oligomycins and Colonic-Neoplasms

We investigated the role of mitochondrial function in the invasiveness of human colorectal cancer (CRC) cell lines, using paired primary SW480 and metastatic SW620 cells, and appraised the clinical relevance of the alteration of mtDNA copy number in 33 pairs of CRC specimens after surgical resection. Suppression of mitochondrial function was achieved by the exposure of cells to oligomycin A (OA) or by knockdown of mitochondrial transcriptional factor A (TFAM) to evaluate their effects on energy metabolism, reactive oxygen species, protein expression levels of epithelial-mesenchymal transition (EMT) markers and invasive activity of CRC cells. We found that SW620 cells expressed higher levels of TFAM and mitochondrial DNA (mtDNA)-encoded NADH dehydrogenase subunit 6 (ND6) and cytochrome c oxidase subunit II (COX-II) and nuclear DNA-encoded NADH ubiquinone oxidoreductase subunit A9 (NDUFA9), iron-sulfur protein subunit B of succinate dehydrogenase (SDHB), ubiquinol‑cytochrome c reductase core protein I/II (UQCRC1/2) and cytochrome c oxidase subunit IV (COX-IV) when compared with the SW480 cells. The mtDNA copy number, ADP-triggered oxygen consumption rate (OCR) and respiratory control ratio (RCR) of succinate-supported respiration in the SW620 cells were higher than those noted in the SW480 cells. The intracellular levels of H2O2 and O2-• in the SW620 cells were lower than levels noted in the SW480 cells. Moreover, SW620 cells displayed lower protein levels of hexokinase II (HK-II), glucose 6-phosphate isomerase (GPI) and lactate dehydrogenase (LDH), and lower lactate production rate, and expressed higher levels of EMT markers N-cadherin, vimentin and Snail, and showed higher Transwell migration and invasion activities as compared with the SW480 cells. After OA treatment, SW620 cells exhibited a decrease in OCR and RCR of succinate-supported respiration, an increase in lactate production rate and intracellular levels of H2O2 and O2-•. Moreover, the level of vimentin and Transwell migration activity of the SW620 cells were decreased. After TFAM knockdown, the protein levels of TFAM, ND6 and COX-II, and mtDNA copy number, OCR and RCR of succinate-supported respiration in the SW620-KD#4 and SW620-KD#5 cells were all lower than those noted in the SW620‑Control cells. By contrast, the protein level of HK-II, lactate production rate, the intracellular levels of H2O2 and O2-• in the SW620-KD#4 and SW620-KD#5 cells were all higher than those noted in the SW620-Contr

Topics: Cell Line, Tumor; Colonic Neoplasms; DNA-Binding Proteins; DNA, Mitochondrial; Energy Metabolism; Gene Dosage; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Mitochondria; Mitochondrial Proteins; Neoplasm Invasiveness; Oligomycins; Reactive Oxygen Species; Transcription Factors

5-Fluorouracil (5-FU) is widely used for treatment of advanced colorectal cancer. However, it is common for such patients to develop resistance to 5-FU, and this drug resistance becomes a critical problem for chemotherapy. The mechanisms underlying this resistance are largely unknown. To screen for proteins possibly responsible for 5-FU resistance, cells resistant to 5-FU were derived from human colon cancer cell lines and two-dimensional gel electrophoresis-based comparative proteomics was done. Two-dimensional gel electrophoresis data showed there was lower expression of the alpha subunit of mitochondrial F(1)F(0)-ATP synthase (ATP synthase) in 5-FU-resistant cells compared with parent cells. Western blotting showed that expression of other ATP synthase complex subunits was also lower in 5-FU-resistant cell lines and that these resistant cells also showed decreased ATP synthase activity and reduced intracellular ATP content. The ATP synthase inhibitor, oligomycin A, strongly antagonized 5-FU-induced suppression of cell proliferation. When 5-FU sensitivity was compared with ATP synthase activity in six different human colon cancer cell lines, a positive correlation has been found. Furthermore, suppressed ATP synthase d-subunit expression by siRNA transfection increased cell viability in the presence of 5-FU. Bioenergetic dysfunction of mitochondria has been reported as a hallmark of many types of cancers (i.e., down-regulation of ATP synthase beta-subunit expression in liver, kidney, colon, squamous oesophageal, and lung carcinomas, as well as in breast and gastric adenocarcinomas). Our findings show that ATP synthase down-regulation may not only be a bioenergetic signature of colorectal carcinomas but may also lead to cellular events responsible for 5-FU resistance.

Topics: Antimetabolites, Antineoplastic; Aurovertins; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Down-Regulation; Drug Resistance, Neoplasm; Energy Metabolism; Enzyme Inhibitors; Fluorouracil; Humans; Mitochondria; Mitochondrial Proton-Translocating ATPases; Oligomycins; RNA, Small Interfering; Transfection

The uptake kinetics of alpha-linolenic acid (18:3(n - 3)), an essential fatty acid, were investigated in the human intestinal cell line Caco-2. Four clones (PD10, PF11, PD7 and TC7) from the heterogeneous parental Caco-2 cells population were used. After a screening step using isolated cells, the TC7 clone was selected for the study of alpha-linolenic acid uptake. [1-(14)C]linolenic acid dissolved in 10 mM taurocholate was presented to the microvillus plasma membrane (apical side) of TC7 differentiated cells, grown on a semi-permeable polycarbonate membrane. The results show that the initial rate of uptake is not a linear function of the 18:3(n- 3) monomer concentration in the incubation medium. In the monomer concentration range studied (0.2 to 36 microM) apical uptake was saturable and followed Michaelis-Menten kinetics (V(max) = 15.4 +/- 0.6 nmol/mg protein per min, K(m) = 14.3 +/- 1.3 microM). In addition, it was temperature- and energy-dependent but was apparently unaffected by the sodium gradient and intracellular metabolic fate of 18:3(n - 3). Excess of unlabeled saturated or unsaturated long chain fatty acids (C16 to C22) led to a 27-68% reduction of [1-(14)C]linolenic acid uptake. Likewise basolateral uptake was saturable (V(max) = 4.9 +/- 0.7 nmol/mg protein per min, K(m) = 8.7 +/- 2.9 microM). These facts argue in favour of the existence in these human intestinal cells of a carrier-mediated transport system for alpha-linolenic acid and probably other long chain fatty acids as well.

Topics: Adenocarcinoma; alpha-Linolenic Acid; Antimetabolites; Caco-2 Cells; Carbon Radioisotopes; Cell Division; Clone Cells; Colonic Neoplasms; Deoxyglucose; Enzyme Inhibitors; Fatty Acids; Glucose; Humans; Intestinal Mucosa; Intestines; Ionophores; Kinetics; Lipids; Monensin; Oligomycins; Osmolar Concentration; Serine Endopeptidases; Substrate Specificity; Temperature; Time Factors

N-(4-Methylphenylsulfonyl)-N'-(4-chlorophenyl)urea (MPCU) is a new agent that exhibits high therapeutic activity against human and rodent tumor models. Initial studies indicated that in vitro [3H]MPCU was concentrated 4- to 6-fold in GC3/c1 human colon adenocarcinoma cells in an azide-sensitive manner. In this study the dependence of uptake and concentrative accumulation of MPCU upon temperature, plasma membrane potential, and the electrochemical potential of mitochondria has been examined. Accumulation and efflux of MPCU were temperature dependent. At 3.6 microM MPCU, initial rates of uptake (15 s) were 1.4, 38.0, and 84.2 pmol/min/10(6) cells at 2 degrees C, 23 degrees C, and 37 degrees C, respectively. The rate of uptake and concentrative accumulation within GC3/c1 cells was not altered in high K+ buffer or by 1 mM ouabain, indicating that plasma membrane potential was not significant in these processes. Concentrative accumulation, but not initial uptake, was inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, 2,4-dinitrophenol, and sodium azide. Glucose partially antagonized the inhibition of these agents which uncouple oxidative phosphorylation. Oligomycin, an inhibitor of mitochondrial ATP synthase, did not inhibit uptake or concentrative accumulation of MPCU. However, oligomycin in the presence of 2-deoxyglucose significantly inhibited concentrative accumulation of MPCU. These results suggested that concentrative accumulation of MPCU was dependent upon the mitochondrial transmembrane gradient rather than ATP, although direct implication of ATP could not be excluded. To examine which component of this gradient was predominant in causing MPCU sequestration, the ionophores valinomycin and nigericin were used. Valinomycin, which collapses the charge gradient across the mitochondrial matrix membrane, caused only slight inhibition of MPCU accumulation, and the effect was similar at 2 or 10 mumol. In contrast, nigericin (which collapses the pH gradient and increases mitochondrial membrane potential) inhibited by approximately 90% concentrative accumulation of MPCU. These data suggested that MPCU was being concentrated in mitochondria and that this was dependent upon the pH gradient across mitochondrial membrane. In cells exposed to MPCU or the analogue N-(5-indanylsulfonyl)-N'-(4-chlorophenyl)urea, enlargement of mitochondria was observed within 24 h and appeared to be the initial morphological change associated with drug treatment. These res

Topics: Adenocarcinoma; Antineoplastic Agents; Biological Transport; Cell Compartmentation; Colonic Neoplasms; Humans; Hydrogen-Ion Concentration; Membrane Potentials; Microscopy, Electron; Mitochondria; Nigericin; Oligomycins; Ouabain; Sulfonylurea Compounds; Uncoupling Agents; Valinomycin