Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels.
Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels.
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DOI：
10.1016/j.redox.2016.01.002
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发表时间：
2016-08
期刊：
影响因子：
11.4
通讯作者：
Landar A
中科院分区：
文献类型：
--
作者：
Smith MR;Vayalil PK;Zhou F;Benavides GA;Beggs RR;Golzarian H;Nijampatnam B;Oliver PG;Smith RA;Murphy MP;Velu SE;Landar A
许多癌细胞遵循一种异常的代谢程序来维持细胞快速增殖所需的能量。代谢重编程通常涉及谷氨酰胺水解的上调，以产生电子传递链的还原等价物和蛋白质合成的氨基酸。参与代谢的关键酶具有活性硫酸酯基团，它可以被某些氧化剂修饰。在目前的研究中，我们发现一种模型化合物碘丁基三苯膦（IBTP）修饰线粒体蛋白硫醇，在初始24小时治疗后6天内，MDA-MB 231 （MB231）乳腺腺癌细胞的线粒体代谢和ATP降低。与非硫醇修饰类似物相比，线粒体硫醇修饰也以剂量依赖的方式降低了氧气消耗率（OCR），这表明硫醇反应性是抑制癌细胞代谢的重要因素。在非致瘤性MCF-10A细胞中，IBTP也降低了OCR；然而，除了最高浓度（10µM）的IBTP外，细胞外酸化率均显著增加，这表明巯基修饰对致瘤细胞和非致瘤细胞的生物能量学有显著不同的影响。巯基修饰后，MB231细胞的ATP和其他腺苷水平也下降至治疗后6天，表明MB231细胞的整体能量状态下降。治疗后6天，MB231细胞的增殖也受到抑制，但细胞活力变化不大。目标代谢组学分析显示，巯基修饰导致克雷布斯循环和谷氨酰胺解中间产物的消耗。进一步的实验表明，克雷布斯循环酶（乌头酸酶）的活性随着硫醇的修饰而减弱。此外，抑制谷氨酰胺水解与谷氨酰胺酶C （GAC）蛋白水平降低相对应，尽管其他蛋白水平不受影响。这项研究首次证明线粒体硫醇修饰通过抑制乳腺癌细胞模型中的乌头酸酶和GAC来抑制代谢。IBTP依赖性巯基修饰降低了MB231和MCF-10A细胞的生物能量学。IBTP治疗后1 ~ 6天会降低ATP和其他腺核苷酸。IBTP治疗不会导致明显的细胞毒性。IBTP治疗降低了生物能量相关代谢物的水平。IBTP处理降低了乌头酸酶活性和谷氨酰胺酶蛋白水平。
Many cancer cells follow an aberrant metabolic program to maintain energy for rapid cell proliferation. Metabolic reprogramming often involves the upregulation of glutaminolysis to generate reducing equivalents for the electron transport chain and amino acids for protein synthesis. Critical enzymes involved in metabolism possess a reactive thiolate group, which can be modified by certain oxidants. In the current study, we show that modification of mitochondrial protein thiols by a model compound, iodobutyl triphenylphosphonium (IBTP), decreased mitochondrial metabolism and ATP in MDA-MB 231 (MB231) breast adenocarcinoma cells up to 6 days after an initial 24 h treatment. Mitochondrial thiol modification also depressed oxygen consumption rates (OCR) in a dose-dependent manner to a greater extent than a non-thiol modifying analog, suggesting that thiol reactivity is an important factor in the inhibition of cancer cell metabolism. In non-tumorigenic MCF-10A cells, IBTP also decreased OCR; however the extracellular acidification rate was significantly increased at all but the highest concentration (10 µM) of IBTP indicating that thiol modification can have significantly different effects on bioenergetics in tumorigenic versus non-tumorigenic cells. ATP and other adenonucleotide levels were also decreased by thiol modification up to 6 days post-treatment, indicating a decreased overall energetic state in MB231 cells. Cellular proliferation of MB231 cells was also inhibited up to 6 days post-treatment with little change to cell viability. Targeted metabolomic analyses revealed that thiol modification caused depletion of both Krebs cycle and glutaminolysis intermediates. Further experiments revealed that the activity of the Krebs cycle enzyme, aconitase, was attenuated in response to thiol modification. Additionally, the inhibition of glutaminolysis corresponded to decreased glutaminase C (GAC) protein levels, although other protein levels were unaffected. This study demonstrates for the first time that mitochondrial thiol modification inhibits metabolism via inhibition of both aconitase and GAC in a breast cancer cell model. IBTP dependent thiol modification decreases bioenergetics in MB231 and MCF-10A cells. IBTP treatment decreases ATP and other adenonucleotides after 1 to 6 days. IBTP treatment does not result in overt cellular toxicity. IBTP treatment decreases levels of bioenergetically-linked metabolites. IBTP treatment decreases aconitase activity and glutaminase protein levels.