Marko Klobučar

Novel Coumarin Derivatives Containing 1,2,4-Triazole, 4,5-Dicyanoimidazole and Purine Moieties: Synthesis and Evaluation of Their Cytostatic Activity

We report here on the synthesis and in vitro anti-tumor effects of a series of novel 1,2,4-triazole (compounds 3–6), 4,5-dicyanoimidazole (compound 7), and purine (compounds 8–13) coumarin derivatives and their acyclic nucleoside analogues 14–18. Structures of novel compounds 3–18 were deduced from their 1H- and 13C-NMR and corresponding mass spectra. Results of anti-proliferative assays performed on a panel of selected human tumor cell lines revealed that compound 6 had moderate cytostatic activity against the HeLa cell line (IC50 = 35 µM), whereas compound 10 showed moderate activity against the HeLa (IC50 = 33 µM), HepG2 (IC50 = 25 µM) and SW620 (IC50 = 35 µM) cell lines. These compounds showed no cytotoxic effects on normal (diploid) human fibroblasts.

UV-induced retinal proteome changes in the rat model of age-related macular degeneration

Age-related macular degeneration (AMD) is characterized by irreversible damage of photoreceptors in the central posterior part of the retina, called the macula and is the most common cause of vision loss in those aged over 50. A growing body of evidence shows that cumulative long-term exposure to UV radiation may be harmful to the retina and possibly leads to AMD irrespective of age. In spite of many research efforts, cellular and molecular mechanisms leading to UV-induced retinal damage and possibly retinal diseases such as AMD are not completely understood. In the present study we explored damage mechanisms accounting for UV-induced retinal phototoxicity in the rats exposed to UVA and UVB irradiation using a proteomics approach. Our study showed that UV irradiation induces profound changes in the retinal proteomes of the rats associated with the disruption of energy homeostasis, oxidative stress, DNA damage response and structural and functional impairments of the interphotoreceptor matrix components and their cell surface receptors such as galectins. Two small leucine-rich proteoglycans, biglycan and lumican, were identified as phototoxicity biomarkers associated with UV-induced disruption of interphotoreceptor matrix (IPM). In addition, UVB induced activation of Src kinase, which could account for cytoskeletal rearrangements in the retina was observed at the proteomics level. Pharmacological intervention either to target Src kinase with the aim of preventing cytoskeletal rearrangements in the retinal pigment epithelium (RPE) and neuronal retina or to help rebuild damaged IPM may provide fresh avenues of treatment for patients suffering from AMD.

Discovery of New Acid Ceramidase-Targeted Acyclic 5-Alkynyl and 5-Heteroaryl Uracil Nucleosides.

A series of novel N-acyclic uracil analogs with linear, branched, aromatic, and cyclopropyl-alkynyl as well as heteroaryl moieties at C-5 were prepared using palladium catalyzed Sonogashira and Stille cross-coupling and evaluated against malignant tumor cell lines. C-5-Furan-2-yl uracil derivative 6 was shown to be more potent against MCF-7 than the reference drug 5-fluorouracil (5-FU), while C-5-alkynyl uracil derivatives 9c and 9e exhibited antibreast cancer activities comparable to 5-FU. Selected compounds induced cell death, partially due to apoptosis, of MCF-7 breast cancer cells. Abrogation of acid ceramidase (ASAH1) expression of 9c and 9e indicated that these compounds could perturb ASAH1-mediated sphingolipid signaling. The selective activity of 9c and 9e against breast cancer cells via the ASAH1-mediated signaling, as a molecular target, might have a great advantage for potential future therapeutic use.

Basement membrane protein ladinin-1 and the MIF-CD44-β1 integrin signaling axis are implicated in laryngeal cancer metastasis

Laryngeal squamous cell carcinoma (LSCC) is the most common form of malignant disease in the head and neck region characterized by frequent occurrence of metastases in the neck lymph nodes early in the disease onset. In the presented study, we performed quantitative proteomic profiling of patient-matched primary tumor and adjacent non-tumorous tissues derived from metastatic LSCC as to identify new protein candidates with potential diagnostic and therapeutic significance. Obtained results revealed for the first time involvement of the basement membrane protein ladinin-1 in laryngeal cancer metastases. Alterations in the cellular microenvironment that propel metastatic events in laryngeal cancer include activation of MIF-CD44-β1 integrin signal transduction pathway and induction of downstream signaling mediated by NF-κB and Src tyrosine kinase, which ultimately impinge on cytoskeletal dynamics and architecture resulting in increased cellular motility and invasiveness. In this context, particularly interesting finding is upregulation of several actin-binding proteins novel to laryngeal cancer pathogenesis including coronin-1C and plastin-2, whose functional significance in laryngeal carcinogenesis has yet to be established. We also detected for the first time a complete loss of afamin in metastatic laryngeal cancer tissues, which warrants further studies into its use as a possible marker for monitoring disease progression and/or treatment outcome.

Affinity chromatography on monolithic supports for simultaneous and high-throughput isolation of immunoglobulins from human serum

Posttranslational modifications of immunoglobulins have been a topic of great interest and have been repeatedly reported as a major factor in disease pathology. Cost‐effective, reproducible, and high‐throughput (HTP) isolation of immunoglobulins from human serum is vital for studying the changes in protein structure and the following understanding of disease development. Although there are many methods for the isolation of specific immunoglobulin classes, only a few of them are applicable for isolation of all subtypes and variants. Here, we present the development of a scheme for fast and simultaneous affinity purification of α (A), γ (G), and μ (M) immunoglobulins from human serum through affinity monolith chromatography. Affinity‐based monolithic columns with immobilized protein A, G, or L were used for antibody isolation. Monolithic stationary phases have a high surface accessibility of binding sites, large flow‐through channels, and can be operated at high flow rates, making them the ideal supports for HTP isolation of biopolymers. The presented method can be used for HTP screening of human serum in order to simultaneously isolate all three above‐mentioned immunoglobulins and determine their concentration and changes in their glycosylation pattern as potential prognostic and diagnostic disease biomarkers.

Expression of polysialic acid in primary laryngeal squamous cell carcinoma

Aims: Expression of polySia is associated with metastatic dissemination and progression of various malignant diseases. In particular, it may contribute to tumorigenesis by a negative modulatory effect on cellular signaling cascades responsible for cellular migration, differentiation and proliferation. In this study, we investigated the expression of polySia in primary metastatic and non‐metastatic laryngeal squamous cell carcinoma (LSCC) tumor tissues and its potential impact on the LSCC progression. Main methods: The expression of polySia in metastatic and non‐metastatic primary laryngeal squamous cell carcinoma (LSCC) tumor biopsy specimens was investigated by immunohistochemistry, while the expression of polysialyltransferase IV (ST8SiaIV)(), fibroblast growth factor receptor 1 (FGFR1), extracellular signal regulated kinases 1 and 2 (Erk 1/2) and c‐Raf was tested in metastatic and non‐metastatic primary tumor tissues (including the corresponding non‐tumor control tissues) by Western blot analysis. Key findings: The expression of polySia was detected in LSCC biopsies specimens with generally stronger immunoreactivity in non‐metastatic tumor LSCC sections and in histologically undifferentiated tumors. Also, increased polySia expression was observed in adjacent histologically unaltered laryngeal tumor‐associated tissue of the metastatic sections. In addition, we provide an evidence of increased polysialyltransferase IV (ST8SiaIV) expression, involved in polySia synthesis in both metastatic and non‐metastatic primary tumors which is accompanied by decreased levels of FGFR1, Erk 1/2 and c‐Raf. Significance: We present for the first time the evidence for the polySia expression in LSCC biopsies specimens which suggests its potential impact on initial steps of LSCC malignant transformation.