Ornella Fantappiè

Cyclooxygenase-2 Activation Mediates the Proangiogenic Effect of Nitric Oxide in Colorectal Cancer

Purpose: Up-regulation of both inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) enzymes has been reported in colorectal cancer. We aimed at evaluating the possible interaction between the nitric oxide and COX-2 pathways, and its effect on promoting tumor angiogenesis. Experimental Design: Expression of iNOS, COX-2, vascular endothelial growth factor (VEGF), and CD31 was analyzed in tumor samples and corresponding normal mucosa obtained from 46 surgical specimens. We also evaluated iNOS activity, prostaglandin E2 (PGE2), cyclic GMP and cyclic AMP production in the same specimens. Nitrite/nitrate levels, and PGE2 and VEGF production were assessed in HCT116 and HT29 colon cancer cell lines after induction and selective inhibition of the two enzyme pathways. Results: A significant correlation was found between iNOS and COX-2 immunohistochemical expression. PGE2 production significantly correlated with iNOS activity and cGMP levels. A significant correlation was also found among PGE2 production, microvessel density, and VEGF expression. Coinduction of both iNOS and COX-2 activities occurred after lipopolysaccharide (LPS) and epidermal growth factor (EGF) treatment in HCT116 and HT29 cells. Inhibition of iNOS by 1400W significantly reduced both LPS- and EGF-induced PGE2 production. Treatment with LPS, EGF, and arachidonic acid significantly increased VEGF production in the iNOS-negative/COX-2-positive HT29 cells. This effect was completely reversed by treatment with the selective COX-2 inhibitor celecoxib. Conclusions: Our data showed a prominent role of nitric oxide in stimulating COX-2 activity in colorectal cancer. This interaction is likely to produce a cooperative effect in promoting angiogenesis through PGE2-mediated increase in VEGF production.

Inducible Nitric Oxide Synthase Expression in Human Colorectal Cancer : Correlation with Tumor Angiogenesis

To investigate the potential involvement of the nitric oxide (NO) pathway in colorectal carcinogenesis, we correlated the expression and the activity of inducible nitric oxide synthase (iNOS) with the degree of tumor angiogenesis in human colorectal cancer. Tumor samples and adjacent normal mucosa were obtained from 46 surgical specimens. Immunohistochemical expression of iNOS, vascular endothelial growth factor (VEGF), and CD31 was analyzed on paraffin-embedded tissue sections. iNOS activity and cyclic GMP levels were assessed by specific biochemical assays. iNOS protein expression was determined by Western blot analysis. iNOS and VEGF mRNA levels were evaluated using Northern blot analysis. Both iNOS and VEGF expressions correlated significantly with intratumor microvessel density (r(s) = 0.31, P = 0.02 and r(s) = 0.67, P < 0.0001, respectively). A significant correlation was also found between iNOS and VEGF expression (P = 0.001). iNOS activity and cyclic GMP production were significantly higher in the cancer specimens than in the normal mucosa (P < 0.0001 and P < 0.0001, respectively), as well as in metastatic tumors than in nonmetastatic ones (P = 0.002 and P = 0.04, respectively). Western and Northern blot analyses confirmed the up-regulation of the iNOS protein and gene in the tumor specimens as compared with normal mucosa. NO seems to play a role in colorectal cancer growth by promoting tumor angiogenesis.

P-glycoprotein mediates celecoxib-induced apoptosis in multiple drug-resistant cell lines.

In several neoplastic diseases, including hepatocellular carcinoma, the expression of P-glycoprotein and cyclooxygenase-2 (COX-2) are often increased and involved in drug resistance and poor prognosis. P-glycoprotein, in addition to drug resistance, blocks cytochrome c release, preventing apoptosis in tumor cells. Because COX-2 induces P-glycoprotein expression, we evaluated the effect of celecoxib, a specific inhibitor of COX-2 activity, on P-glycoprotein-mediated resistance to apoptosis in cell lines expressing multidrug resistant (MDR) phenotype. Experiments were done using MDR-positive and parental cell lines at basal conditions and after exposure to 10 or 50 micromol/L celecoxib. We found that 10 micromol/L celecoxib reduced P-glycoprotein, Bcl-x(L), and Bcl-2 expression, and induced translocation of Bax from cytosol to mitochondria and cytochrome c release into cytosol in MDR-positive hepatocellular carcinoma cells. This causes the activation of caspase-3 and increases the number of cells going into apoptosis. No effect was shown on parental drug-sensitive or on MDR-positive hepatocellular carcinoma cells after transfection with MDR1 small interfering RNA. Interestingly, although inhibiting COX-2 activity, 50 micromol/L celecoxib weakly increased the expression of COX-2 and P-glycoprotein and did not alter Bcl-x(L) and Bcl-2 expression. In conclusion, these results show that relatively low concentrations of celecoxib induce cell apoptosis in MDR cell lines. This effect is mediated by P-glycoprotein and suggests that the efficacy of celecoxib in the treatment of different types of cancer may depend on celecoxib concentration and P-glycoprotein expression.

Hepatocyte Growth Factor and Inducible Nitric Oxide Synthase Are Involved in Multidrug Resistance–Induced Angiogenesis in Hepatocellular Carcinoma Cell Lines

Based on literature, it is possible to hypothesize that multidrug resistance (MDR) and angiogenic phenotypes are linked to each other in human liver cancer cells. Our goal is to assess whether MDR cells trigger angiogenesis and to study the possible molecular mechanisms involved. Conditioned medium from parental drug-sensitive P5 cells (P5-CM) and MDR-positive P1(0.5) cells [P1(0.5)-CM] stimulated human umbilical vein endothelial cells (HUVEC) survival, proliferation, migration, and microtubular structure formation, but P1(0.5)-CM had a significantly greater effect than P5-CM. Cell implants were done in the rabbit avascular cornea to measure angiogenesis in vivo: P1(0.5) cells induced an important neovascular response in rabbit cornea after 1 week, whereas P5 cells had no effect. P1(0.5) and P5 cells produced vascular endothelial growth factor, but only P1(0.5) secreted hepatocyte growth factor (HGF) into the medium, and small interfering RNA specific for MDR1 clearly reduced HGF production in P1(0.5) cells. The transcription factor Ets-1 and the HGF receptor c-Met were up-regulated in P1(0.5) cells and in HUVEC cultured in P1(0.5)-CM. Inducible nitric oxide synthase (iNOS) seemed to play a major role in the proangiogenic effect of P1(0.5), and its inhibition by 1400W blunted the capacity of P1(0.5) cells to stimulate HUVEC proliferation, migration, and Ets-1 expression. In conclusion, these data show that development of MDR and angiogenic phenotypes are linked to each other in MDR cells. HGF production, Ets-1 and c-Met up-regulation, and iNOS expression can be part of the molecular mechanisms that enhance the angiogenic activity of the MDR-positive hepatocellular carcinoma cell line.

Heme Oxygenase-1 and the Ischemia-Reperfusion Injury in the Rat Heart

Carbon monoxide (CO) is a signaling gas produced intracellularly by heme oxygenase (HO) enzymes using heme as a substrate. During heme breakdown, HO-1 and HO-2 release CO, biliverdin, and Fe2+. In this study, we investigated the effects of manipulation of the HO-1 system in an in vivo model of focal ischemia–reperfusion (FIR) in the rat heart. Male Wistar albino rats, under general anesthesia and artificial ventilation, underwent thoracotomy, the pericardium was opened, and a silk suture was placed around the left descending coronary artery; ischemia was induced by tightening the suture and was monitored for 30 min. Subsequently, the ligature was released to allow reperfusion lasting for 60 min. The first group of rats was sham operated and injected intraperitoneally (ip) with saline. The second group underwent FIR. The third group was treated ip 18 hr before FIR with hemin (4 mg/kg). The fourth group was pretreated ip 24 hr before FIR and 6 hr before hemin with zinc protoporphyrin IX (ZnPP-IX, 50 μg/kg). Specimens of the left ventricle were taken for determination of HO expression and activity, infarct size, malonyldialdehyde (MDA) production, and tissue calcium content. FIR led to a significant increase in the generation of MDA and notably raised tissue calcium levels. Induction of HO-1 by hemin significantly decreased infarct size, incidence of reperfusion arrhythmias, MDA generation, and calcium overload induced by FIR. These effects were prevented by the HO-1 inhibitor ZnPP-IX. The present experiments show that the concerted actions of CO, iron, and biliverdin/bilirubin modulate the FIR-induced myocardial injury.

Mitochondrial Expression and Functional Activity of Breast Cancer Resistance Protein in Different Multiple Drug-Resistant Cell Lines

The multidrug resistance (MDR) phenotype is characterized by the overexpression of a few transport proteins at the plasma membrane level, one of which is the breast cancer resistance protein (BCRP). These proteins are expressed in excretory organs, in the placenta and blood-brain barrier, and are involved in the transport of drugs and endogenous compounds. Because some of these proteins are expressed in the mitochondria, this study was designed to determine whether BCRP is expressed at a mitochondrial level and to investigate its function in various MDR and parental drug-sensitive cell lines. By using Western blot analysis, immunofluorescence confocal and electron microscopy, flow cytometry analysis, and the BCRP (ABCG-2) small interfering RNA, these experiments showed that BCRP is expressed in the mitochondrial cristae, in which it is functionally active. Mitoxantrone accumulation was significantly reduced in mitochondria and in cells that overexpress BCRP, in comparison to parental drug-sensitive cells. The specific inhibitor of BCRP, fumitremorgin c, increased the accumulation of mitoxantrone significantly in comparison with basal conditions in both whole cells and in mitochondria of BCRP-overexpressing cell lines. In conclusion, this study shows that BCRP is overexpressed and functionally active in the mitochondria of MDR-positive cancer cell lines. However, its presence in the mitochondria of parental drug-sensitive cells suggests that BCRP can be involved in the physiology of cancer cells.

Haeme oxygenase‐1 and cardiac anaphylaxis

Haeme oxygenase (HO) is an enzyme mainly localized in the smooth endoplasmic reticulum and involved in haeme degradation and in the generation of carbon monoxide (CO). Here we investigate (1) whether the inducible isoform of HO (HO‐1) is expressed in the isolated heart of the guinea‐pig and (2) the functional significance of HO‐1 on the response to antigen in isolated hearts taken from actively sensitized guinea‐pigs. Both the HO‐1 expression and activity are consistently increased in hearts from guinea‐pigs pretreated with hemin, an HO‐1 inducer (4 mg kg−1 i.p., 18 h before antigen challenge). The administration of the HO‐1 inhibitor zinc‐protoporphyrin IX (ZnPP‐IX, 50 μmol kg−1, i.p., 6 h before hemin) abolished the increase of both the HO‐1 expression and activity. In vitro challenge with the specific antigen of hearts from actively sensitized animals evokes a positive inotropic and chronotropic effect, a coronary constriction followed by dilation and an increase in the amount of histamine in the perfusates. In hearts from hemin‐pretreated animals, antigen challenge did not modify the heart rate and the force of contraction; the coronary outflow was significantly increased and a diminution of the release of histamine was observed. The patterns of cardiac anaphylaxis were fully restored in hearts from animals treated with ZnPP‐IX 6 h before hemin. In isolated hearts perfused with a Tyrode solution gassed with 100% CO for 5 min and successively reoxygenated, the response to antigen was similar to that observed in hearts from hemin‐pretreated animals. Pretreatment with hemin or the exposure to exogenous CO were linked to an increase in cardiac cyclic GMP levels and to a decrease of tissue Ca2+ levels. The study demonstrates that overexpression of HO‐1 inhibits cardiac anaphylaxis through the generation of CO which, in turn, decreases the release of histamine through a cyclic GMP‐ and Ca2+‐dependent mechanism.

Vitamin E protects DNA from oxidative damage in human hepatocellular carcinoma cell lines.

Expression of multiple drug resistant (MDR) phenotype and over-expression of P-glycoprotein (P-gp) in the human hepatocellular carcinoma (HCC) cell clone P1(0.5), derived from the PLC/PRF/5 cell line (P5), are associated with strong resistance to oxidative stress and a significant ( p<0.01) increase in intracellular vitamin E content as compared with the parental cell line. This study evaluates the role of vitamin E in conferring resistance to drugs and oxidative stress in P1(0.5) cells. Parental drug-sensitive cells, P5, were incubated in α-tocopherol succinate (α-TS, 5 μM for 24 h) enriched medium to increase intracellular vitamin E content to levels comparable to those observed in P1(0.5) cells at basal conditions. Susceptibility to lipid peroxidation and oxidative DNA damage were assessed by measuring the concentration of thiobarbituric-reactive substances (TBARS) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) at basal and after experimental conditions. Cell capacity to form colonies and resistance to doxorubicin were also studied. P5 cells, treated with α-TS, became resistant to ADP-Fe3+ and to ionizing radiation-induced lipid peroxidation as P1(0.5) cells. Exposure to ADP-Fe3+ or ionizing radiation increased TBARS and the 8-OHdG content in the P5 cells, while vitamin E enrichment abolished these effects. Irradiation doses at 5 cGy increased TBARS and 8-OHdG. They also inhibited cell capacity to form colonies in the untreated P5 cells. Incubation with α-TS fully reverted this effect and significantly (p<0.01) reduced the inhibitory effect of cell proliferation induced by irradiation doses at >500 cGy. Resistance to doxorubicin was not affected by α-TS. These observations demonstrate the role of vitamin E in conferring protection from lipid peroxidation, ionizing radiation and oxidative DNA damage on the human HCC cell line. They also rule out any role of P-gp over-expression as being responsible for these observations in cells with MDR phenotype expression.

Cyclooxygenase-2 and inflammation mediators have a crucial role in reflux-related esophageal histological changes and Barrett's esophagus.

BackgroundGastroesophageal reflux (GER) causes injury of the esophageal squamous epithelium, a condition called reflux esophagitis. The sequence reflux-esophagitis-intestinal metaplasia-dysplasia-invasive cancer is widely accepted as the main adenocarcinogenetic pathway in the esophagus; however, the mechanisms of this progression need to be better defined.AimsWe evaluated COX-2 expression and activity in biopsies from patients affected with GER, and these parameters have been correlated with the stage of the disease, ceramide expression, apoptotic process, and angiogenesis. The effects of celecoxib on bile acid- and EGF-induced mucosal proliferation, apoptosis and angiogenesis have been also investigated.MethodsFour groups of patients were distinguished: non esophagitis, non erosive esophagitis, erosive esophagitis, and Barrett’s esophagus. COX-2 expression, basal PGE2 levels, proliferative activity, VEGF expression and apoptosis were evaluated in esophageal biopsies.ResultsCOX-2 expression, basal PGE2 levels, proliferative activity, VEGF expression and apoptosis progressively increase from non esophagitis patients to patients with non erosive and erosive esophagitis, to those with BE. Incubation of the cells with DCA/EGF increases PGE2 production, proliferative activity and VEGF production, effects prevented by celecoxib pretreatment. Ceramide expression increased from non esophagitis patients to patients with non erosive and erosive esophagitis, and decreased in BE; caspase-3 activity progressively decreased from non esophagitis to BE patients, suggesting an impairment of the apoptotic process with disease progression.ConclusionThese results stand for a close relationship between progression of initial steps of gastroesophageal reflux disease (GERD) and COX-2, proliferative activity and EGF/VEGF expression and could have implications in GERD treatment in order to prevent its neoplastic evolution.

The effect of losartan treatment on the response of diabetic cardiomyocytes to ATP depletion.

The present work aimed to investigate the effect of losartan treatment of healthy and diabetic rats on cardiomyocyte response to ATP depletion. Cells were isolated from normoglycemic (N) and streptozotocin-injected (55 mg/kg) rats (D) treated or not treated with losartan (20 mg/kg/day in the drinking water; NL and DL, respectively) for 3 weeks. In each group of cells, enzyme activities such as glucose-6-phosphate (G6PDH) and glycerol-3-phosphate dehydrogenases (G3PDH), lactate/pyruvate, glycogen levels and citrate synthase were measured as an index of glycolytic dysregulation and mitochondrial mass, respectively. Cells were then challenged with NaCN (2 mM) in glucose-free Tyrode solution (metabolic intoxication, MI), a protocol to study ischemia at cell level. Under these conditions, the time to contractile failure up to rigor-type hyper-contracture in field-stimulated cells and K(ATP) current activation by patch-clamp recordings were measured. In comparison with N and NL, D cells presented higher G6PDH and cytoplasmic G3PDH activities, lactate/pyruvate, glycogen content but similar levels of citrate synthase, and decay time of contraction. When subjected to MI, D cells showed delayed activation of the K(ATP) current (25.7±7.1 min; p<0.001 vs. N and NL), increased time to contractile failure and rigor-type hyper-contracture (p<0.001 vs. N and NL). In cells from DL rats both functional (time to rigor and to K(ATP) current activation) and metabolic parameters, approached values similar to those measured in N and NL cells. These results demonstrate that diabetic cardiomyocytes from rats treated with losartan, maintain the capacity to respond promptly to ATP depletion reaching contractile failure, rigor-type hypercontracture and K(ATP) opening with a similar timing of N cells.