Francesca Marino-Merlo

Inflammatory and cell death pathways in brain and peripheral blood in Parkinson's disease.

Evidence has been accumulated showing that inflammatory and cell death pathways are altered both in brain and periphery during Parkinson disease (PD). Neuronal loss in PD is associated with chronic neuroinflammation characterized by microglia activation through the release of reactive oxygen radicals, cytokines, and Prostaglandin E2. The release of these inflammatory mediators in addition to deprivation in growth factors and increase of calcium and dopamine seem implicated in triggering apoptosis. The interaction of leucine-rich repeat kinase and Fas- Associated protein with Death Domain has been implicated in the switching-on of the extrinsic apoptotic pathway via caspase-8 activation, while deficiency in PTEN induced putative kinase 1 has been shown to cause Ca2+ accumulation in mitochondria, increased generation of reactive oxygen species and intrinsic cell death. Autophagy/mitophagy appears to be impaired in the brain during PD; this impairment could be related to defective degradation of mutant α-synuclein and consequent apoptotic cell death. Regarding the peripheral blood, reduced amounts of dopamine, reduced levels of immunoreactivity for tyrosine hydroxylase and dopamine active transporter, and alterations of dopamine receptor expression have been detected in mononuclear cells from PD patients. In addition, mononuclear cells from PD patients show mitochondrial, ubiquitin-proteasome system dysfunction and up-regulation of α-synuclein gene, associated to high expression of the Fas molecule, activation of caspase-3 and -9 and proneness to apoptosis. These and other observations reported in this mini-review suggest that a better understanding of molecular dysfunctions in inflammatory and cell death/autophagy pathways, both in the brain and peripheral blood, could provide useful targets for future investigation on drug-discovery and biomarker identification in PD.

Involvement of HVEM receptor in activation of nuclear factor κB by herpes simplex virus 1 glycoprotein D

The UV‐inactivated herpes simplex virus 1 (HSV‐1) and glycoprotein D (gD) of HSV‐1 have been shown to activate nuclear factor κB (NF‐κB) in U937 cells, but mechanisms involved in this activation have not been elucidated. Here we report that: (i) UV‐inactivated HSV‐1 induced an increased NF‐κB activation in cells expressing human HVEM (for herpesvirus entry mediator) at surface level, naturally or following stable transfection, but not in cells in which this receptor was not detected by flow cytometry analysis, (ii) treatment with soluble gD induced a dose‐dependent NF‐κB activation in THP‐1 cells naturally expressing HVEM, and a monoclonal antibody that prevents binding of gD to HVEM significantly reduced NF‐κB activation by soluble gD in the same cells, (iii) coculture with transfectants expressing wild‐type gD on their surface induced an approximately twofold increase in NF‐κB activation in cells naturally expressing HVEM, while coculture with transfectants expressing a mutated form of gD, lacking its capability to bind HVEM, did not induce a similar effect and (iv) treatment with soluble gD induced a dose‐dependent NF‐κB activation in CHO transfectants expressing HVEM, but not in control CHO transfectants lacking any functional gD receptor. Overall, these results establish that HVEM is involved in NF‐κB activation by HSV‐1 gD.

Involvement of gD/HVEM interaction in NF-kB-dependent inhibition of apoptosis by HSV-1 gD.

In the present paper, we aimed to verify whether the interaction of the glycoprotein D (gD) of herpes simplex 1 (HSV-1) with the HSV-1 receptor HVEM is involved in NF-kappaB-dependent protection against apoptosis by gD. To this purpose, first we utilized MAbs that interfere with gD/HVEM interaction and U937 cells that naturally express human HVEM on their surface. Pre-incubation with these MAbs, but not with a control antibody, partially reverted the protection of infectious HSV-1 towards anti-Fas induced apoptosis in U937 cells. Similarly, pre-incubation of UV-inactivated HSV-1 (UV-HSV-1) or recombinant gD with the same MAbs, significantly reduced the inhibition of Fas-mediated apoptosis by UV-HSV-1 or gD, respectively, in U937 cells. Moreover, coculture with stable transfectants expressing at surface level wild type gD protected U937 cells against Fas-induced apoptosis, while coculture with transfectants expressing a mutated form of gD, incapable to bind HVEM, did not protect. Finally, UV-HSV-1 protected against staurosporine-induced apoptosis in U937 cells as well as in the CHO transfectants expressing human HVEM on their surface, but not in the control CHO transfectants, which did not express HVEM. These results suggest that signaling triggered by binding of gD to HVEM could represent an additional mechanism of evasion from premature apoptotic death exerted by HSV-1-gD in HVEM-expressing cells, disclosing new opportunities of cell death manipulation by using gD preparations.

Signaling Pathway Used by HSV-1 to Induce NF-κB Activation

Abstract:  We have previously demonstrated that wild‐type herpes simplex virus type 1 (HSV‐1), as well as nonreplicating UV‐inactivated HSV‐1, promptly activates the nuclear factor‐κB (NF‐κB) in U937 monocytoid cells and that glycoprotein D (gD) of HSV‐1 is sufficient by itself to exert a similar effect.We then investigated the signaling pathway used by HSV‐1 to initiate NF‐κB activation and, particularly, whether our observation could be related to the capability of HSV‐1‐gD to directly stimulate NF‐κB through its interaction with the herpes virus entry receptor A (HveA). Here we report that: (a) co‐cultivation of U937 cells with an adherent cell line expressing wild‐type gD on its surface led to increased NF‐κB activation, while co‐cultivation with the same adherent cell line expressing a mutated form of gD, lacking the capability to bind HveA, did not cause the same effect; (b) exposure to UV‐inactivated HSV‐1 induced the activation of NF‐κB in HveA‐expressing U937 and THP‐1 cells, but not in non‐HveA‐expressing HEp‐2 cells; and (c) activation of NF‐κB in U937 and THP‐1 cells exposed to soluble gD was inhibited by an antibody able to interfere with gD–HveA interaction. These results suggest that HSV‐1‐gD–HveA interaction initiates a signal transduction pathway leading to NF‐κB activation.

D (−)lentiginosine-induced apoptosis involves the intrinsic pathway and is p53-independent

We have recently found that D(−)lentiginosine, a synthetic iminosugar exerting glucosidase inhibitory activity, but not its natural enantiomer lentiginosine, is endowed with an unexpected, pro-apoptotic activity. Here, we investigated mechanisms involved in apoptosis induced by D(−)lentiginosine in MOLT-3, HT-29 and SH-SY5Y tumour cell lines. The results showed that D(−)lentiginosine increased caspase 9 expression at 18 h in all the cell lines from 1.5–3.1 folds. Cytochrome c in the cytoplasm was found to be increased from 2.3–2.6 folds in treated cells with respect to control cells. These effects were accompanied by a remarkable collapse of the mitochondrial membrane potential and by the downregulation of anti-apoptotic genes, as well as the upregulation of pro-apoptotic genes of the Bcl-2 family. U937Bcl-2 transfectants, highly expressing Bcl-2, were reluctant to undergo apoptosis even following treatment with 500 μM D(−)lentiginosine, whereas apoptosis by D(−)lentiginosine was induced also in U937 cells, naturally deficient in P53. Thus, our study establishes that the enantiomer of a natural iminosugar is endowed with a possible anti-tumorigenic effect that might be ascribed not only to their capacity to inhibit glycosidases but also to other unknown mechanisms. These data encourage further investigation on similar compounds to make them an interesting platform for the generation of new anticancer drugs.

Synthesis and biological evaluation of a new class of glycoconjugated disulfides that exhibit potential anticancer properties.

A synthetic strategy, based on the in situ generation of sulfenic acids and their thermolysis in the presence of thiols, was developed for obtaining a collection of polyvalent disulfides in which a benzene scaffold accommodates two or three flexible arms connecting saccharide moieties. Targeting carbohydrate metabolism or carbohydrate-binding proteins may constitute important approaches in the discovery process of new therapeutic anticancer agents. Therefore, a preliminary screening to ascertain the cytostatic/cytotoxic potential of this new class of enantiopure glycoconjugated disulfides has been conducted. Among them, products with two disulfide arms, harbouring galactose rings, induced high levels of apoptosis on U937 histiocytic lymphoma cells, but lower levels of cell death on peripheral blood mononuclear cells from healthy donors. Further experiments indicated that apoptosis induced by these glycoconjugated bis(disulfides) in U937 cells corresponds to the Bcl-2-sensitive, intrinsic form of apoptotic cell death. The bioinvestigation was extended to a panel of human cancer cell lines with different levels of malignancy and resistance to chemotherapeutic agents. Compounds under study proved to induce detectable levels of cell death towards all the tested cancer cell lines.

Inhibition of NF-κB activation sensitizes U937 cells to 3'-azido-3'-deoxythymidine induced apoptosis.

In this study, we investigated molecular mechanisms underlying low susceptibility to apoptosis induced by the nucleoside analog azidothymidine (AZT) and the role of nuclear factor-κB (NF-κB) activation in these phenomena. A preliminary screening in different cell lines indicated U937 monocytic cell line as suitable to this purpose. Treatment of U937 cells even with suprapharmacological concentrations of AZT induced only moderate levels of apoptosis. Surprisingly, SuperArray analysis showed that AZT induced the transcriptional activity of both pro- and anti-apoptotic genes. Interestingly, moreover, several genes upregulated by AZT were NF-κB related. In fact, AZT, after an initial inhibition of NF-κB activation with respect to control, induced a transient, but consistent, increase in NF-κB-binding activity. Inhibition of NF-κB activation in U937 cells, stably transfected with a dominant-negative IκBα or by pharmacological treatment, sensitized them to apoptosis induced by AZT and impaired the upregulation of anti-apoptotic genes in response to AZT treatment, with respect to control cells. These results indicate that NF-κB activation by AZT has a role in protecting target cells from apoptotic cell death, improving our understanding of the toxicology and the therapeutic usage of this drug.

Synthesis and biological testing of thioalkane- and thioarene-spaced bis-β-d-glucopyranosides

A three-step synthesis of bis-beta-D-glucopyranosides containing thioalkane or thioarene spacers of different length and flexibility is described. The key-step reaction allows an easy modulation of final saccharidic products so that a library of molecules with different glycosidic residues and spacers can be obtained. Two of the new thioarene-spaced bis-beta-D-glucopyranosides endow with a specific cytotoxic potential. A more detailed investigation of one of the two compounds ascertains that this effect is attributable to induction of cell death by apoptosis.

Role of inflammation and apoptosis in multiple sclerosis: Comparative analysis between the periphery and the central nervous system

Multiple sclerosis (MS) is a complex, multifactorial disease associated with damage to the axonal myelin sheaths and neuronal degeneration. The pathognomonic event in MS is oligodendrocyte loss accompanied by axonal damage, blood-brain barrier leakage, inflammation and infiltration of immune cells. The etiopathogenesis of MS is far from being elucidated. However, increasing evidence suggests that the inflammatory and apoptotic responses, occurring in patients either at the peripheral level or the central nervous system (CNS), can play a role. In this review, we give a comprehensive picture of general aspects of inflammation and apoptosis in MS, with special emphasis on the until now not well highlighted possible links between phenomena relevant to these aspects occurring in either the periphery or in the CNS during MS.

Characterization of the enhanced apoptotic response to azidothymidine by pharmacological inhibition of NF-kB.

AIMS The present study addresses the issue of enhanced apoptotic response to AZT following co-treatment with an NF-kB inhibitor. MAIN METHODS To investigate this issue, different cell lines were assayed for susceptibility to AZT-mediated apoptosis without or with the addition of the NF-kB inhibitor Bay-11-7085. For further investigation, U937 cells were selected as good-responder cells to the combination treatment with 32 or 128 μM AZT, and 1 μM Bay-11-7085. Inhibition of NF-kB activation by Bay-11-7085 in cells treated with AZT was assayed through Western blot analysis of p65 expression and by EMSA. Involvement of the mitochondrial pathway of apoptosis in mechanisms underlying the improved effect of AZT following Bay-11-7085 co-treatment, was evaluated by assaying the cytochrome c release and the mitochondrial membrane potential (MMP) status using the JC-1 dye. Moreover, the transcriptional activity of both anti- and pro-apoptotic genes in U937 cells after combination treatment was quantitatively evaluated through real-time PCR. KEY FINDINGS We found that the combined treatment induced high levels of cytochrome c release and of MMP collapse in association with evident changes in the expression of both anti- and pro-apoptotic genes of the Bcl-2 family. Overexpression of Bcl-2 significantly suppressed the sensitization of U937 cells to an enhanced apoptotic response to AZT following co-treatment with the NF-kB inhibitor. SIGNIFICANCE The new findings suggest that a combination regimen based on AZT plus an NF-kB inhibitor could represent a new chemotherapeutic tool for retrovirus-related pathologies.