Chlorogenic acids – their properties, occurrence and analysis

Chlorogenic acids (CQAs), the esters of caffeic acid and quinic acid, are biologically important phenolic compounds present in many plant species. Nowadays much is known from their pro-health properties, including anti-cancer activity. Yet, the supposition that they may be helpful in fighting obesity and modify glucose-6-phosphatase involved in glucose metabolism have led to a revival of research on CQAs properties and their natural occurrence. Much attention is also paid to the proper analysis of CQAs content in plants and plant products due to the fact that the main CQAs representative in nature i.e. 5-O-caffeoylquinic acid (5-CQA) is commonly employed as a quality marker in the control of various natural products.The common procedures used so far for CQAs determination in plants involve conventional long lasting solvent extraction realized at higher temperatures prior to chromatographic analysis. The drawbacks associated to the conventional extraction techniques have led to the search for new alternative extraction processes that additionally could improve extracts quality. The latter is particularly important as regards CQAs applications, and the fact that these compounds easily transform/degrade to others. According to reports, the conventional heating of 5-CQA in the presence of water causes its isomerization and transformation. The reports prove that 5-CQA not only isomerizes to 3- and 4-O-caffeoylquinic acid, but also undergoes other transformations such as esterification and reactions with water. Hence, in the attempt to improve the process of chlorogenic acids extraction and to make it more effective and environmentally friendly, innovative so-called "enhanced" techniques of extraction have been recently developed and applied. To guarantee the proper analysis of compounds with very similar properties forming the chlorogenic acids family and their numerous transformation/degradation products, hyphenated techniques, in particular LC-MS, are currently being used

Carbohydrate moieties of N-cadherin from human melanoma cell lines

Expression of N-cadherin an adhesion molecule of the cadherin family, in tumor cells is associated with their increased invasive potential. Many studies suggested the role of N-linked oligosaccharides as important factors that contribute to metastasis by influencing tumor cell invasion and adhesion. N-cadherin is a heavily glycosylated protein. We have analysed the carbohydrate profile of this protein synthesized in human melanoma cell lines: WM35 from the primary tumor site and WM239, WM9, and A375 from different metastatic sites. N-cadherin was immunoprecipitated with anti-human N-cadherin polyclonal antibodies. Characterisation of its carbohydrate moieties was carried out by SDS/PAGE electrophoresis and blotting, followed by immunochemical identification of the N-cadherin polypeptides and analysis of their glycans using highly specific digoxigenin or biotin labelled lectins. The positive reaction of N-cadherin from the WM35 cell line with Galanthus nivalis agglutinin (GNA), Datura stramonium agglutinin (DSA) and Sambucus nigra agglutinin (SNA) indicated the presence of high-mannose type glycans and biantennary complex type oligosaccharides with α2-6 linked sialic acid. N-cadherin from WM239, WM9, and A375 cell lines gave a positive reaction with Phaseolus vulgaris leukoagglutinin (L-PHA) and lotus Tetragonolobus purpureas agglutinin (LTA). This indicated the presence of tri- or tetra-antennary complex type glycans with α-fucose. In addition, N-cadherin from WM9 (lymphomodus metastatic site) and A375 (solid tumor metastatic site) contained complex type chains with α2-3 sialic acid (positive reaction with Maackia amurensis agglutinin - MAA). The results demonstrated that N-glycans of N-cadherin are altered in metastatic melanomas in a way characteristic for invasive tumor cells

mTOR inhibitor everolimus reduces invasiveness of melanoma cells

The mammalian target of rapamycin (mTOR) plays a key role in several cellular processes: proliferation, survival, invasion, and angiogenesis, and therefore, controls cell behavior both in health and in disease. Dysregulation of the mTOR signaling is involved in some of the cancer hallmarks, and thus the mTOR pathway is an important target for the development of a new anticancer therapy. The object of this study is recognition of the possible role of mTOR kinase inhibitors-everolimus single and in combination with selected downstream protein kinases inhibitors: LY294002 (PI3 K), U0126 (ERK1/2), GDC-0879 (B-RAF), AS-703026 (MEK), MK-2206 (AKT), PLX-4032 (B-RRAF) in cell invasion in malignant melanoma. Treatment of melanoma cells with everolimus led to a significant decrease in the level of both phosphorylated: mTOR (Ser2448) and mTOR (Ser2481) as well as their downstream effectors. The use of protein kinase inhibitors produced a significant decrease in metalloproteinases (MMPs) activity, as well as diminished invasion, especially when used in combination. The best results in the inhibition of both MMPs and cell invasiveness were obtained for the combination of an mTOR inhibitor- everolimus with a B-RAF inhibitor-PLX-4032. Slightly less profound reduction of invasiveness was obtained for the combinations of an mTOR inhibitor-everolimus with ERK1/2 inhibitor-U126 or MEK inhibitor-AS-703026 and in the case of MMPs activity decrease for PI3 K inhibitor-LY294002 and AKT inhibitor-MK-2206. The simultaneous use of everolimus or another new generation rapalog with selected inhibitors of crucial signaling kinases seems to be a promising concept in cancer treatment

mTOR inhibitor Everolimus-induced apoptosis in melanoma cells

Melanoma is the most aggressive, therapy-resistant skin cancer. The mammalian target of rapamycin (mTOR), the serine/ threonine kinase which integrates both intracellular and extracellular signals, plays a crucial role in coordinating the balance between the growth and death of cells. The object of this study is a comparison of the influence of mTOR inhibitor everolimus in the concentration range between 20 nM and 10 μM, used individually and in combination with selected downstream protein kinases inhibitors: LY294002 (PI3K), U0126 (ERK1/2), AS-703026 (MEK) and MK-2206 (AKT) on the expression of prosurvival proteins: p-Bcl-2 (S70), p-Bcl-2 (T56), Bcl-2, Bcl-xL, Mcl-1, activity of caspase-3, proliferation and induction of apoptosis in melanoma cells. Current results clearly show that the nanomolar concentration of the mTOR inhibitor everolimus in combination with the inhibitor of MAP kinase (AS-703026) or AKT kinase (MK-2206) is effective in inducing apoptosis and reducing proliferation of melanoma cells. The herein research results confirm the hypothesis on the important role of mTOR signaling in cancer progression, and gives hope that implementation of successful combination of its inhibitors will find recognition and application in cancer treatment in the near future

Integrin linked kinase regulates endosomal recycling of N-cadherin in melanoma cells

Malignant transformation is characterized by a phenotype "switch" from E- to N-cadherin - a major hallmark of epithelial to mesenchymal transition (EMT). The increased expression of N-cadherin is commonly followed by a growing capacity for migration as well as resistance to apoptosis. Integrin Linked Kinase (ILK) is a key molecule involved in EMT and progression of cancer cells. ILK is known as a major signaling mediator involved in cadherin switch, but the specific mechanism through which ILK modulates N-cadherin expression is still not clear. Studies were carried out on human melanoma WM793 and 1205Lu cell lines. Expression of proteins was analyzed using PCR and Western Blot; siRNA transfection was done for ILK. Analysis of cell signaling pathways was monitored with phospho-specific antibodies. Subcellular localization of protein was studied using the ProteoExtract Subcellular Kit and Western blot analysis. Our data show that ILK knockdown by siRNA did suppress N-cadherin expression in melanoma, but only at the protein level. The ILK silencing-induced decrease of N-cadherin membranous expression in melanoma highlights the likely crucial role of ILK in the coordination of membrane trafficking through alteration of Rab expression. It is essential to understand the molecular mechanism of increased N-cadherin expression in cancer to possibly use it in the search of new therapeutic targets

Integrin-linked kinase regulates cadherin switch in bladder cancer

Cadherin switch is specific of epithelial-mesenchymal transition (EMT) and is closely related to tumor cell invasion. However, the molecular mechanism that promotes the phenotypic changes remains unclear and elusive. We found that integrin-linked kinase (ILK) is a key factor involved in cadherin switch. The expression and activity of ILK are elevated in a variety of cancers but its mechanisms are not exactly understood. In this report, we studied the role and mechanism of ILK in EMT of human bladder cancer. We showed that silencing of ILK expression by small interfering RNA (siRNA) significantly abolished the nuclear translocation or the presence of markers associated with EMT like Snail, Twist, Zeb, and beta-catenin. ILK knockdown by siRNA suppressed N-cadherin expression and increased re-expression of E-cadherin in bladder cancer cells. We suggest that ILK is a major signaling factor involved in EMT. It is essential to understand the molecular mechanism of EMT in aim to possibly use it in search for new therapeutic targets. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13277-016-5354-x) contains supplementary material, which is available to authorized users

Philosophers, scandalists and ethnarchs : the authors of the Montenegrin enlightenment literature

This present article is devoted to an issue of assimilation and adaptation of the philosophical tendencies and literary patterns of the European enlightenment in Montenegrin literature which was established in 18th century in difficult internal circumstances and during constant threat to the independence of the country. Key advocates of the rationalistic, democratic and anticleri-cal demands were mainly descended from the romanized Adriatic communities and obtained their education in Western Europe – transplanting into native ground the ideas of the free mind and conscience, religious tolerance, just politics, reform of the state or general education. In the context of the above-mentioned and similar categories the works of Stefan Zanović, Jovan Baljević, Katerina Radonjić and other authors were considered. The special example of taking advantage of the enlightenment paradigm are texts of the bishops and political rulers from the Petrović Njegoš dynasty – their attitude was formed through the necessity of the defence of the legal-state interest and fidelity to the Christian ethic. As a case of such a meaning of the civilizing mission in the environment of disintegrated Montenegrin tribes Petar I was presented. Especially his messages to the clan communities contain many didactic appeals which are faith-ful to the enlightenment vision of the man and socie