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.

Parkinson's disease is associated with oxidative stress: comparison of peripheral antioxidant profiles in living Parkinson's, Alzheimer's and vascular dementia patients.

Antioxidant profiles in Parkinson's disease (PD; n = 15), dementias of Alzheimer's type (DAT; 18) and Vascular (VD; 15), and control subjects (C; 14) were studied. Cu-Zn superoxide dismutase (SOD), catalase (CAT), glutathione system (GLU) and thiobarbituric acid reactive substances (TBARS) were measured in erythrocytes; antioxidant capacity (TRAP) in plasma. Biochemical variables were analyzed simultaneously using multi-variate and non-parametric methods. Clinical diagnostic resulted associated with the main source of variability in antioxidant variables (Kruskal-Wallis: H = 32.58, p = 0.000001). Comparison of PD and C resulted highly significant (z = 4.47, p = 0.000047), demonstrating an association between oxidative stress and PD. SOD and TBARS were significantly higher in pathological groups against C (p = 0.0000001, p = 0.051); TRAP resulted lower (p = 0.00015). Discriminant functions constructed using biochemical variables separated pathological groups (93% success) from C, and DAT (88.9%) from VD (73.3%); but not PD from DAT or VD. Antioxidant profiles of PD patients showed characteristics overlapping with DAT (60%) and with VD (40%), suggesting biochemical similarities between them.

Comparison of the determination of superoxide dismutase and antioxidant capacity in neurological patients using two different procedures.

As oxidative stress in relation with neurological diseases has become an important point in recent research, simple methods to be used in epidemiological studies and clinical practice are required. The hypothesis that the analytical methods used in research laboratories (RLM) can be used interchangeably with commercial kits (CKM) for SOD and TRAP is tested. Both methods were compared using linear transformations of the RLM measurements into the CKM scales. Data were obtained from Alzheimer's, Parkinson's, and vascular dementia patients and controls. The lack of fit and the run's test of residuals were not significant, but the same sign method detected significant nonlinearities (P<0.000001 for SOD, P<0.01 for TRAP). The intragroup CVs of both methods were comparable for TRAP, while in the RLM determinations of SOD resulted in <50% of those obtained with the CKM. The ANCOVA comparison of the regression parameters across the clinical groups resulted significant for SOD (P<0.0001) and not significant for TRAP. Both methods agree in describing the features of the clinical groups, but the degree of agreement at the individual concentration was poor and they could not be readily intercalibrated. Normal and pathological values should be obtained independently for the CKM to insure their applicability to large populations.

Peripheral markers of oxidative stress in probable Alzheimer patients.

BACKGROUND: The current research on Alzheimer's disease is mainly focused in the post-mortem characterization of pathological and biochemical alterations in the brain. The finding of peripheral markers that could be associated with the changes observed in the Alzheimer's brain would be of interest in this field. The aim of the present study was to evaluate the state of different peripheral markers of oxidative stress in probable Alzheimer patients and compare them with a group of healthy individuals. DESIGN: The determinations made include the plasma total antioxidant capacity (TRAP) and tert-butyl hydroperoxide-initiated chemiluminescence and catalase activity in erythrocytes from 18 patients with probable Alzheimer's disease and 18 matched control subjects with normal cognitive function. RESULTS: TRAP was decreased in Alzheimer patients by 24% (control group 308 micromol L-1 Trolox, SEM 34, n = 18). tert-Butyl hydroperoxide-initiated chemiluminescence and catalase activity showed an increase in erythrocytes from Alzheimer patients by 52% (control group 116 700 cps mg-1 haemoglobin, SEM 6690) and 75% (control group 2.55 pmol mg-1 protein, SEM 0.39, n = 18) respectively. CONCLUSION: Oxidative stress in the blood of probable Alzheimer patients could be a reflection of the brain condition and suggests that oxygen free radicals could be partially responsible of the damage observed in this disease.

Differential nitric oxide release and sensitivity to injury in different murine mammary tumor cell lines.

The purpose of this study was to determine whether nitric oxide (NO) production by different mammary tumor cell lines correlated with their sensitivity to NO mediated injury. Three mammary tumor cell lines LM2, LM3 and LMM3 syngeneic to BALB/c mice were cultured in vitro with IFNgamma + LPS. Different levels of NO production among the three lines were detected in culture supernatants. The only tumor cell line which did not produce NO (LM2) showed the highest sensitivity to SNP-derived NO cytotoxicity (87%), while LM3 and LMM3 which both produced higher levels of NO than LM2, showed lower cytotoxicity by SNP (39% and 22% respectively). Spleen cells (SC) from M2 tumor bearing mice (TBM) were able to lyse LM2 cells by NO-dependent mechanisms. SC from M3-TBM exerted cytotoxicity against LM3 cells mainly by NO-independent mechanisms. Thus, we postulate an inverse correlation between NO production and NO mediated cytotoxicity in the three mammary tumor cell lines. It is possible that tumor cells producing NO develop mechanisms to resist NO injury.

Downregulation of fibronectin transcription in highly metastatic adenocarcinoma cells.

Silencing of fibronectin (FN) expression seems to be one of the key mechanisms underlying metastatic behaviour. An inverse correlation exists between FN expression levels and the metastatic potential of two related murine mammary adenocarcinomas, M3 and MM3. Primary cultures of M3 tumour, which is moderately metastatic to lung (40% incidence), show a conspicuous FN extracellular matrix (ECM) and high levels of FN mRNA, while primary cultures of the highly metastatic MM3 tumour (95% lung incidence) are negative for FN in immunofluorescence and show at least 40-fold lower levels of FN mRNA, only detectable by RT-PCR, with a different pattern of alternatively spliced EDI isoforms compared to M3 cells. We show that the FN promoter sequence is not altered in MM3 cells. Transfection experiments with CAT constructs indicate that silencing occurs at the transcriptional level, involving the 220-bp proximal promoter region.

Excessive urokinase-type plasminogen activator activity in the euglobulin fraction of patients with Alzheimer-type dementia.

We examined the activity of the serine protease urokinase-type plasminogen activator (uPA) present in the euglobulin fraction of plasma from 17 demented patients with probable Alzheimer's disease (AD), 12 patients with vascular dementia (VD) and 10 healthy controls. Euglobulin protein fractions were separated by electrophoresis and gels were incubated at the surface of plasminogen-rich casein-agarose underlays. The degradative activity of uPA in this system was measured by densitometric analysis. In 8/17 (47%) patients with AD we observed an excessive uPA activity (> 200 mIU/ml). In contrast, only 2/12 (16%) patients with VD and 1/10 (10%) control subjects revealed a comparable increase in circulating uPA activity. Further evaluation of dementia stage in patients with AD allow us to associate high levels of uPA activity with severity of disease. uPA levels were significantly elevated (2.8-fold increase) in AD patients with severe cognitive and memory impairments (Alzheimer Disease Assessment Scale) with respect to controls, VD patients or AD patients with moderate cognitive and memory impairments (P < 0.001, ANOVA). Our data suggest that the anormalities in circulating fibrinolytic enzymes could be correlated with the severity of dementia. In light of this findings, the free uPA activity in euglobulin plasma fraction should be considered a marker of serious damage in patients with AD.

Inhibition of lymphocyte-induced angiogenesis by free radical scavengers.

Solid tumors induce an angiogenic response by the host blood vessels to form a new vascular network for the supply of fresh nutrients and oxygen responsible for tumor growth. Furthermore, tumor growth and metastatic spread is abrogated or markedly reduced in the absence of neovascularization. Spleen T lymphocytes from tumor-bearing mice elicit a strong neovascular response. It is well known that certain T cell responses require the presence of active oxygen radicals. Because these metabolites are produced during tumor growth, we studied whether oxygen free radicals play a role in the angiogenesis induction by lymphocytes. In this study, we demonstrated that the administration of a free radical scavenger (EGb-761) to tumor-bearing mice, blocked the angiogenic response and decreased the lung metastatic incidence. On the other hand, when normal lymphocytes were incubated with the xanthine-xanthine oxidase system (X-XO), a known superoxide anion generator, this elicited a dose-response positive angiogenic reaction in normal recipient mice. No angiogenic response was observed in the absence of X-XO, or when EGb-761 or superoxide dismutase (SOD) plus catalase (CAT) were added to the incubation medium. These results suggest that free radicals are involved in some step of the angiogenic process, and that the EGb-761 treatments block this response due to the free radical scavenging activity of this compound.

Peripheral markers and diagnostic criteria in Alzheimer's disease: critical evaluations.

This review analyzes recent developments in diagnostic criteria and peripheral markers used clinically in the definitive diagnosis of Alzheimer's disease (AD), comparing past and current views, together with a discussion of their shortcoming and difficulties of implementation. Consideration is given to studies on the presence of amyloid substances outside the central nervous system: in cerebrospinal fluid, in plasma, in primary cultures, and in continuous cultures of cell lines of neuronal and glial origin. We discuss alterations of cholinesterases and noradrenaline in red blood cells (RBC) in AD and, with relation to the infectious theory, the presence of spirochaetes in patients. The activities of the enzymes leading to the formation of amyloid substances and those reflecting more general alterations of metabolic processes are considered, both in respect to their role in the pathogenesis of the neurodegenerative disorders of AD and of their potential use as markers. Enzymatic changes have been studied comparing AD patients with non AD controls as well as with AD relatives: proteases and their inhibitors; plasminogen activators; transketolases; increases in the activity of Cu-Zn superoxide dismutase in AD patients' RBC, serum, fibroblasts and cortical neurons, pointing to alterations in oxidative processes; and apolipoprotein E epsilon 4 allele, linked to late-onset AD and familial cases. This review presents reasons why the involvement of peripheral markers in AD should advance from hypothesis to accepted fact.

Copper-zinc superoxide dismutase activity in red blood cells in probable Alzheimer's patients and their first-degree relatives.

The activity of the enzyme copper-zinc superoxide dismutase (Cu-Zn SOD) has been investigated in red blood cell (RBC) homogenate obtained from demented patients with probable Alzheimer's disease (DAT), from their first-degree relatives (sisters/brothers and sons/daughters), and from healthy control families of the same age. A statistically significant increase in SOD activity (P < 0.01) was found in RBC's homogenate between families of DAT patients (not including the demented individual) and control families. Variability in SOD activity due to differences between families was not significant for DAT relatives; a significant variance component (P < 0.05) was found between control families. Additionally, a statistically significant increase in SOD activity (P < 0.001) with age in DAT patients up to 70 years and a significant decrease above this age were found, confirming a previously found relation. No changes in SOD activity with age were detected in healthy controls nor in DAT relatives. The increased levels of Cu-Zn SOD, probably represent a general alteration of the oxidative processes characteristic of this dementia and support the proposal that the enzyme could be used as an early diagnostic peripheral marker of the Alzheimer's disease (AD), and to determine to which subgroup the patient belongs, as well as a risk factor in non-demented first-degree relatives.

Effect of host-organ environment on the in vivo and in vitro behavior of a murine mammary adenocarcinoma.

We investigated the role that organ environment may play in determining the homing of disseminated cells from a murine mammary adenocarcinoma moderately metastatic to lung (M3). Conditioned medium (CM) from normal lung was able to enhance both local and metastatic growth. It increased the number of lung colonies when inoculated together with tumor cells via intravenous or separately via intraperitoneal route. Several in vitro studies were performed in order to elucidate possible mechanisms. It was shown that lung CM stimulated the in vitro growth and the migration of M3 cells. Normal kidney and liver CM lacked all these capacities.

Copper-zinc superoxide dismutase activity in red blood cells and serum in demented patients and in aging.

The activity of the enzyme copper-zinc superoxide dismutase (Cu-Zn SOD) has been investigated in serum and red blood cells (RBC) homogenate obtained from demented patients with associated vascular lesions (VD), demented patients with probable Alzheimer's disease (DAT) and healthy controls (CG) of the same age. The increase in SOD activity was statistically significant (P < 0.01) in RBCs homogenate of DAT and VD patients, when compared to controls, but no differences appear between the two diseases groups. Additionally, a statistically significant increase in SOD activity (P < 0.01) in DAT patients above 70 years as compared to those 50-70 years old, and a relation between SOD and age were found. No changes in SOD activity with age in healthy controls nor in vascular dementia group were detected. A statistically significant increase in Circulating SOD activity (P < 0.01) was observed in vascular patients compared to controls. The observed increase in DAT Circulating SOD activity (against CG) was not significant. The increased levels of Cu-Zn SOD, probably represent a general alteration of the oxidative processes characteristic of these dementias and suggest that the enzyme might be used as a marker.

[Modulation of angiogenesis induced by lymphocytes by extracellular matrix proteins]. / Modulación de la angiogénesis inducida por linfocitos por proteínas de la matriz extracelular.

The neovascularization is a known event in the de development of tumors. The progressive growth of solid tumors is strictly dependent on angiogenesis. Furthermore, shedding of tumor cells into the circulation is not observed in a prevascular phase of tumors. Therefore, the inhibition of angiogenesis could be a good target for cancer control. Lymphocytes from, tumor bearing-mice were capable of inducing neovascular response in the skin of syngeneic mice. This response was named syngeneic lymphocyte-induced angiogenesis (SLIA). This work was an attempt to study if two proteins present in extracellular matrix, collagen and fibronectin (FN), could modulate lymphocyte-induced angiogenesis. The angiogenic response induced by lymphocytes from S13 tumor bearing-nice in the skin of BAL/c mice was blocked by treatment with FN and Gly. Arg. Gly. Asp. peptide. On the contrary, collagen and Gly. Arg. Gly. Asp. Ser did not modify SLIA response.

Modulación de la angiogénesis inducida por linfocitos por proteínas de la matriz extracelular. / [Modulation of angiogenesis induced by lymphocytes by extracellular matrix proteins]

The neovascularization is a known event in the de development of tumors. The progressive growth of solid tumors is strictly dependent on angiogenesis. Furthermore, shedding of tumor cells into the circulation is not observed in a prevascular phase of tumors. Therefore, the inhibition of angiogenesis could be a good target for cancer control. Lymphocytes from, tumor bearing-mice were capable of inducing neovascular response in the skin of syngeneic mice. This response was named syngeneic lymphocyte-induced angiogenesis (SLIA). This work was an attempt to study if two proteins present in extracellular matrix, collagen and fibronectin (FN), could modulate lymphocyte-induced angiogenesis. The angiogenic response induced by lymphocytes from S13 tumor bearing-nice in the skin of BAL/c mice was blocked by treatment with FN and Gly. Arg. Gly. Asp. peptide. On the contrary, collagen and Gly. Arg. Gly. Asp. Ser did not modify SLIA response.

Modulation of tumor-induced angiogenesis by proteins of extracellular matrix.

The formation of new vessels is a known event in enlarging tumors. Furthermore, the metastatic potential is abrogated or reduced markedly in the absence of neovascularization. Shedding of tumor cells into the circulation is not observed until vascularization has occurred. As a result, the interruption of neovascularization could be a good target for cancer control. This research was an attempt to see if two proteins present in extracellular matrix, collagen and fibronectin (FN), could modify the tumor-induced angiogenesis. The strong angiogenic response induced by S13 tumor cells in the skin of BALB/c mice was blocked by treatment with FN and FN-derived peptides. In contrast, collagen did not modify tumor-induced angiogenesis.

Localization of tissue copper in mouse mammary tumors.

The purpose of this study was to determine the copper deposition and localization during the evolution of two murine mammary adenocarcinomas. In the normal tissue, the copper was located within the cytoplasm, whereas it was intra- and perinuclear in the tumors. The more angiogenic and metastatic tumor showed the higher percentage of copper-positive cells. In the tumor, copper deposits correlated well with its angiogenic and metastatic ability, but additional factors would be required for the process to be induced.

Copper histochemistry of 5 murine tumors and their respective metastases.

Tissue copper content has been evaluated in 4 murine mammary adenocarcinomas, 1 murine lung adenocarcinoma and their respective metastases. Histochemical techniques have been used to analyze copper distribution in tumor tissues. It is observed that the degree of copper staining is inversely related to tumor differentiation. As the copper level reflects one of the metabolic changes in the host carrying the tumor, it is suggested that it could be used as a good marker for tumor differentiation.