项。