养阴解毒方调控EGFR/PI3K/AKT信号通路克服T790M获得性耐药的作用机制

With the rate of incidence and mortality of non-small-cell lung cancer increasing annually, epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are effective in advanced lung adenocarcinoma patients with EGFR mutations, but the acquired resistance to EGFR-TKIs is inevitable, T790M mutation is the main mechanism. Although the 2nd or 3rd generation of EGFR-TKIs are active to some extent, each has its limitations, meanwhile it is meanless for the 1st line therapy with EGFR-TKIs. T790M mutation can reactivate the EGFR/PI3K/AKT signal pathway to promote the proliferation and metastasis of tumors. Therefore, it is important to regulate the EGFR/PI3K/AKT signal pathway to overcome the resistance induced by T790M mutation and prolong the acivities of the 1st generation of EGFR-TKIs. Traditional Chinese Medicine believes that yin deficiency with internal heat is the main pathogenesis with EGFR-TKIs therapy, so the principle of therapy is nourishing yin to moisten lung and clearing heat to remove toxin. The results of our former study exerted Yang yin jie du decoction could download the expressions of p-EGFR, PI3K, p-AKT in H1975 resistant cell line and finally suppressed its growth. Small sample clinical observation also showed that the PFS of treatment group was prolonged. Based on the results above, we believe that Yangyin Jiedu Decoction can regulate the EGFR/PI3K/AKT signal pathway to overcome the acquired resistance to EGFR-TKIs induced by T790M mutation. Our study intends to culture cells, build animal models and use CCK-8, flow cytometry, siRNA, qPCR, Western blot assay to further explore the mechanism of Yangyin Jiedu decoction regulating the EGFR/PI3K/AKT signal pathway to overcome T790M acquired resistance in vitro and vivo. These results would be helpful for further clinical practice.

非小细胞肺癌发病与死亡率逐年升高，虽EGFR-TKIs对晚期肺腺癌EGFR突变者疗效肯定，但获得性耐药不可避免，主要机制为T790M突变。二、三代EGFR-TKIs对耐药有一定疗效，但各有局限，一线用药现意义不大。T790M突变可重新激活EGFR/PI3K/AKT通路促肿瘤增殖转移，因此，调控此通路克服T790M耐药，延长一代疗效十分重要。中医认为服用EGFR-TKIs后，患者多为阴虚内热证，故本课题以养阴润肺、清热解毒为则。前期研究表明，养阴解毒方下调H1975耐药株p-EGFR、PI3K、p-AKT水平，抑制肿瘤生长；小样本临床观察也见治疗组PFS延长。由此我们认为：该方可调控此信号通路克服T790M耐药。本研究从体内外途径，采用CCK-8、流式细胞术、siRNA、qPCR、蛋白印迹等方法，研究养阴解毒方调控EGFR/PI3K/AKT通路克服T790M耐药的机制，为临床提供依据。

表皮生长因子受体酪氨酸激酶抑制剂（epidermal growth factor receptor tyrosine kinase inhibitors, EGFR-TKIs）已成为治疗晚期非小细胞肺癌EGFR突变患者的主要药物之一。但是耐药不可避免，主要原因是T790M突变。中药与一代EGFR-TKIs联合治疗是克服这一局限的策略。本研究通过体外和体内实验，探讨养阴解毒汤是否能克服T790M耐药及其机制。首先，我们运用高效液相色谱法（High Performance Liquid Chromatography, HPLC）技术对养阴解毒方进行了质控，发现5种主要有效成分：橙皮苷、去乙酰基车叶草苷酸甲酯、薯蓣皂苷、金松双黄酮、党参炔苷。体外研究中，因H1975细胞含有EGFR 21外显子L858R及20外显子T790M突变，故为研究对象，结果表明： 养阴解毒方能显著增强吉非替尼对H1975细胞的抗增殖作用，且二者呈中度协同作用。与单用吉非替尼相比，联合用药后细胞凋亡率升高，能够促进PARP、Caspase-3的剪切活化。联合用药可明显降低p-PI3K/PI3K和p-AKT/AKT的相对水平。与LY294002共培养后，细胞活力和p-AKT/AKT比值进一步降低，细胞凋亡明显增加。体内实验显示，联合用药组移植瘤组织体积和重量明显缩小，瘤体组织内TUNEL阳性率显著升高，Ki-67水平升高，PI3K/AKT信号通路相关蛋白表达降低。综上所述，养阴解毒方可以通过下调PI3K/AKT信号通路来克服T790M耐药，为临床应用提供了一种新的策略。