扶正抗癌方对吉非替尼治疗非小细胞肺癌增敏作用的研究

It was found in previous clinical trials that the efficacy of FuZhengKangAi decoction (FZKA) together with EGFR-TKI (Gefitinib as a representitive) in treatment of advanced non-small cell lung cancer (NSCLC) was better than current reports on efficacy of single drug gefitinib. Therefore we predict and raise the hypothesis that FZKA is effective in sensitizing NSCLC cells towards gefitinib. To further understand the effects and mechanism of FZKA in sensitizing EGFR-TKI, we would use 3 NSCLC cell lines (PC9, A549 and H1650) which have different sensitivity towards EGFR-TKI, to undergo in vitro and in vivo experiments, together with signal transduction pathway investigation. Flow cytometry would be used to study the effect of the 2 drugs on cell cycle and apoptosis; Transwell compartment method for effect on tumor cell invasion and migration; Western blot for the effect of drugs on expression of EGFR, MAPK, AKT and their phosporylated products in the 3 normal NSCLC cell lines，those with EGFR gene silenced by designed siRNA,or EGFR gene over-expression by over-expression vector. Therefore we can clairfy if FZKA has sensitizing effect on gefitinib, explore the effect of the drugs on EGFR, Ras/Raf/Erk pathway and PI3K/AKT pathway, and thus understand the mechanism of FZKA in sensitizing tumor cells towards gefitinib.

基于我们前期在临床中发现中药扶正抗癌方联合酪氨酸激酶抑制剂（TKI，代表药物吉非替尼）治疗晚期非小细胞肺癌的疗效优于目前文献报道的单药吉非替尼疗效，我们推测并提出扶正抗癌方对TKI药物有增敏作用的假说。为进一步明确该方对TKI药物的增敏作用与机制，本研究拟采用3种对TKI药物敏感度不同的细胞株（PC9, A549, H1650）开展细胞、动物及信号转导通路研究。利用流式细胞仪检测两药联合对肺癌细胞周期改变及细胞凋亡的作用，Transwell小室法检测两药联合对细胞迁移和侵袭的影响，Western Blot检测两药对上述细胞株及经过siRNA处理后EGFR基因沉默或过表达质粒处理后基因过表达的细胞株的EGFR及下游分子ERK及AKT的表达，从而明确扶正抗癌方对吉非替尼是否具有增敏作用，探索扶正抗癌方对EGFR及下游分子ERK及AKT信号转导通路的影响，揭示中医药对TKI增敏的分子机制。

前期临床研究显示，与吉非替尼单药相比，扶正抗癌方联合吉非替尼能显著提高患者的中位疾病无进展生存时间和中位生存时间，为进一步在分子层面探索其作用机制，本课题以A549、PC9、H1650为研究对象展开研究。MTS实验发现，扶正抗癌方能显著抑制三株细胞株的增殖，且协同吉非替尼的抑制效应优于吉非替尼单药；transwell和划痕试验发现，扶正抗癌方能显著抑制三株细胞株的侵袭和迁移；流式细胞术发现扶正抗癌方能显著促进三株细胞株的凋亡。我们进一步选择蛋白质印迹法、MMP-9活力检测法、QPCR、启动子活力检测、瞬时转染等实验方法研究发现，扶正抗癌方能通过抑制STAT3-MMP9通路，并逆转EMT过程，抑制A549、PC9和H1650的转移；通过激活Caspase通路促进A549、PC9、H1650的凋亡。最重要的是，我们发现扶正抗癌方能够下调A549、PC9、H1650中p-EGFR（Tyr1068）及其下游p-Akt的表达，进一步研究发现，扶正抗癌方在A549、H1650细胞株中能通过抑制Akt-p65-MUC1通路抑制细胞增殖，而扶正抗癌方与吉非替尼的协同效应亦能够通过Akt-p65-MUC1通路来实现。这证明了在吉非替尼原发和继发性耐药细胞株中，扶正抗癌方联合吉非替尼具有协同效应，这与我们的临床实验结果是一致的，即扶正抗癌方可以对耐药的细胞株增敏。而在另一项研究中，我们也发现扶正抗癌方在A549、PC9细胞株中能通过激活AMPKα-IGFBP1-FOXO3α通路抑制细胞增殖，这表明复方的作用机制是多靶点多通路的，而对于不同的细胞株，其作用机制亦不相同。本课题对于挖掘扶正抗癌方的优势人群，在基因层面上指导中医中药的个体化治疗具有重要意义。