Maria Cristina Caroleo

Human monocyte/macrophages activate by exposure to LPS overexpress NGF and NGF receptors

Monocyte/macrophages (M/M) represent the main cellular component of the immune system involved in the inflammatory response. In the present study we investigate whether NGF is produced by M/M and is involved in this event. The results show that unstimulated human M/M produce NGF and its synthesis is stimulated by LPS. The increase of NGF is associated with enhanced expression of high affinity NGF receptor on M/M and with no changes of low affinity NGF receptors (p75). The neutralization of endogenous NGF by NGF antibody in LPS-activated M/M, leads to overexpression of p75 receptor causing apoptosis. These findings provide new insight in the mechanisms governing monocyte survival in the inflamed tissue, representing a crucial aspect of host defence and maintenance of homeostasis.

Primary macrophages infected by human immunodeficiency virus trigger CD95-mediated apoptosis of uninfected astrocytes

Infection of macrophages (M/M) by human immunodeficiency virus (HIV) is a main pathogenetic event leading to neuronal dysfunction and death in patients with AIDS dementia complex. Alteration of viability of neurons and astrocytes occurs in vivo even without their infection, thus it is conceivable that HIV‐infected M/M may affect viability of such cells even without direct infection. To assess this hypothesis, we studied the effects of HIV‐infected M/M on an astrocytic cell‐line lacking CD4‐receptor expression. Exposure to supernatants of HIV‐infected M/M triggers complete disruption and apoptotic death of astrocytic cells. This effect is not related to HIV transmission from infected M/M, because HIV‐DNA and p24 production in astrocytic cells remained negative. Apoptotic death of astrocytes is mainly mediated by Fas ligand released in supernatants of HIV‐infected M/M (as demonstrated by complete reversal of such phenomenon by adding neutralizing antibodies against CD95 receptor). Treatment of astrocytic cells with recombinant (biologically active) Tat induces <10% apoptosis, and gp120 was totally ineffective. Treatment of HIV‐infected M/M with AZT completely reverses the proapoptotic effect of their supernatants on astrocytes, thus demonstrating that productive virus replication within M/M is required for the induction of astrocytic cell death.

Doxorubicin loaded magneto-niosomes for targeted drug delivery

In chemotherapy the magnetic drug targeting to a specific organ or tissue is proposed on the assumption that magnetic fields are harmless to biological systems. In this light we have vehiculated doxorubicin as model drug by novel magneto-niosomes in order to evaluate the physico-chemical properties of the obtained formulations and the in vitro release profile. Tween 60 and Pluronic L64 have been used as surfactants and the formulation cytotoxicity has been performed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolum bromide and trypan blue dye esclusion tests. Results show that niosome dimensions and doxorubicin entrapment efficiencies are influenced by bilayer composition. In addition, formulations are able to control the deliver and release of the drug active form in a retarded manner. No additional toxicity, due to the encapsulation of ferrofluid into niosomes core, has been detected.

Metabolic remodeling of the tumor microenvironment: Migration stimulating factor (MSF) reprograms myofibroblasts toward lactate production, fueling anabolic tumor growth

Migration stimulating factor (MSF) is a genetically truncated N-terminal isoform of fibronectin that is highly expressed during mammalian development in fetal fibroblasts, and during tumor formation in human cancer-associated myofibroblasts. However, its potential functional role in regulating tumor metabolism remains unexplored. Here, we generated an immortalized fibroblast cell line that recombinantly overexpresses MSF and studied their properties relative to vector-alone control fibroblasts. Our results indicate that overexpression of MSF is sufficient to confer myofibroblastic differentiation, likely via increased TGF-b signaling. In addition, MSF activates the inflammation-associated transcription factor NFκB, resulting in the onset of autophagy/mitophagy, thereby driving glycolytic metabolism (L-lactate production) in the tumor microenvironment. Consistent with the idea that glycolytic fibroblasts fuel tumor growth (via L-lactate, a high-energy mitochondrial fuel), MSF fibroblasts significantly increased tumor growth, by up to 4-fold. Mechanistic dissection of the MSF signaling pathway indicated that Cdc42 lies downstream of MSF and fibroblast activation. In accordance with this notion, Cdc42 overexpression in immortalized fibroblasts was sufficient to drive myofibroblast differentiation, to provoke a shift towards glycolytic metabolism and to promote tumor growth by up to 2-fold. In conclusion, the MSF/Cdc42/NFκB signaling cascade may be a critical druggable target in preventing “Warburg-like” cancer metabolism in tumor-associated fibroblasts. Thus, MSF functions in the metabolic remodeling of the tumor microenvironment by metabolically reprogramming cancer-associated fibroblasts toward glycolytic metabolism.

Localization of nerve growth factor (NGF) receptors in the mitochondrial compartment: characterization and putative role.

BACKGROUND The neurotrophin NGF receptors trkA and p75NTR are expressed in the central and peripheral nervous system as well as in non-neuronal tissues; originally described to localize to the plasma membrane, recent studies have suggested other intracellular localizations for both NGF receptors. SCOPE OF REVIEW In order to determine whether NGF receptors localize to the mitochondrial compartment mitochondria isolated from human kidney, rat tissues and a human podocyte as cell line before and after differentiation were used. MAJOR CONCLUSIONS Our results demonstrate that NGF receptors are localized in the mitochondrial compartment of undifferentiated human podocytes and in all tissues analyzed including rat central nervous system. In mitochondria p75NTR, but not trkA, co-immunoprecipitates with the adenine nucleotide translocator (ANT) and the phosphodiesterase 4 isoform A5 (PDE4A5). Moreover, NGF, via trkA, protects isolated mitochondria of rat brain cortex from mitochondrial permeability transition induced by Ca(2+). GENERAL SIGNIFICANCE Although NGF receptors have been described as mainly citoplasmatic so far, we proved evidence of their expression at the mitochondrial level and their interaction with specific proteins. Our results demonstrating the expression of NGF receptors in the mitochondria provide new insights into the role of NGF at subcellular level, in different areas of the organism, including CNS.

Nerve Growth Factor and Its Monocyte Receptors Are Affected in Kidney Disease

Nerve growth factor (NGF) plays a critical role in both physiological and pathological conditions. Their biological effects are mediated by two receptors (NGF-R): TrkA and p75. We previously reported NGF and NGF-R overexpression in various renal disorders. The aim of the study was to determinate NGF levels and NGF-R expression in peripheral blood mononuclear cells from subjects affected by glomerulonephritis (GN) and by end-stage renal disease before and after hemodialysis (HD). We enrolled 48 patients with biopsy-proven diagnosis of GN and 16 patients undergoing chronic HD. 25 subjects were enrolled as controls (C). Quantification of NGF in the serum samples was performed using NGF immunoassay. We demonstrated, for the first time, an increased NGF concentration in GN and HD patients compared to C. HD is able to restore serum NGF concentration. In GN, TrkA is overexpressed, whereas p75 did not show any difference versus C. By contrast in HD, TrkA expression is associated with increased p75 levels. In conclusion, NGF can act as protective factor against cytotoxic injuries. p75 plays a role in both survival and death of cells, depending on absence of ligand, cytoplasmic/ligand interaction and interaction with TrkA. The present findings suggest that cell survival during cellular damage is dependent on co-expression of TrkA and p75 and independent of NGF concentration. Further studies are required to confirm these observations.

Old Drug Scaffold, New Activity: Thalidomide‐Correlated Compounds Exert Different Effects on Breast Cancer Cell Growth and Progression

Thalidomide was first used for relief of morning sickness in pregnant women and then withdrawn from the market because of its dramatic effects on normal fetal development. Over the last decades, it has been used successfully for the treatment of several pathologies, including cancer. Many analogues with improved activity have been synthesized and tested. Herein we report some effects on the growth and progression of MCF‐7 and MDA‐MB‐231 breast cancer cells by a small series of thalidomide‐correlated compounds, which are very effective at inducing cancer cell death by triggering TNFα‐mediated apoptosis. The most active compounds are able to drastically reduce the migration of breast cancer cells by regulation of the two major proteins involved in epithelial–mesenchymal transition (EMT): vimentin and E‐cadherin. Moreover, these compounds diminish the intracellular biosynthesis of vascular endothelial growth factor (VEGF), which is primarily involved in the promotion of angiogenesis, sustaining tumor progression. The multiple features of these compounds that act on various key points of the tumorigenesis process make them good candidates for preclinical studies.

Human kidney podocyte cell population as a novel biological target of nerve growth factor.

Abstract Human podocytes are highly specialized cells with a key role in kidney physiology. Alteration of their structure as a consequence of injury or developmental failure leads to severe renal diseases. Although several studies have tried to elucidate the molecular framework of this cellular system, the functional bases for the maintenance of podocytes in their specialized state to sustain kidney barrier filtration are not completely understood. In this study, the capability of podocytes to produce and secrete the nerve growth factor (NGF) has been demonstrated via a validated in vitro model. During the process of cell differentiation, NGF and its receptors are modulated in human podocytes just as NGF-responsive neurons. Blockade of NGF biological activity results in severe changes of cell morphology. Collectively, our results outline a novel function of the neurotrophin and add a new cellular target in the complex biological framework of NGF.

9-cis Retinoic acid modulates myotrophin expression and its miR in physiological and pathophysiological cell models

ABSTRACT Functional studies indicate that essential cellular processes are controlled by Vitamin A derivatives. Among these the retinoic acid isoforms, all‐trans‐ and 9‐cis (9cRA), regulate the expression of various genes in both physiological and pathological conditions. Using several in vitro experimental models such as pancreatic &bgr;‐cells, pre‐adipocytes and breast cancer cells with different phenotypes, we demonstrated the capability of 9cRA to modulate myotrophin (Mtpn) and miR‐375 expressions. The 9cRA effect in pancreatic &bgr;‐cells line INS‐1 832/13 point out a decreased expression of Mptn at both mRNA and protein levels associated to a concomitant increase of miR‐375. We also studied the effect of this molecule on 3T3‐L1 pre‐adipocytes cells demonstrating a down‐regulation of Mtpn and a dramatic increase of miR‐375. Moreover, in the in vitro breast cancer model such as MDA‐MB‐231 and MCF‐7 cells, 9cRA showed different effect on both Mtpn and miR‐375 expression. In INS‐1 832/13, 3T3‐L1 pre‐adipocytes and MCF‐7 but not in MDA‐MB‐231, the effect of 9cRA on Mptn gene expression and its miR was under the control of RARs and RXRs receptors, as revealed by the exposure of these cell line to LE540 or HX603 receptor antagonists. In our findings 9cRA emerges has a hormone with a regulatory action on miR‐375 that in most cases interfere with Mtpn expression.

Crystallographic Study and Biological Evaluation of 1,4-dimethyl- N -alkylcarbazoles†

The 9-(bromoalkyl)-1,4-dimethyl-9H-carbazole (2a-d) derivatives, characterized by the presence of five or seven methylenic spacer groups bonded to the carbazole nitrogen, have been synthesized from the corresponding 1,4- dimethyl-9H-carbazole and appropriate dibromoalkane following a general synthetic method. All the prepared species have been fully characterized by means of IR, and (1)H and (13)C NMR spectroscopy, GC-MS and Elemental analysis. Good crystals of the 2c have been obtained and the crystal structure has been solved by means of X-ray diffractometry. In order to study the cytotoxic effect of 2a, 2b, 2c, 2d carbazole derivatives on A2780 ovarian cancer cells, we performed MTT assay after exposure of this cell population to those compounds in a concentration range from 1 to 10μM. Finally, we want to verify whether the cytotoxic effect of the 2c carbazole is mediated by apoptotic mechanisms, by performing chromatin condensation assay on the A2780 cell cultures upon the carbazole treatment at concentration of 10 μM for 72h. All together our data demonstrate that carbazole derivatives exert inhibitory effects on ovarian cancer cell growth, highlighting a stronger and a dose-dependent anti proliferative activity displayed by 2c carbazole, designating this compound, as a better candidate in the treatment of human ovarian cancer.