Chiara Dianzani

Interaction of aldehydes derived from lipid peroxidation and membrane proteins.

A great variety of compounds are formed during lipid peroxidation of polyunsaturated fatty acids of membrane phospholipids. Among them, bioactive aldehydes, such as 4-hydroxyalkenals, malondialdehyde (MDA) and acrolein, have received particular attention since they have been considered as toxic messengers that can propagate and amplify oxidative injury. In the 4-hydroxyalkenal class, 4-hydroxy-2-nonenal (HNE) is the most intensively studied aldehyde, in relation not only to its toxic function, but also to its physiological role. Indeed, HNE can be found at low concentrations in human tissues and plasma and participates in the control of biological processes, such as signal transduction, cell proliferation, and differentiation. Moreover, at low doses, HNE exerts an anti-cancer effect, by inhibiting cell proliferation, angiogenesis, cell adhesion and by inducing differentiation and/or apoptosis in various tumor cell lines. It is very likely that a substantial fraction of the effects observed in cellular responses, induced by HNE and related aldehydes, be mediated by their interaction with proteins, resulting in the formation of covalent adducts or in the modulation of their expression and/or activity. In this review we focus on membrane proteins affected by lipid peroxidation-derived aldehydes, under physiological and pathological conditions.

Characterization of ionotropic glutamate receptors in human lymphocytes.

The effect of L‐glutamate (Glu) on human lymphocyte function was studied by measuring anti‐CD3 monoclonal antibody (mAb) or phytohaemagglutinin (PHA)‐induced intracellular Ca2+ ([Ca2+]i) rise (Fura‐2 method), and cell proliferation (MTT assay). Glu (0.001 – 100 μM) did not modify basal lymphocyte [Ca2+]i, but significantly potentiated the effects of anti‐CD3 mAb or PHA. Maximal [Ca2+]i rises over resting cells were: 165±8 and 247±10 nM at 3.0×10−2 mg ml−1 anti‐CD3 mAb; 201±4 and 266±9 nM at 5.0×10−2 mg ml−1 PHA, in the absence or presence of 1 μM Glu, respectively. The Glu effect showed a bell‐shape concentration‐dependent relationship, with a maximum (+90±3% for anti‐CD3 mAb and +57±2% for PHA over Glu‐untreated cells) at 1 μM. Non‐NMDA receptor agonists (1 μM) showed a greater efficacy (+76±2% for (S)‐AMPA; +78±4% for KA), if compared to NMDA (+46±2%), or Glu itself. Ionotropic Glu receptor antagonists completely inhibited the effects of the corresponding specific receptor agonists (1 μM). The IC50 values calculated were: 0.9 μM for D‐AP5; 0.6 μM for (+)‐MK801; 0.3 μM for NBQX. Both NBQX and KYNA were able to abolish Glu effect. The IC50s calculated were: 3.4 μM for NBQX; 0.4 μM for KYNA. Glu (0.1 – 1 mM) did not change the resting cell proliferation, whereas Glu (1 mM) significant inhibited (−27±4%) PHA (1.0×10−2 mg ml−1)‐induced lymphocyte proliferation at 72 h. In conclusion, human lymphocytes express ionotropic Glu receptors functionally operating as modulators of cell activation.

TREATMENT WITH THE GLYCOGEN SYNTHASE KINASE-3β INHIBITOR, TDZD-8, AFFECTS TRANSIENT CEREBRAL ISCHEMIA/REPERFUSION INJURY IN THE RAT HIPPOCAMPUS

The serine/threonine glycogen synthase kinase 3&bgr; (GSK-3&bgr;) is abundant in the central nervous system, particularly in the hippocampus, and plays a pivotal role in the pathophysiology of a number of diseases, including neurodegeneration. This study was designed to investigate the effects of GSK-3&bgr; inhibition against I/R injury in the rat hippocampus. Transient cerebral ischemia (30 min) followed by 1 h of reperfusion significantly increased generation of reactive oxygen species and modulated superoxide dismutase activity; 24 h of reperfusion evoked apoptosis (determined as mitochondrial cytochrome c release and Bcl-2 and caspase-9 expression), resulted in high plasma levels of TNF-&agr; and increased expression of cyclooxygenase-2, inducible nitric oxide synthase, and intercellular adhesion molecule-1. The selective GSK-3&bgr; inhibitor, 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), was administered before and after ischemia or during reperfusion alone to assess its potential as prophylactic or therapeutic strategy. Prophylactic or therapeutic administration of TDZD-8 caused the phosphorylation (Ser9) and hence inactivation of GSK-3&bgr;. Infarct volume and levels of S100B protein, a marker of cerebral injury, were reduced by TDZD-8. This was associated with a significant reduction in markers of oxidative stress, apoptosis, and the inflammatory response resulting from cerebral I/R. These beneficial effects were associated with a reduction of I/R-induced activation of the mitogen-activated protein kinases JNK1/2 and p38 and nuclear factor-&kgr;B. The present study demonstrates that TDZD-8 protects the brain against I/R injury by inhibiting GSK-3&bgr; activity. Collectively, our data may contribute to focus the role of GSK-3&bgr; in cerebral I/R.

Drug delivery nanoparticles in skin cancers.

Nanotechnology involves the engineering of functional systems at nanoscale, thus being attractive for disciplines ranging from materials science to biomedicine. One of the most active research areas of the nanotechnology is nanomedicine, which applies nanotechnology to highly specific medical interventions for prevention, diagnosis, and treatment of diseases, including cancer disease. Over the past two decades, the rapid developments in nanotechnology have allowed the incorporation of multiple therapeutic, sensing, and targeting agents into nanoparticles, for detection, prevention, and treatment of cancer diseases. Nanoparticles offer many advantages as drug carrier systems since they can improve the solubility of poorly water-soluble drugs, modify pharmacokinetics, increase drug half-life by reducing immunogenicity, improve bioavailability, and diminish drug metabolism. They can also enable a tunable release of therapeutic compounds and the simultaneous delivery of two or more drugs for combination therapy. In this review, we discuss the recent advances in the use of different types of nanoparticles for systemic and topical drug delivery in the treatment of skin cancer. In particular, the progress in the treatment with nanocarriers of basal cell carcinoma, squamous cell carcinoma, and melanoma has been reported.

Substance P increases neutrophil adhesion to human umbilical vein endothelial cells

Adhesion of neutrophils (PMNs) to vascular endothelial cells (EC) is a critical step in recruitment and infiltration of leukocytes into tissues during inflammation. Substance P (SP), a neuropeptide released from sensory nerves, evoked PMN adhesion to EC. The NK receptor subtype(s) and the cell type(s) involved were investigated. SP was coincubated with human PMNs and EC from the human umbilical vein (HUVEC); adhesion was quantitated by computerised microimaging fluorescence analysis. The proadhesive effects of SP (range 10−18–10−6 M) were illustrated in a biphasic dose–response curve, with a maximum at 10−15 M (276±16% adhesion vs control; P<0.01) and another one at 10−10 M (200±18% adhesion vs control; P<0.01). Neurokinin A was less active and neurokinin B was inactive. The adhesion molecules LFA‐1 and OKM‐1, but not selectins, were involved according to results with selective mAbs. The NK1 agonist [Sar9,Met(O2)11]SP reproduced the effects of SP, whereas the NK2 agonist [βAla8]‐neurokininA (4–10) acted at 10−13–10−8 M only. The NK3 agonist, senktide, was ineffective. The NK1 antagonists, CP 96,345 and L 703,606 (both 10−6 M), abolished the effect of 10−15 M SP and inhibited that of 10−10 M SP by 56±5% (P<0.01). By comparison, the NK2 antagonist, SR 48,968 (10−7 M), partially antagonised the adhesion evoked by 10−10 M SP (% inhibition: 61±6; P<0.05). Since preincubation of PMNs and HUVEC with SP gave the same results it is clear that both cell types contributed to its proadhesive effects. These results indicate that SP induced a proadhesive effect during inflammatory processes, which was mediated by NK1 and NK2 receptors.

Celecoxib decreases expression of the adhesion molecules ICAM‐1 and VCAM‐1 in a colon cancer cell line (HT29)

We investigated the ability of celecoxib, a selective cyclooxygenase‐2 (COX‐2) inhibitor, to modulate expression of ICAM‐1 and VCAM‐1 in the colon cancer cell line HT29.

Substance P-induced cyclooxygenase-2 expression in human umbilical vein endothelial cells

Substance P (SP) is a neuropeptide involved in neurogenic inflammation and an agonist for NK1, NK2, and NK3 receptors. SP induces prostaglandin (PG) production in various cell types, and these eicosanoids are responsible for numerous inflammatory and vascular effects. Cyclooxygenase (COX) are needed to convert arachidonic acid to PGs. The study evaluated the effect of SP on COX expression in human umbilical vein endothelial cells (HUVEC). COX‐2 protein expression was upregulated by SP with a peak at 100 nM and at 20 h; in the same experimental conditions COX‐1 protein expression was unchanged. A correlation between COX‐2 expression and PGI2 and PGE2 release was detected. Dexamethasone (DEX) inhibited SP‐mediated COX‐2 expression. Mitogen‐activated protein kinases (MAPK) p38 and p42/44 were activated by SP, whereas SB202190 and PD98059, inhibitors of these kinases, blocked COX‐2 expression. 5,5‐dimethyl‐3‐(3‐fluorophenyl)‐4‐(4‐methylsulphonyl)phenyl‐2(5H)‐furanone (DFU), an experimental selective COX‐2 inhibitor, blocked SP‐induced PG release. By RT–PCR and Western blot analysis, we demonstrated that NK1 and NK2 but not NK3 receptors are present on HUVEC. Selective NK1 and NK2 agonists, namely [Sar9, Met(O2)11]SP and [β‐Ala8] NKA(4–10), upregulated COX‐2 protein expression and PG production, whereas senktide (Suc–Asp–Phe–MePhe–Gly–Leu–Met–NH2), a selective NK3 agonist, was ineffective in this respect. The NK1 selective antagonist L703,606 ((cis)‐2‐(diphenylmethyl)‐N‐((2‐iodophenyl)‐methyl)‐1‐azabicyclo(2.2.2)octan‐3‐amine) and the NK2 selective antagonist SR 48,968 ((S)‐N‐methyl‐N‐(4‐(4‐acetylamino‐4‐phenylpiperidino)‐2‐(3,4 dichlorophenyl)butyl) benzamide) competitively antagonised SP‐induced effects. The study shows HUVEC to possess functional NK1 and NK2 receptors, which mediate the ability of SP to induce expression of COX‐2 in HUVEC, thus showing a previously‐undetected effect of SP on endothelial cells.

Group I mGlu receptor stimulation inhibits activation-induced cell death of human T lymphocytes.

1 The effects of L‐glutamate on activation‐induced cell death (AICD) of human activated (1 μg ml−1 phytohemagglutinin plus 2 U ml−1 interleukin‐2; 8 days) T lymphocytes were studied by measuring anti‐CD3 monoclonal antibody (10 μg ml−1; 18 h)‐induced cell apoptosis (Annexin V and propidium iodide staining). 2 L‐Glutamate (1 × 10−8–1 × 10−4 M) significantly (P0.01) inhibited AICD in a concentration‐dependent manner (EC50=6.3 × 10−8 M; maximum inhibition 54.8±6.3% at 1 × 10−6 M). 3 The L‐glutamate inhibitory effect was pharmacologically characterized as mediated by group I mGlu receptors, since mGlu receptor agonists reproduced this effect. The EC50 values were: 3.2 × 10−7 M for (1S,3R)‐ACPD; 4.5 × 10−8 M for quisqualate; 1.0 × 10−6 M for (S)‐3,5‐DHPG; 2.0 × 10−5 M for CHPG. 4 Group I mGlu receptor antagonists inhibited the effects of quisqualate 1.0 × 10−6 M. The IC50 values calculated were: 8.7 × 10−5, 4.3 × 10−6 and 6.3 × 10−7 M for AIDA, LY 367385 and MPEP, respectively. 5 L‐Glutamate (1 × 10−6 M; 18 h) significantly (P0.05) inhibited FasL expression (40.8±11.3%) (cytofluorimetric analysis), whereas it did not affect Fas signalling. 6 Expression of both mGlu1 and mGlu5 receptor mRNA by T lymphocytes and T‐cell lines, as demonstrated by reverse transcriptase–PCR analysis, suggests that L‐glutamate‐mediated inhibition of AICD was exerted on T cells. 7 These data depict a novel role for L‐glutamate in the regulation of the immune response through group I mGlu receptor‐mediated mechanisms.

Role of 4-Hydroxynonenal-Protein Adducts in Human Diseases

SIGNIFICANCE Oxidative stress provokes the peroxidation of polyunsaturated fatty acids in cellular membranes, leading to the formation of aldheydes that, due to their high chemical reactivity, are considered to act as second messengers of oxidative stress. Among the aldehydes formed during lipid peroxidation (LPO), 4-hydroxy-2-nonenal (HNE) is produced at a high level and easily reacts with both low-molecular-weight compounds and macromolecules, such as proteins and DNA. In particular, HNE-protein adducts have been extensively investigated in diseases characterized by the pathogenic contribution of oxidative stress, such as cancer, neurodegenerative, chronic inflammatory, and autoimmune diseases. RECENT ADVANCES In this review, we describe and discuss recent insights regarding the role played by covalent adducts of HNE with proteins in the development and evolution of those among the earlier mentioned disease conditions in which the functional consequences of their formation have been characterized. CRITICAL ISSUES Results obtained in recent years have shown that the generation of HNE-protein adducts can play important pathogenic roles in several diseases. However, in some cases, the generation of HNE-protein adducts can represent a contrast to the progression of disease or can promote adaptive cell responses, demonstrating that HNE is not only a toxic product of LPO but also a regulatory molecule that is involved in several biochemical pathways. FUTURE DIRECTIONS In the next few years, the refinement of proteomical techniques, allowing the individuation of novel cellular targets of HNE, will lead to a better understanding the role of HNE in human diseases.

Priming effects of substance P on calcium changes evoked by interleukin-8 in human neutrophils

The neurokinin (NK) substance P (SP), which is a mediator of neurogenic inflammation, has been reported to prime human polymorphonuclear neutrophils (PMNs). The priming effects of SP on PMNs activated by recombinant interleukin‐8 (rIL‐8) were investigated. SP enhanced, in a dose‐ and time‐dependent way, the rise in cytosolic free‐calcium concentration, [Ca2+]i, evoked by the chemokine. The priming effects of SP were abolished by exposing PMNs to a calcium‐free medium supplemented with EGTA. The C‐terminal peptides SP(4–11) and SP(6–11) but not the N‐terminal peptide SP(1–7) shared the priming effects of SP. The selective NK‐1 receptor agonist [Sar‐9, Met(O)2‐11]SP mimicked the effects of SP, which were not reproduced by the selective NK‐2 receptor agonist [βAla‐8]‐NKA(4–10) or the selective NK‐3 agonist senktide. Two selective NK‐1 antagonists, CP96,345 and L703,606, dose dependently inhibited SP priming effects. These results demonstrated that SP primes PMNs exposed to rIL‐8 and suggested that SP priming effects are receptor mediated.