The synthetic molecule stauprimide impairs cell growth and migration in triple-negative breast cancer.

Stauprimide, a semi-synthetic derivative of staurosporine, is known mainly for its potent differentiation-enhancing properties in embryonic stem cells. Here, we studied the effects of stauprimide in cell growth and migration of triple-negative breast cancer cells in vitro, evaluating its potential antitumoral activity in an orthotopic mouse model of breast cancer in vivo. Our results from survival curves, EdU incorporation, cell cycle analysis and annexin-V detection in MDA-MB-231 cells indicated that stauprimide inhibited cell proliferation, arresting cell cycle in G2/M without induction of apoptosis. A decrease in the migratory capability of MDA-MB-231 was also assessed in response to stauprimide. In this work we pointed to a mechanism of action of stauprimide involving the modulation of ERK1/2, Akt and p38 MAPK signalling pathways, and the downregulation of MYC in MDA-MB-231 cells. In addition, orthotopic MDA-MB-231 xenograft and 4T1 syngeneic models suggested an effect of stauprimide in vivo, increasing the necrotic core of tumors and reducing metastasis in lung and liver of mice. Together, our results point to the promising role of stauprimide as a putative therapeutic agent in triple-negative breast cancer.

A re-evaluation of the mitogenic effect of serotonin on vascular endothelial cells.

Serotonin is an extracellular mediator recognized by seven different types of receptors, thus giving rise to pleiotropic intracellular responses. One of these responses is the activation of proliferation for a number of cell types. The induction of proliferation of otherwise quiescent endothelial cells is a key step of angiogenesis. Previously published work concerning the effect of serotonin on endothelial cell proliferation is controversial. The present work is aimed to re-evaluate the mitogenic role of serotonin on endothelial cells, since a pro-angiogenic role for serotonin could be hypothesized if its mitogenic potential on these cells were confirmed. By using three different types of endothelial cells and three experimental approaches, we demonstrate that serotonin cannot be considered a general mitogen for endothelial cells.

Contribution by host tissues to circulating glutamine in mice inoculated with Ehrlich ascites tumor cells.

Twenty-four h after tumor transplantation increases of free glutamine in plasma, liver, and kidney occurred simultaneously with the exponential phase of tumor growth. Kidney and muscle glutamine synthetase also increased in the first 2 days following tumor transplantation, while kidney and liver glutaminases decreased. The levels of free glutamine in plasma and tissues, and the activities of glutamine synthetase and glutaminase, tended to approach normal values in the last days of life of the tumor-transplanted animals. Eleven days after transplantation, liver glutamine synthetase activity diminished. The results are discussed in terms of a glutamate/glutamine intercellular cycle which could augment the circulating glutamine, the main source of nitrogen for tumor cells.

Novel application assigned to toluquinol: inhibition of lymphangiogenesis by interfering with VEGF-C/VEGFR-3 signalling pathway.

BACKGROUND AND PURPOSE: Lymphangiogenesis is an important biological process associated with the pathogenesis of several diseases, including metastatic dissemination, graft rejection, lymphoedema and other inflammatory disorders. The development of new drugs that block lymphangiogenesis has become a promising therapeutic strategy. In this study, we investigated the ability of toluquinol, a 2-methyl-hydroquinone isolated from the culture broth of the marine fungus Penicillium sp. HL-85-ALS5-R004, to inhibit lymphangiogenesis in vitro, ex vivo and in vivo. EXPERIMENTAL APPROACH: We used human lymphatic endothelial cells (LECs) to analyse the effect of toluquinol in 2D and 3D in vitro cultures and in the ex vivo mouse lymphatic ring assay. For in vivo approaches, the transgenic Fli1:eGFPy1 zebrafish, mouse ear sponges and cornea models were used. Western blotting and apoptosis analyses were carried out to search for drug targets. KEY RESULTS: Toluquinol inhibited LEC proliferation, migration, tubulogenesis and sprouting of new lymphatic vessels. Furthermore, toluquinol induced apoptosis of LECs after 14 h of treatment in vitro, blocked the development of the thoracic duct in zebrafish and reduced the VEGF-C-induced lymphatic vessel formation and corneal neovascularization in mice. Mechanistically, we demonstrated that this drug attenuates VEGF-C-induced VEGFR-3 phosphorylation in a dose-dependent manner and suppresses the phosphorylation of Akt and ERK1/2. CONCLUSIONS AND IMPLICATIONS: Based on these findings, we propose toluquinol as a new candidate with pharmacological potential for the treatment of lymphangiogenesis-related pathologies. Notably, its ability to suppress corneal neovascularization paves the way for applications in vascular ocular pathologies.

Cell cycle arrest of human hematopoietic progenitors induced by medroxyprogesterone acetate.

Clinical studies have shown that continuous or intermittent scheduled administration of medroxyprogesterone acetate (MPA) reduces the bone marrow toxicity induced by antitumor drugs. This MPA myeloprotection has been attributed to an arrest of hematopoietic progenitors in a quiescent phase, although no in vitro studies have demonstrated such an effect of MPA. Human bone marrow cells were preincubated for 3 days with MPA (100 ng/mL) and then exposed to sublethal doses of adriamycin; LD50 was significantly increased in MPA-preincubated cells (896 +/- 172 ng/mL) vs. control cells (162 +/- 37 ng/mL); this protective effect of MPA was shown to be more efficient against S-phase-specific drugs such as 5-fluorouracil (5-FU) than against non-phase-specific drugs such as cisplatin. MPA did not protect several human leukemic cell lines from the cytotoxic action of adriamycin. "Suicide" assays showed that the percentage of myeloid progenitor cells (granulocyte-macrophage colony-forming units [CFU-GM]) in S-phase was significantly reduced from 67 +/- 2.5% (control cells) to 38 +/- 5.5% (24-hour MPA-preincubated cells). These results demonstrate in vitro that MPA exerts a cell cycle arrest of hematopoietic precursors, protecting them from the toxicity of chemotherapy.

Evaluation of fluorometric and zymographic methods as activity assays for stromelysins and gelatinases.

To measure matrix metalloproteinase (MMP) activity in a large number of samples it is advisable to use easily automated methods. We have evaluated and compared the activity of stromelysin-1 (MMP-3), matrilysin (MMP-7), 72 kDa gelatinase A (MMP-2) and 92 kDa gelatinase B (MMP-9) by zymogram analysis and fluorescent substrate degradation assays. FITC-casein and the fluorogenic peptide Dnp-Pro-beta-cyclo-hexyl-Ala-Gly-Cys(Me)-His-Ala-Lys-(N-Me-Abz)-NH 2 were used as fluorescent substrates. FITC-casein was more efficiently degraded than the fluorogenic peptide by all MMPs tested except MMP-9. MMP-2 was not significantly able to degrade the fluorogenic peptide. Gelatin zymography was the most sensitive method to detect the activity of both gelatinases but quantitation problems compromise its use. The degradation of fluorogenic substrates by MMPs could be inhibited by the chelating agent EDTA and by the tissue inhibitor of metalloproteinases 2 (TIMP-2), an MMP-specific inhibitor. Fluorometric methods represent a good alternative for MMP activity measurement, especially when a large number of samples must be processed.

Effect of palmitate, acetate and glucose on glutamine metabolism in Ehrlich ascites tumor cells.

Acetate and the long chain free fatty acid palmitate provoked a decrease in the rates of glutamine utilization and glutamate production in Ehrlich ascites tumor cells incubated with 0.5 mM glutamine. There was a cumulative effect with glucose on glutamine metabolism.

Malate-citrate cycle during glycolysis and glutaminolysis in Ehrlich ascites tumor cells.

The malate-citrate cycle was studied during aerobic glycolysis and glutaminolysis in a strain of Ehrlich ascites tumor cells which showed a very low malate-aspartate shuttle system activity. The experimental approach includes: estimation of mitochondrial NAD[P]+-dependent malic enzyme activity; respiratory activity of freshly harvested or fasted cells, and of isolated mitochondria; and determination of the metabolites involved in the glycolytic and glutaminolytic pathways. The results suggest that in this strain, the malate-citrate shuttle is not an effective pathway for transferring glycolytic reducing equivalents from cytosol to mitochondria. Less than 15% of the glucose uptake was affected by the 1,2,3-benzenetricarboxylate inhibition of the malate-citrate shuttle. Moreover, in the presence of glucose, the malate-citrate cycle did not appear to play an important role in the glutaminolytic process. The present work supports and extends the finding of previous studies, since the results showed that the glucose metabolism depressed the oxidative processes in Ehrlich ascites tumor mitochondria, not only alone, but also in the presence of glutamine. Interestingly, the high glutamine uptake was maintained in the presence of glucose.

Reversal of P-glycoprotein-mediated multidrug resistance in vitro by AV200, a new ardeemin derivative.

The activity of AV200, a synthetic ardeemin derivative, in reversing the multidrug resistance phenotype has been investigated. At non-toxic doses, AV200 was able to completely restore vincristine and paclitaxel toxicities and partially restore that of doxorubicin in multidrug-resistant cells. The potency of AV200 as a modulator of the resistance to doxorubicin, vincristine and paclitaxel resulted to be seven-, 59 and 12-fold, respectively, higher than that of verapamil. In vitro measurements of rhodamine 123 accumulation in human resistant cells suggest that AV200 reverses multidrug resistance by directly inhibiting the P-glycoprotein-mediated drug efflux. This work underscores the possibility of utilizing ardeemin derivatives as a source of non-toxic modulators of the multidrug resistance phenotype.

Mobilization of glutamine and asparagine in mouse kidney during Ehrlich cell carcinoma development.

Glutamine, glutamate, asparagine, and aspartate contents in mouse kidney during Ehrlich ascites carcinoma development were determined. Significant changes in the concentrations of these amino acids were observed only 24 h after tumour inoculation, and they were highest during the exponential phase of tumour growth. These data agree with other previously reported studies and point to a potential of tumour cells to modulate host metabolism for its benefit. Discussed under this hypothesis, the new data reported here seem to indicate that there is an increase in the mobilization of the amino acids studied in mice kidney to provide Ehrlich tumour cells with sources of nitrogen (asparagine and glutamine) which they consume avidly.

Chemosensitization and drug accumulation assays as complementary methods for the screening of multidrug resistance reversal agents.

Two methods based on the reversion of adriamycin-resistance o the increase of Rhodamine 123 accumulation in a multidrug resistant (MDR) cell line have been simplified and adapted for the screening of MDR reversal agents. Both methods are carried out in microtiter plates, are highly sensitive and can be easily automated. In both assays verapamil and the cyclosporine derivative PSC 833 could be detected at concentrations lower than 1 and 0.05 microM, respectively. Depending on the MDR cell line used, drugs exhibiting the collateral sensitivity phenomenon can be selected in the cytotoxicity assay, while interferences due to sample toxicity are easily avoided in the dye accumulation assay.

Histamine, polyamines, and cancer.

Mammalian ornithine decarboxylase and histidine decarboxylase present common structural and functional features, and their products also share pharmacological and physiological properties. Although accumulated evidence pointed for years to a direct involvement of polyamines and histamine in tumour growth, it has been only in the last few years that new molecular data have contributed to the clarification of this topic. The aim of this commentary is to review the molecular grounds of the role of histamine and polyamines in cancer and to point to possible directions for future research in emerging areas of interest.

Expression and purification of human matrilysin produced in baculovirus-infected insect cells.

The baculovirus expression system was used to produce recombinant human matrilysin. Expression of promatrilysin reached a peak at 72 h post-infection. Most of the recombinant protein remained in the intracellular fraction in an insoluble form, which after renaturation was purified by S-Sepharose and Green A Dyematrex chromatography in order to remove host proteases. Active recombinant matrilysin degraded casein, type I and type IV collagens and fibronectin. Expression of recombinant human matrilysin using the baculovirus system represents a useful tool for obtaining large amounts of this metalloproteinase in order to carry out further biochemical studies and to screen for inhibitors.

Altered ornithine metabolism in tumor-bearing mice.

A flux of ornithine from the host tissues to the tumor was deduced from the concentrations of ornithine in plasma, ascitic liquid, liver and tumor cells during tumor growth. The activities of arginase and ornithine decarboxylase in both liver and tumor cells confirmed this proposed ornithine supply. Moreover, "in vitro" incubations of tumor cells showed that glutamine could be an additional source of ornithine for tumors. Finally, shortly before death, when tumor cell proliferation had ceased, altered hepatic ornithine metabolism was also detected.

A re-evaluation of fumagillin selectivity towards endothelial cells.

We have re-evaluated the selectivity of fumagillin against endothelial cell proliferation and compared it to the reported selectivity of its potent analog TNP-470. We showed that fumagillin does not inhibit endothelial cell proliferation in a specific manner, but on the contrary it inhibits the proliferation of other cell types at the same range of concentrations. Furthermore, the IC50 values of fumagillin for endothelial cells are two orders of magnitude higher than those values reported for TNP-470 on endothelial cells; on the contrary, the IC50 value of fumagillin for human breast cancer MDA-MB231 cells is four orders of magnitude lower than the value reported for TNP-470 on the same cell line.

Effects of genistein and 2-methoxyestradiol on matrix metalloproteinases and their inhibitors secreted by Ehrlich ascites tumor cells.

BACKGROUND: Ehrlich ascites tumor is an experimental tumor model very suitable for performing comparative studies relating its growth in vitro and in vivo. We used this tumor model to study the potential modulatory effects of genistein and 2-methoxyestradiol, two anti-angiogenic compounds, on the proteolytic balance MMP/TIMP. Ehrlich cells grown in vitro secreted MMP-9, MMP-2 and two TIMPs; the treatment with either of the anti-angiogenic compounds here tested stimulated all these activities, but the increase in TIMPs activities of genistein-treated cells were higher than those of MMPs, thus inducing a decrease in the proteolytic balance. On the other hand, Ehrlich cells growing in vivo did not produce any detectable TIMP activity, but accumulated MMP-9 and MMP-2 during tumor growth. Both compounds induced significant decrease of MMPs activity when tumor cells were actively proliferating. It was concluded that both genistein and 2-methoxyestradiol could shift the proteolytic balance MMP/TIMP towards antiproteolysis in media or ascitic fluid conditioned by actively growing Ehrlich cells.

Evaluation of the anti-angiogenic effect of aloe-emodin.

The present study identified aloe-emodin (AE, a hydroxyanthraquinone from Aloe vera and other plants) as a new anti-angiogenic compound with inhibitory effects in an in vivo angiogenesis assay and evaluates its effects on specific key steps of the angiogenic process. AE inhibits endothelial cell proliferation, but this effect is not cell specific, since AE also inhibits tumor cell proliferation. Cell migration and invasion are not remarkably affected by AE. On the other hand, AE has different effects on endothelial and tumor cell gelatinases. Two main targets of the pharmacological action of AE as an anti-angiogenic compound seem to be urokinase secretion and tubule formation of endothelial cells. Finally, AE produces a remarkable photocytotoxic effect on tumor cells. Taken together, our data indicate that AE can behave both as an anti-tumor and an anti-angiogenic compound and suggest that AE could be a candidate drug for photodynamic therapy.

A rapid method for the functional reconstitution of amino acid transport systems from rat liver plasma membranes. Partial purification of System A.

A rapid method for the functional reconstruction of amino acid transport from liver plasma-membrane vesicles using the neutral detergent decanoyl-N-glucamide ('MEGA-10') is described. The method is a modification of that previously employed in this laboratory for reconstitution of amino acid transport systems from kidney brush-border membranes [Lynch & McGivan (1987) Biochem. J. 244, 503-508]. The transport activities termed 'System A', 'System N', and 'System L' are all reconstituted. The reconstitution procedure is rapid and efficient and is suitable as an assay for transport activity in studies involving membrane fractionation. By using this reconstitution procedure, System A transport activity was partially purified by lectin-affinity chromatography.

Angiogenesis and signal transduction in endothelial cells.

Endothelial cells receive multiple information from their environment that eventually leads them to progress along all the stages of the process of formation of new vessels. Angiogenic signals promote endothelial cell proliferation, increased resistance to apoptosis, changes in proteolytic balance, cytoskeletal reorganization, migration and, finally, differentiation and formation of a new vascular lumen. We aim to review herein the main signaling cascades that become activated in angiogenic endothelial cells as well as the opportunities of modulating angiogenesis through pharmacological interference with these signaling mechanisms. We will deal mainly with the mitogen-activated protein kinases pathway, which is very important in the transduction of proliferation signals; the phosphatidylinositol-3-kinase/protein kinase B signaling system, particularly essential for the survival of the angiogenic endothelium; the small GTPases involved in cytoskeletal reorganization and migration; and the kinases associated to focal adhesions which contribute to integrate the pathways from the two main sources of angiogenic signals, i.e. growth factors and the extracellular matrix.

L-glutamine transport in native vesicles isolated from Ehrlich ascites tumor cell membranes.

Native vesicles isolated from Ehrlich ascites tumor cells accumulate glutamine by means of Na(+)-dependent transport systems; thiocyanate seems to be the more effective anion. The apparent affinity constant for the process was 0.38 mM. The Arrhenius plot gave an apparent activation energy of 12.3 kJ/mol. The structural analogs of glutamine, acivicin (2.5 mM) and azaserine (2.5 mM), inhibited the net uptake by 67 and 70%, respectively. The sulfhydryl reagents mersalyl, PCMBS, NEM, and DTNB also inhibited net uptake, suggesting that sulfhydryl groups may be involved in the activity of the carrier protein. A strong inhibition was detected when the vesicles were incubated in the presence of alanine, cysteine, or serine; in addition, histidine, but not glutamate or leucine, had a negative effect on glutamine transport.