Even a mild anemia is related to tumor aggressiveness mediated by angiogenic factors.

UNLABELLED: Esophagogastric cancers have high recurrence rates with lymph nodes being a common pattern. Pre-treatment anemia has been reported an independent prognostic factor of treatment failure regardless of treatment strategy, particularly associated with poor locoregional control. A causative relationship between anemia - tumor hypoxia - tumor aggressiveness mediated by angiogenesis up-regulation is advocated, yet remains controversial. AIM: To determine whether and how the pre-treatment anemia is associa-ted with various aspects of disease aggressiveness and to evaluate the possible involvement of angiogenesis mediators. METHODS: In 111 esophagogastric cancer patients we investigated the association of pre-treatment hemoglobin concentration and anemia presence with cancer-related, patients-related features and laboratory parameters including angiogenic factors: vascular endothelial growth factors A and C, interleukin-8 and midkine. Serum levels of angiogenic factors were assessed with immunoenzymatic tests. RESULTS: Histology, disease stage, regional metastasis and dissemination in general, malnutrition and angiogenesis represented by midkine were found to correlate with anemia presence and hemoglobin concentration, while tumor extension, patient's age and sex accounted only for anemia presence. A tendency towards hemoglobin correlation with VEGF-A and Il-8 was also observed. Midkine, tumor histology and malnutrition were found to exert an independent effect on pre-treatment hemoglobin concentration and anemia presence in esophagogastric cancer patients. Hemoglobin level of 12 g/dL was found an optimal cut-off value for discrimination between localized and disseminated cancers. CONCLUSIONS: Even a mild pre-treatment anemia is associated with cancers metastasizing especially to regional lymph nodes, which seems to be mediated by some of studied angiogenic factors.

Respiratory insufficiency related to COPD accelerates systemic inflammation, under-nutrition, and angiogenesis in esophageal malignancies.

UNLABELLED: A number of esophageal cancer patients suffer from respiratory insufficiency due to the coexistence of chronic obstructive pulmonary disease (COPD). AIM: To test the hypothesis that COPD-related systemic hypoxemia may result in accelerated inflammation, malnutrition, and angiogenesis in esophageal cancer patients. METHODS: Serum levels of C-reactive protein (CRP), albumin, transferrin, interleukin-1, interleukin-6, interleukin-8, TNF-alpha, platelet-derived growth factor (PDGF-BB), and midkine and patient BMI and weight-loss rate were determined and compared with blood oxygenation status (pO(2), SaO(2)) in 35 esophageal cancer patients and 42 controls. RESULTS: The incidence of cachexia tended to be higher in patients with systemic hypoxemia (67% vs 40%, p = 0.169). Mean SaO(2) level was also significantly decreased in cachectic patients (90.3 vs 93.3%, p = 0.026) and pO(2) exhibited a similar trend (58.0 vs 63.4 mmHg, p = 0.120). Transferrin (234 vs 316 mg/dl, p = 0.005) and albumin (31.9 vs 37.1 mg/dl, p= 0.002) concentrations were reduced and CRP was elevated (129.9 vs 54.7 mg/l, p = 0.004) in hypoxemic patients and correlated with pO(2) (r = 0.47, p = 0.016; r= 0.48, p = 0.012; r = -0.37, p = 0.064) and SaO(2) (r = 0.52, p = 0.006; r = 0.53, p = 0.006; r = -0.40, p= 0.042). Interleukin-6 (9.97 vs 2.21 pg/ml, p = 0.005) and midkine (2101 vs 944 pg/ml, p < 0.001) were elevated and PDGF-BB was decreased (12.2 vs 17.3 pg x 10(-6)/PLT, p = 0.014) in hypoxemic compared with normoxemic patients. Interleukin-6 and midkine negatively correlated with pO(2) (r = -0.44, p = 0.016; r = -0.42, p = 0.011) and SaO(2) (r = -0.54, p = 0.003; r = -0.57, p < 0.0001) and PDGF-BB correlated positively (r = 0.53, p = 0.003; r = 0.44, p = 0.020). Interleukin-8 level was affected by pO(2) (r = -0.55, p = 0.015) and SaO(2) (r= -0.55, p = 0.018) only in hypoxemic patients. CONCLUSIONS: COPD-related systemic hypoxemia negatively affects the status of esophageal cancer patients by accelerating inflammation, under-nutrition, and angiogenesis.

Impact of systemic hypoxemia on cancer aggressiveness and circulating vascular endothelial growth factors A and C in gastroesophageal cancer patients with chronic respiratory insufficiency.

AIM: Due to the common etiologic factor, a considerable number of esophagogastric cancer patients suffer from respiratory insufficiency in course of chronic obstructive pulmonary disease, primary to cancer. Systemic hypoxemia may account for poor oxygenation of tumor tissue-a main driving force of tumor neoangiogenesis. We hypothesized that in cancer patients with respiratory insufficiency, systemic hypoxemia may be related to enhanced aggressiveness of cancer on one side and to the elevation of angiogenic factors on the other. METHODS: The levels of vascular endothelial growth factors A and C were determined with immunoenzymatic methods in patients diagnosed with esophagogastric cancer with or without co-existing respiratory insufficiency in course of chronic obstructive pulmonary disease and in healthy controls. Blood gasometry and hemoglobin levels of cancer patients were related to cancer histology and TNM status, and to circulating vascular endothelial growth factors A and C. RESULTS: Patients with systemic hypoxemia had higher incidence rates of locally advanced tumors. Partial oxygen pressure and blood oxygen saturation were significantly lowered in patients with T4 cancers as compared to less advanced ones. Circulating vascular endothelial growth factor A, but not C, was more elevated in esophagogastric cancer patients with co-existing respiratory insufficiency, as compared to those without respiratory insufficiency. Vascular endothelial growth factor A was also strongly related to the extension of primary tumor. CONCLUSION: Our results show that systemic hypoxemia in esophagogastric cancer patients is associated with the extension of primary tumor and that this effect might be mediated by the up-regulation of circulating vascular endothelial growth factor A.

Serum midkine depends on lymph node involvement and correlates with circulating VEGF-C in oesophageal squamous cell carcinoma.

Lymph node metastasis (LNM) is a key factor for selection of treatment method and patients' prognosis in oesophageal squamous cell carcinoma (ESCC). However, no biomarkers able to support the clinical detection of LNM have been reported. Recently, vascular endothelial growth factor C (VEGF-C) was found to be a more accurate marker of LNM in lung cancer than computed tomography. Midkine is a multifunctional cytokine involved in cancer development. We investigated circulating midkine levels in ESCC patients (n=73) compared with those in healthy subjects (n=42) with double-antibody-sandwich indirect enzyme-linked immunosorbent assay (DASI-ELISA). We found that midkine was elevated in ESCC and involved in metastatic disease. Serum midkine (sMK) was a good marker of LNM, evaluated both clinically and pathologically, as revealed by ROC analysis. It also correlated with serum levels of VEGF-C. The increase of sMK was related to cancer cells, although a weak correlation was observed between sMK and platelet and leucocyte counts.

Enolase from Candida albicans--purification and characterization.

This paper describes isolation of electrophoretically homogenous enolase from Candida albicans. The purification involved: disintegration of C. albicans cells in a Braun's mill (67-100%) ammonium sulfate precipitation, chromatography on DEAE-Sephadex A-50 at pH 9.0 and chromatography on CM-Sephadex A-50 at pH 6.2. The procedure resulted in a 30-fold purification of the enzyme with a recovery rate of 6% and a specific activity 35 U mg-1. The subunit molecular weight was 46 kDa and the pH optimum of the enzyme was 6.8. Km and Vmax values for the 2PGA-->PEP reaction were determined (Km = 0.95 mM, Vmax = 4200 mumol min-1 mumol-1). In the absence of orthophosphate, inhibition by fluoride was competitive, which became noncompetitive in the presence of phosphate. It was confirmed that Mg2+ is the most potent activator (Km = 0.286 mM); Mn2+ gave less activity and Zn2+ less still. It was also demonstrated that the presence of two types of cations in the reaction mixture nullified the activatory effect of the stronger agent. Properties of the enzyme from C. albicans are compared with those of enolases from other sources.

Effect of proteases on the activity of enolase from muscle of carp (Cyprinus carpio) and pig.

The treatment of enolase from pig and carp (Cyprinus carpio) with proteases resulted in a decrease of enzymatic activity, which depended on the kind of protease used. The most active were trypsin and subtilisin. Substrate and magnesium ions protected enolase against inactivation. The enolase from pig muscle was much more resistant to protease action than this enzyme from carp muscle. Some differences in the structure between the two enolases are suggested.

Chemical modification of histidine, tyrosine, tryptophan and cysteine residues in carp (Cyprinus carpio) muscle enolase.

Enolase from carp (Cyprinus Carpio) muscle was modified by diethylpyrocarbonate, tetranitromethane, N-bromosuccinimide and 5,5'-dithiobis(2-nitrobenzoic acid). The extent and rate of modification and its effect on the enzyme activity were determined. Modification of histidine, tyrosine and tryptophan residues caused complete inactivation of the enzyme; Mg2+ as well as 2-phosphoglycerate markedly altered the rates of modification and inactivation. The above-mentioned amino acid residues seem to be essential for the functioning of muscle enolases. Modification of cysteine residues had no effect on the enolase activity.

Studies on immunological properties of enolase from carp muscles (Cyprinus carpio) after chemical modification of some amino-acid residues.

After modification of histidine residues, enolase from carp muscles lost the ability to precipitate with antibodies against native enzyme. This ability remained after nitration of tyrosine residues, but the immunoelectrophoretic pattern--localisation and height of arcs--differed from that of native enolase. Minor changes of this pattern were found in enolase with modified tryptophan residues.

Effect of some chemical modifications of basic pancreatic trypsin inhibitor (BPTI) on its reaction with specific antibody.

Anti-BPTI-antibody inactivated the antitrypsin activity of basic pancreatic trypsin inhibitor. Esterification of BPTI with methanol did not affect its antitrypsin activity and precipitate formation with antibody. Acetylation, maleylation and hexa-S-carboxylation of BPTI completely inactivated the inhibitor reactivity and markedly diminished its precipitating ability. Performic acid oxidized BPTI and thermolysin digested BPTI lost its antitrypsin as well as antigenic activities. The both preparations as well as oxidized N-acetyl-L-cysteinyl-L-lysyl-L-alanylglycylglycyl-L-cysteine amide did not affect the complex formation between the inhibitor and antibody.

Purification and properties of enolase from carp (Cyprinus carpio). Comparison with enolases from mammals' muscles and yeast.

The enolase (2-phospho-D-glycerate hydrolyase E.C. 4.2.1.11) from carp muscle was obtained, the specific activity--88 U/mg of protein. Km for 2-phosphoglycerate was 0.313 mM and for phosphoenolpyruvate--0.76 mM. The enzyme is active only in the presence of divalent metal ions, Mg2+ being the best activator. The phosphate and fluoride decreased the activity of enzyme. The molecular weight of the dimeric form of enzyme was found to be 93,000. The enzyme is immunologically different from pig muscle enolase.

Molecular forms of bovine gamma-glutamyltransferase and their enzyme immunoassay.

Light form of bovine kidney gamma-glutamyl transferase was isolated from heavy form of the enzyme after digestion with bromelain. Its apparent molecular weight was 95,000 and in SDS solution it dissociated into two non-identical subunits with molecular weights 26,000 and 69,000. No substantial differences between both forms in activation, kinetic parameters and inhibition with anthglutin and its isomers were noted. Using enzyme immunoassay it was possible to determine one enzyme form in the presence of the other. This was applied for studies of gamma-glutamyltransferase forms in cow serum and colostrum.