Maria Antonietta Belisario

IKKγ protein is a target of BAG3 regulatory activity in human tumor growth

BAG3, a member of the BAG family of heat shock protein (HSP) 70 cochaperones, is expressed in response to stressful stimuli in a number of normal cell types and constitutively in a variety of tumors, including pancreas carcinomas, lymphocytic and myeloblastic leukemias, and thyroid carcinomas. Down-regulation of BAG3 results in cell death, but the underlying molecular mechanisms are still elusive. Here, we investigated the molecular mechanism of BAG3-dependent survival in human osteosarcoma (SAOS-2) and melanoma (M14) cells. We show that bag3 overexpression in tumors promotes survival through the NF-κB pathway. Indeed, we demonstrate that BAG3 alters the interaction between HSP70 and IKKγ, increasing availability of IKKγ and protecting it from proteasome-dependent degradation; this, in turn, results in increased NF-κB activity and survival. These results identify bag3 as a potential target for anticancer therapies in those tumors in which this gene is constitutively expressed. As a proof of principle, we show that treatment of a mouse xenograft tumor model with bag3siRNA-adenovirus that down-regulates bag3 results in reduced tumor growth and increased animal survival.

A chemical-biological study reveals C9-type iridoids as novel heat shock protein 90 (Hsp90) inhibitors.

The potential of heat shock protein 90 (Hsp90) as a therapeutic target for numerous diseases has made the identification and optimization of novel Hsp90 inhibitors an emerging therapeutic strategy. A surface plasmon resonance (SPR) approach was adopted to screen some iridoids for their Hsp90 α binding capability. Twenty-four iridoid derivatives, including 13 new natural compounds, were isolated from the leaves of Tabebuia argentea and petioles of Catalpa bignonioides. Their structures were elucidated by NMR, electrospray ionization mass spectrometry, and chemical methods. By means of a panel of chemical and biological approaches, four iridoids were demonstrated to bind Hsp90 α. In particular, the dimeric iridoid argenteoside A was shown to efficiently inhibit the chaperone in biochemical and cellular assays. Our results disclose C9-type iridoids as a novel class of Hsp90 inhibitors.

Metal-ion catalyzed oxidation affects fibrinogen activity on platelet aggregation and adhesion.

Exposure of fibrinogen to the Fe3+/ascorbate oxidative system resulted in structural modifications and altered functionality of the glycoprotein. The overnight treatment of fibrinogen by oxidants caused a 20-fold increase of carbonyl content with respect to the native protein. Formation of dityrosines as well as loss of tryptophan following fibrinogen oxidation were observed. The occurrence of conformational changes of the fibrinogen molecule as a consequence of the oxidative treatment was also established. Oxidized fibrinogen showed a distinct capability from the native molecule to mediate platelet aggregation and adhesion. The percentage of ADP-induced platelet aggregation decreased as a function of fibrinogen oxidative damage. Further, both unstimulated platelets and ADP-activated platelets showed a reduced ability to adhere to oxidized fibrinogen than to the native protein. These results suggest that oxidative treatment alters fibrinogen domains involved in the recognition and the binding of this molecule by the platelet receptor GP IIb/IIIa.

Induction of adaptive response in human blood lymphocytes exposed to 900 MHz radiofrequency fields: Influence of cell cycle

Abstract Purpose: To investigate the influence of cell cycle on the adaptive response (AR) induced by the exposure of human blood lymphocytes to radiofrequency fields (RF). Materials and methods: Human peripheral blood lymphocytes in G0-, G1- or S-phase of the cell cycle were exposed for 20 hours to an adaptive dose (AD) of 900 MHz RF at an average specific absorption rate of 1.25 W/kg and then treated with a challenge dose (CD) of 100 ng/ml mitomycin C (MMC). Un-exposed and sham-exposed controls as well as cells treated with MMC alone were included in the study. The incidence of micronuclei (MN) was evaluated to determine the induction of AR. Results: The results indicated that the cells which were exposed to AD of RF in G0- and G1-phase of the cell cycle did not exhibit AR while such a response was observed when the cells were exposed to AD of RF in S-phase of the cell cycle. Conclusions: These results confirmed the observations reported in our previous investigation where AR was observed in human blood lymphocytes exposed to AD of RF in S-phase of the cell cycle and further suggested that the timing of AD exposure of RF is important to elicit AR.

Structural characterization of tetranortriterpenes from Pseudrocedrela kotschyi and Trichilia emetica and study of their activity towards the chaperone Hsp90

Investigation of roots extracts Pseudrocedrela kotschyi and Trichilia emetica led to identification of 5 limonoid derivatives, Kotschyins D-H, and 11 known compounds. Their structures were elucidated by extensive 1D and 2D NMR experiments in conjunction with mass spectrometry. A surface plasmon resonance (SPR) approach was adopted to screen their Hsp90 binding capability and kotschyin D showed a significant affinity for the chaperone. Therefore, the characterization of the biological activity of kotschyin D by means of a panel of chemical and biological approaches, including limited proteolysis, molecular docking and biochemical and cellular assays, was performed. Our result indicated this compound as a type of client selective Hsp90 inhibitor, directly binding to the middle domain of the protein and possibly preventing its interaction with the activator of Hsp90 ATPase 1 (Aha1).

1-Methoxy-canthin-6-one induces c-Jun NH2-terminal kinase-dependent apoptosis and synergizes with tumor necrosis factor-related apoptosis-inducing ligand activity in human neoplastic cells of hematopoietic or endodermal origin.

We investigated the effects of 1-methoxy-canthin-6-one, isolated from the medicinal plant Ailanthus altissima Swingle, on apoptosis in human leukemia (Jurkat), thyroid carcinoma (ARO and NPA), and hepatocellular carcinoma (HuH7) cell lines. Cultures incubated with the compound showed >50% of sub-G1 (hypodiploid) elements in flow cytometry analysis; the apoptosis-inducing activity was evident at <10 micromol/L and half-maximal at about 40 micromol/L 1-methoxy-canthin-6-one. The appearance of hypodiploid elements was preceded by mitochondrial membrane depolarization, mitochondrial release of cytochrome c, and Smac/DIABLO and procaspase-3 cleavage. We subsequently investigated the effect of 1-methoxy-canthin-6-one in combination with human recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in the four cell lines. Suboptimal concentrations (10 micromol/L 1-methoxy-canthin-6-one and 0.25 ng/mL TRAIL, respectively) of the two agents, unable to elicit apoptosis when used alone, induced mitochondrial depolarization, activation of caspase-3, and 45% to 85% of sub-G1 elements when added together to the cells. The synergism seemed to rely partly on the enhanced expression of TRAIL receptor 1 (TRAIL-R1; DR4), analyzed by immunofluorescence, by 1-methoxy-canthin-6-one. Cell incubation with 1-methoxy-canthin-6-one resulted in activating c-Jun NH2-terminal kinase (JNK), as revealed by Western blotting; induction of apoptosis and TRAIL-R1 up-regulation by 1-methoxy-canthin-6-one were >80% prevented by the addition of the JNK inhibitor (JNKI) SP600125JNKI, indicating that both effects were almost completely mediated by JNK activity. On the other hand, synergism with TRAIL was reduced by about 50%, suggesting that besides up-regulating TRAIL-R1, 1-methoxy-canthin-6-one could influence other factor(s) that participated in TRAIL-induced apoptosis. These findings indicate that 1-methoxy-canthin-6-one can represent a candidate for in vivo studies of monotherapies or combined antineoplastic therapies.

H2O2 activity on platelet adhesion to fibrinogen and protein tyrosine phosphorylation

Platelets represent a target of reactive oxygen species produced under oxidative stress conditions. Controversial data on the effect of these species on platelet functions have been reported so far. In this study we evaluated the effect of a wide range of H(2)O(2) concentrations on platelet adhesion to immobilized fibrinogen and on pp72(syk) and pp125(FAK) tyrosine phosphorylation. Our results demonstrate that: (1) H(2)O(2) does not affect the adhesion of unstimulated or apyrase-treated platelets to immobilized fibrinogen; (2) H(2)O(2) does not affect pp72(syk) phosphorylation induced by platelet adhesion to fibrinogen-coated dishes; (3) H(2)O(2) reduces, in a dose-dependent fashion, pp125(FAK) phosphorylation of fibrinogen-adherent platelets; (4) concentrations of H(2)O(2) near to physiological values (10-12 microM) are able to strengthen the subthreshold activation of pp125(FAK) induced by epinephrine in apyrase-treated platelets; (5) H(2)O(2) doses higher than 0.1 mM inhibit ADP-induced platelet aggregation and dense granule secretion. The ability of H(2)O(2) to modulate pp125(FAK) phosphorylation suggests a role of this molecule in physiological hemostasis as well as in thrombus generation.

Biological availability of mutagenic compounds adsorbed onto diesel exhaust particulate

Diesel particles were collected from the exhaust of a VW Golf diesel car by electrostatic precipitation. The particulate and its DCM extract were highly mutagenic in the Salmonella/microsome test in the presence and absence of metabolic activation; the highest response was observed with TA98 and TA1538 tester strains. The biological availability of particulate-associated mutagenic compounds was demonstrated by administering powder to rats and assaying, in vitro, the urine excreted within 24 h after treatment. The highest activity was obtained with TA98 in the presence of metabolic activation. Typical dose-effect responses were evident in urine of animals treated by all the administration routes tested (i.p., s.c. and per os), both in the presence and absence of a suspending vehicle. Concentration of mutagenic compounds present in urine of treated animals could be achieved by chromatography on Amberlite XAD-2 and XAD-7 resins. This study provides direct evidence for bioavailability to animal tissues of mutagens adsorbed onto diesel particulate, although part of the activity might be ascribed to nitroaromatic compounds formed during the collection of the powder. The present study is part of a more comprehensive work on diesel exhaust particulate, and results have to be considered in this light before any final conclusion can be drawn.

Thioredoxin System Modulation by Plant and Fungal Secondary Metabolites

Thioredoxin (Trx) is the major cellular protein disulfide reductase in a broad range of organisms, including humans. Trx, together with glutaredoxin (Grx), plays critical roles in the regulation of cellular protein redox homeostasis. Reduced thioredoxin transfers reducing equivalents to disulphides in target proteins, leading to reversible oxidation of its active centre dithiol to a disulphide. The resulting disulphide bridge is, in turn, reduced to a dithiol by thioredoxin reductase (TrxR). Increasing attention has been paid to the role of Trx, as it has been shown to be a signalling intermediate beyond its intrinsic antioxidant activity. Indeed, this protein acts as a growth factor, activates a number of transcription factors regulating cell growth and survival, acts as cofactor for ribonucleotide reductase, and promotes angiogenesis. In addition, Trx have been demonstrated to cooperatively inhibit programmed cell death. Because of the multiple roles of Trx system in tumorigenesis, this protein represents an emerging target for anti-cancer drugs. Several Trx system modulators have been identified: a semi-synthetic Trx inhibitor, PX-12 (1-methylpropyl 2-imidazolyl disulfide), has been placed in a clinical trial. However, there is a growing interest in finding new selective ones. Natural products continue to provide structurally complex, but highly original lead structures for drug discovery programs: polyphenols, quinones, and terpenoids showed to affect the Trx/TrxR system at different levels. The purpose of this review is to provide an overview of the plant and fungal secondary metabolites interfering with Trx and TrxR activities, paying particularly attention on their mechanism of action. Among polyphenols, curcumin and some flavonoids such as myricetin and quercetin, have been identified as potential anticancer agents with a mechanism of action that may be mediated by the Trx system.

Effect of sesquiterpene lactone coronopilin on leukaemia cell population growth, cell type-specific induction of apoptosis and mitotic catastrophe

Objectives:  The aim of this study was to investigate anti‐leukaemic potential of coronopilin, a sesquiterpene lactone from Ambrosia arborescens, and to characterize mechanism(s) underlying its activity.