Identification of compound CA-5f as a novel late-stage autophagy inhibitor with potent anti-tumor effect against non-small cell lung cancer

Currently, particular focus is placed on the implication of autophagy in a variety of human diseases, including cancer. Discovery of small-molecule modulators of autophagy as well as their potential use as anti-cancer therapeutic agents would be of great significance. To this end, a series of curcumin analogs previously synthesized in our laboratory were screened. Among these compounds, (3E,5E)-3-(3,4-dimethoxybenzylidene)-5-[(1H-indol-3-yl)methylene]-1-methylpiperidin-4-one (CA-5f) was identified as a potent late-stage macroautophagy/autophagy inhibitor via inhibiting autophagosome-lysosome fusion. We found that CA-5f neither impaired the hydrolytic function nor the quantity of lysosomes. Use of an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic screen in combination with bioinformatics analysis suggested that treatment of human umbilical vein endothelial cells (HUVECs) with CA-5f for 1 h suppressed the levels of cytoskeletal proteins and membrane traffic proteins. Subsequent studies showed that CA-5f exhibited strong cytotoxicity against A549 non-small cell lung cancer (NSCLC) cells, but low cytotoxicity to normal human umbilical vein endothelial cells (HUVECs), by increasing mitochondrial-derived reactive oxygen species (ROS) production. Moreover, CA-5f effectively suppressed the growth of A549 lung cancer xenograft as a single agent with an excellent tolerance in vivo. Results from western blot, immunofluorescence, and TdT-mediated dUTP nick end labeling (TUNEL) assays showed that CA-5f inhibited autophagic flux, induced apoptosis, and did not affect the level of CTSB (cathepsin B) and CTSD (cathepsin D) in vivo, which were consistent with the in vitro data. Collectively, these results demonstrated that CA-5f is a novel late-stage autophagy inhibitor with potential clinical application for NSCLC therapy. Abbreviations: 3-MA, 3-methyladenine; ANXA5, annexin A5; ATG, autophagy related; CA-5f, (3E,5E)-3-(3,4-dimethoxybenzylidene)-5-[(1H-indol-3-yl)methylene]-1-methylpiperidin-4-one; CQ, chloroquine; CTSB, cathepsin B; CTSD, cathepsin D; DMSO, dimethyl sulfoxide; DNM2, dynamin 2; EBSS, Earle’s balanced salt solution; GFP, green fluorescent protein; HCQ, hydroxyl CQ; HEK293, human embryonic kidney 293; HUVEC, human umbilical vein endothelial cells; LAMP1, lysosomal associated membrane protein 1; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry; LDH, lactic acid dehydrogenase; LMO7, LIM domain 7; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; NAC, N-acetyl cysteine; MYO1E, myosin IE; NSCLC, non-small cell lung cancer; PARP1, poly(ADP-ribose) polymerase 1; PI, propidium iodide; RFP, red fluorescent protein; ROS, reactive oxygen species; SQSTM1, sequestosome 1; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.目前，学界对自噬（autophagy）在包括癌症在内的多种人类疾病中的作用机制给予了高度关注。开发自噬小分子调节剂及其作为抗癌治疗剂的潜在用途具有重要意义。为此，本实验室此前合成的一系列姜黄素类似物被用于筛选。在这些化合物中，(3E,5E)-3-(3,4-二甲氧基苯亚甲基)-5-[(1H-吲哚-3-基)亚甲基]-1-甲基哌啶-4-酮（CA-5f）被鉴定为一种强效的晚期巨自噬/自噬抑制剂，其通过抑制自噬体-溶酶体融合发挥作用。研究发现，CA-5f既不会损害溶酶体的水解功能，也不会影响其数量。基于同量异位素标签相对和绝对定量（isobaric tag for relative and absolute quantitation, iTRAQ）的蛋白质组学筛选结合生物信息学分析显示，用CA-5f处理人脐静脉内皮细胞（human umbilical vein endothelial cells, HUVECs）1小时后，细胞骨架蛋白与膜转运蛋白的水平受到抑制。后续研究表明，CA-5f通过诱导线粒体来源的活性氧（reactive oxygen species, ROS）生成，对A549非小细胞肺癌（non-small cell lung cancer, NSCLC）细胞表现出强烈的细胞毒性，而对正常人脐静脉内皮细胞（HUVECs）的细胞毒性较低。此外，CA-5f作为单一药剂可有效抑制A549肺癌异种移植瘤的生长，且在体内具有优异的耐受性。蛋白质免疫印迹（western blot）、免疫荧光及TdT介导的dUTP缺口末端标记（terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, TUNEL）实验结果显示，CA-5f在体内抑制自噬流、诱导细胞凋亡，且不影响组织蛋白酶B（cathepsin B）与组织蛋白酶D（cathepsin D）的表达水平，这与体外实验结果一致。综上，上述结果表明CA-5f是一种新型晚期自噬抑制剂，在非小细胞肺癌治疗中具有潜在的临床应用价值。缩写说明：3-MA，3-甲基腺嘌呤；ANXA5，膜联蛋白A5；ATG，自噬相关；CA-5f，(3E,5E)-3-(3,4-二甲氧基苯亚甲基)-5-[(1H-吲哚-3-基)亚甲基]-1-甲基哌啶-4-酮；CQ，氯喹；CTSB，组织蛋白酶B；CTSD，组织蛋白酶D；DMSO，二甲基亚砜；DNM2，动力蛋白2；EBSS，Earle平衡盐溶液；GFP，绿色荧光蛋白；HCQ，羟氯喹；HEK293，人胚肾293细胞；HUVEC，人脐静脉内皮细胞；LAMP1，溶酶体相关膜蛋白1；LC-MS/MS，液相色谱-串联质谱；LDH，乳酸脱氢酶；LMO7，LIM结构域7；MAP1LC3B/LC3B，微管相关蛋白1轻链3β；NAC，N-乙酰半胱氨酸；MYO1E，肌球蛋白IE；NSCLC，非小细胞肺癌；PARP1，多聚ADP-核糖聚合酶1；PI，碘化丙啶；RFP，红色荧光蛋白；ROS，活性氧；SQSTM1，自噬底物结合蛋白1；TUNEL，末端脱氧核苷酸转移酶介导的dUTP缺口末端标记。