Antiproliferative and apoptotic effects in rat and human hepatoma cell cultures of the orally active iron chelator ICL670 compared to CP20: a possible relationship with polyamine metabolism.

OBJECTIVE: Iron loading has been observed to have a hyperproliferative effect on hepatocytes in vitro and on tumour cells in vivo; removal of this iron being required to induce antitumour activity. MATERIAL AND METHODS: Antiproliferative effects of orally active tridentate iron chelator ICL670 (deferasirox) and bidentate iron chelator CP20 (deferiprone), mediated through the chelation of intracellular iron, were compared in rat hepatoma cell line FAO and human hepatoma cell line HUH7. RESULTS: In FAO cell cultures, we have shown that ICL670 decreased cell viability and DNA replication and induced apoptosis more efficiently than an iron-binding equivalent concentration of CP20. Moreover, ICL670 decreased significantly the number of the cells in G(2)-M phase. In the HUH7 cell cultures, ICL670 and a four-time higher iron-binding equivalent concentration of CP20, decreased cell viability and DNA replication in the same range. CP20 increased the number of the cells in G(2)-M phase. However, ICL670 inhibited polyamine biosynthesis by decreasing ornithine decarboxylase mRNA level; in contrast, CP20 increased polyamine biosynthesis, particularly putrescine level, by stimulating spermidine-spermine N(1)-acetyl transferase activity that could activate the polyamine retro-conversion pathway. By mass spectrometry, we observed that ICL670 cellular uptake was six times higher than CP20. CONCLUSIONS: These results suggest that ICL670 has a powerful antitumoural effect and blocks cell proliferation in neoplastic cells by a pathway different from that of CP20 and may constitute a potential adjuvant drug for anticancer therapy.

Biochemical, genetic and immunoblot analyses of 17 patients with an isolated cytochrome c oxidase deficiency.

Mitochondrial respiratory chain defects involving cytochrome c oxidase (COX) are found in a clinically heterogeneous group of diseases, yet the molecular basis of these disorders have been determined in only a limited number of cases. Here, we report the clinical, biochemical and molecular findings in 17 patients who all had isolated COX deficiency and expressed the defect in cultured skin fibroblasts. Immunoblot analysis of mitochondrial fractions with nine subunit specific monoclonal antibodies revealed that in most patients, including in a patient with a novel mutation in the SURF1 gene, steady-state levels of all investigated COX subunits were decreased. Distinct subunit expression patterns were found, however, in different patients. The severity of the enzymatic defect matched the decrease in immunoreactive material in these patients, suggesting that the remnant enzyme activity reflects the amount of remaining holo-enzyme. Four patients presented with a clear defect of COX activity but had near normal levels of COX subunits. An increased affinity for cytochrome c was observed in one of these patients. Our findings indicate a genetic heterogeneity of COX deficiencies and are suggestive of a prominent involvement of nuclear genes acting on the assembly and maintenance of cytochrome c oxidase.

Clinical relevance of genetic instability in prostatic cells obtained by prostatic massage in early prostate cancer.

We investigated whether genetic lesions such as loss of heterozygosity (LOH) are detected in prostatic cells obtained by prostatic massage during early diagnosis of prostate cancer (CaP) and discussed their clinical relevance. Blood and first urine voided after prostatic massage were collected in 99 patients with total prostate-specific antigen (PSA) between 4 and 10 ng ml(-1), prior to prostate biopsies. Presence of prostatic cells was confirmed by quantitative RT-PCR analysis of PSA mRNA. Genomic DNA was analysed for LOH on six chromosomal regions. One or more allelic deletions were found in prostatic fluid from 57 patients analysed, of whom 33 (58%) had CaP. Sensitivity and specificity of LOH detection and PSA free to total ratio <15% for positive biopsy were respectively 86.7 and 44% (P=0.002) for LOH, and 55 and 74% (P=0.006) for PSA ratio <15%. Analysis of LOH obtained from prostatic tumours revealed similar patterns compared to prostatic fluid cells in 86% of cases, confirming its accuracy. The presence of LOH of urinary prostatic cells obtained after prostatic massage is significantly associated with CaP on biopsy and may potentially help to identify a set of patients who are candidates for further prostate biopsies.

Expression study of genes involved in iron metabolism in human tissues.

Iron is required in all organisms for crucial functions, as a number of proteins need iron for activity. Mutations of the genes encoding proteins involved in iron uptake, transport, and utilization result in various human disorders or animal models with very different clinical presentations and organ involvement. However, little is known concerning the expression of iron metabolism genes in various human tissues and their eventual concerted regulation. We therefore examined the expression levels of various genes involved in iron uptake, reduction, and storage, in Fe-S protein biogenesis, in mitochondrial electron transport chain, plus the two SOD genes, in human adult tissues by Northern blot analysis. We observed that most of these genes were ubiquitously expressed, but that their transcript showed strongly different levels in the various tissues investigated denoting different mechanisms for iron utilization in various organs. However, surprisingly, no correlation could be made between expression pattern of these genes and the clinical presentation resulting in their mutations.

Characterization of the murine gene for subunit VIIaL of cytochrome c oxidase.

Mammalian cytochrome c oxidase consists of thirteen subunits, ten encoded by the nuclear genome and three by the mitochondrial DNA. In several species, two isoforms have been isolated for nuclear-encoded subunits VIa, VIIa and VIII: an ubiquitous L (liver) form and a heart- and skeletal-muscle specific H form. The gene for murine cytochrome c oxidase subunit VIIa-L (Cox7aL) and its promoter region were isolated, sequenced and analysed. The coding region is split in four exons spanning 4.1 kbp and the promoter carries potential binding sites for Sp1, NRF1 and NRF2 transcription factors. Transcriptional activity of the promoter in reporter assays suggested an ubiquitous expression in mouse tissues.

Disabled early recruitment of antioxidant defenses in Friedreich's ataxia.

Friedreich's ataxia (FRDA) results from a generalized deficiency of mitochondrial iron-sulfur protein activity ascribed to mitochondrial iron overload. However, iron overload appears to be a late event in the disease. Here we show that neither superoxide dismutases nor the import iron machinery was induced by an endogenous oxidative stress in FRDA patients' fibroblasts in contrast to control cells. Superoxide dismutase activity was not induced in the heart of conditional frataxin-KO mice either. This suggests that continuous oxidative damage to iron-sulfur clusters, resulting from hampered superoxide dismutase signaling, is causative of the mitochondrial deficiency and long term mitochondrial iron overload occurring in FRDA.

Effect of idebenone on cardiomyopathy in Friedreich's ataxia: a preliminary study.

BACKGROUND: Friedreich's ataxia is caused by a deficiency of frataxin, a protein involved in regulation of mitochondrial iron content. We have reported a combined deficiency of a Krebs-cycle enzyme, aconitase, and three mitochondrial respiratory-chain complexes in endomyocardial biopsy samples from patients with this disorder. All four enzymes share iron-sulphur cluster-containing proteins that are damaged by iron overload through generation of oxygen free radicals. We used an in-vitro system to elucidate the mechanism of iron-induced injury and to test the protective effects of various substances. On the basis of these results, we assessed the effect of idebenone (a free-radical scavenger) in three patients with Friedreich's ataxia. METHODS: Heart homogenates from patients with valvular stenosis were tested for respiratory-chain complex II activity, lipoperoxidation, and aconitase activity by spectrophotometric assays, in the presence of reduced iron (Fe2+), oxidised iron (Fe3+), desferrioxamine, ascorbic acid, and idebenone. The Friedreich's ataxia patients (aged 11 years, 19 years, and 21 years) underwent ultrasonographic heart measurements at baseline and after 4-9 months of idebenone (5 mg/kg daily). FINDINGS: Fe2+ (but not Fe3+) decreased complex II activity and increased lipoperoxidation in heart homogenate. Addition of ascorbate or desferrioxamine increased some of the iron-induced adverse effects. Idebenone protected against these effects. In the three patients, left-ventricular mass index decreased from baseline to 4-9 months of idebenone treatment (patient 1, 145 g to 114 g; patient 2, 215 g to 151 g; patient 3, 408 g to 279 g). INTERPRETATION: Our in-vitro data suggest that both iron chelators and antioxidant drugs that may reduce iron are potentially harmful in patients with Friedreich's ataxia. Conversely, our preliminary findings in patients suggest that idebenone protects heart muscle from iron-induced injury.