Biomedical subjects

Dario Ghigo

Publications and source records attributed to Dario Ghigo.

At least 19 recordsLinked to original sources

PubMed2006

Statins revert doxorubicin resistance via nitric oxide in malignant mesothelioma.

Human malignant mesothelioma (HMM) is resistant to many anticancer drugs, including doxorubicin. Mevastatin and simvastatin, 2 inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase, potentiated the intracellular accumulation and the cytotoxicity of doxorubicin in HMM cells constitutively expressing P-glycoprotein and multidrug resistance-associated protein 3. This effect of statins was nitric oxide (NO)-dependent, since it was reverted by either an NO synthase inhibitor or an NO scavenging system. The NO synthase up-regulation in HMM and other cells is known to be associated with the activation of the transcription factor NF-kappaB: in HMM cells statins increased the NF-kappaB translocation into the nucleus, decreased the level of the NF-kappaB inhibitor IkBalpha and increased the phosphorylation/activation of IkB kinase alpha (IKKalpha). IKKalpha is under the negative control exerted by RhoA in its prenylated (active) form: incubation of HMM cells with statins lowered the amount of active RhoA and the level of Rho-associated kinase activity. All statins' effects were reverted by mevalonic acid, thus suggesting that they were mediated by the inhibition of HMGCoA reductase and were likely to be subsequent to the reduced availability of precursor molecules for RhoA prenylation. Both the Rho kinase inhibitor Y27632 and the RhoA inhibitor toxin B (from Clostridium difficile) mimicked the statins' effects, enhancing doxorubicin accumulation, NO synthesis and IKKalpha phosphorylation and decreasing the amount of IkBalpha in HMM cells. Simvastatin, Y27632 and toxin B elicited tyrosine nitration in the P-glycoprotein, thus providing a likely mechanism by which NO reverts the doxorubicin resistance in HMM cells.

Amides↗

PubMed2006

Cycling of NADPH by glucose 6-phosphate dehydrogenase optimizes the spectrophotometric assay of nitric oxide synthase activity in cell lysates.

The measurement of nitric oxide synthase activity in cell lysates is often performed by radiochemical assay that quantifies the conversion of L-[3H]arginine to L-[3H]citrulline. We have developed a spectrophotometric procedure which continuously recycles NADPH through the addition of glucose 6-phosphate dehydrogenase to the cell lysate. This allows nitric oxide synthase to operate linearly for hours, so that nitric oxide-derived nitrite accumulates at amounts sufficient to be detected with the Griess assay. The incorporation of cycling of NADPH also improves the radiochemical assay for nitric oxide synthase activity.

Animals↗

PubMed2005

The NADPH oxidase inhibitor apocynin (acetovanillone) induces oxidative stress.

Apocynin (acetovanillone) is often used as a specific inhibitor of NADPH oxidase. In N11 glial cells, apocynin induced, in a dose-dependent way, a significant increase of both malonyldialdehyde level (index of lipid peroxidation) and lactate dehydrogenase release (index of a cytotoxic effect). Apocynin evoked also, in a significant way, an increase of H(2)O(2) concentration and a decrease of the intracellular glutathione/glutathione disulfide ratio, accompanied by augmented efflux of glutathione and glutathione disulfide. Apocynin induced the activation of both pentose phosphate pathway and tricarboxylic acid cycle, which was blocked when the cells were incubated with glutathione together with apocynin. The cell incubation with glutathione prevented also the apocynin-induced increase of malonyldialdehyde generation and lactate dehydrogenase leakage. Apocynin exerted an oxidant effect also in a cell-free system: indeed, in aqueous solution, it evoked a faster oxidation of the thiols glutathione and dithiothreitol, and elicited the generation of reactive oxygen species, mainly superoxide anions. Our results suggest that apocynin per se can induce an oxidative stress and exert a cytotoxic effect in N11 cells and other cell types, and that some effects of apocynin in in vitro and in vivo experimental models should be interpreted with caution.

Acetophenones↗

PubMed2005

Na+/H+ exchanger activity is increased in doxorubicin-resistant human colon cancer cells and its modulation modifies the sensitivity of the cells to doxorubicin.

Multidrug resistant (MDR) tumor cells exhibit an altered pH gradient across different cell compartments, which favors a reduced intracellular accumulation of antineoplastic drugs and a decreased therapeutic effect. In our study, we have observed that the activity and expression of Na+/H+ exchanger (NHE), which is involved in the homeostasis of intracellular pH (pHi), are increased in doxorubicin-resistant (HT29-dx) human colon carcinoma cells in comparison with doxorubicin-sensitive HT29 cells. The pH(i) was significantly higher in HT29-dx cells, which accumulated less doxorubicin than HT29 cells. The NHE inhibitor 5-(N-ethyl-N-isopropyl)amiloride (EIPA) significantly reduced the pHi value and increased the intracellular accumulation of doxorubicin in both cell populations: in the presence of EIPA HT29-dx cells accumulated as much drug as control HT29 cells. On the other hand, monensin, a Na+/H+ ionophore mimicking NHE activation, and phorbol 12-myristate 13-acetate (PMA), which stimulates NHE, significantly increased the pHi and decreased the drug accumulation in HT29 cells to values similar to those observed in control HT29-dx cells. EIPA potentiated the cytotoxic effect of doxorubicin in HT29 cells, and made HT29-dx cells as sensitive to the cytotoxic effect of the drug as control HT29 cells. Instead, PMA and monensin made HT29 cells as insensitive to doxorubicin as HT29-dx cells. These results suggest that in MDR cells the higher cytosolic pH is likely to decrease drug accumulation, and that such resistance can be reverted by inhibiting the NHE activity. This result opens the possibility to revert MDR with the clinical use of NHE inhibitors.

Amiloride↗

PubMed2005

Different cellular responses evoked by natural and stoichiometric synthetic chrysotile asbestos.

The carcinogenic potency of asbestos, including chrysotile, is well established. Several physico-chemical features of the fibers appear implied, such as fibrous habit, size, crystallinity, morphology, and surface active metal ions, where free radical generation may take place. In contrast to other asbestos forms, iron is not a stoichiometric component of chrysotile, but is only present together with other extraneous ions as a magnesium- and silicon-replacing contaminant. To determine the role played by contaminating ions and morphological features of the fibers, a stoichiometric chrysotile with constant structure and morphology was synthesized in hydrothermal conditions. Free radical generation and the effects of these fibers on human lung epithelial A549 cells have been compared to that elicited by a well known toxic natural chrysotile (UICC A, from Rhodesia). After a 24-h incubation, the natural, but not the synthetic, form exerted a cytotoxic effect, detected as leakage of lactate dehydrogenase. Homolytic rupture of a C-H bond and lipoperoxidation in A549 cells took place in the presence of the natural, but not of the synthetic, chrysotile. Antioxidant systems were also affected differently. The pentose phosphate pathway and its regulatory enzyme glucose 6-phosphate dehydrogenase were markedly inhibited only by the natural specimen, which also caused a depletion of intracellular reduced glutathione in A549 cells. These results suggest that metal ions, fiber size and state of the surface play a crucial role in the oxidative stress caused by chrysotile asbestos. Stoichiometric synthetic fibers may thus be proposed as a reference standard (negative control) for toxicological studies.

Antioxidants↗

PubMed2005

Nitric oxide reverts the resistance to doxorubicin in human colon cancer cells by inhibiting the drug efflux.

Multidrug resistance (MDR) is a phenomenon by which cancer cells evade the cytotoxic effects of chemotherapeutic agents. It may occur through different mechanisms, but it often correlates with the overexpression of integral membrane transporters, such as P-glycoprotein (Pgp) and MDR-associated proteins (MRPs), with resulting decrease of drug accumulation and cellular death. Doxorubicin is a substrate of Pgp; it has been suggested that its ability to induce synthesis of nitric oxide (NO) could explain, at least in part, its cytotoxic effects. Culturing the human epithelial colon cell line HT29 in the presence of doxorubicin, we obtained a doxorubicin-resistant (HT29-dx) cell population: these cells accumulated less intracellular doxorubicin, were less sensitive to the cytotoxic effects of doxorubicin and cisplatin, overexpressed Pgp and MRP3, and exhibited a lower NO production (both under basal conditions and after doxorubicin stimulation). The resistance to doxorubicin could be reversed when HT29-dx cells were incubated with inducers of NO synthesis (cytokines mix, atorvastatin). Some NO donors increased the drug accumulation in HT29-dx cells in a guarosine-3':5'-cyclic monophosphate-independent way; this effect was associated with a marked reduction of doxorubicin efflux rate in HT29 and HT29-dx cells, and tyrosine nitration in the MRP3 protein. Our results suggest that onset of MDR and impairment of NO synthesis are related; this finding could point to a new strategy to reverse doxorubicin resistance in human cancer.

Antibiotics, Antineoplastic↗

PubMed2005

Potential toxicity of nonregulated asbestiform minerals: balangeroite from the western Alps. Part 1: Identification and characterization.

In the Italian western Alps, asbestos mineralization (both chrysotile and tremolite amphibole) takes place from serpentinites, together with other less common asbestiform minerals not regulated by the current legislation. In the context of a study on the evaluation of the asbestos risk in this area, the possible role played by the associated asbestiform minerals in the overall toxicity of the airborne fraction has been examined. The first mineral investigated was balangeroite [(Mg,Fe2+,Fe3+,Mn2+)42Si16O54(OH)36], an iron-rich asbestiform contaminant of chrysotile from the Balangero mine (Piedmont), which crystallizes as rigid and brittle fibers. In order to prepare a sample in a form appropriate for chemical and cellular tests, the fibers were separated from the rock and comminuted without damage to their crystalline structure and surface state (as confirmed by X-ray diffraction [XRD] and ultraviolet-visible [UV-Vis] spectroscopy). The first properties examined were durability in simulated body fluids (Gamble's solution) and toxicity to epithelial cells. When compared to UICC crocidolite (the amphibole blue asbestos, regarded as the most pathogenic form), balangeroite appears even more durable than crocidolite. Balangeroite and UICC crocidolite showed a similar in vitro cytotoxic effect on a human epithelial cell line, as evidenced by leakage of intracellular lactate dehydrogenase (LDH) activity, which, observed after a 24-h incubation, was dose dependent and maximal at 12 microg/cm2 for each fiber type. Data show that chemical composition, form, durability, and cell toxicity indicate balangeroite as a potentially harmful fibrous mineral that needs to be examined by further chemical and cellular tests.

Asbestos, Serpentine↗

PubMed2005

Potential toxicity of nonregulated asbestiform minerals: balangeroite from the western Alps. Part 2: Oxidant activity of the fibers.

The asbestiform mineral balangeroite [(Mg,Fe2+,Fe3+,Mn2+)42Si16O54(OH)36], whose toxic potential is unknown, is associated with chrysotile asbestos in the western Alps (Balangero mine, Piedmont, Italy). In order to examine whether such fibers may contribute to the oxidative damage produced by local asbestos dusts when inhaled, balangeroite was studied by means of both cell-free and cellular tests, comparing the results with those concerning the most pathogenic asbestos form, crocidolite. Similarly to the crocidolite surface, iron was mobilized from balangeroite by chelators, to a different extent: deferoxamine > ascorbic acid > ferrozine. Poorly coordinated surface ions, as evaluated from the adsorption of NO as a probe molecule (by both calorimetry and infrared spectroscopy), are even more abundant on balangeroite than on crocidolite. The spin trapping technique shows that surface iron-derived Fenton activity (HO* from H2O2) is similar for the two fiber types, while a pretreatment in ascorbic acid, by reducing previously oxidized surface iron, activates the potential to cleave a C-H bond (yielding *CO2- from formate anion). Balangeroite, like crocidolite, produces nitrite accumulation, lipid peroxidation, and NO synthase activation in a human lung epithelial cell line (A549). All these findings, regarded as features related to the toxic potential of asbestos, suggest that balangeroite may be a potentially hazardous fiber per se and could be partly responsible for lung diseases reported in epidemiological studies in exposed miners.

Adsorption↗

PubMed2005

Potential toxicity of nonregulated asbestiform minerals: balangeroite from the western Alps. Part 3: Depletion of antioxidant defenses.

The asbestiform fibrous silicate balangeroite exhibits cytotoxic and oxidative properties similar to those exerted by crocidolite asbestos. In human lung epithelial cells A549, balangeroite, like crocidolite, inhibited the pentose phosphate pathway (PPP), one of the main antioxidant intracellular tools; this inhibition was exerted also when PPP was activated by the redox-cycling compound menadione. PPP inhibition may be accounted for by the inhibition of its rate-limiting enzyme, glucose-6-phosphate dehydrogenase (G6PD). Reduced glutathione (GSH), the most important intracellular antioxidant molecule, was decreased by both balangeroite and crocidolite incubation. This effect was not related to any increased content of oxidized glutathione, or to any enhanced efflux of glutathione, suggesting that balangeroite fibers, like crocidolite, might favor the reaction of GSH with other molecules.

Antioxidants↗

PubMed2004

Diphenyleneiodonium inhibits the cell redox metabolism and induces oxidative stress.

Diphenyleneiodonium (DPI) and the structurally related compound diphenyliodonium (DIP) are widely used as inhibitors of flavoenzymes, particularly NADPH oxidase. Here we report further evidence that DPI and DIP are not specific flavin binders. A 3-h incubation of N11 glial cells with DPI significantly inhibited in a dose-dependent way both the pentose phosphate pathway and the tricarboxylic acid cycle. In parallel, we observed a dose-dependent increase of reactive oxygen species generation and lipoperoxidation and increased leakage of lactate dehydrogenase activity in the extracellular medium. The glutathione/glutathione disulfide ratio decreased, whereas the efflux of glutathione out of the cells increased. This suggests that DPI causes an augmented oxidative stress and exerts a cytotoxic effect in N11 cells. Indeed, the cells were protected from these events when loaded with glutathione. Similar results were observed using DIP instead of DPI and also in other cell types. We suggest that the DPI-elicited inhibition of the pentose phosphate pathway and tricarboxylic acid cycle may be mediated by the blockade of several NAD(P)-dependent enzymes, such as glucose 6-phosphate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, and lactate dehydrogenase. In light of these results, we think that some effects of DPI or DIP in in vitro and in vivo experimental models should be interpreted with caution.

Animals↗

PubMed2004

Simian virus 40 infection down-regulates the expression of nitric oxide synthase in human mesothelial cells.

The cytotoxic effects of asbestos are partly mediated by the production of free radicals, including nitric oxide (NO). SV40 has been suggested to synergize with asbestos in the pathogenesis of malignant mesothelioma. Crocidolite asbestos fibers induced in human mesothelial and malignant mesothelioma cells a significant increase of NO synthase activity and expression, which was absent in SV40-infected cells. Furthermore, SV40 infection prevented the NF kappa B activation elicited by crocidolite in both mesothelial and mesothelioma cells. These data suggest that SV40, by inhibiting the synthesis of NO, could favor the survival of transformed, potentially neoplastic cells.

Asbestos, Crocidolite↗

PubMed2003

Long and short fiber amosite asbestos alters at a different extent the redox metabolism in human lung epithelial cells.

The mechanism by which asbestos fibers are fibrogenic and tumorigenic is still matter of debate. The higher pathogenicity of longer fibers has been traditionally associated with their slower clearance in respiratory airways. However, short amosite fibers, obtained by grinding longer ones, exhibited a lower potential to damage nude DNA and a lower in vitro cytotoxicity. We have thus revisited the two sets of long and short fibers in order to compare their surface properties to their activity in cell systems. In this study we report that, in human lung epithelial cells A549, long amosite fibers, more effectively than the short ones, initiate free radical reactions, inhibit the glucose 6-phosphate dehydrogenase activity and the pentose phosphate pathway, decrease the intracellular level of reduced glutathione, and increase the generation of thiobarbituric acid reactive substances and the leakage of lactate dehydrogenase in the extracellular medium. These results suggest that the shortening of fibers by prolonged milling affects not only their biopersistence, but also their surface properties, hence their interaction with cellular metabolism. Our data provide also a mechanism by which asbestos fibers inhibit the pentose phosphate pathway, i.e., via the oxidative inhibition of glucose 6-phosphate dehydrogenase, which is prevented by reduced glutathione.

Animals↗

PubMed2003

Nitroarginine methyl ester and canavanine lower intracellular reduced glutathione.

In rat glial cells, arginine analogs N(G)-nitroarginine methyl ester (both D- and L-stereoisomer) and L-canavanine lower the intracellular levels of reduced glutathione, stimulate the pentose phosphate pathway, increase the level of malonyldialdehyde, and increase the leakage of lactate dehydrogenase. These effects are not related to the inhibition of nitric oxide synthase and depend on the oxidation of intracellular thiols; indeed, there are no signs of lipoperoxidation and cytotoxicity in cells previously loaded with glutathione. Furthermore, these arginine analogs elicit an oxidative burst in N11 cells and decrease the detectable level of both glutathione and dithiothreitol in cell-free experiments. These effects were not observed with the arginine analog N(G)-monomethyl-L-arginine, suggesting that the substituting moiety in (or near) the guanidine group could modify the reactivity of the arginine analogs with thiol compounds.

Animals↗

PubMed2003

Artemisinin inhibits inducible nitric oxide synthase and nuclear factor NF-kB activation.

Artemisinin is a natural product used as an alternative drug in the treatment of severe and multidrug-resistant malaria. In the present work we show that artemisinin shares with other sesquiterpene lactones the ability to inhibit the activation of the nuclear factor NF-kB: by this mechanism, artemisinin, as well as parthenolide, inhibits nitric oxide synthesis in cytokine-stimulated human astrocytoma T67 cells. These results suggest that artemisinin, in addition to its antiparasitic properties, could also exert a therapeutic effect on neurological complications of malaria.

Animals↗

PubMed2003

Nitric oxide synthesis in human nonpregnant myometrium and uterine myomas.

OBJECTIVE: To clarify whether nitric oxide (NO) synthesis in myomas differs from that in parental human myometrium. DESIGN: Prospective study. SETTING: Academic research institution. PATIENT(S): Twenty-one patients undergoing laparoscopy or laparotomy for uterine myoma. MAIN OUTCOME MEASURE(S): Measurement of NO synthase activity in homogenates from myoma and parental myometrium biopsies, and NO synthesis assessment in cultured smooth-muscle cells. RESULT(S): Nitric oxide synthase activity in homogenates did not significantly differ between myoma and healthy myometrium. The medium taken from myoma cultures showed a significant increase in nitrite concentration compared with that taken from control myometrium cultures, but 24-hour incubation of both cell types with physiologic concentrations of 17beta-estradiol or progesterone did not significantly modify nitrite production. CONCLUSION(S): The maximal activity of NO synthase does not differ in myoma cells and in normal myometrial cells, but basal NO synthesis seems to be enhanced by an unknown signaling pathway that is not controlled by 17beta-estradiol or progesterone.

Adult↗

PubMed2002

Doxorubicin induces an increase of nitric oxide synthesis in rat cardiac cells that is inhibited by iron supplementation.

Doxorubicin is an anthracycline antibiotic generally used in the treatment of solid tumors, but its use is limited by a severe cardiotoxicity, which has been related to the generation of oxygen- and nitrogen-derived free radicals. We have demonstrated that doxorubicin induces nitric oxide (NO) synthesis in the rat cardiac cells H9c2: the drug, after a 24-h incubation, evoked a dose-dependent increase of both NO synthase (NOS) activity in the cells and nitrite levels in the culture supernatant; the accumulation of nitrite (a stable derivative of NO) was prevented by different NOS inhibitors. The increase of NO production was associated with an increased expression of the inducible NOS isoform gene. These effects were significantly inhibited by the coincubation of doxorubicin with iron nitrilotriacetate, a compound that releases iron into the cells. Our results suggest that doxorubicin could induce NO generation in cardiac cells by modifying the iron homeostasis.

Animals↗

PubMed2002

Crocidolite asbestos inhibits pentose phosphate oxidative pathway and glucose 6-phosphate dehydrogenase activity in human lung epithelial cells.

The cytotoxicity of asbestos has been related to its ability to increase the production of reactive oxygen species (ROS), via the iron-catalyzed reduction of oxygen and/or the activation of NADPH oxidase. The pentose phosphate pathway (PPP) is generally activated by the cell exposure to oxidant molecules. Contrary to our expectations, asbestos (crocidolite) fibers caused a dose- and time-dependent inhibition of PPP and decreased its activation by an oxidative stress in human lung epithelial cells A549. In parallel, the intracellular activity of the PPP rate-limiting enzyme, glucose 6-phosphate dehydrogenase (G6PD), was significantly diminished by crocidolite exposure. This inhibition was selective, as the activity of other PPP and glycolysis enzymes was not modified, and was not attributable to a decreased expression of G6PD. On the opposite, the incubation with glass fibers MMVF10 did not modify PPP and G6PD activity. PPP and G6PD inhibition did not correlate with the increased nitric oxide (NO) production elicited by crocidolite in A549 cells. Experiments with the purified enzyme suggest that crocidolite inhibits G6PD by directly interacting with the protein. We propose here a new mechanism of asbestos-evoked oxidative stress, wherein fibers increase the intracellular ROS levels also by inhibiting the main antioxidant pathway of the cell.

Asbestos, Crocidolite↗

PubMed2002

Guanylate cyclase activity and sperm function.

In species with external fertilization, the guanylate cyclase family is responsible for the long-distance interaction between gametes, as its activation allows sperm chemotaxis toward egg-derived substances, gamete encounter, and fertilization. In species with internal fertilization, guanylate cyclase-activating substances, which are secreted by several tissues in the genital tracts of both sexes, deeply affect sperm motility, capacitation, and acrosomal reactivity, stimulating sperm metabolism and promoting the ability of the sperm to approach the oocyte, interact with it, and finally fertilize it. A complex system of intracellular pathways is activated by guanylate cyclase agonists in spermatozoa. Sperm motility appears to be affected mainly through an increase in intracellular cAMP, whereas the acrosome reaction depends more directly on cyclic GMP synthesis. Both cyclic nucleotides activate specific kinases and ion signals. A complex cross-talk between cAMP- and cyclic GMP-generating systems occurs, resulting in an upward shift in sperm function. Excessive amounts of certain guanylate cyclase activators might exert opposite, antireproductive effects, increasing the oxidative stress on sperm membranes. In view of the marked influence exerted by guanylate cyclase-activating substances on sperm function, it seems likely that guanylate cyclase activation or inhibition may represent a new approach for the diagnosis and treatment of male and/or female infertility.

Animals↗