A young researcher’s guide to NME/Nm23/NDP Kinase

Nucleoside diphosphate kinases (NDPKs) catalyze the exchange of the terminal phosphate from trinucleotides to dinucleotides through a high-energy phosphohistidine intermedier. They are encoded by NME genes and have been found, with a few exceptions, in all living beings. Besides their well-known function as key regulators of the cellular nucleotide homeostasis, they have been appointed numerous additional biochemical and biological functions. The discovery of NME1/NDPK A as the first metastasis suppressor opened new avenues in cancer research. Although the precise role of NME genes/proteins in cancer dissemination is not yet revealed, it seems that further intensive research in this field may lead to new advances in cancer diagnosis and prognosis, as well as encourage new therapeutic strategies.</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

Antiinflammatory and Antiproliferative Activity of Naphthoxazole, Fused Hetero-benzoxazole and Bridged Benzobicyclic Photoproducts

Biological activity of naphthoxazoles, fused hetero-benzoxazoles and benzobicyclo[3.2.1]-derivatives was investigated in proliferation and inflammation based assays. The tested compounds were prepared by photocylization or photocycloaddition reactions. Effect of compounds on proliferation of several cancer cell lines was determined by measuring cell metabolic activity through time. Lipopolysaccharide (LPS) stimulation of peripheral blood mononuclear cells (PBMC) was used to investigate antiinflammatory properties of the compounds. Several naphthoxazoles and fused hetero-benzoxazoles inhibited TNFα protein expression in LPS stimulated PBMC, indicating possible antiinflammatory role which would be interesting to further investigate. Physico-chemical properties of tested compounds have been also studied using chromatographic lipophilicity measure, chrom logD and logP was calculated as the importance of physico-chemical properties of compounds at early stage of discovery of new drugs is well established. The similarities in structure and activity of some representative compounds affirm the need to further address their antiinflammatory properties. This work is licensed under a Creative Commons Attribution 4.0 International License

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

Characterization of Vemurafenib-Resistant Melanoma Cell Lines Reveals Novel Hallmarks of Targeted Therapy Resistance

Regardless of the significant improvements in treatment of melanoma, the majority of patients develop resistance whose mechanisms are still not completely understood. Hence, we generated and characterized two melanoma-derived cell lines, primary WM793B and metastatic A375M, with acquired resistance to the RAF inhibitor vemurafenib. The morphology of the resistant primary WM793B melanoma cells showed EMT-like features and exhibited a hybrid phenotype with both epithelial and mesenchymal characteristics. Surprisingly, the vemurafenib-resistant melanoma cells showed a decreased migration ability but also displayed a tendency to collective migration. Signaling pathway analysis revealed the reactivation of MAPK and the activation of the PI3K/AKT pathway depending on the vemurafenib-resistant cell line. The acquired resistance to vemurafenib caused resistance to chemotherapy in primary WM793B melanoma cells. Furthermore, the cell-cycle analysis and altered levels of cell-cycle regulators revealed that resistant cells likely transiently enter into cell cycle arrest at the G0/G1 phase and gain slow-cycling cell features. A decreased level of NME1 and NME2 metastasis suppressor proteins were found in WM793B-resistant primary melanoma, which is possibly the result of vemurafenib-acquired resistance and is one of the causes of increased PI3K/AKT signaling. Further studies are needed to reveal the vemurafenib-dependent negative regulators of NME proteins, their role in PI3K/AKT signaling, and their influence on vemurafenib-resistant melanoma cell characteristics

Association of NLRP1 Coding Polymorphism with Lung Function and Serum IL-1β Concentration in Patients Diagnosed with Chronic Obstructive Pulmonary Disease (COPD)

Chronic obstructive pulmonary disease (COPD) is a chronic disease characterized by a progressive decline in lung function due to airflow limitation, mainly related to IL-1β-induced inflammation. We have hypothesized that single nucleotide polymorphisms (SNPs) in NLRP genes, coding for key regulators of IL-1β, are associated with pathogenesis and clinical phenotypes of COPD. We recruited 704 COPD individuals and 1238 healthy controls for this study. Twenty non-synonymous SNPs in 10 different NLRP genes were genotyped. Genetic associations were estimated using logistic regression, adjusting for age, gender, and smoking history. The impact of genotypes on patients’ overall survival was analyzed with the Kaplan–Meier method with the log-rank test. Serum IL-1β concentration was determined by high sensitivity assay and expression analysis was done by RT-PCR. Decreased lung function, measured by a forced expiratory volume in 1 s (FEV1% predicted), was significantly associated with the minor allele genotypes (AT + TT) of NLRP1 rs12150220 (p = 0.0002). The same rs12150220 genotypes exhibited a higher level of serum IL-1β compared to the AA genotype (p = 0.027) in COPD patients. NLRP8 rs306481 minor allele genotypes (AG + AA) were more common in the Global Initiative for Chronic Obstructive Lung Disease (GOLD) definition of group A (p = 0.0083). Polymorphisms in NLRP1 (rs12150220; OR = 0.55, p = 0.03) and NLRP4 (rs12462372; OR = 0.36, p = 0.03) were only nominally associated with COPD risk. In conclusion, coding polymorphisms in NLRP1 rs12150220 show an association with COPD disease severity, indicating that the fine-tuning of the NLRP1 inflammasome could be important in maintaining lung tissue integrity and treating the chronic inflammation of airways