Anti-CCR7 therapy exerts a potent anti-tumor activity in a xenograft model of human mantle cell lymphoma.

BACKGROUND: The chemokine receptor CCR7 mediates lymphoid dissemination of many cancers, including lymphomas and epithelial carcinomas, thus representing an attractive therapeutic target. Previous results have highlighted the potential of the anti-CCR7 monoclonal antibodies to inhibit migration in transwell assays. The present study aimed to evaluate the in vivo therapeutic efficacy of an anti-CCR7 antibody in a xenografted human mantle cell lymphoma model. METHODS: NOD/SCID mice were either subcutaneously or intravenously inoculated with Granta-519 cells, a human cell line derived from a leukemic mantle cell lymphoma. The anti-CCR7 mAb treatment (3 × 200 µg) was started on day 2 or 7 to target lymphoma cells in either a peri-implantation or a post-implantation stage, respectively. RESULTS: The anti-CCR7 therapy significantly delayed the tumor appearance and also reduced the volumes of tumors in the subcutaneous model. Moreover, an increased number of apoptotic tumor cells was detected in mice treated with the anti-CCR7 mAb compared to the untreated animals. In addition, significantly reduced number of Granta-519 cells migrated from subcutaneous tumors to distant lymphoid organs, such as bone marrow and spleen in the anti-CCR7 treated mice. In the intravenous models, the anti-CCR7 mAb drastically increased survival of the mice. Accordingly, dissemination and infiltration of tumor cells in lymphoid and non-lymphoid organs, including lungs and central nervous system, was almost abrogated. CONCLUSIONS: The anti-CCR7 mAb exerts a potent anti-tumor activity and might represent an interesting therapeutic alternative to conventional therapies.

Angiotensin II differentially modulates cyclooxygenase-2, microsomal prostaglandin E2 synthase-1 and prostaglandin I2 synthase expression in adventitial fibroblasts exposed to inflammatory stimuli.

AIMS: To assess whether angiotensin II (Ang II) modulates key enzymes of the cyclooxygenase (COX)-2/prostanoid pathway, including prostaglandin E synthase-1 (mPGES-1) and prostacyclin synthase (PGIS) in rat aortic adventitial fibroblasts in the presence or absence of an inflammatory stimulus [interleukin (IL)-1ß]. METHODS AND RESULTS: Fibroblasts stimulated with IL-1ß (10 ng/ml, 24 h) and/or Ang II (0.1 µmol/l, 24 h) were used. IL-1ß up-regulated COX-2 and mPGES-1 (protein and mRNA) and increased PGI2 and PGE2 release, without altering PGIS protein expression. Ang II did modify neither COX-2 and mPGES-1 expression nor prostanoid levels, but it induced PGIS expression. Interestingly, Ang II further enhanced IL-1ß-induced COX-2 expression and PGI2 release and concomitantly reduced IL-1ß-induced mPGES-1 expression. The AT1 receptor antagonist losartan prevented the effects of Ang II on IL-1ß-induced COX-2 or mPGES-1 expression. IL-1ß activated p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK)1/2 pathways, and coincubation with Ang II resulted in a higher and more sustained phosphorylation of both MAPK. Inhibition of either p38 MAPK (SB203580) or ERK1/2 (PD98059) reduced COX-2 and mPGES-1 expression in cells treated with IL-1ß or the combination of IL-1ß and Ang II. Ang II did not modify COX-2 transcriptional activity but increased COX-2 mRNA stability in IL-1ß-treated cells; by contrast, it increased PGIS mRNA levels through a transcriptional mechanism. CONCLUSION: Ang II differentially modulates key enzymes involved in prostanoid biosynthesis thereby altering the balance between PGI2/PGE2 in vascular cells exposed to inflammatory stimuli.

p38 MAPK contributes to angiotensin II-induced COX-2 expression in aortic fibroblasts from normotensive and hypertensive rats.

OBJECTIVE: To investigate the effect of angiotensin II on cyclooxygenase-2 (COX-2) expression in aortic adventitial fibroblasts from normotensive [Wistar-Kyoto (WKY)] rats and spontaneously hypertensive rats (SHRs). METHODS: Protein expression was determined by western blot, mRNA levels by real-time PCR, transcriptional activity by luciferase assays, superoxide anion (O2*-) production by dihydroethidine fluorescence and prostaglandin E2 by enzyme immunoassay. RESULTS: Angiotensin II (0.1 micromol/l, 0.5-6 h) time dependently induced COX-2 protein expression, this effect being transient in fibroblasts from WKY rats and maintained over time in SHRs. Angiotensin II effect was abolished by valsartan (1 micromol/l), an angiotensin II type 1 receptor antagonist. Angiotensin II-induced prostaglandin E2 production was reduced by valsartan and the COX-2 inhibitor NS398 (1 micromol/l). Angiotensin II increased O2*- production more in SHR than WKY rats. This increase was reduced by apocynin (30 micromol/l) and allopurinol (10 micromol/l), respective nicotinamide adenine dinucleotide phosphate (NADPH) and xanthine oxidase inhibitors. However, angiotensin II-induced COX-2 expression was unaffected by apocynin, allopurinol, tempol (1 mmol/l) or catalase (1000 U/ml). Angiotensin II (2-30 min) induced p38 mitogen-activated protein kinase (MAPK) phosphorylation, transiently in WKY rats but sustained in SHRs. The p38 inhibitor SB203580 (10 micromol/l) reduced angiotensin II-induced COX-2 protein and mRNA levels. The angiotensin II effect was not prevented by inhibition of mRNA synthesis, and angiotensin II was unable to modulate COX-2 transcriptional activity. CONCLUSIONS: Angiotensin II increases COX-2 expression in aortic fibroblasts through mechanisms including p38 MAPK pathway, independent of reactive oxygen species production and nonmediated by COX-2 transcriptional activity modulation. The sustained angiotensin-induced p38 MAPK activation in SHR cells might be related to the maintained COX-2 expression in this strain.

Ouabain treatment increases nitric oxide bioavailability and decreases superoxide anion production in cerebral vessels.

OBJECTIVE: Chronic administration of ouabain induces hypertension and increases the contribution of nitric oxide to vasoconstrictor responses in peripheral arteries. The aim of this study was to analyse whether ouabain treatment alters the nitric oxide bioavailability in cerebral arteries. METHODS: Basilar arteries from control and ouabain-treated rats ( approximately 8.0 microg/day, 5 weeks) were used. Vascular reactivity was analysed by isometric tension recording, protein expression by western blot, nitric oxide levels by diaminofluorescein-induced fluorescence, superoxide anion (O2) production by ethidium fluorescence and lucigenin chemiluminescence and plasma total antioxidant status by a commercial kit. RESULTS: The relaxations induced by bradykinin (1 nmol/l-10 micromol/l) and L-arginine (0.01-300 micromol/l) and the contractile responses induced by both N-nitro-L-arginine methyl ester (0.1-100 micromol/l) and oxyhaemoglobin (0.01-10 micromol/l) were greater in arteries from ouabain-treated than control rats. However, the relaxation to diethylamine NONOate-nitric oxide (0.1 nmol/l-10 micromol/l) and the contractions to KCl (7.5-120 mmol/l) and 5-hydroxytryptamine (0.01-10 micromol/l) were similar in arteries from both groups. Ouabain treatment increased basal nitric oxide levels but did not modify endothelial and neuronal nitric oxide synthase protein expression. O2 production was lower in cerebral arteries from ouabain-treated rats; however, plasma total antioxidant status and vascular protein expression of Cu/Zn-superoxide dismutase, Mn-superoxide dismutase and extracellular superoxide dismutase were similar in both groups. CONCLUSION: Chronic ouabain treatment increased nitric oxide basal levels in basilar arteries probably due to the decreased O2 levels. This might be an adaptive mechanism of the cerebral vasculature to the increase in blood pressure.

Vascular effects of the Mangifera indica L. extract (Vimang).

The effects of the Mangiferia indica L. (Vimang) extract, and mangiferin (a C-glucosylxanthone of Vimang) on the inducible isoforms of cyclooxygenase (cyclooxygenase-2) and nitric oxide synthase (iNOS) expression and on vasoconstrictor responses were investigated in vascular smooth muscle cells and mesenteric resistance arteries, respectively, from Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Vimang (0.5-0.1 mg/ml) and mangiferin (0.025 mg/ml) inhibited the interleukin-1beta (1 ng/ml)-induced iNOS expression more in SHR than in WKY, and cyclooxygenase-2 expression more in WKY than in SHR. Vimang (0.25-1 mg/ml) reduced noradrenaline (0.1-30 microM)- and U46619 (1 nM-30 microM)- but not KCl (15-70 mM)-induced contractions. Mangiferin (0.05 mg/ml) did not affect noradrenaline-induced contraction. In conclusion, the antiinflammatory action of Vimang would be related with the inhibition of iNOS and cyclooxygenase-2 expression, but not with its effect on vasoconstrictor responses. Alterations in the regulation of both enzymes in hypertension would explain the differences observed in the Vimang effect.

Heparin and low molecular weight heparin decrease nitric oxide production by human polymorphonuclear cells

Background. Heparin and heparin derivatives with low anticoagulant activity exhibit a wide spectrum of biological functions affecting adhesion, activation and trafficking of luekocytes. Methods. We investigated the in vitro effect of heparin and low molecular weight heparin derivative (LMWH) on nitric oxide (NO) production by human polymorphonuclear leukocytes (PMN). Results. N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated NO production was significantly decreased by heparin at doses of 0.5 and 5 µg/mL, while LMWH was only effective at doses of 50 and 200 µg/mL by means of a mechanism not related to No synthase (NOS) activity. Conclusions. These results support the hypothesis that heparin and LMWH derivatives may offet therapeutic benefit for inflammatory diseases where No plays a protagonic role