ABSTRACT
Wilson's disease (Wd) is a genetic disorder resulting in Cu2+ accumulation, and is caused by mutations in the ATP7B gene, the copper transporter. In vivo studies show a correlation between Cu2+ accumulation and malfunction of the heme biosynthesis pathway. In this study, we describe multiple effects of Cu2+ accumulation on heme synthesis, which, in turn, affect proteasomal activity. Cu2+ toxicity was examined in two hepatocellular carcinoma cell lines, HepG2 and Hep3B, with Hep3B cells containing an integrated hepatitis B virus genome. Exposure of HepG2 and Hep3B cells to Cu2+ inhibited the enzymes PBGD and ALAD of the heme synthesis pathway and, in parallel, upregulated heme oxygenase-1 (HO-1). Proto-porphyrin IX (PpIX) and the heme pool were reduced as a result of these processes. PpIX synthesis was found to be lower in cells expressing the mutant ATP7B (P1134P), compared to those expressing the WT enzyme. Proteasomal activity was inhibited under Cu2+ treatment in HepG2 cells; however, Cu2+ induced marked proteosomal acceleration in Hep3B cells. Under these conditions, Ub-conjugated proteins were gradually accumulated, whereas treatment with bathocuproine disulfonic acid (BCS), a Cu2+ chelator, reversed this effect. In conclusion, our data suggest that copper downregulates the heme synthesis pathway in hepatocellular cells and further reduces it in the presence of mutated ATP7B.
Subject(s)
Copper/toxicity , Enzyme Inhibitors/toxicity , Heme/biosynthesis , Hepatocytes/drug effects , Hepatolenticular Degeneration , Proteasome Endopeptidase Complex/drug effects , Adenosine Triphosphatases/biosynthesis , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/metabolism , Cation Transport Proteins/biosynthesis , Cell Line, Tumor , Cell Survival/drug effects , Copper-Transporting ATPases , Disulfiram/pharmacology , Down-Regulation/drug effects , Hepatocytes/metabolism , Humans , Hydroxymethylbilane Synthase/antagonists & inhibitors , Liver Neoplasms/drug therapy , Liver Neoplasms/metabolism , Porphobilinogen Synthase/antagonists & inhibitors , Proteasome Endopeptidase Complex/metabolism , Protoporphyrins/metabolism , Reactive Oxygen Species/metabolism , Ubiquitin/metabolismABSTRACT
The 4-nitroquinoline 1-oxide (4NQO)-induced rat tongue carcinoma, in which the carcinogen is administered systemically in drinking water, is the most comparable animal model to the development of human oral carcinoma. This is the first study to report the ultrastructural changes in this model. The most significant changes were observed in the carcinoma cells at the invasion front and included unique modifications in the basal lamina, presence of micropinocytotic vesicles (plasmalemmal caveolae), and emergence of cytoplasmic microfilaments featuring a parallel arrangement. The microfilaments, in both appearance and organization, were consistent with contractile microfilaments. These observations may be the morphological reflection of the phenotypic modifications occurring within the carcinoma cells, approaching smooth muscle differentiation.
