novobiocin and Liver-Neoplasms

Voltage-dependent anion-selective channel protein 1 (VDAC1) is the most abundant protein in the mitochondrial outer membrane and plays a crucial role in the control of hepatocellular carcinoma (HCC) progress. Our previous research found that cytosolic molecular chaperone heat shock protein 90 (Hsp90) interacted with VDAC1, but the effect of the C-terminal and N-terminal domains of Hsp90 on the formation of VDAC1 oligomers is unclear. In this study, we focused on the effect of the C-terminal domain of Hsp90 on VDAC1 oligomerization, ubiquitination, and VDAC1 channel activity. We found that Hsp90 C-terminal domain inhibitor Novobiocin promoted VDAC1 oligomerization, release of cytochrome c, and activated mitochondrial apoptosis pathway. Atomic coarse particle modeling simulation revealed C-terminal domain of Hsp90α stabilized VDAC1 monomers. The purified VDAC1 was reconstituted into a planar lipid bilayer, and electrophysiology experiments of patch clamp showed that the Hsp90 C-terminal inhibitor Novobiocin increased VDAC1 channel conductance via promoting VDAC1 oligomerization. The mitochondrial ubiquitination proteomics results showed that VDAC1 K274 mono-ubiquitination was significantly decreased upon Novobiocin treatment. Site-directed mutation of VDAC1 (K274R) weakened Hsp90α-VDAC1 interaction and increased VDAC1 oligomerization. Taken together, our results reveal that Hsp90 C-terminal domain inhibition promotes VDAC1 oligomerization and VDAC1 channel conductance by decreasing VDAC1 K274 mono- ubiquitination, which provides a new perspective for mitochondria-targeted therapy of HCC.

Topics: Apoptosis; Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Novobiocin; Ubiquitination; Voltage-Dependent Anion Channel 1

The authors determined the effects of growth inhibition on glutamine transport and metabolism in human hepatoma cells.. Hepatoma cells exhibit markedly higher (10- to 30-fold) glutamine uptake than normal human hepatocytes, via a disparate transporter protein with a higher affinity for glutamine. Currently, little is known about the effects of growth arrest on glutamine transport and metabolism in hepatoma cells.. The authors determined proliferation rates, glutamine transport, and glutaminase activities in the human hepatoma cell lines HepG2, Huh-7, and SK-Hep, both in the presence and absence of the chemotherapeutic agents novobiocin and sodium butyrate. The transport activities for alanine, arginine, and leucine also were determined in both treated and untreated cells. Glutaminase activity was determined in normal human liver tissue and compared with that present in hepatoma cells.. Glutaminase activities were similar in all three cell lines studied, despite differences in proliferation rates, and were sixfold higher than the activity in normal human liver. In contrast to normal hepatocytes, which expressed the liver-specific glutaminase, hepatomas expressed the kidney-type isoform. Sodium butyrate (1 mmol/L) and novobiocin (0.1 mmol/L) inhibited cellular proliferation and reduced both glutamine transport and glutaminase activity by more than 50% after 48 hours in the faster-growing, less differentiated SK-Hep cells. In contrast, the agents required 72 hours to attenuate glutamine uptake by 30% and 50% in the slower-growing, more differentiated HepG2 and Huh-7 cell lines, respectively. Treatment of all three cell lines with novobiocin/butyrate also resulted in a 30% to 60% attenuation of the transport of alanine, arginine, and leucine, and glutamine, indicating that inhibition of cellular proliferation similarly affects disparate amino acid transporters.. Hepatocellular transformation is characterized by a marked increase in glutamine transport and metabolism. Inhibition of cellular proliferation attenuates glutamine transport and metabolism, especially in fast-growing, relatively undifferentiated hepatoma cells. Because the uptake of other amino acids is similarly reduced under cytostatic conditions, plasma membrane amino acid transport activity in hepatoma cells is regulated by the proliferation state of the cells.

Topics: Amino Acid Transport Systems; Amino Acids; Animals; Biological Transport; Butyrates; Butyric Acid; Carcinoma, Hepatocellular; Carrier Proteins; Cell Division; Female; Glutaminase; Glutamine; Humans; Liver Neoplasms; Male; Novobiocin; Rats; Rats, Inbred F344; Time Factors; Tumor Cells, Cultured

A differentiation inducer butyrate and a tumor promoter teleocidin had inhibitory effects on the proliferation of PLC/PRF/5 hepatoma. Both of these reagents stimulated the production of procollagen type III peptide, enhanced the cytokeratin assembly and altered the morphological appearance. Novobiocin, a topoisomerase II inhibitor, enhanced the cytokeratin assembly induced by butyrate but antagonized that induced by teleocidin without changing the expression and the phosphorylation state of cytokeratin proteins. In addition, novobiocin acted synergistically with butyrate but not with teleocidin in stimulating the procollagen production and the acetate uptake. These results suggest that butyrate and teleocidin induce cell differentiation via distinct signaling pathway and that novobiocin and butyrate can be used as subsidiary drugs in preventing the growth of hepatoma.

Topics: Acetylation; Butyrates; Butyric Acid; Carcinogens; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Cytoskeletal Proteins; Drug Synergism; Humans; Keratins; Liver Neoplasms; Lyngbya Toxins; Novobiocin; Peptide Fragments; Procollagen; Tumor Cells, Cultured

Chang liver cells cultured in the simultaneous presence of novobiocin and butyrate stopped proliferating and changed into fibroblast-like cells with remarkably elongated cytoplasm. In these fibroblast-like cells, the cellular content of both protein and DNA was increased 2- to 3-fold. In addition, the production of specific proteins such as type III procollagen, actin, and tubulin was increased and the expression of proliferation-associated nuclear antigen which was reactive with Ki-67 monoclonal antibody was reduced remarkably. Therefore, Chang liver cells cultured with novobiocin and butyrate were considered to be arrested at the premitotic G2 phase of the cell cycle and then enter into the noncycling resting state. These actions of novobiocin and butyrate were not mediated by the pathway of epidermal growth factor action or modulated by the diacylglycerol agonist 1-oleoyl-2-acetylglycerol and the C kinase inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine. On the other hand, novobiocin was disclosed to be a stimulator of [3H]acetate uptake and acted synergistically with butyrate in enhancing nuclear protein acetylation. From these results it can be speculated that novobiocin and butyrate chemically modulate nuclear proteins and thereby alter the gene expression and the differentiation state of Chang liver cells.

Topics: Acetylation; Blotting, Western; Butyrates; Carcinoma, Hepatocellular; Cell Cycle; Cell Differentiation; Cell Division; Collagen; Epidermal Growth Factor; Fibroblasts; Gene Expression; Humans; In Vitro Techniques; Liver; Liver Neoplasms; Molecular Weight; Novobiocin; Nuclear Proteins; Proliferating Cell Nuclear Antigen; Tumor Cells, Cultured