抗肿瘤候选活性化合物L029作用机制研究

Inhibitors of receptor tyrosine kinases might provide targeted treatments for cancer. c-Met is the receptor of hematic growth factor (HGF) and TrkA is the receptor of nerve growth factor (NGF). The ectopic activation of c-Met and Trk pathway are associated with the development and progression of tumor. There is a crosstalk between HGF/c-Met and Trk signal pathway in some tumor cells. In our previous study, using a high throughput sceening model, a novel compound named L029 was identified to show significant anti-tumor activity. We found that L029 inhibits the growth of various tumor cell lines in vitro and in vivo. Using in vitro kinase activity assay, L029 showed specific inhibition activity on c-Met and TrkA kinases. In tumor cells, L029 inhibits HGF induced c-Met phosphorylation and activation of downstream signal pathway. Also the metastasis induced by HGF was also inhibted by L029. In Tpr-Met transformed Ba/F3 cells which show constitutive activation of c-Met activity, L029 inhibits cell growth and induced cell cycle arrest. Furthermore, L029 inhibits the TrkA phosphorylation and ERK activation induced by NGF in A549 cells. These studies suggest that L029 inhibits cell growth of tumor cells propably through specific inhibition of c-Met and TrkA signal pathway. In the present study, we will investigate the molecular mechanims of L029 in anti-tumor in detail and then using L029 as a probe, to illustrate the molecular basis of the crosstalk between c-Met and Trk signal pathway. This study will help to confirm whehter L029 is a leading compound of tumor therapy and provide new strategy to devleop novel anti-tumor drug.

受体型酪氨酸激酶是国际公认的抗肿瘤靶点。c-Met及TrkA激酶分别为肝细胞生长因子HGF和神经生长因子NGF的受体，两者的异常活化均与肿瘤的发生、发展密切相关，并且两者介导的信号通路存在内在交联。本实验室前期利用抗肿瘤化合物高通量筛选模型，对两千种化合物进行筛选，获得一种新结构的抗肿瘤先导化合物L029（已申请中国发明专利）。体内外研究表明L029具有明确的抗肿瘤活性,对其作用机制初步研究表明L029发挥抗肿瘤作用可能通过特异抑制c-Met与Trk通路来进行的。本项目拟深入研究L029对c-Met、Trk信号通路的抑制活性及作用特征，评估L029对c-Met、Trk共异常恶性细胞的作用及特点，以及以L029为分子探针，研究c-Met、Trk信号途径的交联及分子基础。本研究将有助于确认L029是否作为抗肿瘤先导化合物及其作用基础,为开发新型靶向抗肿瘤药物提供新思路。

受体型酪氨酸激酶是国际公认的抗肿瘤靶点。c-Met及TrkA激酶分别为HGF和NGF的受体，两者的异常活化均与肿瘤发生、发展密切相关。本实验室前期获得一种新结构的抗肿瘤先导化合物L029，可特异抑制c-Met与Trk介导的信号通路。本研究系统评价了Met与Trk调控肝癌恶性表型的作用，并深入研究了L029对c-Met、Trk信号通路的抑制活性及作用特征。.首先，我们检测了多种肿瘤细胞中c-Met与Trk的表达情况，并分析了肝癌病人术后生存期，结果发现两者在肝癌病人中异常高表达，并且两者共同高表达的病人预后更差，提示两者共同高表达调控了肝癌恶性表型。敲低c-Met或TrkB可使肝癌细胞的增殖受到抑制，侵袭能力削弱；同时敲低两者抑制作用更加明显。利用c-Met和Trk激酶的特异性抑制剂得到相似结果。此外，我们还发现敲低c-Met可导致Trk激通路活化受到抑制；反之，敲低Trk也抑制了c-Met信号通路的活化，这些结果表明两种激酶介导的信号通路之间存在相互交联作用，同时靶向Met与Trk是治疗肝癌的有效策略。.其次，我们进一步研究了L029对Trk信号通路的影响，结果发现L029可抑制NGF诱导的TrkA及BDNF诱导的TrkB磷酸化及下游信号通路的活化，抑制NGF及BDNF诱导的肝癌细胞侵袭，抑制TrkA转化的NIH3T3细胞增殖及信号通路活化，在体内皮下荷瘤实验中可显著抑制Trk与Met的磷酸化。在肝癌细胞内敲低Met与Trk可显著减弱L029抑制肿瘤细胞增殖的效果。表明L029作为一种选择性的Met与Trk激酶抑制剂，在体外、体内具有明显的抗肝癌细胞活性，可作为抗肿瘤先导化合物。.第三，前期研究我们发现L029原料药的水溶性较差，口服给药很难吸收，经过筛选我们获得L029包合物处方。我们采用肝脏原位接种模型评价了L029体内抗肿瘤活性，发现口服L029包合物对肝脏原位肿瘤具有良好的治疗效果，活性优于阳性对照药物，表明L029具备开发为抗肿瘤药物的潜力。.第四，我们对L029包合物在小鼠体内的生物利用度进行了测定，并初步分析了L029在组织中的分布。.综上，本研究揭示了靶向Met与Trk是抗肝癌治疗的有效途径，小分子化合物可作为新型抗肿瘤先导化合物开展进一步的研究。