Boitier E, Merad-Boudia M, Guguen-Guillouzo C, Defer N, Ceballos-Picot I, Leroux J P, Marsac C
Institut National de la Santé et de la Recherche Médicale U75, Biochimie Pharmacologique et Métabolique, Faculté de Médecine Necker-Enfants Malades, Université Paris, France.
Cancer Res. 1995 Jul 15;55(14):3028-35.
Alterations in the energy metabolism of cancer cells have been reported for many years. However, the deleterious mechanisms involved in these deficiencies have not yet been clearly proved. The main goal of this study was to decipher the harmful mechanisms responsible for the respiratory chain deficiencies in the course of diethylnitrosamine (DENA)-induced rat hepatocarcinogenesis, where mitochondrial DNA abnormalities had been previously reported. The respiratory activity of freshly isolated hepatoma mitochondria, assessed by oxygen consumption experiments and enzymatic assays, presented a severe complex I deficiency 19 months after DENA treatment, and later on, in addition, a defective complex III activity. Since respiratory complex subunits are encoded by both nuclear and mitochondrial genes, we checked whether the respiratory chain defects were due to impaired synthesis processes. The specific immunodetection of complex I failed to show any alterations in the steady-state levels of both nuclear and mitochondrial encoded subunits in the hepatomas. Moreover, in vitro protein synthesis experiments carried out on freshly isolated hepatoma mitochondria did not bring to light any modifications in the synthesis of the mitochondrial subunits of the respiratory complexes, whatever the degree of tumor progression. Finally, Southern blot analysis of mitochondrial DNA did not show any major mitochondrial DNA rearrangements in DENA-induced hepatomas. Because the synthetic processes of respiratory complexes did not seem to be implicated in the respiratory chain impairment, these deficiencies could be partly ascribed to a direct toxic impact of highly reactive molecules on these complexes, thus impairing their function. The mitochondrial respiratory chain is an important generator of noxious, reactive oxygen free radicals such as superoxide and H2O2, which are normally catabolized by powerful antioxidant scavengers. Nineteen months after DENA treatment, a general collapse of the antioxidant enzymatic system was demonstrated in the hepatomas, as recurrently observed in cancer cells. This oxidant versus antioxidant imbalance was characterized by the establishment of oxidative stress in the course of hepatocarcinogenesis, as partly shown by the important decrease of glutamine synthetase activity, an enzyme whose function is highly sensitive to oxidant reactions. This disequilibrium would result in a net increase of the steady-state concentration of superoxide generated between respiratory complexes I and III in the mitochondria. Once generated, superoxide would likely inactivate complexes I and III via oxidant reactions on their superoxide-sensitive [4Fe, 4S] clusters. The role of mitochondrial respiratory chain impairment in chemical carcinogenesis and/or the persistence of the cancerous state is further discussed.
多年来，已有关于癌细胞能量代谢改变的报道。然而，这些缺陷所涉及的有害机制尚未得到明确证实。本研究的主要目的是破解在二乙基亚硝胺（DENA）诱导的大鼠肝癌发生过程中导致呼吸链缺陷的有害机制，此前已报道该过程中存在线粒体DNA异常。通过耗氧实验和酶分析评估新鲜分离的肝癌线粒体的呼吸活性，结果显示在DENA处理19个月后，线粒体呈现严重的复合体I缺陷，随后还出现了复合体III活性缺陷。由于呼吸复合体亚基由核基因和线粒体基因共同编码，我们检查了呼吸链缺陷是否是由于合成过程受损所致。对复合体I进行特异性免疫检测，结果未能显示肝癌中核编码和线粒体编码亚基的稳态水平有任何改变。此外，对新鲜分离的肝癌线粒体进行的体外蛋白质合成实验表明，无论肿瘤进展程度如何，呼吸复合体线粒体亚基的合成均未出现任何变化。最后，对线粒体DNA进行的Southern印迹分析未显示DENA诱导的肝癌中有任何主要的线粒体DNA重排。由于呼吸复合体的合成过程似乎与呼吸链损伤无关，这些缺陷可能部分归因于高反应性分子对这些复合体的直接毒性作用，从而损害了它们的功能。线粒体呼吸链是有害的活性氧自由基（如超氧化物和H2O2）的重要产生源，这些自由基通常由强大的抗氧化清除剂进行分解代谢。在DENA处理19个月后，肝癌中出现了抗氧化酶系统的普遍崩溃，这在癌细胞中经常被观察到。这种氧化剂与抗氧化剂的失衡表现为肝癌发生过程中氧化应激的建立，谷氨酰胺合成酶活性的显著降低部分显示了这一点，该酶的功能对氧化反应高度敏感。这种失衡将导致线粒体中呼吸复合体I和III之间产生的超氧化物稳态浓度净增加。一旦产生，超氧化物可能会通过对其超氧化物敏感的[4Fe, 4S]簇进行氧化反应而使复合体I和III失活。本文进一步讨论了线粒体呼吸链损伤在化学致癌和/或癌状态持续中的作用。
