New insights into IL-12-mediated tumor suppression

During the past two decades, interleukin-12 (IL-12) has emerged as one of the most potent cytokines in mediating antitumor activity in a variety of preclinical models. Through pleiotropic effects on different immune cells that form the tumor microenvironment, IL-12 establishes a link between innate and adaptive immunity that involves different immune effector cells and cytokines depending on the type of tumor or the affected tissue. The robust antitumor response exerted by IL-12, however, has not yet been successfully translated into the clinics. The majority of clinical trials involving treatment with IL-12 failed to show sustained antitumor responses and were associated to toxic side effects. Here we discuss the therapeutic effects of IL-12 from preclinical to clinical studies, and will highlight promising strategies to take advantage of the antitumor activity of IL-12 while limiting adverse effects.     

Nitric oxide modulates interleukin-2-induced proliferation in CTLL-2 cells

The role of the L-arginine-nitric oxide metabolic pathway was explored for interleukin-2-induced proliferation in the cytotoxic T lymphocyte clone CTLL-2. Specific inhibition of nitric oxide synthase significantly diminished, in a concentration-dependent manner, (3)H-thymidine uptake of CTLL-2 cells in response to different concentrations of interleukin 2. Withdrawal of L-arginine from culture medium resulted as potent as the higher inhibition obtained when blocking nitric oxide synthase with L-arginine analogues. Furthermore, intermedial concentrations of Larginine and exogenous nitric oxide donors were found for achieving optimal IL2-induced proliferation of CTLL-2. These findings prompted us to suggest that intra- and/or inter-cellular nitric oxide signalling may contribute to the modulation of the IL2 mitogenic effect upon cytotoxic T lymphocytes.     

Nitric oxide promotes resistance to tumor suppression by CTLs

Many human tumors express inducible NO synthetase (NOS2), but the roles of NO in tumor development are not fully elucidated. An important step during tumor development is the acquisition of apoptosis resistance. We investigated the dose-dependent effects of endogenously produced NO on apoptosis using ecdysone-inducible NOS2 cell lines. Our results show that short-term NOS2 expression enhances CD95-mediated apoptosis and T cell cytotoxicity dose dependently. Furthermore, we could show that during chronic exposure to NO, besides the primary cytotoxic NO effect, there is selection of cell clones resistant to NO that show cross-resistance to CD95-induced apoptosis and the killing by CTLs. We propose that NO production could initially act as an autocrine suicide or paracrine killing mechanism in cells undergoing malignant transformation. However, once failed, the outcome is fatal. NO promotes tumor formation by enhancing the selection of cells that can evade immune attack by acquiring apoptosis resistance.     

Gene-gene interactions between interleukin-12A and interleukin-12B with the risk of brain tumor

Emerging evidence from preclinical and clinical studies has shown that interleukin-12 (IL-12) has some effectiveness against endogenously arising brain tumor. The aim of this study was to investigate interactions of IL-12A and IL-12B polymorphisms on the risk of brain tumor. We analyzed IL-12A rs2243115 and IL-12B rs3212227 polymorphisms in 170 patients with brain tumor and 222 healthy controls in a Chinese population using a polymerase chain reaction-restriction fragment length polymorphism assay and DNA sequencing method. Individuals carrying a G allele of IL-12A rs2243115 had a significantly higher risk of developing brain tumor compared with those carrying a T allele (odds ratio [OR]=2.01, 95% confidence interval [CI], 1.17-3.45). After stratification analysis according to tumor types, a similarly higher risk was detected in patients with glioma (OR=2.56, 95% CI, 1.25-5.21). When gene-gene interactions were examined, carriers at both loci rs2243115 TG/GG and rs3212227 AC/CC had a 2.62-fold increased risk of glioma compared with those with rs2243115 TT and rs3212227 AC/CC genotypes (OR=2.62, 95% CI, 1.05-6.50). This study provides evidence that IL-12A rs2243115 may be associated with the risk of brain tumor. Additionally, gene-gene interactions of IL-12A rs2243115 and IL-12B rs3212227 may contribute to brain tumor susceptibility.     

Nitric oxide mediates laminin-induced neurite outgrowth in PC12 cells

Laminin is a potent stimulator of neurite outgrowth in a variety of primary neurons and neuronal cell lines. Here, we investigate the role of nitric oxide in the signaling mechanism of laminin-mediated neurite outgrowth in the PC12 cell line. Within 8 s of exposure to laminin, PC12 cells produce nitric oxide. Peak laminin-induced nitric oxide levels reach 8 nM within 12 s of exposure to laminin and constitutive nitric oxide production is sustained for 1 min. A neurite outgrowth promoting synthetic peptide (AG73), derived from the laminin-1-alpha globular domain, also stimulated nitric oxide release. The nitric oxide synthase inhibitor, 1-NAME, prevents the formation of nitric oxide and here, 1-NAME inhibited both laminin-mediated and AG73-mediated neurite outgrowth by 88 and 95%, respectively. In contrast, C16, a synthetic peptide derived from the laminin-1-gamma chain, is shown here to promote PC12 cell attachment, but not neurite outgrowth. Interestingly, the C16 peptide did not activate nitric oxide release, suggesting that laminin-induced nitric oxide release in PC12 cells is associated only with neurite outgrowth promoting laminin domains and signals. In addition, the data here show that the nitric oxide released by PC12 cells in response to laminin is required as a part of the mechanism of laminin-mediated neurite outgrowth.     

Nitric oxide

Nitric oxide (NO)--a 1:1 combination of the two most abundant gaseous elements--is a biological mediator of complexity, subtlety and protean effects. The history of its discovery as a mediator is fascinating, and its role in mammalian biology and medicine is proving to be of fundamental importance.     

Nitric oxide mediates tumor necrosis factor-alpha cytotoxicity in endothelial cells.

Tumor necrosis factor alpha (TNF-alpha) exerts multiple actions on endothelial cells including among others the expression of pro-coagulant activity and adhesion molecules, and secretion of cytokines. We now show that TNF-alpha induces a time- and dose-dependent cytotoxic effect on cultured bovine aortic endothelial cells. This TNF-induced cytotoxicity, which is preceded by increased production of nitric oxide (NO), is significantly decreased by the NO synthase inhibitor N-iminoethyl-L-ornithine (L-NIO). Dexamethasone, which prevents the expression of cytokine-induced NO synthase in endothelial cells, also inhibits TNF-alpha-dependent cytotoxicity. The results indicate that NO is involved in the cytotoxic effect of TNF-alpha on endothelial cells.     

Nitric oxide: a possible mediator of ovulation and postovulatory follicle regression in chicken

Nitric oxide (NO) has recently emerged as a regulator of functional and structural regression in mammalian reproductive tissues. However, the role of NO in ovulation and postovulatory follicles (POF) that undergo regression in laying birds is unclear. In the present investigation, the expression profiles of iNOS mRNA, tissue NO levels and the percentage of apoptotic cells were studied in the regressing chicken postovulatory follicle (POF). The postovulatory follicles gradually lost weight during its regression and reached the lowest weight on POF5. The number of apoptotic cells was increased significantly during the regression of POF. The mRNA expression of iNOS was noticed in the second largest preovulatory follicle (F2) that subsequently increased in the largest preovulatory follicle (F1). However, the level of iNOS mRNA was declined immediately after ovulation and thereafter upregulated again to reach a peak in POF3 with a subsequent reduction in POF5 to below the basal level. The tissue NO levels followed a similar pattern except with a peak production in POF4. The gross regression and apoptosis in POFs were well associated with iNOS expression and NO production. In conclusion, NO appears to play a role in ovulation and regression of postovulatory follicle in chicken.     

Nitric oxide donors

Nitric oxide (NO) donors are pharmacologically active substances that release NO in vivo or in vitro. NO has a variety of functions such as the release of prostanoids, inhibition of platelet aggregation, effect on angiogenesis, and production of oxygen free radicals. This report discusses the chemical and pharmacological characteristics of NO donors, their effect on platelet function and cyclooxygenase, their cardiac action including myocardial infarction, and release of superoxide anions. This review stresses NO tolerance and the effect of NO donors on angiogenesis in myocardial infarction and in solid tumors.     

Nitric oxide mediates interleukin-1 induced inhibition of glycosaminoglycan synthesis in rat articular cartilage

Interleek-1beta (IL-1) is a key mediator of cartilage matrix degradation in osteoarthritis and rheumatoid arthritis. It was found that the IL-1-induced suppression of glycosaminoglycan (GAG) synthesis in rat articular cartilage occurred simultaneously with the accumulation of nitrite (a metabolite of nitric oxide (NO) in aqueous milieu) in the culture medium. NO-synthase inhibitors, L-NMMA and L-NIO, inhibited both these IL-1 effects. Dexamethasone suppressed GAG synthesis additively to IL-1, but did not alter nitrite accumulation. Three NO-donors (GEA 3175, SNAP and SIN-1) also had an inhibitory effect on cartilage GAG synthesis. Therefore, it is concluded that IL-1 induced suppression of GAG synthesis in rat articular cartilage is mediated by the production of NO.     

Nitric oxide inducing function and intracellular movement of chicken interleukin-18 in cultured cells

To evaluate the characteristics of chicken interleukin-18 （ChIL-18） in different forms in vitro, the ChIL-18 full-length gene （ChIL-18-F） and the ChIL-18 presumed mature protein gene （ChIL-18-M） were cloned and inserted into the eukaryotic expression vector pCI, to construct recombinant pCI-ChIL-18-F and pCI-ChIL-18-M. The recombinant plasmids were then transferred into chicken splenic lymphocytes （CSLs）. Western blot showed that ChIL-18-F, with a molecular weight of 23.0 kDa, was produced in CSLs transfected by pCI-ChIL-18-F; ChIL-18-M, with a molecular weight of 19.5 kDa, was produced in CSLs transfected by pCI-ChIL-18-M. The nitric oxide （NO） level in the transfected CSLs and the culture medium at different time points was further examined under confocal microscopy using 4,5-diaminofluorescein staining. The results showed that both pCI-ChIL-18-F and pCI-ChIL-18-M groups showed significant increase in intracellular and extracellular NO production compared with pCI transfected control cells. These results suggest that both ChIL- 18-F and ChIL- 18-M could stimulate NO secretion in CSLs. To characterize the intracellular distribution of ChIL-18, ChIL-18-F and ChIL-18-M were each fused to the enhanced green fluorescent protein gene, and expressed in Vero cells. The results showed that the ChIL-18-F tended to the membranous region in Veto cells, while ChIL- 18-M did not. This indicates that the N-terminal 27 amino acid peptide helped ChIL-18 target to Vero cell membranes.     

Nitric oxide synthase-cyclooxygenase interactions are involved in tumor cell angiogenesis and migration

Nitric oxide (NO), produced by distinct nitric oxide synthase (NOS) isoforms, and prostaglandins generated by expression of cyclooxygenases are important mediators in tumor progression. Previous studies have shown that NO can influence the formation of prostaglandin E2 (PGE2). We provide evidence that NO, derived from iNOS and eNOS activity in LMM3 murine mammary adenocarcinoma cell line, is involved in tumor angiogenesis and in tumor cell migration. LMM3 cells that also stimulate their neovascularization activity and migration liberate high basal amounts of PGE2. There is large amount of evidence that postulates positive regulatory interactions between NOS and cyclooxygenase (COX) isoforms. We here show that, in the LMM3 cell line, while PGE2 exerts a positive modulation on NOS activity, NO closes the loop with a negative feed back on COX activity. We also provide evidence of a positive regulatory effect of protein tyrosine kinases on NOS as well as on COX enzymatic functions affecting tumor induced angiogenesis and cell migration.     

Nitric oxide mediates the interleukin-1beta- and nicotine-induced hypothalamic-pituitary-adrenocortical response during social stress.

We investigated the role of nitric oxide (NO) in the interleukin 1beta (IL-1beta) and nicotine induced hypothalamic-pituitary-adrenal axis (HPA) responses, and a possible significance of CRH and vasopressin in these responses under basal and social stress conditions. Male Wistar rats were crowded in cages for 7 days prior to treatment. All compounds were injected i.p., nitric oxide synthase (NOS) inhibitors, alpha-helical CRH antagonist and vasopressin receptor antagonist 15 min before IL-1beta or nicotine. Identical treatment received control non-stressed rats. Plasma ACTH and serum corticosterone levels were measured 1 h after IL-1beta or nicotine injection. L-NAME (2 mg/kg), a general nitric oxide synthase (NOS) inhibitor, considerably reduced the ACTH and corticosterone response to IL-1beta (0.5 microg/rat) the same extent in control and crowded rats. CRH antagonist almost abolished the nicotine-induced hormone responses and vasopressin antagonist reduced ACTH secretion. Constitutive endothelial eNOS and neuronal nNOS inhibitors substantially enhanced the nicotine-elicited ACTH and corticosterone response and inducible iNOS inhibitor, aminoguanidine, did not affect these responses in non-stressed rats. Social stress significantly attenuated the nicotine-induced ACTH and corticosterone response. In crowded rats L-NAME significantly deepened the stress-induced decrease in the nicotine-evoked ACTH and corticosterone response. In stressed rats neuronal NOS antagonist did not alter the nicotine-evoked hormone responses and inducible NOS inhibitor partly reversed the stress-induced decrease in ACTH response to nicotine. These results indicate that NO plays crucial role in the IL-1beta-induced HPA axis stimulation under basal and social stress conditions. CRH and vasopressin of the hypothalamic paraventricular nucleus may be involved in the nicotine induced alterations of HPA axis activity. NO generated by eNOS, but not nNOS, is involved in the stress-induced alterations of HPA axis activity by nicotine.     

Nitric oxide in invertebrates

Nitric oxide (NO) is considered an important signaling molecule implied in different physiological processes, including nervous transmission, vascular regulation, immune defense, and in the pathogenesis of several diseases. The presence of NO is well demonstrated in all vertebrates. The recent data on the presence and roles of NO in the main invertebrate groups are reviewed here, showing the widespread diffusion of this signaling molecule throughout the animal kingdom, from higher invertebrates down to coelenterates and even to prokaryotic cells. In invertebrates, the main functional roles described for mammals have been demonstrated, whereas experimental evidence suggests the presence of new NOS isoforms different from those known for higher organisms. Noteworthy is the early appearance of NO throughout evolution and striking is the role played by the nitrergic pathway in the sensorial functions, from coelenterates up to mammals, mainly in olfactory-like systems. All literature data here reported suggest that future research on the biological roles of early signaling molecules in lower living forms could be important for the understanding of the nervous-system evolution.     

Nitric oxide inhibits LPS-induced tumor necrosis factor synthesis in vitro and in vivo

The effect of nitric oxide (NO) on LPS-stimulated TNF-α synthesis has been studied in vitro and in vivo. The synthesis of TNF-α in J774 macrophages stimulated with LPS (0.l μg/ml) was increased in concentration-related fashion by NO synthase inhibitor L-NMMA (3-30-300 μM) and reduced by either L-arginine (3-30-300 μM) or the NO donor SIN-1 (1-10-1OOμM). The level of TNF-α in the serum of LPS-challenged rats (6mg/kg/i.p.) was increased in animals pre-treated s.c. with L-NMMA (10 and 50mg/kg) and reduced in those given L- arginine (100 and 300mg/kg). These results show a negative feedback mechanism exhibited by NO on TNF-α synthesis suggesting an important regulatory link between NO and TNF-α in pathological processes.     

DNA alkylating therapy induces tumor regression through an HMGB1-mediated activation of innate immunity

Dysregulation of apoptosis is associated with the development of human cancer and resistance to anticancer therapy. We have previously shown in tumor xenografts that DNA alkylating agents induce sporadic cell necrosis and regression of apoptosis-deficient tumors. Sporadic tumor cell necrosis is associated with extracellular release of cellular content such as the high mobility group box 1 (HMGB1) protein and subsequent recruitment of innate immune cells into the tumor tissue. It remained unclear whether HMGB1 and the activation of innate immunity played a role in tumor response to chemotherapy. In this study, we show that whereas DNA alkylating therapy leads to a complete tumor regression in an athymic mouse tumor xenograft model, it fails to do so in tumors deficient in HMGB1. The HMGB1-deficient tumors have an impaired ability to recruit innate immune cells including macrophages, neutrophils, and NK cells into the treated tumor tissue. Cytokine array analysis reveals that whereas DNA alkylating treatment leads to suppression of protumor cytokines such as IL-4, IL-10, and IL-13, loss of HMGB1 leads to elevated levels of these cytokines upon treatment. Suppression of innate immunity and HMGB1 using depleting Abs leads to a failure in tumor regression. Taken together, these results indicate that HMGB1 plays an essential role in activation of innate immunity and tumor clearance in response to DNA alkylating agents.