carbobenzoxy-leucyl-leucyl-norvalinal and Carcinoma--Hepatocellular

Melanoma antigen (MAGE)-A4 is processed to generate a C-terminal fragment with proapoptotic activity. Here we demonstrate that Adriamycin promotes generation of the processed MAGE-A4 by activating the proteasome. The proteasome is known to prevent accumulation of toxic proteins to maintain cellular homeostasis.. Treatment of hepatoma cells expressing MAGE-A4 with a sublethal dose of Adriamycin increased the MAGE-A4 processing and sensitized the cells to Adriamycin-induced apoptosis. The processing of MAGE-A4 was inhibited by the proteasome inhibitors MG115, MG132, lactacystin and epoxamicin. MAGE-A4 was coimmunoprecipitated with the S6 proteasomal ATPase, and present in the fractions containing the proteasome during glycerol gradient centrifugation. Consistent with the notion that the proteasome cleaves MAGE-A4, the 26S proteasome, ubiquitin, and cell lysates were necessary for efficient in vitrocleavage of MAGE-A4.. The present study suggests that a low dose of Adriamycin increases the proteasome activity, which either maintains cellular homeostasis or leads to apoptosis depending, at least under the present conditions, on the expression of MAGE-A4.

Topics: Acetylcysteine; Animals; Antigens, Neoplasm; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Chlorocebus aethiops; COS Cells; Doxorubicin; HEK293 Cells; Humans; Leupeptins; Liver Neoplasms; Neoplasm Proteins; Proteasome Endopeptidase Complex; Ubiquitin

The ubiquitin-proteasome pathway is responsible for regulating cell cycle proteins, tumor-suppressor molecules, oncogenes, transcription factors, and pro- and anti-apoptotic proteins. The aim of this study is to evaluate the effects of proteasome inhibitors on human hepatocellular carcinoma (HCC) cells. HCC cells SK-Hep1, HLE and HepG2 were treated with the proteasome inhibitors MG132 and MG115. Our data showed that both inhibitors induce apoptosis in the three cell types tested in a dose-dependent manner. Moreover, subtoxic levels of MG132 and MG115 sensitized HCC cells to TRAIL-induced apoptosis. To investigate the mechanism of increased TRAIL sensitivity in HCC cells, we first examined surface expression of TRAIL and its receptors. MG132 upregulated TRAIL and its receptors (TRAIL-R1 and -R2) in SK-Hep1 and HLE, whereas MG115 upregulated them in SK-Hep1. MG132 downregulated expression of X-linked inhibitor of apoptosis protein (XIAP) in SK-Hep1 and HLE, and of survivin in all three cell-types. MG115 downregulated expression of XIAP in SK-Hep1, and survivin in SK-Hep1 and HepG2. Furthermore, MG132 downregulated phospho-AKT and its downstream target phospho-BAD, indicating that MG132 activated the mitochondrial apoptosis pathway by inhibiting phosphorylation of AKT and BAD. In conclusion, proteasome inhibitors induced apoptosis and augmented TRAIL sensitivity via both the IAP family and AKT pathways. Thus, combining proteasome inhibitors with a TRAIL agonist may provide a new therapeutic strategy for HCC.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Caspase Inhibitors; Caspases; Down-Regulation; Drug Synergism; Humans; Leupeptins; Mice; Oncogene Protein v-akt; Phosphorylation; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proto-Oncogene Proteins c-akt; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; TNF-Related Apoptosis-Inducing Ligand

Chronic ethanol consumption causes increased oxidative damage in the liver. Induction of CYP2E1 is one pathway involved in how ethanol produces oxidative stress. Ethanol can cause protein accumulation, decreased proteolysis, and decreased proteasome activity. The objective of this study was to investigate the effect of inhibition of the proteasome activity on CYP2E1-dependent toxicity. HepG2 cells over-expressing CYP2E1 (E47 cells) were treated with arachidonic acid (AA) plus iron, agents important in development of alcoholic liver injury and which are toxic to E47 cells by a mechanism dependent on CYP2E1, oxidative stress, and lipid peroxidation. Addition of various proteasome inhibitors was associated with significant potentiation of the loss of cell viability caused by AA plus iron. Potentiation of toxicity was associated with increased oxidative damage as reflected by an increase in lipid peroxidation and accumulation of oxidized and nitrated proteins in E47 cells and an enhanced decline in mitochondrial membrane potential. Antioxidants prevented the loss of viability and the potentiation of this loss of viability by proteasome inhibition. CYP2E1 levels were elevated about 3-fold by the proteasome inhibitors. Inhibition of proteasome activity also potentiated toxicity of AA alone and toxicity after treatment to remove glutathione (GSH). Similar results were found in hepatocytes from pyrazole-treated rats with high levels of CYP2E1. In conclusion, proteasome activity plays an important role in modulating CYP2E1-mediated toxicity in HepG2 cells by regulating CYP2E1 levels and by removal of oxidized proteins. Such interactions may be important in CYP2E1-catalyzed toxicity of hepatotoxins and in alcohol-induced liver injury.

Topics: Antioxidants; Arachidonic Acid; Buthionine Sulfoximine; Carcinoma, Hepatocellular; Cysteine Endopeptidases; Cytochrome P-450 CYP2E1; Drug Synergism; Endopeptidases; Humans; Iron; Leupeptins; Lipid Peroxides; Liver Neoplasms; Lysosomes; Membrane Potentials; Mitochondria, Liver; Multienzyme Complexes; Proteasome Endopeptidase Complex; Tumor Cells, Cultured; Tyrosine