Abstract
Cholangiocellular carcinoma (CCA) of the liver was the target of more interest, recently, due mainly to its increased incidence and possible association to new environmental factors. Somatic mitochondrial DNA (mtDNA) mutations have been found in several cancers. Some of these malignancies contain changes of mtDNA, which are not or, very rarely, found in the mtDNA databases. In terms of evolutionary genetics and oncology, these data are extremely interesting and may be considered a sign of poor fitness, which may conduct in some way to different cellular processes, including carcinogenesis. MitoChip analysis is a strong tool for investigations in experimental oncology and was carried out on three CCA cell lines (HuCCT1, Huh-28 and OZ) with different outcome in human and a Papova-immortalized normal hepatocyte cell line (THLE-3). Real time quantitative PCR, western blot analysis, transmission electron microscopy, confocal laser microscopy, and metabolic assays including L-Lactate and NAD+/NADH assays were meticulously used to identify mtDNA copy number, oxidative phosphorylation (OXPHOS) content, ultrastructural morphology, mitochondrial membrane potential (ΔΨm), and differential composition of metabolites, respectively. Among 102 mtDNA changes observed in the CCA cell lines, 28 were non-synonymous coding region alterations resulting in an amino acid change. Thirty-eight were synonymous and 30 involved ribosomal RNA (rRNA) and transfer RNA (tRNA) regions. We found three new heteroplasmic mutations in two CCA cell lines (HuCCT1 and Huh-28). Interestingly, mtDNA copy number was decreased in all three CCA cell lines, while complexes I and III were decreased with depolarization of mitochondria. L-Lactate and NAD+/NADH assays were increased in all three CCA cell lines. MtDNA alterations seem to be a common event in CCA. This is the first study using MitoChip analysis with comprehensive metabolic studies in CCA cell lines potentially creating a platform for future studies on the interactions between normal and neoplastic cells.