和厚朴酚调控Akt/ERK代偿活化通路逆转依维莫司耐药作用机制研究

Everolimus is a kind of mTOR inhibitors with anti-tumor effects. Evidence from clinical trials demonstrates it prolongs progression-free survivals of patients with late stage breast cancers. But its efficiency is restricted due to drug resistance. Comprehensive overactivation of compensatory pathways is one of the core mechanisms associated with everolimus resistance. Honokiol, a natural small molecule drug, significantly inhibits proliferation of breast cancer cells including everolimus-resistant cells. It also reverses everolimus resistance of breast cancer cells. We find honokiol inhibits phosphorylation and activation of core proteins (such as Akt and 4E-BP1) in some overactivated compensatory pathways, but definite regulatory mechanism is still undefined. In the current study, we will detect and confirm the directly targets of honokiol using a synthesized chemical probe biotin-tagged honokiol. And we will further study the interactions between honokiol targets and core proteins of above overactivated compensatory pathways, in order to demonstrate the molecular mechanism that honokiol modulates overactivated compensatory pathways in everolimus resistant cells. The current study will provide evidence to demonstrate our hypotheses ‘honokiol reverses everolimus resistance though modulating multiple overactivated compensatory pathways’. And it also provides theoretical basis for future clinical trial of honokiol- everolimus combination therapy in breast cancers.

依维莫司是具有抗肿瘤作用的mTOR抑制剂，可延长晚期乳腺癌患者生存期，原发或获得性耐药是制约疗效的重要因素，广泛代偿性通路活化是耐药关键机制。我们发现天然小分子药物和厚朴酚可显著抑制包括依维莫司耐药细胞在内的乳腺癌细胞增殖，并可逆转依维莫司耐药。进一步机制研究发现和厚朴酚可抑制乳腺癌细胞中Akt/ERK相关多条耐药代偿通路关键蛋白(如Akt、4EBP1)磷酸化活化，而其对上述通路具体分子调控机制不清。本研究拟采用化学探针生物素标记和厚朴酚，研究和厚朴酚在耐药细胞中的特异性靶蛋白；揭示和厚朴酚结合胞内靶蛋白形成复合物对上述代偿通路关键蛋白表达与功能的调控作用，以阐明其逆转依维莫司耐药的具体分子机制。本研究将为具有低毒、抗肿瘤机制广泛特点的和厚朴酚“调控多条耐药相关代偿通路逆转依维莫司耐药”的治疗理念提供实验数据依据，为制定低毒、高效的和厚朴酚与依维莫司联合方案奠定理论基础。

依维莫司是具有抗肿瘤作用的mTOR抑制剂，可延长晚期乳腺癌患者生存期，但仅有中等疗效，耐药是重要制约因素，广泛代偿性通路活化是耐药关键机制之一。本研究中我们采用和厚朴酚研究其逆转依维莫司耐药作用及其在细胞内诱发肿瘤细胞死亡、逆转耐药的关键靶蛋白。首先采用分步逐渐诱导法获得依维莫司耐药细胞株BT-549/Eve，而其与母本对和厚朴酚有相似的敏感性。采用低浓度和厚朴酚联合依维莫司处理耐药细胞后发现，其可显著增强耐药细胞对依维莫司的敏感性。而和厚朴酚可显著抑制PI3K/Akt/mTORC等耐药相关代偿性信号通路及下游的过度活化，提示这可能是和厚朴酚逆转耐药的关键作用机制。随后我们希望进一步发掘和厚朴酚胞内关键靶蛋白以明确其逆转耐药及诱导肿瘤细胞死亡的分子机制。设计构建亲和填料偶联和厚朴酚琼脂糖凝胶，用其与肿瘤细胞提取的总蛋白和线粒体蛋白共孵育，捕获可予和厚朴酚结合的胞内蛋白，发现分子量约43kD、55kD丰度最高，并进行蛋白质谱分析，经过分析比对初步确定8个潜在结合蛋白（43kD：TMEM43，STOML2，EDIL3；55kD :eEF1A1，Vimentin，ATP5A，ALDH7A1，ATP5B），并通过Western Blot方法初步确证其可与和厚朴酚结合。随后选取其中STOML2及eEF1A1进一步验证，发现发现和厚朴酚结合的STOML2定位在线粒体，和厚朴酚结合的eEF1A1定位在胞浆；和厚朴酚处理不影响上述蛋白的表达水平，同时过表达上述蛋白并不影响和厚朴酚及依维莫司对细胞的敏感性，上述蛋白可能并非和厚朴酚诱导肿瘤细胞死亡及逆转依维莫司耐药的关键靶蛋白。本研究上述结果提示传统和厚朴酚可能成为增敏逆转依维莫司耐药的重要备选药物，其可通过抑制耐药相关代偿通路发挥作用，同时筛选出和厚朴酚进入胞内后可能的结合蛋白，但他们的靶蛋白地位及作用仍需后续研究进一步揭示。