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Breast cancer is one of the leading causes of cancer-related deaths amongst women in the USA. The tumor microenvironment has been suggested to be an attractive therapeutic target for treatment of cancers. The glycosaminoglycan chondroitin sulfate, as part of the cellular microenvironment, consists of long linear chains of repeating disaccharide units, which are covalently attached to core proteins to form chondroitin sulfate-proteoglycans. In vitro studies have implicated chondroitin sulfate in various aspects of carcinogenesis, whereas the in vivo roles of chondroitin sulfate are less clear. Drastically elevated levels of chondroitin sulfate have been observed within the stromal compartment of many solid tumors, including human breast carcinomas, the significance of which is unknown. We examined the role of tumor-associated chondroitin sulfate in breast cancer progression. Enzymatic elimination of endogenous chondroitin sulfate by intra-tumor injections of chondroitinase ABC leads to the development of secondary tumors and increased lung metastases, while primary orthotopic tumor growth was not affected. These results establish a metastasis-inhibiting effect of primary breast tumor-associated chondroitin sulfate, which may open novel carbohydrate-based therapeutic strategies to combat breast cancer.

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Chondroitin-4-sulfotransferase-1(C4ST-1)/carbohydrate sulfotransferase 11 (CHST11) is a Golgi-bound enzyme involved in the biosynthesis of the glycosaminoglycan chondroitin sulfate. The sulfation pattern of chondroitin is tightly regulated during development, injury and disease, with the temporal and spatial expression of chondroitin sulfotransferase genes believed to be a crucial determinant of the fine balance of chondroitin sulfation. We have previously identified mouse C4st-1 as a target gene of ligands of the TGFbeta superfamily of growth factors, which could positively regulate C4st-1 expression in a number of cell types. We have also shown that a gene trap loss-of-function mutation in C4st-1 leads to severe skeletal abnormalities during mouse embryogenesis. In addition, we described a highly specific temporal and spatial expression pattern of C4st-1 during mouse embryogenesis. However, the transcriptional regulatory mechanisms that control C4st-1 gene expression remain unexplored. In order to gain knowledge on the transcriptional regulation of C4ST-1, we used a bioinformatical approach to identify conserved putative long-range cis-regulatory modules in a region of 120 kb spanning the 5' end of the C4ST-1 gene. Luciferase reporter assays in human HEK293T and mouse NmuMG cells identified a functional C4ST-1 promoter, as well as a number of cis-regulatory modules able to positively and negatively regulate C4ST-1 expression. Moreover, we identified TGFbeta- responsive regulatory modules that can function in a cell type-specific fashion. Taken together, our results identify TGFbeta-dependent and -independent cis-regulatory modules of the C4ST-1 gene.

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The inhibition by citrinin (CTN) of lipid peroxidation of mitochondria, sub-mitochondrial particles (SMP) and microsomes was studied. This effect was reversed by the presence of high concentrations of Fe3+ (0.4 and 0.5 mM), suggesting chelation of the mycotoxin with iron or interference in the reduction of Fe3+.

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The effects of amiodarone (AMD) on lipid peroxidation of rat liver mitochondria, the formation of superoxide anions at the respiratory chain level, and the cytosolic and mitochondrial enzymatic protective mechanisms of oxidative stress were studied. An attempt of classify AMD according to its toxic ability to interfere with the integrated function of electron transport enzymes was also investigated. The results confirm the effects of AMD on complex I and permit the placing of this drug in class A of the classification of Knobeloch, together with rotenone, amytal and chaotropic agents. AMD has no effect on the activity of the enzymes superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase, nor on glucose 6-phosphate dehydrogenase. AMD did not promote an increase in the formation of anion superoxide at the respiratory chain level. Pre-incubation with AMD (16.6 microM) inhibited about 70 per cent of lipid peroxidation. The results suggest a protective effect of AMD against lipid peroxidation in mitochondrial membranes by iron-dependent systems.

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The effects of citrinin in the maintenance of the homeostasis of the reactive oxygen species in rat liver cells were evaluated. Citrinin (CTN) modifies the antioxidant enzymatic defences of cells through the inhibition of GSSG-reductase and transhydrogenase. No effect was observed on GSH-peroxidase, catalase, glucose 6-phosphate and 6 phosphogluconate dehydrogenases, and superoxide dismutase. The mycotoxin increased the generation of reactive oxygen species, stimulating the production of the superoxide anion in the respiratory chain. The results suggest that oxidative stress is an important mechanism, side by side with other effects previously shown, in the establishment of the cytotoxicity and cellular death provoked by CTN in several tissues.

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The activities of the enzymes NADH dehydrogenase, NADH cytochrome e reductase, succinate dehydrogenase, succinate cytochrome e reductase, cytochrome c oxidase and citrate synthase in normal and sick human skeletal muscle mitochondria were determined. A control group was formed by 13 normal people and without using continuous medication. The patient group was formed by 10 people whose pathological diagnosis indicated suspicion of mitochondrial myopathy. A decrease in the activity of the enzymes in all patient was observed: 7 with abnormality in all the tested enzymes; 2 with deficiencies in all the enzymes except cytochrome e oxidase; and 1 with dysfunction only in the activities of succinate dehydrogenase and succinate cytochrome e reductase. The results indicate multiple or combined deficiencies in the respiratory chain, besides dysfunction of citrate synthase in 9 patients. In one exceptional case, the enzymatic deficiency was restricted to complex II. It is possible to conclude that the methodology used herein is adequate and easily applicable to clinical objectives, and that the results obtained allow characterization of the deficient mitochondrial enzymatic complexes, thus showing that the origin of the diseases is an energetic metabolic dysfunction.

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The effects of methotrexate (MTX) on oxygen uptake by permeabilized HeLa cells were evaluated. MTX did not inhibit state III respiration when the oxidizable substrate was succinate, but when the substrates were 2-oxoglutarate or isocitrate the respiration decreased about 50 per cent at 1.0 mM concentration of the drug. This effect was explained by inhibition of 2-oxoglutarate and isocitrate dehydrogenases by MTX. No effect was observed on succinate dehydrogenase. An evaluation of the effects of MTX on malic enzyme activity as measured by pyruvate plus lactate production in intact cells supplied with malate showed a decrease of about 40 per cent in metabolite production using 0.4 mM MTX. HeLa cell malic enzyme, as observed for other tumour cells, is compartmentalized in mitochondria and cytosol, and is another example of a dehydrogenase inhibited by MTX.

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The effects of citrinin on energy production along the respiratory chain and on glycolytic lactate production were examined in BHK-21 cultured cells. Citrinin inhibited the oxygen consumption rate by about 45 per cent. The respiratory rate of digitonin-treated cells energized with succinate, in the presence of ADP, was reduced by about 39 per cent. The mycotoxin inhibited the glucose utilization of BHK-21 cells by about 86 per cent. Cells treated with citrinin produced a small quantity of pyruvate, but were unable to produce lactate. It is concluded that BHK-21 cells cannot generate lactate when oxidative metabolism is inhibited by citrinin. The perturbations in BHK-21 cells caused by citrinin are due to alterations in mitochondrial function and in the glycolytic anaerobic pathway.

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The effects of the mycotoxin citrinin on renal cortical and liver mitochondrial swelling were studied. Citrinin decreases the rate of swelling induced by the valinomycin-K+ complex, suggesting that the mycotoxin interferes with the mitochondrial membrane fluidity. Citrinin promotes reduction of the amplitude of swelling in the presence of Na+ ions. This alteration reflects interference with complex I of the respiratory chain and ATP synthase complex activity without disarranging the inner mitochondrial membrane, in view of the fact that the shrinkage was not affected. The effect increases with citrinin concentration. Renal tissue is more susceptible than hepatic tissue.

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The effect of citrinin on Ca2+ transport was studied in isolated kidney cortex and liver mitochondria, and baby hamster kidney cultured cells. The mycotoxin significantly inhibited the activity of 2-oxoglutarate and pyruvate dehydrogenases in both kidney cortex and liver mitochondria. Citrinin promoted a decrease in the velocity and in the total capacity of Ca2+ uptake, in both mitochondria. Apparently, citrinin acts by a mechanism similar to ruthenium red. In intact cultured cells, citrinin also had a preferential effect on mitochondrial Ca2+ fluxes. Citrinin promoted a marked decrease in the Ca2+ level in the mitochondrial matrix, whereas that of the extramitochondiral fraction became less affected. All the observed effects were dependent on the citrinin concentration.

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The cytotoxicity of citrinin was evaluated in an established cell line of baby hamster kidney cells. The primary effect of the mycotoxin was on the adherence of the cells to the culture bottles. Microscopic evaluation of morphological alterations indicated that the cells which were originally elongated and flattened, became swollen and round. Electron microscopic examination showed that citrinin (0.1, 0.5 and 1.0 mM) incubated for 10 hours with cultured cells, promoted drastic alterations of normal mitochondria, with swelling and cell death. Transplasma membrane redox system is inhibited by citrinin (81%). This effect is dependent not only on the toxin concentration, but also on the time of exposure to the cells.

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Enalapril maleate (EM) is the salt of N-[(S)-1-ethoxycarbonyl)-3-phenylpropyl]-L-alanyl-L-proline, used therapeutically as an anti-hypertensive agent. The effects of EM on some aspects of the energy metabolism and membrane properties of mitochondria from rat liver and kidney cortex were studied, but only the latter were significantly affected. With 0.8 mM of EM and 2-oxoglutarate as oxidizable substrate for isolated mitochondria from rat kidney cortex, the findings were: (a) inhibition of the respiratory rate in state III (37 per cent) and decrease (45 per cent) in respiratory control ratio (RCR), with only one addition of ADP; (b) reinforcement of the inhibition when a second addition of ADP was made; (c) no significant effect either on the rate of respiration in state IV or on the ADP/O ratio; (d) no effect on the ATPase activity of mitochondria from liver or kidney cortex; (e) inhibition of the transmembrane potential (delta psi) after a second addition of ADP; (f) inhibition of the 2-oxoglutarate dehydrogenase complex. It is suggested that in kidney mitochondria, EM interferes in the gluconeogenesis dependence of at least five substrates: 2-oxoglutarate, glutamine, glutamate, lactate, and pyruvate. Also, EM may inhibit Na+/H+ exchange causing natriuresis.

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The mycotoxin citrinin, depressed the phosphorylation efficiency of liver mitochondria as deduced from a decrease of respiratory coefficient and of the ADP/O ratio. Citrinin (1.0 mM) inhibited some enzymes linked to the respiratory chain, namely NADH oxidase and NADH cytochrome c reductase involved with complex I. The activities of enzymes related with other enzymatic complexes of the respiratory chain were either unaffected or enhanced. ATPase activity was inhibited by the mycotoxin. Malate, glutamate, and 2-oxoglutarate dehydrogenases were also inhibited. The transmembrane potential (delta psi), developed by energized mitochondria and depolarization on the addition of ADP, was decreased. The results suggest that citrinin promotes a partial dissipation of the transmembrane potential, different from that resulting from a classical uncoupler such as 2,4-dinitrophenol.

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Citrinin depresses the phosphorylation efficiency of rat renal cortical mitochondria, as inferred from the decrease of the respiratory control coefficient (RC) and ADP/O ratios. The transmembrane potential (delta psi) developed by energized mitochondria and the depolarization upon ADP addition are also decreased. Citrinin (1.0 mM) inhibits almost all enzymes linked to the respiratory chain and increases the activity of succinate cytochrome c reductase and succinate oxidase (coupled). Malate and glutamate dehydrogenases are also inhibited. The inhibitory action of citrinin on phosphorylation efficiency could be related to the following findings: the effect on complex I; the action on the ATP synthetase complex; the partial inhibition of the transmembrane potential.

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1. We studied mitochondria of digitonin-permeabilized HeLa cells, since digitonin (60 micrograms/10(6) cells) increases plasma membrane Ca2+ permeability, in order to avoid problems such as low mitochondrial yield and the possibility of obtaining damaged or uncoupled mitochondria from tumor cells. 2. Addition of Ca2+ to digitonin-permeabilized HeLa cells gave rise to a cycle of respiratory stimulation. Ca2+ uptake was almost totally inhibited by antimycin A (6.0 micrograms/ml). 3. Ca2+ release occurred upon addition of carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP) to digitonin-permeabilized HeLa cells, under steady-state conditions. An antimycin A- and FCCP-insensitive Ca2+ uptake was also detected in this preparation when ATP was added, which reflects Ca2+ capture by other organelles. 4. The characteristics of the mitochondrial Ca2+ transport system in HeLa cells are similar to those of other previously studied tumor cells. Mitochondria from HeLa cells are resistant to the deleterious effects of massive Ca2+ loads.

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We studied mitochondria of digitonin-permeabilized Hela cells, since digitonin (60 ug/10 6 cells) increases plasma membrane Ca2+ permeability, in order to avoid problems such as low mitochondrial yield and the possibility of obtaining damaged or uncoupled mitochondria from tumor cells. Addition of Ca2+ to digitonin-permeabilized Hela cells gave rise to a cycle of respiratory stimulation. Ca2+ uptake was almost totally inhibited by antimycin A (6.0 ug/ml). Ca2+ release occurred upon addition of carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP) to digitonin-permeabilized Hela cells, under state conditions. An antimycin A-and FCCP-insensitive Ca2+ uptake was also detected in this preparation when ATP was added, which reflects Ca2+ capture by other organelles. The characteristics of the mitochondrial Ca2+ transport system in Hela cells are similar to those of other previously studied tumor cells. Mitochondria from Hela cells are resistant to the deleterious effects of massive Ca2+ loads

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The effect of methotrexate (MTX) on the mitochondrial oxidation of cytosolic-reducing equivalents in HeLa cells was studied. MTX inhibited (100 per cent) malate dehydrogenase activity, but no effect was observed on that of GOT. MTX (0.5 mM) inhibited (100 per cent) the activity of reconstituted enzymatic system MDH-GOT, probably as a consequence of inhibition of malate dehydrogenase activity. MTX decreased pyruvate production (54 per cent), demonstrating its inhibitory action on the malate-aspartate shuttle. Blockage of the malate-aspartate shuttle by MTX accounts for the decrease in cellular energetic gain. The results obtained are consistent with the view that in HeLa cells, as well as in other tumour cells, the transport of reducing equivalents from cytoplasmic NADH into the respiratory chain of mitochondria is via the malate-aspartate shuttle.

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Effects of methotrexate (MTX) on mitochondrial oxidative metabolism and ion transport were studied. MTX decreases the membrane potential (delta psi) upon energization of the mitochondrial membrane by NAD+-linked substrates and decreases the amplitude and velocity of swelling induced by glutamate and alpha-ketoglutarate. MTX also has an inhibitory effect on the activities of the oxidation enzymes of NAD+-linked substrates without interfering with the oxidation systems of FAD-linked substrates. The effects of MTX could be interpreted as a consequence of a decrease in the ionic conductivity of the mitochondrial inner membrane.

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Effect of methotrexate (MTX) on mitochondrial oxygen uptake, oxidative phosphorylation and on the activity of several enzymes linked to respiratory chain was studied. MTX was able to inhibit state III respiration activated by ADP and to decrease the respiratory coefficient with the substrates alpha-ketoglutarate and glutamate; these effects became pronounced when mitochondria were pre-incubated with MTX for 10 min. No effect was observed on ATPase activity of undamaged or broken mitochondria; the same was true for NADH-oxidase, NADH-dehydrogenase, NADH-cytochrome c reductase, succinate oxidase, and cytochrome c oxidase activity. The effect on the steady-state of cytochrome b, as well as, the inhibitory effect on state III of respiration with NAD+-linked substrates, offers a reasonable possibility to suggesting that the inhibition site of MTX could be in a place anterior to cytochrome b region, and not linked to respiratory chain.

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