Alessandra Carcereri de Prati

Extracorporeal Shock Wave Therapy in Inflammatory Diseases: Molecular Mechanism that Triggers Anti-Inflammatory Action

Shock waves (SW), defined as a sequence of single sonic pulses characterised by high peak pressure (100 MPa), a fast rise in pressure (< 10 ns) and a short lifecycle (10 micros), are conveyed by an appropriate generator to a specific target area at an energy density ranging from 0.03 to 0.11 mJ/mm(2). Extracorporeal SW (ESW) therapy was first used on patients in 1980 to break up kidney stones. During the last ten years, this technique has been successfully employed in orthopaedic diseases such as pseudoarthosis, tendinitis, calcarea of the shoulder, epicondylitis, plantar fasciitis and several inflammatory tendon diseases. In particular, treatment of the tendon and muscle tissues was found to induce a long-time tissue regeneration effect in addition to having a more immediate anthalgic and anti-inflammatory outcome. In keeping with this, an increase in neoangiogenesis in the tendons of dogs was observed after 4-8 weeks of ESW treatment. Furthermore, clinical observations indicate an immediate increase in blood flow around the treated area. Nevertheless, the biochemical mechanisms underlying these effects have yet to be fully elucidated. In the present review, we briefly detail the physical properties of ESW and clinical cases treated with this therapy. We then go on to describe the possible molecular mechanism that triggers the anti-inflammatory action of ESW, focusing on the possibility that ESW may modulate endogenous nitric oxide (NO) production either under normal or inflammatory conditions. Data on the rapid enhancement of endothelial NO synthase (eNOS) activity in ESW-treated cells suggest that increased NO levels and the subsequent suppression of NF-kappaB activation may account, at least in part, for the clinically beneficial action on tissue inflammation.

Nitric oxide mediates anti‐inflammatory action of extracorporeal shock waves

Here, we show that extracorporeal shock waves (ESW), at a low energy density value, quickly increase neuronal nitric oxide synthase (nNOS) activity and basal nitric oxide (NO) production in the rat glioma cell line C6. In addition, the treatment of C6 cells with ESW reverts the decrease of nNOS activity and NO production induced by a mixture of lipopolysaccharides (LPS), interferon‐γ (IFN‐γ) plus tumour necrosis factor‐α (TNF‐α). Finally, ESW treatment efficiently downregulates NF‐κB activation and NF‐κB‐dependent gene expression, including inducible NOS and TNF‐α. The present report suggests a possible molecular mechanism of the anti‐inflammatory action of ESW treatment.

STAT1 as a new molecular target of anti-inflammatory treatment.

Cyclooxygenase (COX) is widely considered as the molecular target of non-steroid anti-inflammatory drugs (NSAIDs). However, due to the harmful side effect frequently observed following chronic use, the development of new anti-inflammatory agents is the matter of many studies. Signal transducers and activators of transcription (STAT) are a family of nuclear proteins mediating the action of a number of cytokines. Among them, STAT1 plays a critical role in the signal transduction pathway of interferon-gamma (IFN-gamma) and growth hormone. STAT1 cascade is one major signalling pathway converting the IFN-gamma signal into gene expression, such as inducible nitric oxide synthase (iNOS), COX, vascular cell adhesion molecules (VCAM) and intercellular cell adhesion molecules (ICAM), critically involved in different pathologies correlated to the inflammatory process. This review focuses the attention on an alternative approach to the development of novel drugs based on inhibition of STAT1 pathway. In this context, a growing interest has been focused on natural compounds. We have recently reported a several data indicating that green tea extract (GTE), St. Johns Wort extract and epigallocatechin-3-gallate (EGCG) exhibit a specific and strong anti-STAT1 activity which is independent of their acclaimed strong anti-oxidant activity. More recently, GTE has been shown to protect heart damage from ischaemia/reperfusion in rats, suggesting that the protective effect of green tea might be correlated to its anti-STAT1 activity. The present review is aimed at providing data that STAT1 may potentially be claimed as a new molecular target of an anti-inflammatory treatment and that among natural compounds there are a number of anti-STAT1 substances.

Targeting STAT1 by myricetin and delphinidin provides efficient protection of the heart from ischemia/reperfusion-induced injury.

Flavonoids exhibit a variety of beneficial effects in cardiovascular diseases. Although their therapeutic properties have been attributed mainly to their antioxidant action, they have additional protective mechanisms such as inhibition of signal transducer and activator of transcription 1 (STAT1) activation. Here, we have investigated the cardioprotective mechanisms of strong antioxidant flavonoids such as quercetin, myricetin and delphinidin. Although all of them protect the heart from ischemia/reperfusion‐injury, myricetin and delphinidin exert a more pronounced protective action than quercetin by their capacity to inhibit STAT1 activation. Biochemical and computer modeling analysis indicated the direct interaction between STAT1 and flavonoids with anti‐STAT1 activity.

Two Naturally Occurring Terpenes, Dehydrocostuslactone and Costunolide, Decrease Intracellular GSH Content and Inhibit STAT3 Activation

The main purpose of the present study is to envisage the molecular mechanism of inhibitory action ofdehydrocostuslactone (DCE) andcostunolide (CS), two naturally occurring sesquiterpene lactones, towards the activation of signal transducer and activator of transcription 3 (STAT3). We report that, in human THP-1 cell line, they inhibit IL-6-elicited tyrosine phosphorylation of STAT3 and its DNA binding activity with EC50 of 10 µM with concomitantdown-regulation ofthe phosphorylation of the tyrosine Janus kinases JAK1, JAK2 and Tyk2. Furthermore, these compounds that contain an α-β-unsatured carbonyl moiety and function as potent Michael reaction acceptor, induce a rapid drop in intracellular glutathione (GSH) concentration by direct interaction with it, thereby triggering S-glutathionylation of STAT3. Dehydrocostunolide (HCS), the reduced form of CS lacking only the α-β-unsaturated carbonyl group, fails to exert any inhibitory action. Finally, the glutathione ethylene ester (GEE), the cell permeable GSH form, reverts the inhibitory action of DCE and CS on STAT3 tyrosine phosphorylation. We conclude that these two sesquiterpene lactones are able to induce redox-dependent post-translational modification of cysteine residues of STAT3 protein in order to regulate its function.

Protective effect of Arbutus unedo aqueous extract in carrageenan-induced lung inflammation in mice.

In the present study, we show that an aqueous extract of Arbutus unedo L. (AuE), a Mediterranean endemic plant widely employed as an astringent, diuretic and urinary anti-septic, in vitro down-regulates the expression of some inflammatory genes, such as those encoding inducible nitric oxide synthase (iNOS) and intracellular adhesion molecule-(ICAM)-1, exerting a inhibitory action on both IFN-gamma-elicited STAT1 activation and IL-6-elicited STAT3 activation. To evaluate further the effect of AuE in animal models of acute inflammation, we examined whether AuE administration attenuates inflammatory response of murine induced by intrapleural injection of carrageenan. For this purpose we studied: (1) STAT1/3 activation, (2) TNF-alpha, IL-1beta and IL-6 production in pleural exudate, (3) lung iNOS, COX-2 and ICAM-1 expression, (4) neutrophil infiltration, (5) the nitration of cellular proteins by peroxynitrite, (6) lipid peroxidation, (7) prostaglandin E2 and nitrite/nitrate levels and (8) lung injury. We show that AuE strongly down-regulates STAT3 activation induced in the lung by carrageenan with concomitant attenuation of all parameters examined associated with inflammation, suggesting that STAT3 should be a new molecular target for anti-inflammatory treatment. This study demonstrates that acute lung inflammation is significantly attenuated by the treatment with AuE.

Pro-apoptotic activity of α-bisabolol in preclinical models of primary human acute leukemia cells

BackgroundWe previously demonstrated that the plant-derived agent α-bisabolol enters cells via lipid rafts, binds to the pro-apoptotic Bcl-2 family protein BID, and may induce apoptosis. Here we studied the activity of α-bisabolol in acute leukemia cells.MethodsWe tested ex vivo blasts from 42 acute leukemias (14 Philadelphia-negative and 14 Philadelphia-positive B acute lymphoid leukemias, Ph-/Ph+B-ALL; 14 acute myeloid leukemias, AML) for their sensitivity to α-bisabolol in 24-hour dose-response assays. Concentrations and time were chosen based on CD34+, CD33+my and normal peripheral blood cell sensitivity to increasing α-bisabolol concentrations for up to 120 hours.ResultsA clustering analysis of the sensitivity over 24 hours identified three clusters. Cluster 1 (14 ± 5 μM α-bisabolol IC50) included mainly Ph-B-ALL cells. AML cells were split into cluster 2 and 3 (45 ± 7 and 65 ± 5 μM IC50). Ph+B-ALL cells were scattered, but mainly grouped into cluster 2. All leukemias, including 3 imatinib-resistant cases, were eventually responsive, but a subset of B-ALL cells was fairly sensitive to low α-bisabolol concentrations. α-bisabolol acted as a pro-apoptotic agent via a direct damage to mitochondrial integrity, which was responsible for the decrease in NADH-supported state 3 respiration and the disruption of the mitochondrial membrane potential.ConclusionOur study provides the first evidence that α-bisabolol is a pro-apoptotic agent for primary human acute leukemia cells.

Mild oxidative stress induces S-glutathionylation of STAT3 and enhances chemosensitivity of tumoural cells to chemotherapeutic drugs.

STAT3 is a transcription factor constitutively activated in a variety of cancers that has a critical role in the inhibition of apoptosis and induction of chemoresistance. Inhibition of the STAT3 signaling pathway suppresses cell survival signals and leads to apoptosis in cancer cells, suggesting that direct inhibition of STAT3 function is a viable therapeutic approach. Herein, we identify the naturally occurring sesquiterpene lactone cynaropicrin as a potent inhibitor of both IL-6-inducible and constitutive STAT3 activation (IC50=12 μM). Cynaropicrin, which contains an α-β-unsaturated carbonyl moiety and acts as potent Michael reaction acceptor, induces a rapid drop in intracellular glutathione (GSH) concentration, thereby triggering S-glutathionylation of STAT3. Furthermore, glutathione ethylene ester, the cell permeable form of GSH, reverts the inhibitory action of cynaropicrin on STAT3 tyrosine phosphorylation. These findings suggest that this sesquiterpene lactone is able to induce redox-dependent post-translational modification of cysteine residues of STAT3 protein to regulate its function. STAT3 inhibition led to the suppression of two anti-apoptotic genes, Bcl-2 and survivin, in DU145 cells that constitutively express active STAT3. This event may be responsible for the decline in cell viability after cynaropicrin treatment. As revealed by PI/annexin-V staining, PARP cleavage, and DNA ladder formation, cynaropicrin cytotoxicity is mediated by apoptosis. Finally, cynaropicrin displayed a slight to strong synergism with two well-established chemotherapeutic drugs, cisplatin and docetaxel. Taken together our studies suggest that cynaropicrin suppresses the STAT3 pathway, leading to the down-regulation of STAT3-dependent gene expression and chemosensitization of tumour cells to chemotherapy.

Nitric oxide in the liver: Physiopathological roles

Many of the known roles of arginine (e.g. in immune function, wound healing, and protection against ammonia intoxication) are mediated by a metabolic pathway synthesising nitric oxide (NO) in the liver. Contrary to some of the current views, liver-produced NO may be basically beneficial, as it exerts both protective actions against tissue injury and cytotoxic effects on invading microorganisms, parasites, or tumor cells. An ongoing equilibrium between NO and other NO-reactive compounds (e.g. O2 and non-heme iron-sulphur-containing moieties) appears to be important in this respect, even under critical conditions. Thus, NO may prevent liver tissue harm from oxidant stress. Only when this putative counterbalance is upset by an uncontrolled, prolonged and/or massive production of NO, liver tissue damage may occur leading to hepatic inflammation or even tumor development. Moreover, the currently available data support the working hypothesis that hepatocytes partake not only to immunoregulatory processes, but even to immune defence mechanisms. Thus, the liver constitutes an excellent model for investigations into the crosstalks regulating the production of NO which take place among not only the various networks operating inside a single hepatic cell, but even the individual types of liver cells.

Differential expression of poly(ADP-ribose) polymerase and DNA polymerase β in rat tissues

The activities of two DNA repair-related enzymes, poly(ADP-ribose) polymerase and DNA polymerase beta, and their mRNA levels were measured in 17 tissues of Wistar rats. A large variety in enzyme activity values could be detected in the tissues examined; the highest levels of activity for both enzymes were found in the testis. A good correlation between poly(ADP-ribose) polymerase activity and the level of the transcript of the gene coding for the enzyme was observed in many tissues. A less satisfactory correlation could be evidenced for DNA polymerase beta. The almost parallel amounts of the mRNAs for poly(ADP-ribose) polymerase and DNA polymerase beta in the tissues examined suggest a possible coexpression of the genes coding for these enzymes. Additional studies have been carried out in testis and liver by immunohistochemical techniques and by in situ hybridization analyses. While in the testis the spermatocytes were shown to contain both enzymes and their transcripts, in other types of cells this could not be observed. In the liver mRNAs and enzymes were only found in 20% of the hepatocytes. This may in part explain both the low levels of the mRNAs and the modest activities of the two enzymes in that tissue.