leupeptins and Hepatoblastoma

This report is focused on the apoptotic effect induced by MG132, an inhibitor of 26S proteasome, in human hepatoma HepG2 cells. The results were compared with those obtained with non-transformed human Chang liver cells. MG132 reduced the viability of HepG2 cells in a time- and dose-dependent manner. The effect was in tight connection with the induction of apoptosis, as indicated by fluorescence microscopy and cytometric analysis, and was accompanied by a remarkable increase in the production of H2O2 and a reduction in mitochondrial transmembrane potential (Deltapsim). In addition cell death was prevented by antioxidants such as GSH, N-acetylcysteine or catalase. Western blot analysis showed that HepG2 cells contain a very low level of Bcl-2 and a much higher level of Bcl-XL, another antiapoptotic factor of the same family. When the cells were exposed to MG132 the level of Bcl-XL diminished, while a new band, corresponding to the expression of the proapoptotic protein Bcl-XS was detected. MG132 also caused the release of cytochrome c from mitochondria and the activation of caspase-3 with the consequent degradation of poly-ADP ribose polymerase (PARP). The observation that the broad spectrum caspase inhibitor z-VAD markedly reduced the apoptotic effect of the drug clearly demonstrated that caspases play an important role in MG132-induced apoptosis. MG132 exerted a modest effect on the viability of Chang liver cells which primarily depended on the G2/M arrest of cell cycle while only a small percentage of apoptotic cells was found. The remarkable differences in the effects induced by MG132 in Chang liver cells and HepG2 cells made us hypothesise the potential use of proteasome inhibitors in hepatocarcinoma therapy.

Topics: Antineoplastic Agents; Apoptosis; bcl-X Protein; Blotting, Western; Caspase 3; Caspases; Cell Cycle; Cell Survival; Cysteine Endopeptidases; Cytosol; Enzyme Activation; Flow Cytometry; G2 Phase; Hepatoblastoma; Humans; Hydrogen Peroxide; Leupeptins; Liver Neoplasms; Membrane Potentials; Mitochondria; Mitosis; Multienzyme Complexes; Oxidative Stress; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-bcl-2; Time Factors; Tumor Cells, Cultured

In a cultured human hepatoblastoma cell line, Hep G2, chenodeoxycholic acid (CDCA) induced LDL receptor mRNA levels approximately 4 fold and mRNA levels for HMG-CoA reductase and HMG-CoA synthase two fold. In contrast, the mRNA levels for mevalonate kinase, farnesyl pyrophosphate synthase and squalene synthase were not changed significantly. The pattern of the induction of the sterol-sensitive genes was similar to the induction by N-acetyl-leucyl-leucyl-norleucinal (ALLN), an SREBP degradation inhibitor, suggesting that CDCA may increase mature SREBPs. CDCA could inhibit the 25-hydroxycholesterol mediated inactivation of SREBP without affecting mRNA levels of SREBPs. These results suggest that CDCA can affect sterol metabolism by a novel mechanism involving the inhibition of the oxysterol-mediated inactivation of SREBP.

Topics: Alkyl and Aryl Transferases; Base Sequence; Cell Line; Chenodeoxycholic Acid; Codon; DNA-Binding Proteins; Farnesyl-Diphosphate Farnesyltransferase; Gene Expression; Geranyltranstransferase; Hepatoblastoma; Humans; Hydroxycholesterols; Hydroxymethylglutaryl CoA Reductases; Hydroxymethylglutaryl-CoA Synthase; Leupeptins; Liver Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Protease Inhibitors; Receptors, LDL; Regulatory Sequences, Nucleic Acid; RNA, Messenger; Transferases; Tumor Cells, Cultured