海绵生物碱AP-7增敏化疗药物抗肺腺癌干细胞作用及Chk1依赖机制

Checkpoint kinase 1 (Chk1) mediated DNA damage repair responses is essential for drug resistance in lung cancer stem cells. The cytotoxic activities of 20 sponge-derived alkaloids were evaluated against lung adenocarcinoma stem cells with ectopic expression of Oct4 and Nanog, revealing that AP-7 in combination with cisplatin dramatically reduced stem cells survival in vitro(IC50<5μM). AP-7 has shown high affinity to Chk1 protein by surface plasmon resonance (SPR) technology (Kd value 0.31μM), therefore inhibiting the expression of cyclin B and cisplatin induced upregulation of Cdc2 phosphorylation. These results suggest that AP-7 enhance the sensitivity of chemotherapy drugs to the human lung adenocarcinoma stem cells by targeting Chk1..The activity of AP-7 in combination with cisplatin, paclitaxel and gemcitabine against lung adenocarcinoma stem cells is proposed to detected by flow cytometry sorting CD44+ALDH+ lung adenocarcinoma stem cells. This study aims to investigate the effects of AP-7 on chemotherapy induced DNA damage, cell cycle arrest and apoptosis using high-content cell analysis，RNA interference technology and xenograft implantation for understanding the Chk1-dependent mechanisms of AP-7 sensitizing lung adenocarcinoma stem cells to chemotherapy drugs. Eventually we provide a theoretical basis for obtaining new Chk1 inhibitors through exploration of its cell signal pathway.

细胞周期检查点激酶1(Chk1)介导的DNA损伤修复与肺癌干细胞的耐药性密切相关。申请者利用成球实验富集和构建Oct4+Nanog+的两种肺腺癌干细胞，从20种海绵生物碱中发现AP-7能显著提高顺铂对肺腺癌干细胞的抑制活性(IC50<5μM)；表面等离子共振(SPR)检测显示，AP-7能与Chk1特异性结合(Kd值0.31μM)；AP-7显著抑制CyclinB的表达和顺铂引起的Cdc2磷酸化水平上调。提示AP-7靶向Chk1增加肺腺癌干细胞对化疗药物的敏感性。.本课题拟利用流式细胞技术分选CD44+ALDH+肺腺癌干细胞，检测AP-7联合顺铂、紫杉醇和吉西他滨抑制肺腺癌干细胞的活性。采用高内涵、RNA干扰等技术和体内成瘤实验，明确AP-7增敏化疗药物抗肺腺癌干细胞的Chk1依赖机制，研究其对化疗药物诱导的DNA损伤、细胞周期阻滞和凋亡的作用及信号通路，为获得新型Chk1抑制剂提供理论依据。

肿瘤干细胞(cancer stem cells，CSCs)是存在于肿瘤组织中的一小部分具有干细胞特征的细胞亚群。由于肿瘤干细胞具有DNA损伤修复能力强、高表达多药耐药蛋白、抗凋亡等特性，易对传统的临床治疗方案产生抵抗，导致肿瘤复发和转移。因此靶向肺癌干细胞的药物研究将成为中晚肿瘤治疗领域的重要方向和研究热点。本项目通过慢病毒转染获得稳定表达Oct4-GFP和Nanog-GFP的干细胞样肿瘤干细胞，成功构建了肺癌干细胞和乳腺癌干细胞体外筛选模型。. 海绵共生体漫长的共生互作生存演化必然会导致活性物质的高识别性和高选择性，是新颖结构、新靶标原创药物的重要来源。本项目利用高表达Oct4-GFP和Nanog-GFP的肿瘤干细胞为筛选模型，对海绵来源的100多种化合物进行了体外抗肿瘤干细胞活性筛选，获得一个Chk1抑制剂aaptamine生物碱AP-7，显著增加化疗药物对肺癌干细胞的杀伤作用；发现一个通过激活p38 MAPK/AMPKα信号通路选择性诱导乳腺癌干细胞凋亡的倍半萜类化合物Sme。对于微量活性分子AP-7，采用绿色、经济的药物先导分子全合成新技术，制备样品达到克级，解决了先导化合物的供应问题。. 本项目构建了多种肿瘤干细胞模型，对海绵来源的化合物进行了体外抗肿瘤干细胞活性筛选。从细胞和动物水平阐明高效低毒的活性化合物对肿瘤干细胞自我更新、存活、凋亡、耐药及其介导的信号通路的影响与作用机制，最终获得2个新型的靶向肿瘤干细胞的抗肿瘤先导化合物。通过本课题的实施不但能促进对肿瘤干细胞调控机制的认识，而且为靶向肿瘤干细胞的药物开发提供药源分子。