Eugenia Pittaluga

Thiol Redox Transitions in Cell Signaling: a Lesson from N-Acetylcysteine

The functional status of cells is under the control of external stimuli affecting the function of critical proteins and eventually gene expression. Signal sensing and transduction by messengers to specific effectors operate by post-translational modification of proteins, among which thiol redox switches play a fundamental role that is just beginning to be understood. The maintenance of the redox status is, indeed, crucial for cellular homeostasis and its dysregulation towards a more oxidized intracellular environment is associated with aberrant proliferation, ultimately related to diseases such as cancer, cardiovascular disease, and diabetes. Redox transitions occur in sensitive cysteine residues of regulatory proteins relevant to signaling, their evolution to metastable disulfides accounting for the functional redox switch. N-acetylcysteine (NAC) is a thiol-containing compound that is able to interfere with redox transitions of thiols and, thus, in principle, able to modulate redox signaling. We here review the redox chemistry of NAC, then screen possible mechanisms to explain the effects observed in NAC-treated normal and cancer cells; such effects involve a modification of global gene expression, thus of functions and morphology, with a leitmotif of a switch from proliferation to terminal differentiation. The regulation of thiol redox transitions in cell signaling is, therefore, proposed as a new tool, holding promise not only for a deeper explanation of mechanisms, but indeed for innovative pharmacological interventions.

N-acetyl-l-cysteine fosters inactivation and transfer to endolysosomes of c-Src

The non-receptor-protein tyrosine kinase c-Src is overexpressed and activated in a large number of human cancers, in which it is associated with tumor development and progression. Canonical regulation takes place by means of an alternative phosphorylation of tyrosine residues -- Tyr419 for activation and Tyr530 for inactivation. An independent redox regulation mechanism, involving cysteine residues, has also been proposed, in which oxidation activates the enzyme. Here we present a kinetic analysis of the effect of N-acetyl-l-cysteine (NAC) on c-Src, demonstrating that reduction reverts the oxidation-driven activation. In cancer cells, we show that NAC treatment produces an increase in specifically labeled reduced thiols of c-Src cysteines, thus confirming a redox transition. In addition to a decrease in Tyr419 phosphorylation, this leads to a massive shift of c-Src from plasma membranes -- where its active form is located -- to endolysosomal compartments. With the objective of deciphering the complex issue of c-Src regulation and of devising new strategies to revert its activation in cancers, redox regulation thus emerges as a promising area for study.

Intervillous circulation in intra-uterine growth restriction. Correlation to fetal well being

Magnetic resonance imaging requested for a potentially serious indication, provided a unique opportunity to explore the intervillous circulation of placentas from pregnancies complicated by Intra Uterine Growth Restriction (IUGR) and to compare them to normal cases. This allowed an innovative characterization of in vivo utero-placental blood flow, correlating a compromised intervillous circulation in IUGR to the deterioration of fetal condition. MR imaging was requested to rule out suspected posterior placental adhesive disorders in 26 patients. Twelve patients had fetuses appropriate for gestational age, while in 14 patients fetuses were affected by severe IUGR. Multiphasic dynamic contrast-enhanced sagittal sequences were acquired and a quantitative analysis of signal intensity and enhancement kinetics was performed for both the entire placenta and for selected regions. Images disclosed a homogeneous perfusion overall the placenta in normal cases, while IUGR placentas displayed a slow intervillous blood flow, along with many patchy unperfused areas. Intermittent stops worsen the perfusion dynamics of the intervillous mostly in IUGR cases with an elevated ductus venosus pulsatility index. In conclusion, we proved that in IUGR placenta maternal placental blood flow is extremely compromised and that superimposed dynamic phenomena concur to worsen the intervillous circulation leading to an end-stage fetal decompensation.

More than antioxidant: N-acetyl-L-cysteine in a murine model of endometriosis

N-acetyl-L-cysteine exerts a complex action on endometrial cells, involving regulation of gene expression and protein activity and location, all converging into a decreased proliferation and a switch toward a differentiating, less invasive, and less inflammatory phenotype. Also considering the lack of undesired side effects, including unaffected fertility potential, this suggests a beneficial use of NAC in endometriosis clinical treatment.

A Promise in the Treatment of Endometriosis: An Observational Cohort Study on Ovarian Endometrioma Reduction by N-Acetylcysteine

Urged by the unmet medical needs in endometriosis treatment, often with undesirable side effects, and encouraged by N-acetylcysteine (NAC) efficacy in an animal model of endometriosis and by the virtual absence of toxicity of this natural compound, we performed an observational cohort study on ovarian endometriosis. NAC treatment or no treatment was offered to 92 consecutive Italian women referred to our university hospital with ultrasound confirmed diagnosis of ovarian endometriosis and scheduled to undergo laparoscopy 3 months later. According to patients acceptance or refusal, NAC-treated and untreated groups finally comprised 73 and 72 endometriomas, respectively. After 3 months, within NAC-treated patients cyst mean diameter was slightly reduced (−1.5 mm) versus a significant increase (+6.6 mm) in untreated patients (P = 0.001). Particularly, during NAC treatment, more cysts reduced and fewer cysts increased their size. Our results are better than those reported after hormonal treatments. Twenty-four NAC-treated patients—versus 1 within controls—cancelled scheduled laparoscopy due to cysts decrease/disappearance and/or relevant pain reduction (21 cases) or pregnancy (1 case). Eight pregnancies occurred in NAC-treated patients and 6 in untreated patients. We can conclude that NAC actually represents a simple effective treatment for endometriosis, without side effects, and a suitable approach for women desiring a pregnancy.

Developing drugs that target the Wnt pathway: recent approaches in cancer and neurodegenerative diseases

ABSTRACT Introduction: Wnt/β-catenin signaling is an evolutionarily conserved pathway that has a crucial role in embryonic and adult life. Dysregulation of Wnt/β-catenin pathway has been associated with various diseases, including cancer and neurodegenerative disorders, including Parkinson’s disease (PD). Several molecular components of the signaling have been proposed as innovative targets for cancer therapy, and very recently, some of them have been also evaluated as potential therapeutic targets for PD. Areas covered: This review focuses on the role of Wnt/β-catenin pathway in the pathogenensis of cancer and PD, examining some recent therapeutic approaches that are ongoing in preclinical and clinical studies. The possibilities that this signaling offers for diagnosis and prognosis of neoplastic diseases, and the concerns of targeting this pathway are also discussed. Expert opinion: Despite the stimulating results obtained in preclinical studies on cancer and other disease models, the clinical experience with Wnt modulators is still in its infancy, and is mainly restricted to anticancer therapy. Even with concerns of the safety of drugs targeting Wnt signaling, the attention of researchers worldwide is increasing to this issue in terms of their therapeutic potential for diseases such as PD, for which no cure exists.

Human amniotic glycodelin actively regulates changes in β-catenin immunoreactivity in cultured human umbilical vein endothelial cells (HUVEC).

Objective: Assessment of the capacity of Glycodelin A (GdA) to modulate the aggregation of cultured human umbelical vein endothelial cells. Methods: Highly purified Glycodelin A (GdA) from late first trimester amniotic fluid has been added to cultured cells and its biological activity has been observed with immunofluorescent staining of β-catenin molecules. Results: GdA induces traslocation of β-catenin molecules promoting cell-to-cell adhesion and formation of adherents junctions through cytoskeletal reorganization. Conclusion: These data provide further mechanistic insight into the specificity of cell-to-cell adhesion, thus corroborating the role of GdA in promoting angiogenesis.

Atrial Natriuretic Peptide Acts as a Neuroprotective Agent in in Vitro Models of Parkinson’s Disease via Up-regulation of the Wnt/β-Catenin Pathway

In the last decades increasing evidence indicated a crucial role of the Wnt/β-catenin signaling in development of midbrain dopaminergic (mDA) neurons. Recently dysregulation of this pathway has been proposed as a novel pathomechanism leading to Parkinson’s disease (PD) and some of the molecules participating to the signaling have been evaluated as potential therapeutic targets for PD. Atrial natriuretic peptide (ANP) is a cardiac-derived hormone having a critical role in cardiovascular homeostasis. ANP and its receptors (NPRs) are widely expressed in mammalian central nervous system (CNS) where they could be implicated in the regulation of neural development, synaptic transmission and information processing, as well as in neuroprotection. Until now, the effects of ANP in the CNS have been mainly ascribed to the binding and activation of NPRs. We have previously demonstrated that ANP affects the Wnt/β-catenin signaling in colorectal cancer cells through a Frizzled receptor-mediated mechanism. The purpose of this study was to investigate if ANP is able to exert neuroprotective effect on two in vitro models of PD, and if this effect could be related to activation of the Wnt/β-catenin signaling. As cellular models of DA neurons, we used the proliferating or RA-differentiated human neuroblastoma cell line SH-SY5Y. In both DA neuron-like cultures, ANP is able to positively affect the Wnt/β-catenin signaling, by inducing β-catenin stabilization and nuclear translocation. Importantly, activation of the Wnt pathway by ANP exerts neuroprotective effect when these two cellular systems were subjected to neurotoxic insult (6-OHDA) for mimicking the neurodegeneration of PD. Our data support the relevance of exogenous ANP as an innovative therapeutic molecule for midbrain, and more in general for brain diseases for which aberrant Wnt signaling seems to be involved.

Thymosin α1 modifies podosome architecture and promptly stimulates the expression of podosomal markers in mature macrophages

Background and aims: The immunomodulatory activity of thymosin α1 (Tα1) on innate immunity has been extensively described, but its mechanism of action is not completely understood. We explored the possibility that Tα1-stimulation could affect the formation of podosomes, the highly dynamic, actin-rich, adhesion structures involved in macrophage adhesion/chemotaxis. Methods: The following methods were used: optical and scanning electron microscopy for analyzing morphology of human monocyte-derived macrophages (MDMs); time-lapse imaging for visualizing the time-dependent modifications induced at early times by Tα1 treatment; confocal microscopy and Western blot for analyzing localization and expression of podosome components; and Matrigel Migration Assay and zymography for testing MDM invasive ability and metalloproteinase secretion. Results: We obtained data to support that Tα1 could affect MDM motility, invasion and chemotaxis by promptly stimulating assembly and disassembly of podosomal structures. At very early times after its addition to cell culture medium and within 1 h of treatment, Tα1 induces modifications in MDM morphology and in podosomal components that are suggestive of increased podosome turnover. Conclusions: Since impairment of podosome formation leads to reduced innate immunity and is associated with several immunodeficiency disorders, we confirm the validity of Tα1 as a potent activator of innate immunity and suggest possible new clinical application of this thymic peptide.