Grzegorz Terlecki

Acute-phase response proteins are related to cachexia and accelerated angiogenesis in gastroesophageal cancers

Abstract Background: Accurate outcome prediction in gastroesophageal malignancies is challenging. Acute-phase response proteins (APRPs) have been claimed to be independent prognosticators, although the basis for their association with prognosis remains unexplained. We hypothesized that, similarly to pancreatic and lung cancers, changes in APRPs in gastroesophageal malignancies are associated with cachexia and accelerated angiogenesis. Methods: C-reactive protein (CRP), albumin and transferrin serum levels were evaluated and the Glasgow Prognostic Score (GPS) calculated. These data were compared with concentrations of circulating interleukin (IL)-1, IL-6 and IL-8, tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF)-A, VEGF-C and midkine in 96 gastroesophageal cancer patients (49 with cachexia) and 42 healthy subjects. Results: Albumin and CRP levels were altered in the cancer patients, with further CRP elevation in those with cachexia. Transferrin was decreased only in the cachectic patients. The interrelationships between the APRPs were strengthened in cachexia and only then were APRPs correlated with the cytokines elevated in gastroesophageal cancer-related cachexia: IL-6, IL-8, VEGF-A and midkine. GPS corresponded well to transferrin, IL-1, IL-6, IL-8, TNF-α, VEGF-A and midkine concentrations. Conclusions: Cachexia in gastroesophageal cancers is associated with changes in APRP concentrations. This, together with a direct relationship of APRPs with accelerated angiogenesis, may constitute a foundation for the association of APRPs and GPS with outcome in these malignancies. Clin Chem Lab Med 2008;46:359–64.

Enhanced formation of advanced oxidation protein products in IBD

Background: Advanced oxidation protein products (AOPPs) are new protein markers of oxidative stress with pro‐inflammatory properties, accumulated in many pathological conditions. The issue of their enhanced formation in IBD has not been addressed yet. Methods: The concentration of relative AOPPs (rAOPP; concentration of AOPPs divided by albumin level) were measured in 68 subjects with ulcerative colitis (UC), 50 subjects with Crohns disease (CD) and 45 healthy volunteers, and related to disease phenotype, clinical and biochemical activity, and therapeutic strategy. Diagnostic utility of rAOPP was evaluated by ROC analysis. Results: In comparison with controls (1.367 &mgr;mol/g), rAOPP were increased in inactive (1.778 &mgr;mol/g, P = 0.053) and active (1.895 &mgr;mol/g, P = 0.013) UC and in active (1.847 &mgr;mol/g, P = 0.003) CD. In CD, but not UC, rAOPP correlated with disease activity (r = 0.42, P = 0.013). Significant correlations with the inflammatory/malnutrition indices‐erythrocyte sedimentation rate (ESR) (r = 0.53), leukocytes (r = 0.33), platelets (r = 0.38), IL‐6 (r = 0.36), and transferrin (r = −0.35) were demonstrated in CD. In UC, rAOPP correlated only with ESR (r = 0.35) and IL‐6 (r = 0.30). Instead, associations with antioxidant dismutase (r = 0.29) and catalase (r = 0.22) were observed. The diagnostic power of rAOPP in discriminating diseased from non‐diseased subjects was less than that of C‐reactive protein (CRP). Simultaneous determination of rAOPP and CRP did not significantly improve the power of single CRP determination. Conclusions: IBD was associated with enhanced formation of AOPP, which differed between C and UC with respect to the relationship between rAOPP and disease activity, inflammatory and antioxidant response. These differences may reflect divergent ways that oxidative stress develops in CD and UC. The diagnostic power of rAOPP was insufficient for its clinical application.

Elevation of circulating interleukin-8 is related to lymph node and distant metastases in esophageal squamous cell carcinomas—Implication for clinical evaluation of cancer patient

The presence of lymph node metastasis (LNM) is an important factor in clinical evaluation of esophageal cancer patients. Biological markers able to support detection of metastatic lymph nodes are sought after. Interleukin-8 (IL-8) is overexpressed by many cancers and involved in cancer dissemination. We investigated the relationship between circulating IL-8 and clinicopathological features of esophageal squamous cell carcinoma (ESCC), and evaluated the diagnostic potential of IL-8, with reference to the key angiogenic and lymphangiogenic factors: vascular endothelial growth factors A and C (VEGF-A and VEGF-C). We found elevated IL-8 levels in ESCC patients, correlated with tumor size and cancer dissemination, especially LNM. Circulating IL-8 correlated with lymphangiogenic VEGF-C rather then angiogenic VEGF-A. The association weakened in metastatic cancers, suggesting divergent mechanism of IL-8 involvement in the dissemination process. The cytokine levels correlated with platelets and neutrophils, pointing at these cells as possible sources of circulating IL-8. We demonstrated IL-8 that positively correlated with inflammation status of ESCC patients. Circulating IL-8 was a better indicator of ESCC dissemination than VEGF-A or VEGF-C. Yet, the detection rates were not satisfactory enough to allow for the recommendation of IL-8 determination as an adjunct to the clinical evaluation of lymph node involvement in ESCC patients.

In vitro and in vivo matrix metalloproteinase expression after photodynamic therapy with a liposomal formulation of aminolevulinic acid and its methyl ester

Photodynamic therapy (PDT) is a well-known method for the treatment of malignant tumors, and its principles have been well established over the past 30 years. This therapy involves the application of a chemical called a photosensitizer and its subsequent excitation with light at the appropriate wavelength and energy. Topical photodynamic therapy with aminolevulinic acid (5-ALA) is an alternative therapy for many malignant processes, including nonmelanoma skin cancers such as basal-cell carcinoma (BCC). Our novel approach for this study was to use a liposomal formulation of 5-ALA and its methyl ester (commercially available as metvix) both in vitro and in vivo, and to check whether the liposome-entrapped precursors of photosensitizers can induce the expression of metalloproteinases (MMPs) in animal tumor cells and in other tissues from tumor-bearing rats and in selected cell lines in vitro. We also checked whether the application of tissue inhibitors of matrix metalloproteinases (TIMPs) has any effect on MMPs in the above-mentioned experimental models, and if they can cause complete inhibition of MMP expression. Immunohistochemical studies revealed that after the PDT, the intensity of expression of MMPs in healthy animals was very low and seen in single cells only. After the PDT in tumor-bearing rats, MMP-3 was expressed in the tumor cells with the highest intensity of staining in the tissues directly adjacent to the tumors, while MMP-2 and -9 were not found. In the control groups, there was no observed expression of MMPs. In vitro studies showed that MMP-3 was expressed in MCF-7 cells after PDT, but MMP-9 was not observed and MMP-2 was only seen in single cases. Our studies confirmed that the application of an MMP-3 inhibitor may block an induction of MMP-3 expression which had previously been initiated by PDT. The preliminary data obtained from cancer patients revealed that new precursors are effective in terms of PDT, and that using MMP inhibitors should be considered as a potential enhancing factor in clinical PDT.

Immunocytochemical studies on the nuclear ubiquitous casein and cyclin-dependent kinases substrate following 5-aminolevulinicacid-mediated photodynamic therapy on MCF-7 cells

BACKGROUND Recent data indicates that nuclear ubiquitous casein and cyclin-dependent kinases substrate (NUCKS) may play role in tumor growth. In present study authors examined whether photodynamic therapy with 5-aminolevulinic acid (5-ALA) induces NUCKS expression in breast cancer cell line, MCF-7. METHODS In the experiment concentration of 5-ALA was 6.5mM. Excitation wavelength was 630 ± 20 nm, total light dose of light 5 or 10 J/cm(2) and irradiance 60 mW/cm(2) was used. Cells were collected at established time points and Western blot and immunocytochemical studies were performed using antibody against NUCKS. RESULTS Studies proved strong cytotoxic effects in cells following PDT with 6.5mM of precursor and 10 J/cm(2). Western blot analysis revealed the strongest expression of NUCKS at 7h after PDT. At next time points, 18 and 24h, expression of NUCKS decreased and became similar to that of control group. Further immunocytochemical studies showed very strong expression of NUCKS following PDT with 5-ALA and light irradiation of 5 J/cm(2). Early, at 0 h, that expression was predominantly seen in nuclei, while at 7h expression of NUCKS was observed in disseminated manner within entire cells in both nuclei and cytoplasm, with prevalence of cytoplasmic staining. CONCLUSIONS Authors suggest that NUCKS is involved in cellular responses following PDT, and since parallel induction of NUCKS and proapoptotic marker Bax and inhibition of anti-apoptotic Bcl-2 was observed, this protein might also be involved in induction of apoptosis following PDT.

Interaction of bovine heart pyruvate kinase with phospholipids

The interaction between bovine heart pyruvate kinase and liposomes was investigated for various phospholipids as function of pH, and salt concentration using steady-state kinetics and ultracentrifugation. Liposomes made from erythrocyte total lipid fraction and individual phospholipids were used. Pyruvate kinase specific activity increases upon the interaction with the phospholipids. The activation is specifically sensitive to presence of phosphatidylserine in liposomes. L-serine, and phospho-L-serine which are main components of phosphatidylserine head group show also some activation effect. Efficient adsorption of pyruvate kinase to phosphatidylserine liposomes occurs in the pH range 6.0-8.0 and at low ionic strength. Interaction with phosphatidylserine liposomes results in the change of Vmax and Km values for phospho enol pyruvate without marked effect on Km value for ADP, and Hill coefficients for both substrates. The interaction does not seem to influence the cooperativity between binding sites.

Inhibition of cathepsin B activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin

Abstract2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent toxic isomer in the dioxin-like family. Due to its resistance to metabolic degradation, this ubiquitous environmental pollutant readily accumulates in multiple organs. Cathepsin B is a lysosomal cysteine protease playing an essential role in the intracellular protein turnover. Alterations in its expression, activity, and localization may facilitate the development of many pathologies, including cancer. TCDD, due to its extremely lipophilic nature, may diffuse through biological membranes and affect lysosomal enzymes, including cathepsins. Therefore, in this study we performed two enzymatic assays, spectrofluorimetry and gelatin zymography, in order to evaluate the effect of TCDD on purified bovine cathepsin B. We showed that the dioxin decreases the enzyme’s activity in a dose-dependent manner. The reversibility of TCDD-induced inhibition of the protease was also examined, suggesting that TCDD does not bind covalently to the enzyme’s active site, acting rather as a reversible inhibitor.

ULTRACENTRIFUGATION STUDIES OF THE LOCATION OF THE SITE INVOLVED IN THE INTERACTION OF PIG HEART LACTATE DEHYDROGENASE WITH ACIDIC PHOSPHOLIPIDS AT LOW pH. A COMPARISON WITH THE MUSCLE FORM OF THE ENZYME

Lactate dehydrogenase (LDH) from the pig heart interacts with liposomes made of acidic phospholipids most effectively at low pH, close to the isoelectric point of the protein (pH = 5.5). This binding is not observed at neutral pH or high ionic strength. LDH-liposome complex formation requires an absence of nicotinamide adenine dinucleotides and adenine nucleotides in the interaction environment. Their presence limits the interaction of LDH with liposomes in a concentration-dependent manner. This phenomenon is not observed for pig skeletal muscle LDH. The heart LDH-liposome complexes formed in the absence of nicotinamide adenine dinucleotides and adenine nucleotides are stable after the addition of these substances even in millimolar concentrations. The LDH substrates and studied nucleotides that inhibit the interaction of pig heart LDH with acidic liposomes can be ordered according to their effectiveness as follows: NADH > NAD > ATP = ADP > AMP > pyruvate. The phosphorylated form of NAD (NADP), nonadenine nucleotides (GTP, CTP, UTP) and lactate are ineffective. Chemically cross-linked pig heart LDH, with a tetrameric structure stable at low pH, behaves analogously to the unmodified enzyme, which excludes the participation of the interfacing parts of subunits in the interaction with acidic phospholipids. The presented results indicate that in lowered pH conditions, the NADH-cofactor binding site of pig heart LDH is strongly involved in the interaction of the enzyme with acidic phospholipids. The contribution of the ATP/ADP binding site to this process can also be considered. In the case of pig skeletal muscle LDH, neither the cofactor binding site nor the subunit interfacing areas seem to be involved in the interaction.

3-Bromopyruvate as a potential pharmaceutical in the light of experimental data

3-Bromopyruvate (3-BrPA) is an halogenated analogue of pyruvic acid known for over four decades as an alkylating agent reacting with thiol groups of many proteins. It enters animal cells like a lactate: via monocarboxylic acid transporters. Increasing interest in this compound, in recent times, is mainly due to hopes associated with its anticancer action. It is based on the impairment of energy metabolism of tumor cells by inhibiting enzymes in the glycolysis pathway (hexokinase II, glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase) and the oxidative phosphorylation (succinate dehydrogenase). Two cases of clinical application of this compound in the treatment of advanced cancers were reported. By using 3-BrPA, rheumatoid arthritis in SKG mice has been reduced. This compound has also antiparasitic activity: lowers cell viability of Trypanosoma brucei, decreases intracellular proliferation of Toxoplasma gondii and reduces the metabolic activity of Schistosoma mansoni. It also has antifungal properties; particularly it acts strongly on Cryptococcus neoformans, as well as Saccharomyces cerevisiae. An inhibitory effect on bacterial enzymes was also described on: isocitrate lyase from Escherichia coli, Mycobacterium tuberculosis, Pseudomonas indigofera and 2-methylisocitrate lyase, succinate dehydrogenase and acetohydroxylic acid synthase from Escherichia coli. Wherever undesirable (cancer, parasitic) cells differ from normal by more intense glycolysis and higher energy needs, there is a good chance of successful 3-BrPA use. However, this compound acts on all cells and it, therefore, seems that its future as a pharmaceutical is dependent upon the development of appropriate methods for its effective and safe application.

Dynamic Analysis of Changes of Protein Levels and Selected Biochemical Indices in Rat Serum in the Course of Experimental Pleurisy

A significant role is played in inflammation by the liver, which, stimulated by inflammatory mediators, synthetizes plasma proteins with various dynamics. The purpose of these studies is to generate a detailed dynamic analysis of changes to concentrations of plasma and serum protein fractions and selected acute-phase proteins as well as nonspecific biochemical indices during the course of an induced pleurisy. The studies were conducted on female inbred Buffalo rats, which were divided into two groups: a control group (C) and an experimental group (IP) in which pleurisy was induced. In the IP group, significant changes in biochemical indices were observed between the 48th and 96th hours of pleurisy. A reduction of albumin, transferrin, urea, and creatinine concentrations was observed, while concentrations of the complement components C3 and C4, haptoglobin, and fibrinogen increased. An early increase of IL-1 was observed, while increases of IL-6 and TNF were noted in the later period. The maximum intensity of the processes described above occurred between the 72nd and 96th hours of pleurisy.