[Mechanisms of signaling associated with reactive nitrogen and oxygen in apoptosis]. / Mechanizmy przekazywania sygnalu powiazane z reaktywnymi formami azotu i tlenu w apoptozie.

The knowledge of apoptotic mechanisms is essential in many biologic aspects related to both normal and neoplastic cells. Cell death by apoptosis is a very desirable way to eliminate unwanted cells: prevents release of the cellular content, which, in contrast to necrosis, provides no activation of inflammatory reactions. Apoptosis is a multistep process in where an extremely important role is played by caspases. Functions of caspases and their modifications are fundamental to understanding the signaling pathways responsible for regulation of apoptosis. These enzymes belong to a family of cysteine proteases that have the potential to destroy the enzymatic and structural proteins, and in the final stages of apoptosis, to lead to the disintegration of the cell. Apoptosis can be modulated by certain signaling pathway.

[Reactive oxygen and nitrogen species]. / Reaktywne formy tlenu i azotu.

Reactive oxygen and nitrogen species (ROS and RNS respectively) play an important role in the proper functioning of many cellular processes. Generation of reactive oxygen species is an integral part of aerobic metabolism of cells. Their overproduction and subsequent oxidative stress occurs during pathogenesis of many diseases. Nitrosative stress is very closely linked to oxidative stress. Nitric oxide (NO), can react with molecular oxygen, superoxide anions and metal cations generating consecutive reactive oxygen species. These highly reactive chemical compounds react with proteins impairing their function by oxidation, or nitrosylation of amino acid residues, which may induce apoptosis. Furthermore, nitric oxide enhances the effect induced by cyclooxygenases and becomes a mediator of the inflammatory response. This paper gathers key information on the reactive oxygen and nitrogen species as well as processes in which they participate.

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.

Up-regulation of VEGF-C secreted by cancer cells and not VEGF-A correlates with clinical evaluation of lymph node metastasis in esophageal squamous cell carcinoma (ESCC).

Tissue expression of VEGF-C correlates with lymph node involvement (LNI) in ESCC and serum VEGF-C (sVEGF-C) in a non-small cell lung cancer has been more accurate marker of LNI than chest CT. Despite LNI importance in ESCC, the usefulness of serum VEGF-C (sVEGF-C) as a disease and LNI marker in ESCC has not been investigated yet. We found elevated sVEGF-C in ESCC (17.40 vs. 10.57 ng/ml in controls, p<0.001). It proved to be a better ESCC marker than described elsewhere: CEA, CA19-9 and SCC-Ag, with: sensitivity--70%, specificity--81%, accuracy--83.7%. Analysis of sVEGF-C correlation with clinico-pathological cancer features revealed relation to LNI (N0: 15.77 vs. N1: 21.78 ng/ml, p=0.02), especially in advanced cancers. Serum VEGF-C as a marker of LNI was characterized by: sensitivity--76%, specificity--58%, accuracy--64.4%. No relation was observed between LNI and sVEGF-A or sVEGF-A/platelets (PLT). Because sVEGF-C was higher in N0 cancers (p<0.01), the tumor presence also up-regulates sVEGF-C. We found sVEGF-C correlation with PLT and WBC: R=0.36 and R=0.32 (p<0.01). Nevertheless, analysis of PLT and WBC dependence on cancer features implies that elevation of sVEGF-C in N1 cancers is not related to them.

Impact of weight loss on circulating IL-1, IL-6, IL-8, TNF-alpha, VEGF-A, VEGF-C and midkine in gastroesophageal cancer patients.

OBJECTIVE: Proinflammatory cytokines are involved in cancer-related weight loss, but the involvement of VEGF-A, VEGF-C, IL-8 and midkine in gastroesophageal cancer patients remains unknown. DESIGN AND METHODS: Serum IL-1, IL-6, IL-8, TNF-alpha, VEGF-A, VEGF-C, and midkine were evaluated in 96 cancer patients and 42 controls using ELISAs and were related to the occurrence of weight loss, patient's age, gender and BMI, cancer TNM status and blood cell counts. RESULTS: All cytokines were elevated in cancer patients with further up-regulation of IL-6, IL-8, midkine and VEGF-A in cachexia. Underweight, midkine and VEGF-A were found independent indicators of weight loss. Primary tumor seems to be a major source of pro-cachectic cytokines, yet neutrophils and platelets also contribute to cytokine elevation. CONCLUSIONS: IL-6 and IL-8, and probably midkine and VEGF-A, appear to participate in the development of cancer-related cachexia in gastroesophageal malignancies, although a detailed mechanism underlying cytokine involvement needs to be elucidated.

Cytosolic superoxide dismutase activity after photodynamic therapy, intracellular distribution of Photofrin II and hypericin, and P-glycoprotein localization in human colon adenocarcinoma.

In photodynamic therapy (PDT), a tumor-selective photosensitizer is administered and then activated by exposure to a light source of applicable wavelength. Multidrug resistance (MDR) is largely caused by the efflux of therapeutics from the tumor cell by means of P-glycoprotein (P-gp), resulting in reduced efficacy of the anticancer therapy. This study deals with photodynamic therapy with Photofrin II (Ph II) and hypericin (Hyp) on sensitive and doxorubicin-resistant colon cancer cell lines. Changes in cytosolic superoxide dismutase (SOD1) activity after PDT and the intracellular accumulation of photosensitizers in sensitive and resistant colon cancer cell lines were examined. The photosensitizers' distributions indicate that Ph II could be a potential substrate for P-gp, in contrast to Hyp. We observed an increase in SOD1 activity after PDT for both photosensitizing agents. The changes in SOD1 activity show that photodynamic action generates oxidative stress in the treated cells. P-gp appears to play a role in the intracellular accumulation of Ph II. Therefore the efficacy of PDT on multidrug-resistant cells depends on the affinity of P-gp to the photosensitizer used. The weaker accumulation of photosensitizing agents enhances the antioxidant response, and this could influence the efficacy of PDT.

[Structure and function of midkine, a novel heparin-binding growth factor]. / Budowa i funkcja midkiny, nowego czynnika wzrostu wiazacego heparyne.

Midkine is a multifunctional peptide which, together with pleiotrophin, forms a structurally distinct family of heparin-binding growth factors. The paper presents the structure of midkine together with its amino-acid sequence and the functions of its domains as well as structural differences between the physiological forms of this peptide and those found in tumors. The localization of the midkine gene and its tissue expression both in embryonic life and in mature organisms is described. The available information on midkine receptors is discussed in detail. Most often they seem to be proteoglycans containing heparan sulfate and chondroitin sulfate, which can also be a part of the multimolecular receptor complexes binding midkine. The variety of midkine receptors is probably responsible for the diverse biological activity of this peptide. The paper presents up-to-date views on the biological activity of midkine both at the cellular (mitogenic properties, participation in apoptosis, and cellular migration, adhesion, morphological differentiation, and chemokine synthesis stimulation) and tissue levels (participation in organogenesis, tissue regeneration and protection and in the formation and degradation of extracellular matrix).

Uptake of photofrin II, a photosensitizer used in photodynamic therapy, by tumour cells in vitro.

Photosensitizing dyes are used in fluorescence diagnostics and photodynamic therapy (PDT). These usually hematoporphyrin derivatives (HpD) accumulate preferentially within neoplastic tissues. HpD is a mixture of ether and ester linked porphyrins. Its partially purified form is known under the commercial name of photofrin II (PII). PII emission spectra were studied in a hydrophilic (PBS) and a lipophilic (PC liposomes) environment. Red shift was observed in their emission maxima from 615 nm in buffer solution to 635 nm in lipid. Identical red shift was obtained when the intracellular fluorescence of two cancer cell lines, MCF 7 and Jurkat, incubated with PII was investigated. Thus, intramembrane localization of PII may be suggested. As determined from the total fluorescence intensity, the uptake of PII was only slightly higher for Jurkat than for MCF 7 cells. Nevertheless the kinetics of the uptake was found to be different for both cell lines.

[Neoplastic cell death induced by photodynamic therapy]. / Smierc komórek nowotworowych indukowana terapia fotodynamiczna.

The photodynamic therapy (PDT) is a new therapeutic method aimed at selective destruction of neoplastic cells. In PDT photosensitizing pigments are used, introduced into cells and then activated with laser light. This leads to formation of free radicals in the cell (peroxide aminoradical, hydroxyl radical), or singlet oxygen. The outcome is death of neoplastic cells through apoptosis or necrosis. The effect of PDT on the type of cell death depends on the biochemical properties of the photosensitizer used, and exposure time of neoplastic cells to light. The studies on application of PDT are primarily aimed at directing neoplastic cells towards apoptosis.

Enolase-like protein present on the outer membrane of Pseudomonas aeruginosa binds plasminogen.

Pseudomonas aeruginosa is one of the pathogenic bacteria which utilize binding of the host plasminogen (Plg) to promote their invasion throughout the host tissues. In the present study, we confirmed that P. aeruginosa exhibits binding affinity for human plasminogen. Furthermore, we showed that the protein detected on the cell wall of P. aeruginosa and binding human plasminogen is an enolase-like protein. The hypothesis that alpha-enolase, a cytoplasmatic glycolytic enzyme, resides also on the cell surface of the bacterium was supported by electron microscopy analysis. The plasminogen-binding activity of bacterial cell wall outer membrane enolase-like protein was examined by immunoblotting assay.

Oxidative alterations induced in vitro by the photodynamic reaction in doxorubicin-sensitive (LoVo) and -resistant (LoVoDX) colon adenocarcinoma cells.

In photodynamic therapy (PDT) a tumor-selective photosensitizer is administered and then activated by exposure to a light source of appropriate wavelength. Multidrug resistance (MDR) is largely caused by the drug efflux from the tumor cell by means of P-glycoprotein, resulting in reduced efficacy of the anticancer therapy. This study deals with photodynamic therapy with Photofrin (Ph) on colon cancer cell lines (doxorubicin-sensitive and -resistant). The cells were treated with 15 and 30 microg/mL Ph and then irradiated by a light dose of 3 or 6 J/cm(2) (632.8 nm). After irradiation the cells were incubated for 0, 3 or 18 h. Crucial factors of oxidative stress (thiobarbituric acid reactive substances [TBARS], protein damage, thiazolyl blue tetrazolium bromide [MTT] assay), changes in cytosolic superoxide dismutase (SOD1) activity after photodynamic reaction (PDR), and the intracellular accumulation of photosensitizers in the cells were examined. Moreover, the expressions of glutathione S-transferase (GST)-pi, a marker protein for photochemical toxicity, and secretory phospholipase A(2), a prognostic and diagnostic marker for colon cancers, were determined. After PDR, increases in SOD1 activity and the level of TBARS were observed in both cell lines. The level of protein-associated -SH groups decreased after PDR. Both cell lines demonstrated stronger GST-pi and PLA(2) expression after PDR, especially after 18 h of incubation. The increasing level of reactive oxygen species following the oxidation of sulfhydryl cell groups and lipid peroxidation influence the activity of many transporters and enzymes. The changes in SOD1 activity show that photodynamic action generates oxidative stress in treated cells. Our study presents that PDR caused oxidative alterations in both examined colon adenocarcinoma cell lines. However, the MDR cells reacted more slowly and all oxidative changes occurred in the delay.

Serum sulfatase activity is more elevated in colonic adenomas than cancers.

BACKGROUND AND AIMS: Elucidation of molecular basis of the adenomatous polyps (AP) and colorectal cancer (CRC) development is crucial for their prevention, early detection, and treatment. According to the recent discoveries, sulfatases are implied in extracellular matrix remodeling and degradation and also in the regulation of certain signaling pathways. However, their exact role in carcinogenesis remains unclear. Because the majority of CRCs arise from AP, the aim of our studies was the investigation of sulfatase activity in adenomas and adenocarcinomas and verification of possible usefulness of sulfatase activity determination as an indicator of the presence and discrimination between adenomas and carcinomas. PATIENT-METHODS: One hundred twenty individuals were enrolled in the study. We assayed serum sulfatase activity in 79 patients with colorectal neoplasms (38 CRC and 41 AP) and 41 controls. Enzyme activity was determined colorimetrically. RESULTS: We found statistically higher serum sulfatase activity in patients with colonic neoplasms than in controls (124; 112-139 vs. 79.5; 73-87 U). The activity was more elevated in adenomas (149; 128-173 U) than in cancers (103; 90-112 U). Sulfatase activity exceeded the cutoff value in 71% of AP and 47% of CRC patients. It increased with number of adenomas and tended to decrease with tumor progression. CONCLUSIONS: Sulfatases seem to be involved in the early stages of colonic neoplastic transformation which is reflected in their serum activity. The likelihood of elevated sulfatase activity is almost ten times higher in subjects with than without polyps. Sulfatase upregulation in majority of adenomas and their correlation tendencies warrants reconsideration of sulfatase determination as a possible diagnostic tool.

Evaluation of superoxide dismutase activity and its impact on semen quality parameters of infertile men.

The evaluation of superoxide dismutase (SOD) activity, as one of the most important antioxidative defence enzymes, in seminal plasma of patients consulting for male infertility was presented in the article. The study included also the determination of its influence on selected human semen quality parameters. The material represents semen samples obtained from 15 men, which were divided into two groups: Group I (n=10) including patients consulting for infertility and Group II (n=5) containing healthy sperm donors as a control. All of the semen samples were cryopreserved and stored in liquid nitrogen. The frozen samples were thawed at the same time and then SOD activity was determined spectrophotometrically. The analysis of the investigations results indicates a significantly lower semen SOD activity detected in oligoasthenozoospermic patients, comparing to the activity found in normospermic men. The study showed a positive correlation between SOD activity in seminal plasma and semen quality parameters--sperm concentration and overall motility, which are regarded as the most important for normal fertilizing ability of the spermatozoa. Significantly lower SOD activity in seminal plasma of infertile patients, comparing to healthy sperm donors, as well as positive correlation and beneficial impact of SOD activity on human semen quality parameters seem to confirm the observations, that decreased seminal plasma scavenger antioxidant capacity, particularly in form of low SOD activity, can be responsible for male infertility. This trial shows that SOD activity survey in seminal plasma could be a useful tool for determining sperm fertilization potential and could improve the diagnosis of male infertility.

Molecular evolution of enolase.

Enolase (EC 4.2.1.11) is an enzyme of the glycolytic pathway catalyzing the dehydratation reaction of 2-phosphoglycerate. In vertebrates the enzyme exists in three isoforms: alpha, beta and gamma. The amino-acid and nucleotide sequences deposited in the GenBank and SwissProt databases were subjected to analysis using the following bioinformatic programs: ClustalX, GeneDoc, MEGA2 and S.I.F.T. (sort intolerant from tolerant). Phylogenetic trees of enolases created with the use of the MEGA2 program show evolutionary relationships and functional diversity of the three isoforms of enolase in vertebrates. On the basis of calculations and the phylogenetic trees it can be concluded that vertebrate enolase has evolved according to the "birth and death" model of evolution. An analysis of amino acid sequences of enolases: non-neuronal (NNE), neuron specific (NSE) and muscle specific (MSE) using the S.I.F.T. program indicated non-uniform number of possible substitutions. Tolerated substitutions occur most frequently in alpha-enolase, while the lowest number of substitutions has accumulated in gamma-enolase, which may suggest that it is the most recently evolved isoenzyme of enolase in vertebrates.