Putignani Lorenza, Raffa Salvatore, Pescosolido Roberta, Aimati Laura, Signore Fabrizio, Torrisi Maria Rosaria, Grammatico Paola
Medical Genetics, Experimental Medicine, University La Sapienza, S. Camillo-Forlanini Hospital, Circ. ne Gianicolense 87, Rome, Italy.
Breast Cancer Res Treat. 2008 Aug;110(3):439-52. doi: 10.1007/s10549-007-9738-x. Epub 2007 Sep 26.
Mitochondria are dynamic intracellular organelles playing a central role in cell metabolism by generating ATP, through the oxidative phosphorylation system (OXPHOS). Altered mitochondrial functions have been identified as causative or contributing factors in some degenerative diseases and are becoming crucial to understanding cancer mechanisms. We report on distinct expression differences between mitochondria of normal and breast-infiltrating ductal carcinoma (IDC) cells. Mitochondria isolated from HMC (human mammary carcinoma) and HMEC (human mammary epithelial cell) cultures were assayed for expression levels of the multi-protein OXPHOS complexes using Western blot and densitometric analyses. Depressed expression levels were detected for all HMC OXPHOS complexes. Drastic signal reduction was observed for the succinate-dehydrogenase complex II iron-sulphur protein SDH-B (3.38%), while decreasing was reported for the NADH-ubiquinone oxidoreductase complex I Fe-S protein 3 NDUFS3 (32.78%) and the ubiquinol-cytochrome c reductase complex III protein 2 UQCRC2 (50.34%). A significant signal dropping was detected for the ATP-synthase complex V F(1)beta subunit (18.07%). For the cytochrome-oxidase complex IV (CO), near-depletion of the mitochondrial-encoded COI (4.37%) and no apparent variation of the COIV (97.26%) subunits were observed. CO and ATP-synthase were also assayed by cryo-immunoelectron microscopy (CIEM) on unfractionated HMC and HEMC cell mitochondria. COI and F(1)beta differential expression, invariance of COIV levels were corroborated, while HMC mitochondria morphology deterioration was highlighted. MitoTracker Red and fluorescence immunolabelling merging confirmed CIEM data. MitoTracker Red and Green co-staining showed mitochondria membrane property modulation. These data describe bioenergetic and phenotypic alterations of IDC cell mitochondria, possibly providing new cancer hallmarks.
线粒体是动态的细胞内细胞器，通过氧化磷酸化系统（OXPHOS）产生三磷酸腺苷（ATP），在细胞代谢中发挥核心作用。线粒体功能改变已被确定为某些退行性疾病的致病或促成因素，对于理解癌症机制也变得至关重要。我们报告了正常细胞和乳腺浸润性导管癌（IDC）细胞线粒体之间明显的表达差异。使用蛋白质印迹法和光密度分析，对从人乳腺癌（HMC）和人乳腺上皮细胞（HMEC）培养物中分离出的线粒体进行多蛋白氧化磷酸化复合物表达水平的检测。所有HMC氧化磷酸化复合物的表达水平均被检测到降低。琥珀酸脱氢酶复合物II铁硫蛋白SDH - B的信号大幅降低（3.38%），而烟酰胺腺嘌呤二核苷酸（NADH）-泛醌氧化还原酶复合物I铁硫蛋白3 NDUFS3降低（32.78%），泛醇 - 细胞色素c还原酶复合物III蛋白2 UQCRC2降低（50.34%）。ATP合酶复合物V F(1)β亚基检测到明显的信号下降（18.07%）。对于细胞色素氧化酶复合物IV（CO），观察到线粒体编码的COI亚基几乎耗尽（4.37%），而COIV亚基无明显变化（97.26%）。还通过冷冻免疫电子显微镜（CIEM）对未分级的HMC和HEMC细胞线粒体进行CO和ATP合酶检测。证实了COI和F(1)β的差异表达以及COIV水平不变，同时突出了HMC线粒体形态的恶化。线粒体红色荧光探针（MitoTracker Red）和荧光免疫标记合并证实了CIEM数据。MitoTracker Red和绿色荧光共染色显示线粒体膜特性的调节。这些数据描述了IDC细胞线粒体的生物能量和表型改变，可能提供新的癌症特征。
