a College of Bioengineering , Henan University of Technology , Zhengzhou , China.
b College of Chemistry and Chemical Engineering , Henan University of Technology , Zhengzhou , China.
Autophagy. 2019 Mar;15(3):391-406. doi: 10.1080/15548627.2018.1511503. Epub 2018 Sep 6.
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，人们特别关注自噬在各种人类疾病（包括癌症）中的作用。发现自噬的小分子调节剂及其作为抗癌治疗剂的潜在用途将具有重要意义。为此，我们对以前在实验室中合成的一系列姜黄素类似物进行了筛选。在这些化合物中，（3E，5E）-3-（3，4-二甲氧基苄叉基）-5-[[1H-吲哚-3-基]亚甲基]-1-甲基哌啶-4-酮（CA-5f）被鉴定为一种有效的晚期巨自噬/自噬抑制剂，通过抑制自噬体-溶酶体融合。我们发现 CA-5f 既不损害溶酶体的水解功能，也不损害其数量。使用等压标签相对和绝对定量（iTRAQ）基于蛋白质组学筛选结合生物信息学分析表明，用 CA-5f 处理人脐静脉内皮细胞（HUVECs）1 小时会抑制细胞骨架蛋白和膜运输蛋白的水平。随后的研究表明，CA-5f 通过增加线粒体来源的活性氧（ROS）的产生，对 A549 非小细胞肺癌（NSCLC）细胞表现出很强的细胞毒性，但对正常的人脐静脉内皮细胞（HUVECs）的细胞毒性较低。此外，CA-5f 作为单一药物有效地抑制 A549 肺癌异种移植物的生长，具有良好的体内耐受性。Western blot、免疫荧光和末端转移酶介导的 dUTP 缺口末端标记（TUNEL）检测结果表明，CA-5f 抑制自噬流，诱导细胞凋亡，并且不影响 CTSB（组织蛋白酶 B）和 CTSD（组织蛋白酶 D）的水平体内，这与体外数据一致。总的来说，这些结果表明 CA-5f 是一种新型的晚期自噬抑制剂，具有治疗 NSCLC 的潜在临床应用。缩写：3-MA，3-甲基腺嘌呤；ANXA5，膜联蛋白 A5；ATG，自噬相关；CA-5f，（3E，5E）-3-（3，4-二甲氧基苄叉基）-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 的平衡盐溶液；GFP，绿色荧光蛋白；HCQ，羟基氯喹；HEK293，人胚肾 293；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-串联质谱法；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，自噬体 1；TUNEL，末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记。
