自噬抑制剂羟氯喹对铂耐药卵巢癌化疗敏感性的影响及作用机制研究

Platinum drugs are successful antitumor agents currently used as first-line chemotherapeutic agents for the treatment of ovarian cancer. Although the majority of patients have a good response to the initial therapy, few are cured. The acquired resistance to platinum agents is the major cause of treatment failure, which contributes to disease progression or recurrence. In this regard, sensitization of drug-resistant ovarian cancer cells to platinum agents has become an important strategy in overcoming chemoresistance. Our previous work showed that apoptosis induced by cisplatin is the major cell-death mechanism of ovarian cancer cells; Autophagy is a protective mechanism in response to cisplatin-induced apoptosis and is correlative with cisplatin resistance of ovarian cancer cells; Specific inhibition of early autophagy induction with siRNA targeted to Beclin 1 or pharmacological inhibitor 3-methyladenine could enhance cisplatin-induced apoptosis in chemo-resistant ovarian cancer cells. Based on these findings, We are going to carry out the following studies: Using samples of blood and tumor tissue from patients diagnosed as platinum -sensitive or -resistant ovarian cancer according to the 2011 NCCN guidelines, and a panel of cisplatin-sensitive（A2780） and -resistant(A2780/DDP) cell lines, we investigate the relationship between autophagy and the evolution of cisplatin resistance of ovarian cancer, along with the molecular mechanism of action by comparative analysis of the expression levels of autophagy protein Beclin1、HMGB1 and apoptosis protein P53、Survivin、ERCC1、BRCA1. Furthermore, the effect of autophagy inhibitor hydroxychloroquine (HCQ) to the sensitivity of cisplatin in drug-resistant ovarian cancer is observed using in vitro cell culture and animal experiments models respectively, to explore the feasibility of autophagy inhibitor HCQ in combination with cisplatin in the treatment of drug-resistant ovarian cancer, hoping to provide a theoretical basis for the effective treatment of platinum-resistant ovarian cancer.

铂类是广泛用于卵巢癌的一线化疗药物，然而化疗诱导产生的铂耐药导致卵巢癌患者极易出现肿瘤复发。我们前期研究发现顺铂诱导耐药卵巢癌细胞产生反应性自噬增强与其顺铂耐药性密切相关，使用自噬抑制剂3-甲基腺嘌呤或抑制自噬基因Beclin 1 的表达可通过抑制自噬、促进凋亡提高耐药细胞对顺铂的敏感性。然而其中分子机制尚不清楚。基于此，本项目拟对铂敏感和耐药卵巢癌患者的组织标本中自噬蛋白Beclin1、HMGB1、铂耐药相关蛋白P53、Survivin、ERCC1、BRCA1表达情况以及外周血中自噬信号分子HMGB1的浓度进行检测和比较分析，从分子水平研究自噬激活与铂耐药关系；采用体外细胞培养和构建裸鼠移植瘤模型，从细胞和动物整体水平研究自噬抑制剂羟氯喹对耐药卵巢癌顺铂敏感性的影响。本研究对阐明自噬激活与卵巢癌顺铂耐药的关系及信号调节机制，探索羟氯喹联合顺铂治疗铂耐药卵巢癌的可行性提供研究依据。

卵巢癌是目前死亡率最高的妇科恶性肿瘤之一。尽管手术范围不断扩大以及术后以铂类和紫杉醇为一线化疗方案的广泛应用，但仍有高达75%的卵巢癌患者在不到两年的时间复发。对铂类药物产生获得性耐药是肿瘤复发的主要根源，但其发生机制及自噬在其中的调节作用尚不清楚。大量研究证实自噬通过复杂的调节机制影响肿瘤的发生发展及其对化疗药物的敏感性。本课题旨在研究自噬抑制剂---抗疟疾药物羟氯喹（HCQ）联合铂类治疗卵巢癌的可行性。我们的结果显示（1）铂耐药卵巢癌患者肿瘤细胞的基础自噬活性明显高于铂敏感患者，提示自噬激活可能与卵巢癌顺铂耐药性的形成密切相关.（2）体内及体外研究结果显示自噬抑制剂HCQ本身对耐药卵巢癌细胞无明显抑制作用，但与顺铂联用可显著提高耐药细胞对顺铂的敏感性。其作用机制可能是通过抑制顺铂诱发的具有细胞保护作用的自噬效应，提高肿瘤细胞的凋亡易感性，从而逆转对顺铂耐药性。据此，将自噬抑制剂与顺铂联合应用可能为临床有效治疗卵巢癌，尤其是复发、耐药的卵巢癌提供新的思路和方法。（3）本研究成功构建了卵巢癌人源性异体移植瘤（patient derived xenograft，PDX）小鼠模型进行耐药卵巢癌药物筛选实验研究。我们的结果显示卵巢癌PDX模型高度保持了卵巢癌组织的异质性、分化程度、形态特征以及分子特性，与患者肿瘤相似度极高，是一种理想的具有高保真性的临床前研究模型，可作为卵巢癌患者的替身进行相关药物筛选实验研究。本研究发表论文两篇，其中SCI一篇。待发表一篇。培养硕士研究生5人。