oligomycins and Carcinoma--Hepatocellular

Human hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide particularly in Asia. Deregulation of cellular energetics was recently included as one of the cancer hallmarks. Compounds that target the mitochondria in cancer cells were proposed to have therapeutic potential. Biguanide drugs which inhibit mitochondrial complex I and repress mTOR signaling are clinically used to treat type 2 diabetes mellitus patients (T2DM) and were recently found to reduce the risk of HCC in T2DM patients. However, whether alteration of energy metabolism is involved in regulating the sensitivity of HCC to biguanide drugs is still unclear. In the present study, we treated four HCC cell lines with mitochondrial inhibitors (rotenone and oligomycin) and biguanide drugs (metformin and phenformin), and found that the HCC cells which had a higher mitochondrial respiration rate were more sensitive to these treatments; whereas the HCC cells which exhibited higher glycolysis were more resistant. When glucose was replaced by galactose in the medium, the altered energy metabolism from glycolysis to mitochondrial respiration in the HCC cells enhanced the cellular sensitivity to mitochondrial inhibitors and biguanides. The energy metabolism change enhanced AMP-activated protein kinase (AMPK) activation, mTOR repression and downregulation of cyclin D1 and Mcl-1 in response to the mitochondrial inhibitors and biguanides. In conclusion, our results suggest that increased mitochondrial oxidative metabolism upregulates the sensitivity of HCC to biguanide drugs. Enhancing the mitochondrial oxidative metabolism in combination with biguanide drugs may be a therapeutic strategy for HCC.

Topics: Adenosine Triphosphate; Apoptosis; Carcinoma, Hepatocellular; Energy Metabolism; Glycolysis; Hep G2 Cells; Humans; Liver Neoplasms; Metformin; Mitochondria; Oligomycins; Oxygen Consumption; Phenformin; Rotenone

7-Ketocholesterol (7-KC) is found at an elevated level in patients with cancer and chronic liver disease. The up-regulation of an efflux pump, P-glycoprotein (P-gp) leads to drug resistance. To elucidate the effect of 7-KC on P-gp, P-gp function and expression were investigated in hepatoma cell lines Huh-7 and HepG2 and in primary hepatocyte-derived HuS-E/2 cells. At a subtoxic concentration, 48-h exposure to 7-KC reduced the intracellular accumulation and cytotoxicity of P-gp substrate doxorubicin in hepatoma cells, but not in HuS-E/2 cells. In Huh-7 cells, 7-KC elevated efflux function through the activation of phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway. 7-KC activated the downstream protein synthesis initiation factor 4E-BP1 and induced P-gp expression post-transcriptionally. The stimulation of efflux was reversible and could not be prevented by N-acetyl cysteine. Total cellular ATP content remained the same, whereas the lactate production was increased and fluorescence lifetime of protein-bound NADH was shortened. These changes suggested a metabolic shift to glycolysis, but glycolytic inhibitors did not eliminate 7-KC-mediated P-gp induction. These results demonstrate that 7-KC induces P-gp through PI3K/mTOR signaling and decreased the cell-killing efficacy of doxorubicin in hepatoma cells.

Topics: Acetylcysteine; Antibiotics, Antineoplastic; Antioxidants; ATP Binding Cassette Transporter, Subfamily B, Member 1; Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Cell Survival; Cholesterol; Doxorubicin; Drug Resistance, Neoplasm; Hepatocytes; Humans; Hydroxycholesterols; Ketocholesterols; Lactic Acid; Oligomycins; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Signal Transduction; TOR Serine-Threonine Kinases; Up-Regulation

The aim of this study was to investigate the contribution of mitochondrial dysfunction to chemoresistance and migration of hepatoma cells. We found that inhibition of mitochondrial respiration and mitochondrial DNA (mtDNA) depletion resulted in induction of amphiregulin (AR) expression in HepG2 cells. Upon oligomycin treatment of HepG2 cells, the cytosolic Ca(2+) was significantly raised after 30 min, and the intracellular level of reactive oxygen species (ROS) was elevated 2.2-fold after 4 h. Moreover, the condition medium of oligomycin-treated HepG2 cells was found to stimulate the migration of SK-Hep-1 cells. On the other hand, oligomycin-induced cisplatin-resistance and cell migration of HepG2 cells were attenuated by AR-specific RNA interference (#L-017435, Dharmacon) and a neutralizing antibody (MAB262, R&D Systems), respectively. Together, these findings suggest that mitochondrial dysfunction induced Ca(2+) mobilization, and ROS overproduction, which modulated the chemo-resistance and migration of hepatoma cells through the induction and activation of AR.

Topics: Amphiregulin; Calcium; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Drug Resistance, Neoplasm; EGF Family of Proteins; ErbB Receptors; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Mitochondria, Liver; Oligomycins; Reactive Oxygen Species; Uncoupling Agents; Up-Regulation

Relative content of membrane sectors of ATPase complex in rat liver and Zajdela hepatoma mitochondria and the ability of mitochondrial membrane of the two sources to bind isolated soluble (F1) ATPase were examined. Approximately equal concentrations of oligomycin were required for 50% inhibition of ATPase activity in submitochondrial particles of rat liver and Zajdela hepatoma indicating practically identical content of membrane sectors of ATPase complex in both types of mitochondria. As detected by the increase in oligomycin-sensitive ATPase activity of submitochondrial particles incubated with isolated F1, the submitochondrial particles of Zajdela hepatoma in contrast to those of rat liver were able to bind specifically considerable amounts of exogenously added F1. The results indicate that mitochondria of Zajdela hepatoma contain membrane sectors of ATPase complex unassociated with F1 but capable of association with this enzyme.

Topics: Adenosine Triphosphatases; Animals; Antigens, Neoplasm; Carcinoma, Hepatocellular; Epitopes; Intracellular Membranes; Liver Neoplasms; Mitochondria, Liver; Neoplasms, Experimental; Oligomycins; Rats

The changes in water and electrolyte content of slices of Morris hepatoma 3924A induced by various conditions of incubation have been compared with the ultrastructural appearance of the tissue. Incubation at 1 degrees led to an increase of water, Na+, and Cl- content and to a loss of K+. There was simultaneous increase in size of the cells and intercellular spaces, loss of junctional complexes, increase in the number of microvilli, and fragmentation and dilation of the cytocavitary network. Subsequent incubation at 38 degrees in oxygenated medium led to a substantial reversal of all of these changes of composition and structure, which was well advanced within 10 min and largely complete by 60 min. The presence of 20 mM glucose in the medium somewhat enhanced the degree of recovery. A reduction of cell volume and intercellular spaces was evident both from the electron microscopic observations and measurements of the volume of inulin distribution. The presence of ouabain inhibited the net accumulation of K+ and much of the Na+ extrusion, but permitted about 50% of the net extrusion of water (accompanied by Na+ and Cl-) and had little effect on the ultrastructural recovery. The presence of glucose increased the resistance of volume and structural recovery of ouabain without releasing the inhibition of K+ accumulation. A marked feature of the recovering tissues was the Golgi apparatus, which assumed an appearance suggestive of increased activity when water extrusion was active. In slices using only endogenous substrate, cyanide and (to a lesser extent) oligomycin greatly inhibited the recovery of volume and structure. The presence of glucose permitted some recovery in the presence of cyanide. The control of cell volume in hepatoma 3924A appears to involve two separate components of water transport, one of which is sensitive, and one insensitive to ouabain. The ouabain-insensitive component appears to be especially related to the recovery of cell ultrastructure after incubation at 1 degrees, to be more sensitive to paucity of adenosine 5'-triphosphate, and to proceed by secretion of water, Na+, and Cl- into vesicles that fuse with the Golgi apparatus. This mechanism may be related to that for bile secretion in normal liver. The ouabain-sensitive component of water transport is a function of the mechanism for the coupled transport of Na+ and K+.

Topics: Anaerobiosis; Animals; Carcinoma, Hepatocellular; Cell Line; Cyanides; Extracellular Space; Glucose; Glycolysis; In Vitro Techniques; Intracellular Fluid; Liver Neoplasms; Oligomycins; Ouabain; Rats; Temperature; Time Factors; Water-Electrolyte Balance

Topics: Aminopyrine; Animals; Antimycin A; Biological Transport; Carcinoma, Hepatocellular; Cyanides; Dinitrophenols; Electrons; Liver Neoplasms; Mitochondria, Liver; Neoplasms, Experimental; Oligomycins; Ouabain; Oxygen Consumption; Phenobarbital; Rats; Rotenone; Succinates

Topics: Antimetabolites; Carcinoma, Hepatocellular; Cyclohexanes; Depression, Chemical; Dinitrophenols; Glucose; Glycogen; Glycolysis; In Vitro Techniques; Lactates; Liver Neoplasms; Mitochondria; Oligomycins; Oxygen Consumption; Stimulation, Chemical

Affinity of glucose, fructose and mannose for tumour hexokinase and their rates of phosphorylation at saturation concentration have been correlated with rates of glycogen synthesis by intact tumour cells at different concentrations of the three substrates. Competition experiments with one sugar labelled and the other sugar unlabelled indicate inhibition of glycogen synthesis by the sugar with a low K(m) for hexokinase. Glycogen synthesis from glucose 1-phosphate in aged cells and from nucleoside in freshly prepared cells is stimulated by fructose and inhibited by glucose. The decrease in glycogen formation from glucose 1-phosphate by oligomycin is partially overcome by increased fructose concentrations. These results are explained by an activation of alpha-glucan phosphorylase by fructose and an inhibition of this enzyme by glucose. It is suggested that differences in localization of glucose 6-phosphate, available to the intact cell in various ways, determine its transformation into glycogen by either the UDP-glucose-alpha-glucan glucosyltransferase reaction or by the alpha-glucan phosphorylase reaction.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line; Culture Techniques; Depression, Chemical; Fructose; Glucose; Glucosyltransferases; Glycogen; Hexokinase; Hexosephosphates; Kinetics; Liver Neoplasms; Mannose; Neoplasms, Experimental; Nucleosides; Oligomycins; Rats; Stimulation, Chemical

Topics: Anti-Bacterial Agents; Antimetabolites; Ascites; Carcinoma, Hepatocellular; Cell Respiration; Dinitrophenols; Glucose; Liver Neoplasms; Metabolism; Neoplasms; Neoplasms, Experimental; Oligomycins; Pharmacology; Research