和厚朴酚通过重启自噬性死亡抵抗吉非替尼耐药的作用机制研究及相关靶点确证

Gefitinib-resistance caused by EGFR T790M mutation in non-small cell lung cancer（NSCLC） treatment has become a global challenge. It is of great importance to develop drugs which showed anti-cancer activity on gefitinib-resistance NSCLC cells. Our previous in vitro study found that honokiol, a main component of traditional Chinese medicine “Mangnolia officinalis”, could induce autophagic cell death in NSCLC cells. The in vivo study revealed that honokiol showed excellent anti-cancer activity on gefitinib-resistant cell（H1975）mouse model, with 90.6% tumor growth inhibition at 20 mg/kg. In this project we intend to employ western、siRNA、photoaffinity agarose beads tag pull down and structural biology methods to further study: (1) The mechanism of how honokiol induces autophagic cell death in gefitinib resistant NSCLC cells (2) How classical autophagy-related pathways such as EGFR-dependent pathway: EGFR/Beclin1, EGFR/PI3K/Akt/mTOR, EGFR/Ras/PI3K/Akt/mTOR, Akt-Beclin1 pathway and other EGFR-independent pathways: AMPK/mTOR, HIF-1α/Beclin1 mediate honokiol-induced autophagy. (3) Cellular targets of honokiol associated with autophagy (4) Further confirmation of the anticancer aciticvity of honokiol on NSCLC cells by restarting autophagic cell death based on the identified cellular targets.

EGFR T790M突变引起吉非替尼耐药已成为非小细胞肺癌（NSCLC）治疗全球性难题，寻找在吉非替尼耐药NSCLC细胞上有效的药物具有重要意义。我们前期体外研究首次发现中药厚朴活性成分和厚朴酚能诱导吉非替尼耐药NSCLC细胞产生自噬性死亡，体内实体瘤模型证实和厚朴酚对吉非替尼耐药肿瘤细胞（H1975）显示良好抗肿瘤活性,20 mg/kg抑瘤率达90.6%。为研究其作用机制和靶点,本项目拟用western、siRNA、光亲和磁珠标记pull down及结构生物学等手段深入研究（1）和厚朴酚诱导吉非替尼耐药NSCLC细胞产生自噬性死亡具体作用机制（2）EGFR相关的自噬经典通路及与EGFR无关的其它通路是否参与调控和厚朴酚诱导的自噬（3）确定和厚朴酚与自噬相关的细胞靶蛋白（4）基于鉴定的靶点验证在吉非替尼耐药细胞和动物模型上和厚朴酚通过重启自噬性死亡而达到抗肿瘤效果。

EGFR T790M突变引起的吉非替尼耐药已成为非小细胞肺癌（NSCLC）治疗全球性难题，寻找能克服吉非替尼耐药的药物对NSCLC治疗具有重要意义。和厚朴酚脂质体目前已在准备进行II其临床抗肿瘤实验。本课题主要完成了以下科学问题:(1)确定了和厚朴酚脂质体在NSCLC上的抗肿瘤效果及毒性检测。(2)完成了和厚朴酚对EGFR-RAS-MEK/ERK信号通路的影响。(3)确定了和厚朴酚的靶点为GRP78。(4)发现和厚朴酚在肿瘤细胞上能诱导产生保护性的自噬。(5)发现和厚朴酚联用自噬抑制剂能够具有更好的抗肿瘤效果。我们的研究结果对于后期指导和厚朴酚脂质体的临床实验具有重要意义。