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Pancreatic ductal adenocarcinoma (PDAC) is a therapy recalcitrant disease characterized by the aberrations in multiple genes that drive pathogenesis and drug chemoresistance. In this study, we synthesize a library of seven novel nitric oxide-releasing gemcitabine pro-drugs (NO-GEMs) in order to improve the effectiveness of GEM by exploiting the therapeutic effects of NO. Among these NO-GEM pro-drugs we select 5b as the most effective compound in GEM-resistant PDAC cells. After its encapsulation in liposomes for drug delivery the intracellular NO level increases and nitration associated to activity inhibition of the multidrug resistance associated protein 5 (MRP5; ABCC5) occurs. This results in GEM intracellular accumulation and enhanced apoptotic cell death in GEM-resistant PDAC cells, which express MRP5 at higher levels than GEM-sensitive cells. Our results support the development of a new anti-tumoral strategy to efficiently affect GEM-resistant PDAC cells based on the usage of NO-GEM pro-drugs., Competing Interests: Declaration of competing interest All authors declare that they have no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Treatment of tumor cells with cold atmospheric plasma (CAP) or with plasma-activated medium (PAM) leads to a biochemical imprint on these cells. This imprint is mediated by primary singlet oxygen, which is mainly generated through the interaction between CAP-derived H 2 O 2 and NO 2 - . This imprint is induced with a low efficiency as local inactivation of a few membrane-associated catalase molecules. As sustained generation of secondary singlet oxygen by the tumor cells is activated at the site of the imprint, a rapid bystander effect-like spreading of secondary singlet oxygen generation and catalase inactivation within the cell population is thus induced. This highly dynamic process is essentially driven by NOX1 and NOS of the tumor cells, and finally leads to intercellular RONS-driven apoptosis induction. This dynamic process can be studied by kinetic analysis, combined with the use of specific inhibitors at defined time intervals. Alternatively, it can be demonstrated and quantified by transfer experiments, where pretreated cells are mixed with untreated cells and bystander signaling is determined. These studies allow to conclude that the specific response of tumor cells to generate secondary singlet oxygen is the essential motor for their self-destruction, after a singlet oxygen-mediated triggering process by CAP or PAM.

Inadequate perfusion of solid cancer tissue results in low local nutrient and oxygen levels and accumulation of acidic waste products. Previous investigations have focused primarily on tumor blood vessel architecture, and we lack information concerning functional differences between arteries that deliver blood to solid cancer tissue versus normal tissue. Here, we use isometric myography to study resistance-sized arteries from human primary colon adenocarcinomas and matched normal colon tissue. Vasocontraction of colon cancer feed arteries in response to endothelin-1 and thromboxane stimulation is attenuated compared with normal colon arteries despite similar wall dimensions and comparable contractions to arginine vasopressin and K + -induced depolarization. Acetylcholine-induced vasorelaxation and endothelial NO synthase expression are increased in colon cancer feed arteries compared with normal colon arteries, whereas vasorelaxation to exogenous NO donors is unaffected. In congruence, the differences in vasorelaxant and vasocontractile function between colon cancer feed arteries and normal colon arteries decrease after NO synthase inhibition. Rhythmic oscillations in vascular tone, known as vasomotion, are of lower amplitude but similar frequency in colon cancer feed arteries compared with normal colon arteries. In conclusion, higher NO synthase expression and elevated NO signaling amplify vasorelaxation and attenuate vasocontraction of human colon cancer feed arteries. We propose that enhanced endothelial function augments tumor perfusion and represents a potential therapeutic target. NEW & NOTEWORTHY Local vascular resistance influences tumor perfusion. Arteries supplying human colonic adenocarcinomas show enhanced vasorelaxation and reduced vasocontraction mainly due to elevated nitric oxide-mediated signaling. Rhythmic oscillations in tone, known as vasomotion, are attenuated in colon cancer feed arteries.

Background: Lung adenocarcinoma is the most primary histologic subtype of non-small cell lung cancer (NSCLC). Compound 8b, a novel coumarin derivative with phenylsulfonylfuroxan group, shows significant antiproliferation activity against lung adenocarcinoma cell with low toxicity., Purpose: This study aims to uncover the potential of compound 8b in relation to apoptosis as well as autophagy induction in lung adenocarcinoma cells., Study Design: The cytotoxicity and apoptosis of A549 and H1299 cells induced by compound 8b were detected by MTT, microscope and western blot analysis. Autophagy was determined by TEM, confocal microscopy and western blot analysis. Akt/mTOR and Erk signaling pathway were also examined by western blot analysis., Results: First, significant growth inhibition and caspase-dependent apoptosis were observed in compound 8b-treated A549 and H1299 cells. Then, we confirmed compound 8b-induced autophagy by autophagosomes formation, upregulated expression of autophagy-related protein LC3-II and autophagic flux. Importantly, abolishing autophagy using inhibitors and ATG5 siRNA enhanced the cytotoxicity of compound 8b, indicating the cytoprotective role of autophagy in lung adenocarcinoma. Further mechanistic investigations suggested that Akt/mTOR and Erk signaling pathways contributed to autophagy induction by compound 8b., Conclusion: This results demonstrate that compound 8b induces caspase-dependent apoptosis as well as cytoprotective autophagy in lung adenocarcinoma cells, which may provide scientific evidence for developing this furoxan-based NO-releasing coumarin derivative as a potential anti-lung adenocarcinoma therapeutic agents., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Previously, we have shown that A549, a human lung adenocarcinoma, can be adapted to nitric oxide (NO ● ). NO ● is a nitrogen-based free radical that is synthesized by a family of enzymes known as nitric oxide synthases. NO ● has been shown to be overexpressed in patient populations of different cancers. In addition, it has been observed that patients who express high levels of nitric oxide synthases tend to have poorer clinical outcomes than those with low levels of expression. The original cell line A549 (parent) and the adapted A549-HNO (high nitric oxide) cell line serve as a useful model system to investigate the role of NO ● in tumor progression and prognosis. We have previously shown that the A549-HNO-adapted cells grow aggressively when compared to A549-parent cells. Furthermore, we have shown that the A549-HNO-adapted cells exhibit a higher percentage of cell viability when exposed to ultraviolet and X-ray radiation than the A549-parent cells. Cancer patients who develop resistance to one treatment often become resistant to other previously unencountered forms of treatment. This phenomenon is known as cross-tolerance. To determine whether NO ● is a potential cross-tolerance causing agent, we have expanded our research by conducting parallel studies to a variety of other agents and conditions beyond radiation and ultraviolet exposure. We exposed both cell lines to varying levels of chemotherapeutic drugs (taxol and doxorubicin), temperature, pH, calcium chloride, cadmium chloride, copper chloride, sodium chloride, ferrous chloride, and sodium-R-lipoic acid. Our results show that the A549-HNO cells exhibit greater viability than the A549-parent cells when exposed to each of the various conditions. Therefore, NO ● is one potential driving force that can make tumor cells exhibit cross-tolerance.

Purpose: Soy and its fermented products are considered functional foods. The study objective was to assess three functional food - a non-fermented soy product (NFP), fermented soy product (FSP), fermented soy product enriched with isoflavones (FI) - in terms of their ability to reduce the development of adenocarcinoma in mice, as well their ability on modulating immune system., Methods: It was observed tumor volume and to verify correlations with the immune system it was measured levels of the cytokines IL-1β and TNF-α produced by macrophages as well as IFN-γ produced by lymphocytes using ELISA test, and nitric oxide production by macrophages using Griess reagent., Results: All products showed immunological activity, but FSP showed the most effective tumor containment, resulting in smallest tumor volumes. FI animals expressed larger amounts of nitric oxide and IL-1β and exhibited larger tumor sizes than FSP and NFP animals., Conclusions: The results suggested that the ingestion of FSP was most efficient in tumor containment, possibly due to a positive modulation of the immune system by when Enterococcus faecium and Lactobacillus helveticus are added to the soy product., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Our group and others have determined that immune effector cells from patients with advanced cancers exhibit reduced activation of IFN signaling pathways. We hypothesized that increases in immune regulatory cells termed myeloid-derived suppressor cells (MDSC) could interfere with the host immune response to tumors by inhibiting immune cell responsiveness to IFNs. The C26 murine adenocarcinoma model was employed to study immune function in advanced malignancy. C26-bearing mice had significantly elevated levels of GR1(+)CD11b(+) MDSC as compared with control mice, and splenocytes from tumor-bearing mice exhibited reduced phosphorylation of STAT1 (P-STAT1) on Tyr(701) in response to IFN-α or IFN-γ. This inhibition was seen in splenic CD4(+) and CD8(+) T cells as well as natural killer cells. In vitro coculture experiments revealed that MDSC inhibited the IFN responsiveness of splenocytes from normal mice. Treatment of C26-bearing mice with gemcitabine or an anti-GR1 antibody led to depletion of MDSC and restored splenocyte IFN responsiveness. Spleens from C26-bearing animals displayed elevated levels of iNOS protein and nitric oxide. In vitro treatment of splenocytes with a nitric oxide donor led to a decreased STAT1 IFN response. The elevation in nitric oxide in C26-bearing mice was associated with increased levels of nitration on STAT1. Finally, splenocytes from iNOS knockout mice bearing C26 tumors exhibited a significantly elevated IFN response as compared with control C26 tumor-bearing mice. These data suggest that nitric oxide produced by MDSC can lead to reduced IFN responsiveness in immune cells.

Background/aims: Nitric oxide, a labile compound synthesized by nitric oxide synthase, is a major regulator not only of physiological vascular tonus but also of the abnormal vascularity associated with tumors. Endothelial production of nitric oxide regulates blood flow and angiogenesis and reduces tumor cell adhesion to the endothelium. A high concentration of nitric oxide and its metabolites causes DNA damage during nitration, nitrosation and deamination. Both positive and negative effects on carcinogenesis and tumor growth, apoptosis, and cytotoxic mechanisms may be explained by differential susceptibility of tumor cells to nitric oxide-mediated reactions., Methods: In this study, three major polymorphisms (786T>C, the 27 base pair variable number of tandem repeats in intron 4, and 894G>T) of the endothelial nitric oxide synthase gene were investigated in gastric cancer and normal tissues of 50 patients with gastric cancer and in the peripheral blood of 98 healthy subjects., Results: We found no significant differences in intron 4a/b and 894G>T (Glu298Asp) allele and genotype frequencies between control and patient specimens. Nevertheless, the genotype and allele frequencies of 786T>C polymorphism were found to be significantly different between the healthy controls and tumor tissues., Conclusions: The results suggest that endothelial nitric oxide synthase 786T>C polymorphism may play a role in the development of gastric cancer.

Background: This study determine oxidative stress and survival prospectively in advanced stage non small cell lung cancer (NSCLC) patients following cisplatin based combination chemotherapy., Materials and Methods: The oxidative stress levels (LPO, NO, GSH and SOD) of 144 control subjects and 203 advanced stage (IIIA/IIIB/IV) newly diagnosed NSCLC patients were assessed at pre-treatment (day '0'), and after the 3rd and 6th cycles of chemotherapy. Groups were compared by repeated measures ANOVA while comparison of survival curves was conduced by Kaplan-Meier methods., Results: The pre-treatment mean levels of LPO and NO in patients were significantly (P<0.01) higher while GSH and SOD were significantly (P<0.01) lower as compared to control. The oxidative stress was elevated more significantly (P<0.01) after the chemotherapy and was more evident in higher stage than lower stage patients. The two year overall survival (%) of stage IV patients was significantly lower (P<0.05) as compared to stage III A and III B. The proportional mortality was also maximal in stage IV patients (37.0%) followed by stage III B (31.7%) and III A (20.0%)., Conclusion: Cisplatin based combination chemotherapy induces oxidative stress in NSCLC patients.

Background & Aims: The luminal microenvironment including acid and nitric oxide (NO) has been implicated in Barrett's esophagus carcinogenesis. We investigated the ability of acid and NO to induce DNA damage in esophageal cells., Methods: Transformed and primary Barrett's esophagus and adenocarcinoma cells were exposed to either acid, (pH 3.5), +/- antioxidant or NO from a donor or generated by acidification of nitrite in the presence of ascorbate +/- NO scavenger. Phosphorylation of histone H2AX and the neutral comet assay were used to detect DNA double-strand breaks (DSBs). Intracellular levels of reactive oxygen species and NO were detected with fluorescent dyes. Mitochondrial viability was measured with a rhodamine dye. Long-term survival was assessed by clonogenic assay., Results: Exposure to acid (pH 3.5) for > or =15 minutes induced DSBs in all cell lines (P < .05). There was a concomitant increase in intracellular reactive oxygen species in the absence of mitochondrial damage, and pretreatment with antioxidants inhibited DNA damage. Exposure to physiologic concentrations of NO produced from the NO donor or acidification of salivary nitrite induced DSBs in a dose- (>25 micromol/L) and cell-dependent manner (adenocarcinoma >Barrett's esophagus, P < .05). This occurred preferentially in S-phase cells consistent with stalled replication forks and was blocked with a NO scavenger. NO also induced DSBs in primary Barrett's esophagus cells treated ex vivo. Cells were able to survive when exposed to acid and NO., Conclusions: Both acid and NO have the potential to generate DSBs in the esophagus and via distinct mechanisms.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by rapid tumor progression, high metastatic potential and profound chemoresistance. We recently reported that induction of a chemoresistant phenotype in the PDAC cell line PT45-P1 by long-term chemotherapy involves an increased interleukin 1 beta (IL1beta)-dependent secretion of nitric oxide (NO) accounting for efficient caspase inhibition. In the present study, we elucidated the involvement of L1CAM, an adhesion molecule previously found in other malignancies, in this NO-dependent chemoresistance. Chemoresistant PT45-P1res cells, but not chemosensitive parental PT45-P1 cells, express high levels of L1CAM in an ILbeta-dependent fashion. PT45-P1res cells subjected to short interfering RNA (siRNA)-mediated L1CAM knock-down exhibited reduced inducible nitric oxide synthase expression and NO secretion, as well as a significant increase of anti-cancer drug-induced caspase activation, an effect reversed by the NO donor S-nitroso-N-acetyl-D,L-penicillamine. Conversely, overexpression of L1CAM in PT45-P1 cells conferred anti-apoptotic protection to anti-cancer drug treatment. Interestingly, L1CAM ectodomain shedding, in example, by ADAM10, as reported for other L1CAM-related activities, seemed to be dispensable for anti-apoptotic protection by L1CAM. Neither the shedded L1CAM ectodomain was detected in chemoresistant L1CAM-expressing PT45-P1 cells nor did the administration of various metalloproteinase inhibitors affect L1CAM-dependent chemoresistance. Immunohistochemical analysis revealed L1CAM expression in 80% of pancreatic cancer specimens, supporting a potential role of L1CAM in the malignancy of this tumor. These findings substantiate our understanding of the molecular mechanisms leading to chemoresistance in PDAC cells and indicate the importance of L1CAM in this scenario.

Expression of cyclooxygenase-2 (COX-2) is involved in the chronic inflammation-related development of Barrett's adenocarcinoma and the use of selective COX-2 inhibitors (coxibs) might provide new chemoprevention strategy for Barrett's adenocarcinoma (BA). Despite an excellent gastrointestinal (GI) safety profile of coxibs, their use is limited because of the possible cardiovascular complications. The coupling of NSAIDs with a NO-donating moiety has led to the birth of a new class of anti-inflammatory drugs, called the COX-inhibiting nitric oxide donators (CINODs). The member of this group, NO-aspirin (NO-ASA) retains the anti-inflammatory properties of traditional aspirin (ASA), but the release of NO accounts for anti-thromboembolic effect and better GI safety profile. The role of NO-ASA in the prevention of Barrett's adenocarcinoma (BA) has not been studied so far. Therefore, the aim of the present study was: 1) to analyse the expression of COX-2 in the biopsies obtained from BE; 2) to compare the effect of NO-ASA with that of ASA on proliferation rate in Barrett''s adenocarcinoma cell line (OE-33 cells); 3) to determine the effect of both compounds on the apoptosis rate using FACS analysis and expression of 32-kDa procaspase-3 and active proapoptotic 20-kDa caspase-3 in OE-33 cell line. The expression of COX-2 was assessed in biopsies obtained from the Barrett's mucosa and normal squamous epithelial esophageal mucosa from 20 BE patients by RT-PCR and Western blot analysis, respectively. The BA cell line (OE-33) was incubated with NO-ASA or ASA (10-1000 microM). The cell proliferation and apoptosis rate was measured by BrdU and FACS-analysis, respectively. The expression of caspase-3 (active and inactive form) was analyzed by Western blot. In Barrett's mucosa a significant up-regulation of COX-2 was observed. Compared with traditional ASA, NO-ASA caused a significantly stronger induction of apoptosis (dose-dependently). Inhibition of cell proliferation in OE-33 cells observed under NO-ASA treatment was due to the apoptosis induction. The increase in apoptotic rate was accompanied by the upregulation of active 20-kDa caspase-3. At the highest concentration (1000 microM), a necrotic death of OE-33 cells was observed under NO-ASA treatment. We conclude that: NO-ASA caused induction of apoptosis in BA cell line and slight growth inhibition. These results indicate that this compound may represent a promising chemopreventive agent for Barrett's adenocarcinoma.

Aim: To investigate the extent of oxidative stress in pre-neoplastic and neoplastic gastric mucosa in relation to their pathological criteria and histological subtypes., Methods: A total of 104 gastric adenocarcinomas from 98 patients (88 infiltrative and 16 intraepithelial tumors) were assessed immunohistochemically for expression of iNOS and occurrence of nitrotyrosine (NTYR)-containing proteins and 8-hydroxy-2'-deoxyguanosine (8-OH-dG)-containing DNA, as markers of NO production and damages to protein and DNA., Results: Tumor cells staining for iNOS, NTYR and 8-OH-dG were detected in 41%, 62% and 50% of infiltrative carcinoma, respectively. The three markers were shown for the first time in intraepithelial carcinoma. The expression of iNOS was significantly more frequent in tubular carcinoma (TC) compared to diffuse carcinoma (DC) (54% vs 18%; P = 0.008) or in polymorphous carcinoma (PolyC) (54% vs 21%; P = 0.04). NTYR staining was obviously more often found in TC than that in PolyC (72% vs 30%; P = 0.03). There was a tendency towards a higher rate of iNOS staining when distant metastasis (pM) was present. In infiltrative TC, the presence of oxidative stress markers was not significantly correlated with histological grade, density of inflammation, the depth of infiltration (pT), lymph nodes dissemination (pN) and pathological stages (pTNM)., Conclusion: The iNOS-oxidative pathway may play an important role in TC, but moderately in PolyC and DC. DNA oxidation and protein nitration occur in the three subtypes. Based on the significant differences of NTYR levels, TC and PolyC appear as two distinct subtypes.

The incidence of oesophageal adenocarcinoma is increasing in the UK faster than any other malignancy. Despite its relatively poor prognosis and the limited success of existing treatments, there is enthusiasm that chemopreventive agents might be able to stem the transition from normal squamous epithelium to adenocarcinoma. We discuss gastro-oesophageal reflux as the main risk factor for the development of Barrett's metaplasia, the only known precursor of oesophageal adenocarcinoma. Treatment options for reflux disease are considered with regard to their effects on cancer risk. Recent advances in the molecular and cell biology of Barrett's are outlined, and potential targets for chemoprevention examined. Available treatments for reflux disease have not convincingly altered the likelihood of cancer development. Epidemiological and animal studies support the use of non-steroidal anti-inflammatory drugs as potential chemopreventive agents. Dietary agents, however, have a more favourable side-effect profile and may prove to be an attractive alternative, although more work is needed to fully explore this prospect.

Aim: To detect the presence of inducible nitric oxide synthase (iNOS), nitrotyrosine (NT) and apoptosis in gastric adenocarcinomas and their possible correlations with the clinicopathological characteristics and prognosis of gastric adenocarcinoma., Methods: Sixty-six specimens of gastric adenocarcinoma and corresponding adjacent normal gastric tissues were studied. Immunohistochemistry was employed to localize iNOS and NT protein and an immunohistochemical scoring system was used. The occurrence of apoptotic cell death (apoptotic index (AI)) was analyzed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labeling (TUNEL) method., Results: Results showed that iNOS expression was detected at an intermediate or high level in 41 of 66 (62%) specimens of gastric adenocarcinoma. NT expression was 58%. Neither of them was found in the normal gastric tissues; there were significant positive correlations among iNOS expression, NT expression and AI. Many clinicopathologic characteristics of gastric adenocarcinoma, such as tumor size, depth of invasion, lymph node metastasis and TNM staging, were related to iNOS and NT expressions (P<0.05). In 66 surviving patients, the 5-year survival rate of 41 patients who had tumors with intermediate or high iNOS expressions and high AIs (4.09%; 19.96%) was significantly lower than that of 25 patients who had tumors with negative or low iNOS expressions and low AIs (0.79%; 47.14%) (P = 0.001). COX's multivariate analysis revealed that the iNOS expression was identified as one of the significant independent prognostic factors predictive of a poor survival (relative risk (RR) = 2.69)., Conclusion: NO produced by iNOS may play a stronger role in promoting gastric adenocarcinoma growth than in suppressing its growth. iNOS and NT expressions by gastric adenocarcinoma may correlate with a poor survival.

This article summarizes the present recommendations for the screening, surveillance and treatment of Barrett's oesophagus, and identifies those areas in which change seems likely within the next decade. As a result of economic constraints and emerging data on ethnic variations in the frequency of Barrett's oesophagus, future screening programmes will probably focus on those individuals most likely to develop Barrett's adenocarcinomas: older white men whose gastro-oesophageal reflux symptoms are of long duration. The present surveillance strategy for patients with Barrett's oesophagus relies heavily on random biopsy sampling of the oesophagus to find dysplasia. In the future, biomarkers other than dysplasia may be used to identify patients at high risk for carcinogenesis, and physicians may use endoscopic techniques, such as fluorescence spectroscopy, to identify areas of dysplasia for biopsy sampling. Indirect evidence suggests that super-aggressive antisecretory therapy and treatment with non-steroidal anti-inflammatory drugs may reduce the risk of cancer in Barrett's oesophagus. Well-designed prospective studies will be needed to determine whether these treatments have sufficient efficacy in cancer prophylaxis to justify the large numbers needed to treat. Finally, recent data are reviewed, which suggest that the gastro-oesophageal junction is exposed repeatedly to concentrated acid and to potentially genotoxic concentrations of nitric oxide generated from dietary nitrate. Future studies on carcinogenesis in Barrett's oesophagus may well focus on the combined roles of nitric oxide and gastric acid.

Several epidemiological cohort studies have suggested that duodeno-gastroesophageal reflux per se induces Barrett's esophagus leading to increased risk of the development of esophageal adenocarcinoma (EAC). However, the exact causative factors behind EAC remain unclear. Recently, we designed a new duodenal contents reflux model which retained normal stomach function. In this model, duodenal contents flowed back into the esophagus and stomach resulting in repeated re-entry into the esophagus through the site of esophagojejunostomy. To elucidate the factors underlying the development of EAC, thiazolidine-4-carboxylic acid (thioproline, TPRO) was applied to the new reflux models as a nitrite scavenger and as a probe to detect reactive nitrogen species (RNS). Post-operatively, 31 animals were divided into two groups according to diet. Animals belonging to the control group were given normal diet (n = 18), while the TPRO group was given food containing 0.5% TPRO (n = 13). All esophageal sections in both groups were examined using hematoxylin and eosin staining and immunohistochemical analysis of inducible nitric oxide synthase (iNOS). EACs developed in 7 of 18 rats (38.9%) of the control group, whereas no EACs were detected in the TPRO group (Fisher's exact test, P < 0.05). Conversely, esophageal squamous cell carcinoma (ESCC) was detected in 1 of 18 rats (5.6%) of the control group and in 1 of 13 rats (7.7%) of the TPRO group. The incidence of ESCC was not significantly different between the two groups (P = 0.671). iNOS protein was overexpressed in Barrett's esophagus of both groups. The present results suggest that RNS such as nitric oxide and peroxynitrite and nitroso compounds derived from reflux of duodenal contents play an important role in the development of EAC, and that the primary causes of ESCC and EAC may differ.

Differential expression and activity of constitutive mitochondrial nitric oxide synthase (mtNOS) in the mitochondrial compartment is followed by significant variations in matrix nitric oxide (NO) steady-state concentration. The mitochondrial utilization of NO involves the production of superoxide anion and H(2)O(2), a species freely diffusible outside the mitochondria that participates in the modulation of cell proliferation and apoptosis and in cell transformation and cancer. On these bases, we analyzed the modulation of mtNOS in the frame of cellular redox state in M3, MM3, and P07 murine tumors and their respective cell lines, as compared with normal proliferating and quiescent tissues. The results showed that: (a) tumoral and proliferating mitochondria only retain 10-50% of the activity of complexes I, II-III, and IV and Mn-SOD of quiescent tissues; (b) normal proliferating tissues, like embryonic liver or pregnant mammary gland, have 10-20% of mtNOS expression and activity and mitochondrial H(2)O(2) yield than quiescent nonproliferating tissues; (c) similarly but irrespective of mtNOS expression, tumoral mitochondria have no >5% of mtNOS activity and H(2)O(2) yield of mature tissues; and (d) in opposition to stable tissues, both tumoral and normal proliferating cells exhibit high cyclin D1 expression and low pro-apoptotic p38mitogen-activated protein kinase activity. Dually, H(2)O(2) stimulated tumor cell proliferation (<10 microM) or markedly inhibited it (>10 microM) with parallel variations of cyclin D1, phospho-extracellular-regulated kinase1/2, and phospho-p38mitogen-activated protein kinase. It is surmised that decreased oxidative phosphorylation, defective tumoral mtNOS, and low mitochondrial NO-dependent H(2)O(2) may be a platform to link persistent tumoral growth to embryonic behavior.

The objective of this study was to investigate the role of induced cytokines, tumor infiltrating cells and nitric oxide (NO) in anti-tumor activity upon intratumoral injection of free and condensed plasmid DNA encoding murine interleukin-12 (pmIL-12) into BALB/c mice bearing subcutaneous tumors. Poly[alpha-(4-aminobutyl)-L-glycolic acid] (PAGA) was used for complex formation with pmIL-12 in presence of 5% (w/v) glucose. Upon characterization, PAGA/pmIL-12 (3/1, +/-) complexes were found to be most effective in gene transfer and were used consistently throughout this study. The levels of mIL-12 p70 and induced cytokines were determined by ELISA in the supernatant of the cultured tumors of the CT-26 subcutaneous tumor bearing BALB/c female mice 48 h after intratumoral injection of PAGA/pmIL-12 complexes and naked pmIL-12. The levels of IL-12, IFN-gamma, TNF-alpha and NO were higher for the PAGA/pmIL-12 complexes than those for the naked pmIL-12, PAGA alone and 5% glucose injected groups. The relative presence of natural killer (NK) cells, CD4(+) T cells, and antigen presenting cells, such as macrophages and dendritic cells determined using immunohistochemistry was higher for PAGA/pmIL-12 complexes compared with naked pmIL-12. The presence of CMV promoter in plasmid encoding IL-12 cDNAs did not induce any type I interferon response. There was a significant improvement in the survival rate and the inhibition of tumor growth after repeated injections of PAGA/pmIL-12 complexes.

Investigations on the influence of the parasympathetic nervous system via muscarinic signaling in tumor progression have produced contradictory evidence. We investigated the expression of muscarinic acetylcholine receptors (mAchR) and their intracellular transduction pathways, in two murine mammary adenocarcinoma cell lines, LM3 and LM2 in comparison with the normal murine mammary epithelial cell line: NMuMG. Saturation binding assays with the tritiated muscarinic antagonist quinuclidinyl benzilate ([3H]-QNB) indicate that LM3 cells express higher amounts of mAchR than LM2 cells. Muscarinic receptor activation with carbachol (CARB) enhanced basal production of citrulline to a greater extent in LM3 cells than in LM2 cells. The nitric oxide synthase (NOS) inhibitor, NGmono-methyl-L-arginine (L-NMMA), blunted this effect only in LM3 cells while in LM2 cells the action of CARB was blocked by Nomega hydroxy-L-arginine (L-OH-Arg), which is known to inhibit the arginase pathway. Atropine blocks the action of CARB in both cell lines. Additionally, mAchR activation stimulates prostaglandin E2 (PGE2) synthesis only in LM2 cells. NMuMG cells show detectable basal amounts of nitric oxide and PGE2, but they did not respond to CARB. Binding experiments confirm the absence of mAchR in these cells. The findings indicate that mAchR expression in tumor cells, and its control on arginine metabolism, via NOS/arginase, and on PGE2 synthesis by COX activation, could be a switch on mechanism that might lead mammary cells from normal to malignant phenotype. Moreover, mAchR coupling to distinct effectors might be associated with differences in aggressiveness of tumor cells.

Resveratrol is a dietary phytochemical that has been shown to inhibit proliferation of a number of cell lines, and it behaves as a chemopreventive agent in assays that measure the three stages of carcinogenesis. We tested for its chemopreventive potential against gastric cancer by determining its interaction with signaling mechanisms that contribute to the proliferation of transformed cells. Low levels of exogenous reactive oxygen (H(2)O(2)) stimulated [(3)H]thymidine uptake in human gastric adenocarcinoma SNU-1 cells, whereas resveratrol suppressed both synthesis of DNA and generation of endogenous O(2)(-) but stimulated nitric oxide (NO) synthase (NOS) activity. To address the role of NO in the antioxidant action of resveratrol, we measured the effect of sodium nitroprusside (SNP), an NO donor, on O(2)(-) generation and on [(3)H]thymidine incorporation. SNP inhibited DNA synthesis and suppressed ionomycin-stimulated O(2)(-) generation in a concentration-dependent manner. Our results revealed that the antioxidant action of resveratrol toward gastric adenocarcinoma SNU-1 cells may reside in its ability to stimulate NOS to produce low levels of NO, which, in turn, exert antioxidant action. Resveratrol-induced inhibition of SNU-1 proliferation may be partly dependent on NO formation, and we hypothesize that resveratrol exerts its antiproliferative action by interfering with the action of endogenously produced reactive oxygen. These data are supportive of the action of NO against reactive oxygen and suggest that a resveratrol-rich diet may be chemopreventive against gastric cancer.

The present study investigates the role of nitric oxide and the involvement of nitric oxide synthase II isoform on the invasion of human colorectal adenocarcinoma cell lines HRT-18 and HT-29. HRT-18 cells, which constitutively express nitric oxide synthase II mRNA were three-fold more invasive in a Matrigel invasion assay than nitric oxide synthase II mRNA negative HT-29 cells. Treatment of HT-29 cells with the nitric oxide donor Deta NONOate (50 nM) as well as induction of nitric oxide synthase II mRNA and production of endogenous nitric oxide by inflammatory cytokines (IFN-gamma and IL-1alpha) increased the invasiveness of HT-29 cells by approximately 40% and 75%, respectively. In HT-29 cells nitric oxide synthase II mRNA was also induced in co-culture with human monocytes. The invasiveness of HRT-18 cells and stimulated HT-29 cells was partly inhibited by the nitric oxide synthase II inhibitor 1400 W. These results show that nitric oxide increases the invasion of human colorectal adenocarcinoma cell lines HRT-18 and HT-29, and the involvement of nitric oxide synthase II isoform in tumour cell invasion. Therefore, the production of nitric oxide and secretion of pro-inflammatory cytokines by tumour-associated macrophages, which in turn induce nitric oxide synthase II isoform in tumour cells, promotes tumour cell invasiveness., (Copyright 2002 Cancer Research UK)

Sequential examinations were done on the pulmonary cytokinetics and pulmonary lesions in rats after inhalation exposure to (239)PuO(2) aerosols to investigate the pathogenesis of lung tumors. Total cell yields of lavaged bronchoalveolar cells as well as the estimated numbers of pulmonary alveolar macrophages were significantly reduced from 1 to 3 months after exposure but recovered thereafter to the control levels. The proportions of multinucleated or micronucleated pulmonary alveolar macrophages increased significantly in lavaged cells from 1 month, and the increase was sustained up to 18 months after exposure. Both tumor necrosis factor and nitric oxide were shown to be differentially released from stimulated cultures of pulmonary alveolar macrophages during the period from 6 to 18 months after exposure. The labeling indices of alveolar and bronchiolar epithelial cells treated with 5-bromo-2'-deoxyuridine increased significantly in lungs from 3 months and were sustained up to 18 months after exposure. Histopathological examinations revealed that after the early inflammation, hyperplasia and metaplasia of the lining of the bronchioloalveolar epithelium were predominant from 3 to 6 months, while adenomatous or adenocarcinomatous lesions appeared and developed from 12 months after exposure. The appearance of primary lung tumors, almost all of which were adenomas and adenocarcinomas, was found in the dose range of 1 to 2 Gy from 12 months after exposures. These results indicate that the pathogenetic process initiated by early cellular damage and alterations associated with inflammation is followed by the proliferative and metaplastic lesions of pulmonary epithelium, leading to the appearance and development of pulmonary neoplasms from 1 year after the inhalation exposures in rats that received a minimum lung dose of more than 1 Gy.

We have previously demonstrated that in vivo activation or inhibition of Kupffer cell (KC) cytotoxic function can reduce or enhance, respectively, the hepatic tumor burden in a syngeneic murine colon adenocarcinoma (MCA26) tumor model. In the current study, we have performed in vitro experiments to define the possible mechanisms of KC cytotoxicity against MCA26 cells. Addition of either anti-tumor necrosis factor (TNF) or anti-interleukin-1 alpha (IL-1 alpha) antisera reduced KC cytotoxicity in coculture against MCA26 targets in a dose-dependent fashion; addition of these sera together resulted in approximately additive inhibition, suggesting the existence of parallel pathways for these effector molecules. Nitric oxide as a mediator of cytotoxicity by KCs in coculture with MCA26 cells was evaluated by two approaches. Activated KCs produced detectable levels of nitric oxide; however, activated KC exerted cytotoxicity against MCA26 targets in the absence of exogenous free L-arginine. Thus, TNF and IL-1 play major roles in producing murine KC cytotoxicity against MCA26 colon cancer cells in vitro, whereas reactive nitric oxides do not.

Nitric oxide (NO) has been previously shown to inhibit crude preparations of ribonucleotide reductase, a key enzyme in DNA synthesis, and to destroy the essential tyrosyl free radical in pure recombinant R2 subunit of the enzyme. In R2-overexpressing TA3 cells, a decrease in the tyrosyl radical was observed by whole-cell EPR spectroscopy, as soon as 4 h after NO synthase induction by immunological stimuli. Complete loss of the tyrosyl EPR signal occurred after 7 h in cells cultured at a high density. Disappearance of the tyrosyl radical was prevented by N omega-nitro-L-arginine, a specific inhibitor of NO synthesis, and by oxyhemoglobin, which reacts rapidly with NO. It was reproduced by S-nitrosoglutathione, a NO-releasing molecule. Stable end products of NO synthase metabolism did not affect the radical. Immunoblot analysis of the R2 subunit indicated that expression of the protein was not influenced by NO synthase activity. These results establish that NO, or a labile product of NO synthase, induces the disappearance of the R2-centered tyrosyl radical. Since the radical is necessary for ribonucleotide reductase activity, its destruction by NO would contribute markedly to the antiproliferative action exerted by macrophage-type NO synthase.

The existence and role of an L-arginine:nitric oxide (NO) pathway in two human colorectal adenocarcinoma cell lines, SW-480 and SW-620, were investigated. Both cell lines, which derive from the same patient, SW-480 from the primary tumor and SW-620 from its metastatic lesion, were shown to have a cytosolic, Ca(2+)-independent, NADPH-dependent NO synthase, the activity of which was lower in the cytosol of SW-620. These cells were more potent inducers of platelet aggregation. In contrast, SW-480, which had more NO synthase activity, were less potent inducers of platelet aggregation. Pretreatment of both cell lines with NG-monomethyl-L-arginine, an inhibitor of NO synthase, potentiated their proaggregating effect and made them equally active. Exogenous L-arginine, NO, and related nitrovasodilators all inhibited platelet aggregation induced by SW-620. The antiaggregating activity of NO was further potentiated by prostacyclin and by M&B22948, a selective inhibitor of cyclic GMP phosphodiesterase. We propose that the generation of NO by tumor cells inversely correlates with their metastatic potential. Furthermore, we show that the lower activity of NO synthase in metastatic cells is due to the presence in these cells of a low molecular weight inhibitor of the NO synthase. In addition, agents which modulate platelet function by a cyclic GMP-dependent mechanism may be useful in the prevention of tumor metastasis.