This study was designed to identify the set of functions activated in cultured endothelial cells by the hematopoietic growth factors, granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage-colony-stimulating factor (GM-CSF), and to compare them with those elicited by prototypic cytokines active on these cells. Moreover, indications as to the in vivo relevance of in vitro effects were obtained. G-CSF and GM-CSF induced endothelial cells to proliferate and migrate. In contrast, unlike appropriate reference cytokines (IL-1 and tumor necrosis factor, IFN-gamma), G-CSF and GM-CSF did not modulate endothelial cell functions related to hemostasis-thrombosis (production of procoagulant activity and of platelet activating factor), inflammation (expression of leukocyte adhesion molecule-1 and production of platelet activating factor), and accessory function (expression of class II antigens of MHC). Other colony-stimulating factors (IL-3 and macrophage-colony-stimulating factor) were inactive on all functions tested. In comparison to basic fibroblast growth factor (bFGF), G-CSF and GM-CSF induced lower maximal proliferation of endothelial cells, whereas migration was of the same order of magnitude. G-CSF and GM-CSF stimulated repair of mechanically wounded endothelial monolayers. Exposure to both cytokines induced shape changes and cytoskeletal reorganization consistent with a migratory phenotype. To explore the in vivo relevance of the in vitro effects of these cytokines on endothelium, we studied the angiogenic activity of human G-CSF in the rabbit cornea. G-CSF, but not the heat-inactivated molecule, had definite angiogenic activity, without any sign of inflammatory reactions. G-CSF was less active than bFGF. However, the combination of a nonangiogenic dose of bFGF with G-CSF resulted in an angiogenic response higher than that elicited by either individual cytokines. Thus, G-CSF and GM-CSF induce endothelial cells to express an activation/differentiation program (including proliferation and migration) related to angiogenesis

It is unknown whether zoledronic acid (ZA) at clinically relevant doses is active against tumours not located in bone. Mice transgenic for the activated ErbB-2 oncogene were treated with a cumulative number of doses equivalent to that recommended in human beings. A significant increase in tumour-free and overall survival was observed in mice treated with ZA. At clinically compatible concentrations, ZA modulated the mevalonate pathway and affected protein prenylation in both tumour cells and macrophages. A marked reduction in the number of tumour-associated macrophages was paralleled by a significant decrease in tumour vascularization. The local production of vascular endothelial growth factor and interleukin-10 was drastically down-regulated in favour of interferon-γ production. Peritoneal macrophages and tumour-associated macrophages of ZA-treated mice recovered a full M1 antitumoral phenotype, as shown by nuclear translocation of nuclear factor kB, inducible nitric oxide synthase expression and nitric oxide production. These data indicate that clinically achievable doses of ZA inhibit spontaneous mammary cancerogenesis by targeting the local microenvironment, as shown by a decreased tumour vascularization, a reduced number of tumour-associated macrophages and their reverted polarization from M2 to M1 phenotype.

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 dosedependent 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. Diphenyleneiodonium (DPI) 1 and the structurally related compound diphenyliodonium (DIP) are widely used as uncompetitive inhibitors of flavoenzymes. Firstly identified as a hypoglycemic agent able to block gluconeogenesis and respiration in rat liver (1), DPI has been subsequently shown to inhibit the activity of NADH:ubiquinone oxidoreductase (2, 3), NADPH oxidase (4 -6), nitric-oxide synthase (7), xanthine oxidase (6), and NADPH cytochrome P450 oxidoreductase (8). DPI and other iodonium derivatives have been shown to react via a radical mechanism, whereby an electron is abstracted from FAD or FMN to form a radical, which then adds back to the flavin to form covalent, phenylated adducts (9). Also, heme groups, such as the heme b of NADPH oxidase, have been found to react with DPI and DIP (10).In most experimental works of the last years, DPI or DIP have been used as inhibitors of NADPH oxidase. NADPH oxidases are a group of plasma membrane-associated enzymes found in a variety of cells of mesodermal origin. The most thoroughly studied is the leukocyte isoform, which catalyzes the production of superoxide (O 2 . ) by the one-electron reduction of oxygen, using NADPH as the reducing agent (11). The O 2 .generated by NADPH oxidase serves as the starting material for the production of a vast assortment of reactive oxidants used by phagocytes to kill invading microorganisms or tumor cells (11). A low activity NADPH oxidase is present in a variety of nonphagocytic cells, wherein this enzyme is a source of second messengers. It has been postulated, for instance, that the O 2 . generated by the aorta functions as a blood ...

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.

BackgroundInvasive micropapillary carcinoma (IMPC) of the breast is a distinct and aggressive variant of luminal type B breast cancer that does not respond to neoadjuvant chemotherapy. It is characterized by small pseudopapillary clusters of cancer cells with inverted cell polarity. To investigate whether hypoxia-inducible factor-1 (HIF-1) activation may be related to the drug resistance described in this tumor, we used MCF7 cancer cells cultured as 3-D spheroids, which morphologically simulate IMPC cell clusters.MethodsHIF-1 activation was measured by EMSA and ELISA in MCF7 3-D spheroids and MCF7 monolayers. Binding of HIF-1α to MDR-1 gene promoter and modulation of P-glycoprotein (Pgp) expression was evaluated by ChIP assay and FACS analysis, respectively. Intracellular doxorubicin retention was measured by spectrofluorimetric assay and drug cytotoxicity by annexin V-FITC measurement and caspase activity assay.ResultsIn MCF7 3-D spheroids HIF-1 was activated and recruited to participate to the transcriptional activity of MDR-1 gene, coding for Pgp. In addition, Pgp expression on the surface of cells obtained from 3-D spheroids was increased. MCF7 3-D spheroids accumulate less doxorubicin and are less sensitive to its cytotoxic effects than MCF7 cells cultured as monolayer. Finally, HIF-1α inhibition either by incubating cells with 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (a widely used HIF-1α inhibitor) or by transfecting cells with specific siRNA for HIF-1α significantly decreased the expression of Pgp on the surface of cells and increased the intracellular doxorubicin accumulation in MCF7 3-D spheroids.ConclusionsMCF7 breast cancer cells cultured as 3-D spheroids are resistant to doxorubicin and this resistance is associated with an increased Pgp expression in the plasma membrane via activation of HIF-1. The same mechanism may be suggested for IMPC drug resistance.