a Experimental Neurosurgery , Goethe University Hospital Frankfurt/Main , Germany.
b Experimental Cancer Research in Pediatrics , Goethe University Hospital Frankfurt/Main , Germany.
Autophagy. 2018;14(10):1693-1709. doi: 10.1080/15548627.2018.1476812. Epub 2018 Jul 21.
In most cases, macroautophagy/autophagy serves to alleviate cellular stress and acts in a pro-survival manner. However, the effects of autophagy are highly contextual, and autophagic cell death (ACD) is emerging as an alternative paradigm of (stress- and drug-induced) cell demise. AT 101 ([-]-gossypol), a natural compound from cotton seeds, induces ACD in glioma cells as confirmed here by CRISPR/Cas9 knockout of ATG5 that partially, but significantly rescued cell survival following AT 101 treatment. Global proteomic analysis of AT 101-treated U87MG and U343 glioma cells revealed a robust decrease in mitochondrial protein clusters, whereas HMOX1 (heme oxygenase 1) was strongly upregulated. AT 101 rapidly triggered mitochondrial membrane depolarization, engulfment of mitochondria within autophagosomes and a significant reduction of mitochondrial mass and proteins that did not depend on the presence of BAX and BAK1. Conversely, AT 101-induced reduction of mitochondrial mass could be reversed by inhibiting autophagy with wortmannin, bafilomycin A and chloroquine. Silencing of HMOX1 and the mitophagy receptors BNIP3 (BCL2 interacting protein 3) and BNIP3L (BCL2 interacting protein 3 like) significantly attenuated AT 101-dependent mitophagy and cell death. Collectively, these data suggest that early mitochondrial dysfunction and HMOX1 overactivation synergize to trigger lethal mitophagy, which contributes to the cell killing effects of AT 101 in glioma cells.
ACD, autophagic cell death; ACN, acetonitrile; AT 101, (-)-gossypol; BAF, bafilomycin A; BAK1, BCL2-antagonist/killer 1; BAX, BCL2-associated X protein; BH3, BCL2 homology region 3; BNIP3, BCL2 interacting protein 3; BNIP3L, BCL2 interacting protein 3 like; BP, Biological Process; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; CC, Cellular Component; Con, control; CQ, chloroquine; CRISPR, clustered regularly interspaced short palindromic repeats; DMEM, Dulbecco's Modified Eagle Medium; DTT, 1,4-dithiothreitol; EM, electron microscopy; ER, endoplasmatic reticulum; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; GO, Gene Ontology; HAcO, acetic acid; HMOX1, heme oxygenase 1; DKO, double knockout; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry; LPL, lipoprotein lipase, MEFs, mouse embryonic fibroblasts; mPTP, mitochondrial permeability transition pore; MTG, MitoTracker Green FM; mt-mKeima, mito-mKeima; MT-ND1, mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1; PBS, phosphate-buffered saline; PE, phosphatidylethanolamine; PI, propidium iodide; PRKN, parkin RBR E3 ubiquitin protein ligase; SDS, sodium dodecyl sulfate; SQSTM1/p62, sequestome 1; STS, staurosporine; sgRNA, single guide RNA; SILAC, stable isotope labeling with amino acids in cell culture; TFA, trifluoroacetic acid, TMRM, tetramethylrhodamine methyl ester perchlorate; WM, wortmannin; WT, wild-type.
在大多数情况下，巨自噬/自噬有助于缓解细胞应激，并以促进生存的方式发挥作用。然而，自噬的影响具有高度的背景性，自噬细胞死亡（ACD）正在成为（应激和药物诱导的）细胞死亡的另一种范式。AT 101（（-）-棉酚），一种来自棉花种子的天然化合物，在这里通过 CRISPR/Cas9 敲除 ATG5 证实了它在神经胶质瘤细胞中诱导 ACD，这部分但显著挽救了 AT 101 处理后的细胞存活。对 AT 101 处理的 U87MG 和 U343 神经胶质瘤细胞的全蛋白质组学分析显示，线粒体蛋白簇明显减少，而 HMOX1（血红素加氧酶 1）强烈上调。AT 101 迅速引发线粒体膜去极化，线粒体被自噬体吞噬，线粒体质量和蛋白质显著减少，这与 BAX 和 BAK1 的存在无关。相反，AT 101 诱导的线粒体质量减少可以通过用渥曼青霉素、巴弗洛霉素 A 和氯喹抑制自噬来逆转。沉默 HMOX1 和线粒体自噬受体 BNIP3（BCL2 相互作用蛋白 3）和 BNIP3L（BCL2 相互作用蛋白 3 样）显著减弱了 AT 101 依赖性线粒体自噬和细胞死亡。总的来说，这些数据表明，早期线粒体功能障碍和 HMOX1 过度激活协同触发致命的线粒体自噬，这有助于 AT 101 在神经胶质瘤细胞中的细胞杀伤作用。
