N-Glycome changes reflecting resistance to platinum-based chemotherapy in ovarian cancer.

A number of studies have reported aberrant glycosylation in connection with malignancy. Our investigation further expands on this topic through the examination of N-glycans, which could be associated with the resistance of advanced stage, high-grade non-mucinous ovarian cancer to platinum/taxane based chemotherapy. We used tissue samples of 83 ovarian cancer patients, randomly divided into two independent cohorts (basic and validation). Both groups involved either cases with/without postoperative tumor residue or the cases determined either resistant or sensitive to this chemotherapy. In the validation cohort, preoperative serum samples were also available. N-glycans released from tumors and sera were permethylated and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The MS analysis yielded a consecutive detection of 68 (tissue) and 63 (serum) N-glycan spectral signals. Eight of these were found to be differentially abundant in tissues of both independent cohorts including the cases with a postoperative cancer residue. One of these glycans was detected as differentially abundant in sera of the validation cohort. No statistically significant differences in intensities due to the same N-glycans were found in the cases without postoperative macroscopic residues in either the basic or validation cohort. From the biochemical point of view, the statistically significant N-glycans correspond to the structures carrying bisecting (terminal) GlcNAc residue and tetra-antennary structures with sialic acid and/or fucose residues. Among them, six tissue N-glycans could be considered potential markers connected with a resistance to chemotherapy in ovarian cancer patients. The prediction of primary resistance to standard chemotherapy may identify the group of patients suitable for alternative treatment strategies. SIGNIFICANCE: Drug resistance has become a major impediment to a successful treatment of patients with advanced ovarian cancer. The glycomic measurements related to cancer are becoming increasingly popular in identification of the key molecules as potential diagnostic and prognostic indicators. Our report deals with identification of differences in N-glycosylation of proteins in tissue and serum samples from the individuals showing sensitivity or resistance to platinum/taxane-based chemotherapy. The detection sensitivity to chemotherapy is vitally important for these patients.

Microgradient separation technique for purification and fractionation of permethylated N-glycans before mass spectrometric analyses.

Analysis of N-glycans released enzymatically from patients' sera or other clinical samples may provide diagnostically and prognostically important information on human disease. Permethylation of these biomolecules simultaneously increases their hydrophobicity and substantially improves their detection parameters in the following mass spectrometric analyses. The overall procedure, from the glycan cleavage to the final mass spectrometric determinations, includes several steps involving extraction, derivatization, and purification. During these steps, certain polymeric contaminants that may have been coincidentally introduced could hamper the final measurements. To understand and counter these interferences and further fractionate or preconcentrate these glycans, we introduce here an effective microgradient chromatographic technique that employs a small reversed-phase microcolumn connected to a gas-tight microsyringe delivering a mobile-phase gradient. After loading the glycan fraction onto the microcolumn, three elution steps are recommended: (1) remove polar contaminants; (2) recover permethylated glycans for either liquid chromatography with electrospray ionization mass spectrometry or matrix-assisted laser desorption/ionization mass spectrometry; and (3) remove larger polymeric contaminants and regenerate the precolumn. We further demonstrate that the trapped second fraction can be beneficially preconcentrated and further separated to achieve matrix-assisted laser desorption/ionization mass spectrometric detection of the derivatized N-glycans up to 6300 Da. The enhanced detection capabilities for tetra-antennary N-glycans are of increasing interest in disease biomarker discovery.

Importance of Membrane Proteins in the Treatment of Tumor Diseases and the Possibilities of Their Further Study.

BACKGROUND: The proteins of the cellular cytoplasmic membrane represent a heterogeneous group of proteins with different structures, localizations, and functions. They participate in many cellular processes including cellular signaling and communication with the external environment and communication between cells. Mutations and post-translational modifications alter the chemical-physical properties of membrane proteins and thus significantly affect the process of carcinogenesis. Therefore, membrane proteins represent important targets for the diagnosis and treatment of cancer. Nowadays, treatment in the form of monoclonal antibodies or low molecular weight inhibitors targets mainly receptors of growth factors on the surface of tumor cells and various types of molecules including the targets of the so-called checkpoint inhibitors on the surface of the cells of the immune system. In order to better understand the properties and functions of membrane proteins, especially with the perspective of developing new targeted approaches in therapy, mainly proteomic and molecular biological approaches are currently being used. AIM: The aim of this article is to describe the properties and functions of different groups of membrane proteins and to summarize their current relevance and potential for use in oncology. Attention is focused on those groups that regulate the proliferation of tumor cells, affect the immune response, cause drug resistance and metastasis, and are already used or accepted as potential targets of biological therapy. Glycosylation and phosphorylation are described in detail as the most studied post-translational modification of membrane proteins, and mass spectrometry is presented as an effective tool for the identification and quantification of membrane proteins. Key words: membrane proteins - glycosylation - phosphorylation - proteomic analysis - targeted therapy This work was supported by the project MEYS - NPS I - LO1413. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Accepted: 3. 8. 2018.

Molecular Evidence of Apatococcus, including A. fuscideae sp. nov., as Photobiont in the Genus Fuscidea.

The knowledge of the taxonomy and classification of algae (including lichenized) has recently increased rapidly, but there are still many gaps. We aimed to 1) identify the Fuscidea photobionts by locating their taxonomic positions in the green algal classification, and 2) to resolve their interspecific relationships. The lichenized algae were examined based on morphological observations of axenic isolates as well as molecular studies of 18S and ITS nrDNA sequences. Analysis of the secondary structure of the ITS2 operon complemented these investigations. We found that the Fuscidea photobionts were placed within the Trebouxiophyceae, related to Apatococcus lobatus (Chodat) J.B.Petersen. Phylogenetic analyses revealed one clade nesting free-living and lichenized Apatococcus F.Brand which comprised six different lineages in the ITS phylogeny. The lichenized alga associated with the investigated Fuscidea species, except for F. lightfootii (Sm.) Coppins & James, represents a hitherto unknown lineage within Apatococcus. Fuscidea lightfootii was lichenized with a separate lineage within Apatococcus, together with free-living members, which were already known from Genbank sequences. All retrieved groups within Apatococcus were rather different in their ITS sequences, thus most likely corresponding to different species. The most common photobiont of Fuscidea species, Apatococcus fuscideae A.Beck & Zahradn., was described as new to science.