Apoptosis induced in hepatoblastoma HepG2 cells by the proteasome inhibitor MG132 is associated with hydrogen peroxide production, expression of Bcl-XS and activation of caspase-3.
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.
Emanuele
S
S
Department of Cell and Developmental Biology, Section of Biochemistry, University of Palermo, Policlinico, Palermo, Italy.
Calvaruso
G
G
Lauricella
M
M
Giuliano
M
M
Bellavia
G
G
D'Anneo
A
A
Vento
R
R
Tesoriere
G
G
D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002453:Cell Cycle; D002470:Cell Survival; D003546:Cysteine Endopeptidases / Q000494:pharmacology*; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D005434:Flow Cytometry; D016195:G2 Phase / Q000187:drug effects; D018197:Hepatoblastoma / Q000473:pathology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology*; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology*; D008564:Membrane Potentials; D008928:Mitochondria / Q000378:metabolism; D008938:Mitosis / Q000187:drug effects; D009097:Multienzyme Complexes / Q000494:pharmacology*; D018384:Oxidative Stress; D046988:Proteasome Endopeptidase Complex; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis*; D013997:Time Factors; D014407:Tumor Cells, Cultured; D051020:bcl-X Protein
D000970:Antineoplastic Agents; C494811:BCL2L1 protein, human; D007976:Leupeptins; D009097:Multienzyme Complexes; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein; D006861:Hydrogen Peroxide; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde