自噬抑制剂增强顺铂抗耐药性食管癌的作用及其机制研究

Esophageal cancer is one of the leading causes of cancer-related death in China. Autophagy is a cellular self-catabolic process where cytoplasmic components are engulfed and trafficked to lysosomes for degradation. In cancer therapy, the role of autophagy is paradoxical, in which this cellular process may serve as a pro-survival or pro-death mechanism to counteract or mediate the cytotoxic action of anticancer agents. Notably, the role of autophagy in mediating cellular response to the cytotoxicity of cisplatin in esophageal cancer cells, especially in cisplatin-resistant cancer cells, remains elusive. We recently found that cisplatin induced autophagy in cisplatin-resistant esophageal cancer cells. Moreover, pharmacological inhibitors of autophagy, including chloroquine and bafilomycin A1, enhanced the cytotoxicity of cisplatin. These findings suggest an autophagic response to cisplatin is a survival mechanism that promotes chemo-resistance and recovery and that inhibition of autophagy has the potential to enhance the cytotoxic effect of cisplatin. In relation to signaling pathways involved, we found that cisplatin provoked the activation of ERK in resistant cancer cells. To investigate the influence of this kinase activation on survival of cisplatin-treated cells, we utilized the pharmacologic inhibitor U0126, which acts as a specific inhibitor of ERK kinase. The results showed that treatment of resistant cancer cells with U0126 during exposure to cisplatin antagonized the cytotoxicity of cisplatin. These findings indicate that ERK plays a role in regulating cisplatin-induced cytotoxicity. Whether ERK activation contributes to the autophagic response in resistant cancer cells is unclear. Moreover, the role of cisplatin in the regulation of mTOR and Beclin 1 pathways remains elusive. In this grant application, we propose that further experiments could be done to elucidate the role of autophagic response in mediating cellular resistance to cisplatin-induced cytotoxicity and the mechanisms involved. In addition, we propose that the antitumor activity of cisplatin in combination with autophagy inhibitors should be further investigated in vivo. Findings of this study will help us better understand the role of autophagic response in mediating chemo-resistance and facilitate the identification of novel approaches for overcoming drug resistance in esophageal cancer cells.

食管癌在我国是致死率最高的肿瘤之一。自噬是真核细胞内降解长寿命蛋白和细胞器的一种溶酶体途径。在肿瘤的治疗上，自噬的作用具有双重性。在食管癌细胞，特别是顺铂耐药的食管癌细胞，对顺铂细胞毒作用的反应上，自噬的作用仍未阐明。我们发现顺铂可诱导食管癌耐药细胞发生自噬，并且自噬抑制剂氯喹和bafilomycin A1能够增强顺铂的细胞毒作用。这一结果提示耐药细胞很可能通过发生自噬来耐受顺铂的细胞毒作用，而抑制自噬能够增强顺铂的作用。在信号通路方面，我们发现顺铂激活了ERK，而且ERK激酶抑制剂U0126还能拮抗顺铂的细胞毒作用，但ERK的激活是否参与调控自噬并不清楚。此外，顺铂如何调控自噬关键通路mTOR和Beclin 1也并未阐明。本项目拟研究自噬在顺铂耐药中的作用及其机制，并观察自噬抑制剂对顺铂体内抗肿瘤活性的影响。本研究将有助于揭示自噬在顺铂耐药中的作用机制，为临床上克服顺铂耐药提供新的思路。

顺铂是食管癌化疗的一线药物。肿瘤细胞对顺铂耐药是造成治疗失败的主要原因。自噬是真核细胞内降解蛋白质和细胞器的一种溶酶体途径。化疗药物可诱导肿瘤细胞发生自噬，而自噬也影响到肿瘤细胞对化疗药物的敏感性。在人食管癌细胞，自噬是否参与肿瘤细胞对顺铂的耐药并不清楚。本课题研究了顺铂对敏感以及耐药的人食管癌细胞凋亡，衰老和自噬活性的影响，探讨了顺铂诱导耐药细胞发生自噬的分子机制，并进一步研究了抑制细胞自噬后在体外和体内对顺铂抗肿瘤活性的影响。我们的研究发现顺铂明显的引起敏感细胞发生凋亡和衰老，而顺铂的这些作用在耐药细胞不明显。值得注意的是，顺铂引起耐药细胞发生自噬而没有明显改变敏感细胞的自噬活性。敲低自噬相关基因或自噬抑制剂均能显著的增强顺铂的细胞毒作用。体内的裸鼠移植瘤实验进一步显示自噬抑制剂氯喹能够明显的增强顺铂的体内抗肿瘤活性。机制研究显示，顺铂增加了ERK磷酸化，而MEK抑制剂显著的减弱了顺铂诱导凋亡和衰老的作用。而顺铂通过抑制mTORC1活性引起耐药细胞发生自噬。以上发现提示，自噬在耐药细胞作为保护机制介导了细胞对顺铂的耐药，而通过敲低自噬相关基因或小分子自噬抑制剂氯喹能够增强耐药细胞对顺铂的敏感性。基于自噬对耐药细胞的保护作用，我们进一步的研究发现Bcl-2抑制剂obatoclax通过损伤溶酶体降解功能而阻断细胞的自噬活性，在敏感和耐药细胞显示出同等的细胞毒作用。这一试验结果提示，obatoclax有望用于治疗对顺铂耐药的食管癌患者。以上研究显示，自噬作为耐药细胞的保护机制参与对顺铂的抵抗，阻断自噬活性能够增强顺铂在耐药细胞中的抗肿瘤作用。并且，通过损伤细胞溶酶体功能阻断自噬能够有效的在敏感细胞和耐药细胞发挥同等的细胞毒作用。 以上研究为临床上治疗食管癌，特别是对顺铂耐药的食管癌，提供了单药或联合用药的实验依据以及新的治疗思路。