Extracellular Vesicles in Breast Cancer: From Biology and Function to Clinical Diagnosis and Therapeutic Management.

Breast cancer (BC) is the first worldwide most frequent cancer in both sexes and the most commonly diagnosed in females. Although BC mortality has been thoroughly declining over the past decades, there are still considerable differences between women diagnosed with early BC and when metastatic BC is diagnosed. BC treatment choice is widely dependent on precise histological and molecular characterization. However, recurrence or distant metastasis still occurs even with the most recent efficient therapies. Thus, a better understanding of the different factors underlying tumor escape is mainly mandatory. Among the leading candidates is the continuous interplay between tumor cells and their microenvironment, where extracellular vesicles play a significant role. Among extracellular vesicles, smaller ones, also called exosomes, can carry biomolecules, such as lipids, proteins, and nucleic acids, and generate signal transmission through an intercellular transfer of their content. This mechanism allows tumor cells to recruit and modify the adjacent and systemic microenvironment to support further invasion and dissemination. By reciprocity, stromal cells can also use exosomes to profoundly modify tumor cell behavior. This review intends to cover the most recent literature on the role of extracellular vesicle production in normal and cancerous breast tissues. Specific attention is paid to the use of extracellular vesicles for early BC diagnosis, follow-up, and prognosis because exosomes are actually under the spotlight of researchers as a high-potential source of liquid biopsies. Extracellular vesicles in BC treatment as new targets for therapy or efficient nanovectors to drive drug delivery are also summarized.

APN/CD13 is over-expressed by Psoriatic fibroblasts and is modulated by CGRP and IL-4 but not by retinoic acid treatment.

Psoriasis vulgaris is a common skin inflammatory disease characterized by recurrent flare episodes associated with scaly well-demarcated skin plaques. Skin biopsies from psoriatic patients with high PASI score (22.67 ± 8.67) and from HD were used to study APN/CD13. APN/CD13 is over-expressed in LP and nLP compare to HD skins and fibroblasts. This over-expression is positively correlated with specific enzymatic activity enhancement. However, discrepancies between APN/CD13 expression in LP and nLP prompt us to focus our study on APN/CD13 modulation. Calcitonin Gene Related Peptide (CGRP), a neuropeptide, positively modulated expression and activity of APN/CD13. CGRP consistently induced IL4 secretion, which is also involved in the increase of APN/CD13 expression and activity, which is significantly reversed using IL-4 blocking antibody. Surprisingly, retinoic acid altered the APN/CD13 enzymatic activity only in nLP fibroblasts without modification of APN/CD13 expression. APN/CD13 is over-expressed on psoriatic fibroblasts and exerted high level of activity compare to HD fibroblasts. Taken together, several factors such as CGRP and IL-4 acted on positive regulation of APN/CD13 expression and activity. This study highlighted the interest of APN/CD13 as a new potential target, which should be investigated in psoriasis.

Dense red blood cell and oxygen desaturation in sickle-cell disease.

Production of abnormal hemoglobin (HbS) in sickle-cell disease (SCD) results in its polymerization in deoxygenated conditions and in sickled-RBC formation. Dense RBCs (DRBCs), defined as density >1.11 and characterized by increased rigidity are absent in normal AA subjects, but present at percentages that vary of a patient to another remaining stable throughout adulthood for each patient. Polymerized HbS has reduced affinity for oxygen, demonstrated by the rightward shift of the oxygen-dissociation curve, leading to disturbances in oxygen transport. Ninety-two SCD patients' total RBCs were separated into LightDRBC (LRBC) (d < 1.11 g/mL) and DRBC fractions. Venous blood partial oxygen pressure and RBC-fraction-deoxygenation and -reoxygenation Hb-oxygen-equilibrium curves were determined. All patients took a 6-minute walking test (6MWT); 10 had results before and after >6 months on hydroxyurea. 6MWT time with SpO2 < 88% (TSpO2 < 88) assessed the physiological impact of exertion. Elevated mean corpuscular hemoglobin (Hb) concentrations, decreased %HbF, and 2,3-bisphosphoglycerates in DRBCs modulated Hb-oxygen affinity. Deoxygenation and reoxygenation Hb-oxygen equilibrium curves differed between normal Hb AA and SS RBCs and between LRBCs and DRBCs, with rightward shifts confirming HbS-polymerization's role in affinity loss. In bivariate analyses, 50% Hb saturation correlated positively with %DRBCs (P < 0.0001, r(2) = 0.34) and negatively with %HbF (P < 0.0001, r(2) = 0.25). The higher the %DRBCs, the longer the TSpO2 88 (P = 0.04). Hydroxyurea was associated with significantly shorter TSpO2 < 88 (P = 0.01). We report that the %DRBCs directly affects SCD patients' SpO2 during exertion; hydroxyurea improves oxygen affinity and lowers the %DRBCs. Am. J. Hematol. 91:1008-1013, 2016. © 2016 Wiley Periodicals, Inc.

GSH monoethyl ester rescues mitochondrial defects in cystic fibrosis models.

Cystic fibrosis (CF), a multisystem disease caused by CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations, is associated with an abnormal inflammatory response and compromised redox homeostasis in the airways. Recent evidence suggests that dysfunctional CFTR leads to redox imbalance and to mitochondrial reduced glutathione (mtGSH) depletion in CF models. This study was designed to investigate the consequences of mtGSH depletion on mitochondrial function and inflammatory response. mtGSH depletion was confirmed in colonic epithelium of CFTR-null mice and in CFTR-mutated human epithelial cells. GSH uptake experiments performed on isolated mitochondria suggest that mtGSH depletion is not due to a defective GSH transport capacity by CF mitochondria, despite the decreased expression of two mtGSH carriers, oxoglutarate carrier and dicarboxylate carrier. CM-H(2)DCFDA [5 (and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester] fluorescence and aconitase activity showed an increase in reactive oxygen species levels in CFTR-defective cells and a pro-oxidative environment within CF mitochondria. The activities of respiratory chain complexes were further examined. Results showed a selective loss of Complex I (CI) function in CF models associated with an altered mitochondrial membrane potential (Δψ(m)). CI analysis showed normal expression but an overoxidation of its NADH-ubiquinone oxidoreductase Fe-S protein 1 subunit. GSH monoethyl ester (GSH-EE) significantly enhanced mtGSH levels in the IB3-1/C38 model and reversed CI inhibition, suggesting that mtGSH depletion is responsible for the loss of CI activity. Furthermore, GSH-EE attenuated Δψ(m) depolarization and restored normal IL-8 secretion by CFTR-defective cells. These studies provide evidence for a critical role of a mtGSH defect in mitochondrial dysfunction and abnormal IL-8 secretion in CF cells and reveal the therapeutic potential of mitochondria-targeted antioxidants in CF.

Tempol prevents cardiac oxidative damage and left ventricular dysfunction in the PPAR-α KO mouse.

Peroxisome proliferator-activated receptor (PPAR)-α deletion induces a profound decrease in MnSOD activity, leading to oxidative stress and left ventricular (LV) dysfunction. We tested the hypothesis that treatment of PPAR-α knockout (KO) mice with the SOD mimetic tempol prevents the heart from pathological remodelling and preserves LV function. Twenty PPAR-α KO mice and 20 age-matched wild-type mice were randomly treated for 8 wk with vehicle or tempol in the drinking water. LV contractile parameters were determined both in vivo using echocardiography and ex vivo using papillary muscle mechanics. Translational and posttranslational modifications of myosin heavy chain protein as well as the expression and activity of major antioxidant enzymes were measured. Tempol treatment did not affect LV function in wild-type mice; however, in PPAR-α KO mice, tempol prevented the decrease in LV ejection fraction and restored the contractile parameters of papillary muscle, including maximum shortening velocity, maximum extent of shortening, and total tension. Moreover, compared with untreated PPAR-α KO mice, myosin heavy chain tyrosine nitration and anion superoxide production were markedly reduced in PPAR-α KO mice after treatment. Tempol also significantly increased glutathione peroxidase and glutathione reductase activities (~ 50%) in PPAR-α KO mice. In conclusion, these findings demonstrate that treatment with the SOD mimetic tempol can prevent cardiac dysfunction in PPAR-α KO mice by reducing the oxidation of contractile proteins. In addition, we show that the beneficial effects of tempol in PPAR-α KO mice involve activation of the glutathione peroxidase/glutathione reductase system.

Variation in endogenous oxidative stress in Escherichia coli natural isolates during growth in urine.

BACKGROUND: Uropathogenic strains of Escherichia coli cause symptomatic infections whereas asymptomatic bacteriuria (ABU) strains are well adapted for growth in the human urinary tract, where they establish long-term bacteriuria. Human urine is a very complex growth medium that could be perceived by certain bacteria as a stressful environment. To investigate a possible imbalance between endogenous oxidative response and antioxidant mechanisms, lipid oxidative damage estimated as thiobarbituric acid reactive substances (TBARS) content was evaluated in twenty-one E. coli belonging to various pathovars and phylogenetic groups. Antioxidant defense mechanisms were also analysed. RESULTS: During exponential growth in urine, TBARS level differs between strains, without correlation with the ability to grow in urine which was similarly limited for commensal, ABU and uropathogenic strains. In addition, no correlation between TBARS level and the phylogroup or pathogenic group is apparent. The growth of ABU strain 83972 was associated with a high level of TBARS and more active antioxidant defenses that reduce the imbalance. CONCLUSIONS: Our results indicate that growth capacity in urine is not a property of ABU strains. However, E. coli isolates respond very differently to this stressful environment. In strain ABU 83972, on one hand, the increased level of endogenous reactive oxygen species may be responsible for adaptive mutations. On the other hand, a more active antioxidant defense system could increase the capacity to colonize the bladder.

Genetic background and sex control the outcome of high-fat diet feeding in mice.

The sharp increase in obesity prevalence worldwide is mainly attributable to changes in physical activity and eating behavior but the metabolic and clinical impacts of these obesogenic conditions vary between sexes and genetic backgrounds. This warrants personalized treatments of obesity and its complications, which require a thorough understanding of the diversity of metabolic responses to high-fat diet intake. By analyzing nine genetically diverse mouse strains, we show that much like humans, mice exhibit a huge variety of physiological and biochemical responses to high-fat diet. The strains exhibit various degrees of alterations in their phenotypic makeup. At the transcriptome level, we observe dysregulations of immunity, translation machinery, and mitochondrial genes. At the biochemical level, the enzymatic activity of mitochondrial complexes is affected. The diversity across mouse strains, diets, and sexes parallels that found in humans and supports the use of diverse mouse populations in future mechanistic or preclinical studies on metabolic dysfunctions.

Postresuscitation syndrome: potential role of hydroxyl radical-induced endothelial cell damage.

OBJECTIVE: After out of hospital cardiac arrest, it has been reported that endothelium dysfunction may occur during the postresuscitation syndrome. However, the consequences of the reperfusion phase on endothelial reactive oxygen species production and redox homeostasis have not been explored in out of hospital cardiac arrest patients. DESIGN: Prospective, observational study. SETTING: Medical intensive care unit in a university hospital. PATIENTS: Twenty successfully resuscitated out of hospital cardiac arrest patients, seven septic shock patients, and ten healthy volunteers. INTERVENTION: Plasma was collected from patients at admission and 12, 24, 36, 48, and 72 hrs after cardiac arrest. We studied the production of reactive oxygen species and cell survival during plasma perfusion using perfused endothelial cells (human umbilical vein endothelial cells) as a model. Cell antioxidant response was studied by measuring superoxide dismutase, glutathione peroxidase, and glutathione reductase activities and reduced and oxidized glutathione levels. Mitochondrial respiratory chain activity was assessed by measuring complex I, II, III, and IV activities and anaerobic glycolysis by measuring glucose-6-phosphate dehydrogenase activity. MEASUREMENTS AND MAIN RESULTS: Using perfused endothelial cells as a model, we demonstrate that plasma from out of hospital cardiac arrest patients induced on naive human umbilical vein endothelial cells a significant and massive cell death compared to plasma from septic shock patients and healthy volunteers. An increase of reactive oxygen species production with a decrease in antioxidant defenses (superoxide dismutase, glutathione peroxidase, and glutathione reductase activities, reduced and oxidized glutathione levels) was observed. The metabolic consequence of plasma exposure showed that mitochondrial respiratory chain activity was significantly impaired and anaerobic glycolysis was significantly increased. Inhibiting hydroxyl radical production significantly decreased cell death, suggesting that plasma from out of hospital cardiac arrest induced significant cell death by triggering the Fenton reaction. CONCLUSION: Plasma from out of hospital cardiac arrest induces major endothelial toxicity with an acute pro-oxidant state in the cells and impairment of mitochondrial respiratory chain activity. This toxicity could be due to hydroxyl radical production by activation of the Fenton reaction.

Versatile Functional Energy Metabolism Platform Working From Research to Patient: An Integrated View of Cell Bioenergetics.

Mitochondrial dysfunctions that were not discovered during preclinical and clinical testing have been responsible for at least restriction of use as far as withdrawal of many drugs. To solve mitochondrial machinery complexity, integrative methodologies combining different data, coupled or not to mathematic modelling into systems biology, could represent a strategic way but are still very hard to implement. These technologies should be accurate and precise to avoid accumulation of errors that can lead to misinterpretations, and then alter prediction efficiency. To address such issue, we have developed a versatile functional energy metabolism platform that can measure quantitatively, in parallel, with a very high precision and accuracy, a high number of biological parameters like substrates or enzyme cascade activities in essential metabolism units (glycolysis, respiratory chain ATP production, oxidative stress...) Its versatility (our platform works on either cell lines or small animals and human samples) allows cell metabolism pathways fine tuning comparison from preclinical to clinical studies. Applied here to OXPHOS and/or oxidative stress as an example, it allows discriminating compounds with acute toxic effects but, most importantly, those inducing low noise chronic ones.

Extracellular vesicles: General features and usefulness in diagnosis and therapeutic management of colorectal cancer.

In the world, among all type of cancers, colorectal cancer (CRC) is the third most commonly diagnosed in males and the second in females. In most of cases, (RP1) patients' prognosis limitation with malignant tumors can be attributed to delayed diagnosis of the disease. Identification of patients with early-stage disease leads to more effective therapeutic interventions. Therefore, new screening methods and further innovative treatment approaches are mandatory as they may lead to an increase in progression-free and overall survival rates. For the last decade, the interest in extracellular vesicles (EVs) research has exponentially increased as EVs generation appears to be a universal feature of every cell that is strongly involved in many mechanisms of cell-cell communication either in physiological or pathological situations. EVs can cargo biomolecules, such as lipids, proteins, nucleic acids and generate transmission signal through the intercellular transfer of their content. By this mechanism, tumor cells can recruit and modify the adjacent and systemic microenvironment to support further invasion and dissemination. This review intends to cover the most recent literature on the role of EVs production in colorectal normal and cancer tissues. Specific attention is paid to the use of EVs for early CRC diagnosis, follow-up, and prognosis as EVs have come into the spotlight of research as a high potential source of 'liquid biopsies'. The use of EVs as new targets or nanovectors as drug delivery systems for CRC therapy is also summarized.

Mitochondrial metabolism supports resistance to IDH mutant inhibitors in acute myeloid leukemia.

Mutations in IDH induce epigenetic and transcriptional reprogramming, differentiation bias, and susceptibility to mitochondrial inhibitors in cancer cells. Here, we first show that cell lines, PDXs, and patients with acute myeloid leukemia (AML) harboring an IDH mutation displayed an enhanced mitochondrial oxidative metabolism. Along with an increase in TCA cycle intermediates, this AML-specific metabolic behavior mechanistically occurred through the increase in electron transport chain complex I activity, mitochondrial respiration, and methylation-driven CEBPα-induced fatty acid ß-oxidation of IDH1 mutant cells. While IDH1 mutant inhibitor reduced 2-HG oncometabolite and CEBPα methylation, it failed to reverse FAO and OxPHOS. These mitochondrial activities were maintained through the inhibition of Akt and enhanced activation of peroxisome proliferator-activated receptor-γ coactivator-1 PGC1α upon IDH1 mutant inhibitor. Accordingly, OxPHOS inhibitors improved anti-AML efficacy of IDH mutant inhibitors in vivo. This work provides a scientific rationale for combinatory mitochondrial-targeted therapies to treat IDH mutant AML patients, especially those unresponsive to or relapsing from IDH mutant inhibitors.

Renoprotective potency of amifostine in rat renal ischaemia-reperfusion.

PURPOSE: Kidneys from haemodynamically unstable donors may suffer from renal ischaemia-reperfusion (RIR) injury. RIR is associated with reactive oxygen species production that induces inflammation and activates the arachidonic acid (AA) pathway which converts AA into prostaglandin E(2). Amifostine was investigated for its renoprotective potential in RIR. MATERIALS AND METHODS: The effect of amifostine (25 mg/kg = 910 mg/m(2)) on the COX pathway, enzymatic antioxidant activity, the lipid peroxidation marker MDA, serum creatinine and apoptosis was determined in rats. Kidneys were subjected to 45 min of ischaemia and 1 or 24 h of reperfusion. Control groups (sham: coeliotomy, no ischaemia; r1: 45 min ischaemia/1 h reperfusion; r2: 45 min ischaemia/24 h reperfusion) were administered physiological saline intraperitoneally, and treated groups (E1: 45 min ischaemia/1 h reperfusion; E2: 45 min ischaemia/24 h reperfusion) received amifostine 30 min before reperfusion. RESULTS: Serum creatinine increased in non-treated control rats: r1 vs sham (1.6-fold; P <0.007), r2 vs sham (2-fold; P <0.007). Amifostine decreased serum creatinine levels in treated rats: E1 vs r1 (8%; P <0.0025), E2 vs r2 (44%; P <0.0025). Amifostine reduced acute tubular necrosis (25%) 24 h after reperfusion: E1 vs r1 (P <0.004), E2 vs r2 (P <0.03) and reduced COX-2 and microsomal prostaglandin E synthase expression: E1 vs r1 (P <0.03), E2 vs r2 (P <0.02). Amifostine decreased MDA (P <0.04) and reduced caspase-3 expression but did not alter enzymatic antioxidant activity after RIR. CONCLUSIONS: Amifostine decreased the degree and severity of tubular damage after reperfusion, probably by scavenging oxygen free radicals and attenuating the cytotoxic effects of inflammatory infiltrates and apoptosis.

Should kidney tubular markers be adjusted for urine creatinine? The example of urinary cystatin C.

BACKGROUND: Evaluation of specific urinary markers with respect to urine creatinine (uCreat) is common. However, as uCreat is a function of both glomerular filtration and tubular secretion, using uCreat for specific tubular markers, suggests that glomerular function is normal, and there is no tubular secretion. Thus, adjusting values of any tubular marker to uCreat, especially in patients with acute or even moderate chronic renal failure, can be misleading. METHODS: Using urine cystatin-C (uCST3) as a model tubular marker for following 120 kidney graft recipients daily, we evaluated the utility of either uCST3 alone or the uCST3/uCreat ratio to detect tubular damage. All positive kidney biopsies were always associated with a uCST3>0.18 mg/L. RESULTS: Using the uCST3/uCreat ratio, discrepancies regarding biopsy status were observed in nine patients (4 false positive, 5 false negative results). In two patients, variability of uCreat appeared to be the most important factor causing inconsistent uCST3/uCreat ratios. With a negative predictive value (NPV) of 85.7%, uCST3/uCreat can lead to errors in clinical interpretation. These errors can be avoided when estimates of tubular damage are based on uCST3 concentrations alone (NPV=100%). CONCLUSIONS: We recommend using the uCST3 value to evaluate the extent of renal tubular damage. Indeed, our conflicting results on uCST3/uCreat can be extended to every marker of tubular function. Evaluating a urine marker specific for renal tubular damage to a second urine marker that is itself strongly dependent upon glomerular or other renal or non-renal conditions, impairs its clinical relevance and may lead to incorrect interpretations. Correction with uCreat can be performed only in pure glomerulopathy, when specific markers of glomerular function are measured (i.e., urinary albumin). In all other cases of renal diseases, such correction is inappropriate and should be avoided. Clin Chem Lab Med 2009;47:1553-6.

Pivotal role of glutathione depletion in plasma-induced endothelial oxidative stress during sepsis.

OBJECTIVE: Plasma from septic shock patients can induce production of reactive oxygen species (ROS) by human umbilical vein endothelial cells (HUVEC) in vitro. How endothelial cells defend themselves against ROS under increased oxidative stress has not yet been examined. This study investigates the antioxidant defenses of HUVEC exposed to plasma obtained from either septic shock patients or healthy volunteers. DESIGN: Prospective, observational study. SETTING: Medical intensive care unit in a university hospital. PATIENTS: Twenty-five patients with septic shock and 10 healthy volunteers. INTERVENTIONS: Blood samples were collected within the first 24 hrs of septic shock. In vitro HUVEC production of ROS was studied by spectrofluorimetry using 2',7'-dichlorodihydrofluorescein diacetate fluorescent dye. Reactive nitrogen species were also assessed. Intracellular reduced glutathione (GSH) levels were measured using monochlorobimane fluorescent dye. Activity of catalase and superoxide dismutase in HUVEC were also measured. Cell death was assessed using YOPRO fluorescent dye and the MTT assay. MEASUREMENTS AND RESULTS: On admission, the septic shock population's mean age was 55 yrs old, the mean Sequential Organ Failure Assessment score was 12, mean simplified acute physiology score was 50, and intensive care unit mortality rate was 45%. Evaluation of HUVEC antioxidant defenses showed a significantly decreased GSH level, increased catalase activity, and unchanged superoxide dismutase activity. ROS levels and cell death were significantly reduced when cells were pretreated with N-acetylcysteine or GSH, but no changes in reactive nitrogen species were observed. CONCLUSION: This study demonstrates that plasma-induced ROS production by HUVEC is associated with an intracellular decrease in reduced GSH. Both ROS levels and cell death decreased when N-acetylcysteine or GSH were added before exposing the cells to plasma. These data suggest a pivotal role of alterations in GSH in damage caused by sepsis-generated ROS in endothelial cell.

Postischemic treatment by trans-resveratrol in rat liver ischemia-reperfusion: a possible strategy in liver surgery.

Liver ischemia-reperfusion (I/R) injury occurs in many clinical conditions, including liver surgery and transplantation. Oxygen free radicals generated during I/R reduce endogenous antioxidant systems and contribute to hepatic injury. trans-Resveratrol (trans-3,5,4'-trihydroxystilbene) is reported to have antioxidant properties. We investigated the effect of trans-resveratrol on liver injury induced by I/R. After 1 hour of ischemia, administered 5 minutes before 3 hours of reperfusion, trans-resveratrol was hepatoprotective at a low dose (0.02 mg/kg). It significantly decreased aminotransferase levels by about 40% and improved sinusoidal dilatation. trans-Resveratrol preserved antioxidant defense by preventing total and reduced glutathione depletion caused by I/R. At 0.2 mg/kg, trans-resveratrol significantly increased glutathione reductase, Cu/Zn-superoxide dismutase, and catalase activities. However, at a high dose (20 mg/kg), trans-resveratrol became prooxidant with an aggravation of liver injury evaluated by aminotransferase release and histological analysis and associated with a depletion of total and reduced glutathione levels and a decrease of antioxidant enzyme activities. In conclusion, a prereperfusion treatment by trans-resveratrol only at low doses decreases liver injury induced by I/R by protecting against antioxidant defense failure. This administration protocol could reduce liver damage during surgery or transplantation.

Transient supplementation of superoxide dismutase protects endothelial cells against Plasmodium falciparum-induced oxidative stress.

The pathogenesis of cerebral malaria, a major complication of Plasmodium falciparum infection, relies on mechanisms such as cytokine production and cytoadherence of parasitized red blood cells (PRBCs) on microvascular endothelial cells. In this way parasites avoid spleen clearance by sequestration in post-capillary venules of various organs including the brain. Infected erythrocytes adhesion has also been shown to have molecular signaling consequences providing insight on how tissue homeostasis could be comprised by endothelium perturbation. Our previous work demonstrated that PRBCs adhesion to human lung endothelial cells (HLEC) induces caspases activation, oxidative stress and apoptosis. Cytoplasmic Cu/Zn superoxide dismutase (SOD1), which provides the first line of defense against oxidative stress within a cell, is now used as a treatment of numerous diseases including traumatic brain injury and ischemic stroke. In this report, we demonstrated that transient supplementation of SOD1 protects endothelial cells against P. falciparum induced oxidative stress and apoptosis. We also showed a significant decrease in PRBCs cytoadherence through a downregulation of ICAM-1 and an induction of iNOS. Protection of endothelium via antioxidant delivery may constitute a relevant strategy in cerebral malaria treatment.

Bilirubin decreases nos2 expression via inhibition of NAD(P)H oxidase: implications for protection against endotoxic shock in rats.

We investigated a possible beneficial role for bilirubin, one of the products of heme degradation by the cytoprotective enzyme heme oxygenase-1 in counteracting Escherichia coli endotoxin-mediated toxicity. Homozygous jaundice Gunn rats, which display high plasma bilirubin levels due to deficiency of glucuronyl transferase activity, and Sprague-Dawley rats subjected to sustained exogenous bilirubin administration were more resistant to endotoxin (LPS)-induced hypotension and death compared with nonhyperbilirubinemic rats. LPS-stimulated production of nitric oxide (NO) was significantly decreased in hyperbilirubinemic rats compared with normal animals; this effect was associated with reduction of inducible NO synthase (NOS2) expression in renal, myocardial, and aortic tissues. Furthermore, NOS2 protein expression and activity were reduced in murine macrophages stimulated with LPS and preincubated with bilirubin at concentrations similar to that found in the serum of hyperbilirubinemic animals. This effect was secondary to inhibition of NAD(P)H oxidase since 1) inhibition of NAD(P)H oxidase attenuated NOS2 induction by LPS, 2) bilirubin decreased NAD(P)H oxidase activity in vivo and in vitro, and 3) down-regulation of NOS2 by bilirubin was reversed by addition of NAD(P)H. These findings indicate that bilirubin can act as an effective agent to reduce mortality and counteract hypotension elicited by endotoxin through mechanisms involving a decreased NOS2 induction secondary to inhibition of NAD(P)H oxidase.

Sonic Hedgehog-dependent proliferation in a series of patients with colorectal cancer.

BACKGROUND: The Hedgehog (Hh) gene family is known to regulate development of stem cells. In addition, activation is responsible for the induction of GLI1 proto-oncogene and subsequent cellular proliferation. Sonic Hedgehog (SHh), one of the Hh family members promotes carcinogenesis in airway and pancreatic epithelia, is expressed in colonic stem cells. As differentiated colonic cells arise from constant renewal of Hedgehog-expressing colonic stem cells, SHh could be involved in human colonic carcinogenesis. METHODS: Tissue samples of colorectal adenocarcinoma (T) and adjacent normal colon tissue (NT) were sampled from each of 44 consecutive patients with colorectal cancer. Specific transcription of SHh, GLI1, and the GLI1 downstream target FOXM1 were evaluated using semiquantitative reverse transcriptase polymerase chain reaction. Similar in vitro measurements of mRNA of GLI1 and FOXM1 transcription levels after specific induction by SHh-Np were performed in the HT-29 colorectal tumor cell line to confirm the in vivo results. RESULTS: SHh mRNA was overexpressed in colorectal adenocarcinomas in 38 of 44 (86%) patients. Expression of transcription levels of GLI1 and FOXM1 correlated with SHh expression (SHh vs GLI1, r = 0.77, P < .0001; GLI1 vs FOXM1, r = 0.68, P < .0001; SHh vs FOXM1, r = 0.79, P < .0001). SHh overexpression did not appear to correlate with the patient characteristics evaluated. Similarly, when studied in the HT-29 colorectal cell line, exogenous SHh promoted cell proliferation, while inhibition of SHh expression decreased proliferation. Expression of GLI1 and FOXM1 mRNA increased with exogenous exposure to SHh. CONCLUSIONS: We demonstrated increased expression of SHh mRNA in human colonic adenocarcinomas and in a colorectal cell line with downstream increased expression of GLI1 and FOXM1 mRNA known to promote cell proliferation. This upregulation within human colorectal adenocarcinoma tissue confirms the potential role of the Hh pathway in colorectal carcinogenesis and suggests a potential therapeutic target of Hh blockade in colorectal cancer.

Protective effect of prior administration of magnesium on delayed hyperalgesia induced by fentanyl in rats.

PURPOSE: Magnesium exerts a physiological block of the ion channel on the N-methyl-D-aspartate receptor, and may therefore prevent the induction of central sensitization. The purpose of this study was to assess whether systemic magnesium can prevent long-lasting hyperalgesia induced by sc fentanyl administration in uninjured rats. METHODS: Long-lasting hyperalgesia was induced in male Sprague Dawley rats with sc fentanyl (four injections, 60 microg x kg(-1) per injection at 15-min intervals). Magnesium sulphate (100 mg x kg(-1)) was injected ip 30 min prior to the first sc fentanyl injection. Sensitivity to nociceptive stimuli (paw-pressure test) was assessed for several days after injections. RESULTS: Subcutaneous fentanyl led to delayed hyperalgesia associated with a decrease in the nociceptive threshold lasting two days (35% decrease for the maximum effect). Intraperitoneal magnesium sulphate partially but significantly (P < 0.05) prevented the delayed decrease in the nociceptive threshold following sc administration of fentanyl. CONCLUSIONS: This study shows that magnesium may prevent the delayed and prolonged hyperalgesia following fentanyl administration in rats.

Antioxidant and anti-inflammatory properties of a Cucumis melo LC. extract rich in superoxide dismutase activity.

The present study was conducted to evaluate in vitro and in vivo the antioxidant and anti-inflammatory properties of a cantaloupe melon (Cucumis melo LC., Cucurbitaceae) extract (CME) selected for its high superoxide dismutase activity. Peritoneal macrophages were pre-activated in vitro with 300 IU of interferon-gamma (IFN-gamma) and were then challenged in culture with IgGl/anti-IgG1 immune complexes (IgG1IC) in presence of various CME extracts. The subsequent production of free radicals (superoxide anion, nitric oxide, and peroxynitrite) and of pro-(TNF-alpha) and anti-(IL-10) inflammatory cytokines was evaluated. The CME inhibited in a dose-dependent manner the production of superoxide anion with a maximal effect at 100 microg/ml. This inhibitory effect of CME appeared to be closely linked to the SOD activity because it was dramatically decreased after heat inactivation of the SOD activity (HI-CME). In addition, the CME inhibited the production of peroxynitrite strengthening the antioxidant properties of this CME rich in SOD activity. The production of the pro- and anti-inflammatory cytokines, namely TNF-alpha and IL-10, being conditioned by the redox status of macrophages we also evaluated the effect of CME and HI-CME on the IgG1IC-induced cytokine production. When the SOD activity was present in the CME it promoted the IgG1IC-induced production of IL-10 instead of TNF-alpha. These data demonstrated that, in addition to its antioxidant properties, the anti-inflammatory properties of the CME extract were principally related to its capacity to induce the production of IL-10 by peritoneal macrophages. The particular properties of wheat gliadin (Triticum vulgare, Poaceae) for the oral delivery of functional proteins led us to test it in a new nutraceutical formula based on its combination with the CME thus monitoring the SOD activity release during the gastro-intestinal digestive process. In these experiments C57BL/6 mice were supplemented orally everyday during 28 days with: (1) the placebo, (2) the CME extract alone, (3) the gliadin, (4) the CME/gliadin combination, or (5) the HI-CME/gliadin combination (SOD inactivated). At the end of the supplementation period all the animals were injected intra-peritoneal (i.p.) with the pro-inflammatory cytokine IFN-gamma (300 IU) and peritoneal macrophages were harvested 24 h after to test their capacities to produce free radicals, TNF-alpha and IL-10 after triggering with IgG1IC. We demonstrated that animals supplemented during 28 days with the CME/gliadin combination were protected against the pro-inflammatory properties of IFN-gamma while the other products were inefficient. These data did not only indicate that the SOD activity is important for the antioxidant and anti-inflammatory properties of the CME extract, but also demonstrated that when the SOD activity is preserved during the digestive process by its combination with wheat gliadin it is possible to elicit in vivo the pharmacological effects of this antioxidant enzyme.