Metastasis – recent scientific insights and challenging new therapeutic approaches

Majority of cancer patients never die from the original disease – primary tumor – but from the metastasis that disseminate throughout the body. Although they disseminate in millions only few of them succeeded in their journey and achieve their main goal – form a distant metastatic colony which is able to thrive in an inhospitable environment of an unrelated tissue. This review will summarize in brief some of the recent advances in cancer invasion and metastasis investigations and possibilities in using these findings for the benefit of cancer patients

Reversibility of Membrane N-Glycome of HeLa Cells upon Treatment with Epigenetic Inhibitors

Glycans are essential regulators of protein function and are now in the focus of research in many physiological and pathophysiological processes. There are numerous modes of regulating their biosynthesis, including epigenetic mechanisms implicated in the expression of glyco- genes. Since N-glycans located at the cell membrane define intercellular communication as well as a cellular response to a given environment, we developed a method to preferentially analyze this fraction of glycans. The method is based on incorporation of living cells into polyacrylamide gels, partial denaturation of membrane proteins with 3 M urea and subsequent release of N-glycans with PNGase F followed by HPLC analysis. Using this newly developed method, we revealed multiple effects of epigenetic inhibitors Trichostatin A, sodium butyrate and zebularine on the composition of N- glycans in human cells. The induced changes were found to be reversible after inhibitor removal. Given that many epigenetic inhibitors are currently explored as a therapeutic strategy in treatment of cancer, wherein surface glycans play an important role, the presented work contributes to our understanding of their efficiency in altering the N-glycan profile of cancer cells in culture

The utilization of pEGFP reporter system in cell-cycle analysis of adherent cells

Background and Purpose: GFP (green fluorescent protein) is widely used in a variety of fluorescent methods aimed at revealing the fate of proteins in the cell, intracellular transport, transfection efficiency and is also recommended for cell-cycle analysis purposes. In our attempt to evaluate the role of nm23 genes in proliferation of head and neck tumor cells in culture we have decided to use EGFP reporter system and analyze the DNA content by flow cytometry. Materials and Methods: To optimize the method we either transiently transfected the cells with pEGFPC1-nm23 constructs or cotransfected the cells with an nm23 carrying constructs and pEGFPC1 as a reporter system. We established stable clones with pEGFPC1-nm23 constructs and analyzed them by flow cytometry, as well. Results and Conclusions:We report our experience for the use of pEGFP reporter system and flow cytometry for determining cell-cycle distribution of transiently and stably transfected adherent tumor cells. We discuss, in brief, the protocol we used and the problems that appeared during our experiments – GFP bleaching, cell clumping and degradation and insufficient number of cells to be analyzed. In conclusion, we suggest useful tips how to avoid or minimize the technical problems of this method and improve the results and analysis

Sponge non-metastatic Group I Nme gene/protein - structure and function is conserved from sponges to humans

<p>Abstract</p> <p>Background</p> <p>Nucleoside diphosphate kinases NDPK are evolutionarily conserved enzymes present in Bacteria, Archaea and Eukarya, with human Nme1 the most studied representative of the family and the first identified metastasis suppressor. Sponges (Porifera) are simple metazoans without tissues, closest to the common ancestor of all animals. They changed little during evolution and probably provide the best insight into the metazoan ancestor's genomic features. Recent studies show that sponges have a wide repertoire of genes many of which are involved in diseases in more complex metazoans. The original function of those genes and the way it has evolved in the animal lineage is largely unknown. Here we report new results on the metastasis suppressor gene/protein homolog from the marine sponge <it>Suberites domuncula</it>, NmeGp1Sd. The purpose of this study was to investigate the properties of the sponge Group I Nme gene and protein, and compare it to its human homolog in order to elucidate the evolution of the structure and function of Nme.</p> <p>Results</p> <p>We found that sponge genes coding for Group I Nme protein are intron-rich. Furthermore, we discovered that the sponge NmeGp1Sd protein has a similar level of kinase activity as its human homolog Nme1, does not cleave negatively supercoiled DNA and shows nonspecific DNA-binding activity. The sponge NmeGp1Sd forms a hexamer, like human Nme1, and all other eukaryotic Nme proteins. NmeGp1Sd interacts with human Nme1 in human cells and exhibits the same subcellular localization. Stable clones expressing sponge NmeGp1Sd inhibited the migratory potential of CAL 27 cells, as already reported for human Nme1, which suggests that Nme's function in migratory processes was engaged long before the composition of true tissues.</p> <p>Conclusions</p> <p>This study suggests that the ancestor of all animals possessed a NmeGp1 protein with properties and functions similar to evolutionarily recent versions of the protein, even before the appearance of true tissues and the origin of tumors and metastasis.</p

Metastasis – recent scientific insights and challenging new therapeutic approaches

Majority of cancer patients never die from the original disease – primary tumor – but from the metastasis that disseminate throughout the body. Although they disseminate in millions only few of them succeeded in their journey and achieve their main goal – form a distant metastatic colony which is able to thrive in an inhospitable environment of an unrelated tissue. This review will summarize in brief some of the recent advances in cancer invasion and metastasis investigations and possibilities in using these findings for the benefit of cancer patients

Platelet serotonin concentration and monoamine oxidase activity in hypothyroid patients

BACKGROUND/AIM: The relationship between the hypothalamic-pituitary-thyroid (HPT) axis and the serotonergic (5-hydroxytryptamine, 5-HT) system is not clear. The aim of the study was to determine platelet biochemical markers (5-HT concentration and monoamine oxidase B, MAO-B, activity) in hypothyroid patients. ----- METHODS: The study included 25 medication-free female hypothyroid patients in postoperative follow-up after total thyroidectomy due to papillary thyroid carcinoma, who had not been treated with synthetic thyroxine (T(4)) for 4 weeks, and 44 age-matched euthyroid healthy women. The platelet 5-HT concentration, platelet MAO-B activity, total T(4) and thyroid-stimulating hormone (TSH) levels were determined using spectrofluorimetric methods, radioimmunoassay and fluoroimmunoassay, respectively. ----- RESULTS: Hypothyroid patients had significantly higher TSH, significantly lower T(4) levels and platelet 5-HT concentrations, and unchanged platelet MAO-B activity than healthy subjects. A positive correlation was found between the 5-HT concentration and platelet MAO-B activity, and between the platelet MAO-B activity and T(4) in control subjects. ----- CONCLUSIONS: Reduced platelet 5-HT concentrations in hypothyroid patients suggests a complex interaction between the 5-HT system and HPT axis activity, which could be related to the frequent occurrence of depressive symptoms in hypothyroid patients. The determination of platelet 5-HT concentrations should be considered a diagnostic tool for the evaluation of depressive symptoms in hypothyroid patients during the hormone withdrawal procedure

The lack of association between monoamine oxidase (MAO) intron 13 polymorphism and platelet MAO-B activity among men

Monoamine oxidase (MAO), a mitochondrial flavine containing enzyme, exists in two isoenzymes, MAO-A and MAO-B. Platelets contain MAO-B subtype, proposed to be a biomarker for different personality characteristics and vulnerability for substance abuse. The most common polymorphism of MAO-B gene, a single base change (A or G) occurs in intron 13. It has been proposed to be a functional polymorphism, controlling the activity of MAO-B in platelets. The aim of the study was to determine the association between platelet MAO-B activity and MAO-B intron 13 polymorphism in 225 racially and ethnically uniform healthy Caucasian men of the Croatian origin. Our results showed that platelet MAO-B activity did not differ between subjects subdivided into those with A-allele or G-allele. This polymorphism of the MAO-B gene did not control the activity of the MAO-B in platelets. Platelet MAO-B activity was associated only with the smoking status, and it was significantly decreased in smokers when compared to nonsmokers. No significant association was found between MAO-B polymorphism and smoking status. In healthy individuals of the Croatian origin, the studied MAO-B polymorphism showed a lack of functional importance in regulating MAO-B activity in platelets. Since different populations may vary in the association between functional polymorphism and the MAO-B activity, and the genotype of transcription factor AP-2β was reported to be associated with altered platelet MAO-B activity, and with specific personality traits, further studies on different populations should be conducted to elucidate the molecular mechanism/s regulating platelet MAO-B activity

Platelet serotonin and monoamine oxidase in Alzheimer's disease with psychotic features

Post mortem brain studies indicate that alterations in serotonergic and catecholaminergic systems might be associated with Alzheimer's disease (AD). The aim of the study was to determine serotonin (5-HT) levels and monoamine oxidase type B (MAO-B) activity in platelets of psychotic and non-psychotic patients with AD, established according to the NINCDS-ADRDA and DSM-IV-TR criteria. Cognitive impairment and psychotic features were evaluated using Mini Mental Status Examination and Neuropsychiatric Inventory. Platelet 5-HT concentration and MAO-B activity were determined spectrofluorimetrically in 116 (51 male, 65 female) healthy subjects and 70 psychotic (10 male, 60 female) and 151 non-psychotic (32 male, 119 female) patients. Psychotic and non-psychotic female and psychotic male patients had significantly lower platelet 5-HT concentration than corresponding sex matched control subjects. Platelet MAO-B activity was significantly increased in both male and female non-psychotic patients compared to the sex matched controls. Non-psychotic female patients had significantly higher platelet MAO-B activity than psychotic female patients. Our data suggest that platelet MAO-B activity, but not platelet 5-HT concentration, could differentiate between psychotic and non-psychotic subtypes of AD