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Background/Aims: Proline availability for proline dehydrogenase/proline oxidase (PRODH/POX) may represent switching mechanism between PRODH/POX-dependent apoptosis and autophagy. The aim of the study was to evaluate the impact of overexpression of prolidase (proline releasing enzyme) on apoptosis/autophagy in breast cancer MCF-7 cells. Methods: The model of MCF-7 cells with prolidase overexpression (MCF-7PL) was obtained. In order to targeting proline for PRODH/POX-dependent pathways substrate for prolidase, glycyl-proline (GP) was provided and proline utilization for collagen biosynthesis was blocked using 2-methoxyestradiol (MOE). Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. DNA, collagen and total protein biosynthesis were determined by radiometric method. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry. Results: Prolidase overexpression in MCF-7PL cells contributed to 10-fold increase in the enzyme activity, 3-fold increase in cytoplasmic proline level and decrease in cell viability and DNA biosynthesis compared to wild type MCF- 7 cells. In MCF-7PL cells MOE and GP significantly decreased the number of living cells. MOE inhibited DNA biosynthesis in both cell lines while GP evoked inhibitory effect on the process only in MCF-7PL cells. In both cell lines, MOE or MOE+GP inhibited DNA and collagen biosynthesis. Although GP in MCF-7 cells stimulated collagen biosynthesis, it inhibited the process in MCF-7PL cells. The effects of studied compounds in MCF-7PL cells were accompanied by increase in the expression of Atg7, LC3A/B, Beclin-1, HIF-1α and decrease in the expression of PRODH/POX, active caspases-3 and -9. Conclusion: The data suggest that overexpression of prolidase in MCF-7 cells contributes to increase in intracellular proline concentration and PRODH/POX-dependent autophagic cell death. [ABSTRACT FROM AUTHOR]

The project entitled "Study of serum metabolic profiles of patients with non-small cell lung cancer with special emphasis on the smoking status of patients" is a study based on metabolomics, which is the latest of the "omics" technologies and involves a comprehensive analysis of small molecule metabolites of a specific biological sample. High-throughput and sensitive analytical techniques used in metabolomic investigations are powerful tools in the fi eld of oncology and aids understanding what is happening in cancer cells and searching for new cancer markers. The aim of the project is to determine whether lung cancer patients have a distinct serum metabolic profile and whether this profile is associated with patients' smoking status. The application of liquid chromatography-high-resolution mass spectrometry-based methodology along with advanced statistical methods will enable to select potential molecules that can be useful in early lung cancer detection. [ABSTRACT FROM AUTHOR]

The aim of this study was to quantitate 42 serum-free amino acids, propose the biochemical explanation of their role in tumor development, and identify new ovarian cancer (OC) biomarkers for potential use in OC screening. The additional value of this work is the schematic presentation of the interrelationship between metabolites which were identified as significant for OC development and progression. The liquid chromatography-tandem mass spectrometry technique using highly-selective multiple reaction monitoring mode and labeled internal standards for each analyzed compound was applied. Performed statistical analyses showed that amino acids are potentially useful as OC biomarkers, especially as variables in multi-marker models. For the distinguishing metabolites the following metabolic pathways involved in cancer growth and development were proposed: histidine metabolism; tryptophan metabolism; arginine biosynthesis; arginine and proline metabolism; and alanine, aspartate and glutamine metabolism. The presented research identifies histidine and citrulline as potential new OC biomarkers. Furthermore, it provides evidence that amino acids are involved in metabolic pathways related to tumor growth and play an important role in cancerogenesis. [ABSTRACT FROM AUTHOR]

The synthetic resveratrol analogue DMU-212 (3,4,4′,5-tetramethoxystilbene) has been shown to possess stronger anticancer activity than resveratrol in a variety of tumour cells. To date, there has been no appropriate procedure that would ensure a reliable data about levels of metabolic products of DMU-212 in cancer cell lines. The purpose of this study was to develop a new procedure for determination of DMU-212 and its three metabolites (DMU-214, DMU-281, DMU-291) in cell lines. Analyses were performed using an HPLC system coupled with a triple quadrupole mass spectrometer operating in multiple reaction monitoring mode. Separation was conducted using a C18 column at a flow rate 800 μL/min with a mobile phase consisting of 5 mM ammonium acetate with 0.1% formic acid (solvent A) and acetonitrile (solvent B). The new methodology is fast, simple and has excellent specificity. Moreover, it showed good linearity in two matrices – cell lysates and culture media. Accuracy values for analytes evaluated at different concentration levels ranged from 0.43 to 18% (%bias). The intra-day and inter-day precision, expressed as CV, was in a range 0.49–5.5% and 0.83–13%, respectively. The validated procedure was successfully applied to quantify the resveratrol analogues in the human ovarian cancer cell line SKOV3. [ABSTRACT FROM AUTHOR]

The aim of the study was to identify significant abnormalities in angiogenic factor profiles occurring at early-stage non-small cell lung cancer (NSCLC). Contrary to the previous studies, our research included patients with only stage I NSCLC, which allowed for estimating the utility of circulating angiogenic factors in early NSCLC detection. The investigation was performed in serum samples collected from individuals with untreated NSCLC (n = 41) and a matched control group of healthy individuals (n = 61). All patients had histopathologically-confirmed stage IA or IB NSCLC. Serum concentrations of 16 angiogenesis markers comprising growth factors, receptors, cytokines, chemokines and hormones, were measured using a bead-based multiplex immunoassay. Among the determined proteins, osteopontin, platelet-derived growth factor-AB and −BB (PDGF-AB/BB) and hepatocyte growth factor (HGF) demonstrated the largest increase in concentration in NSCLC patients as compared with the non-cancer group. ROC curve analysis confirmed their high discriminatory abilities in detection of early NSCLC (AUC = 0.809, 95%CI 0.725–0.881). Serum levels of the studied angiogenic factors differed slightly between patients with lung adenocarcinoma and squamous cell carcinoma. The study demonstrated that osteopontin, PDGF-AB/BB, and HGF might add up to the methods used in NSCLC diagnosis. Future research should address the question about their specificity and performance in early NSCLC detection in a combination with various putative lung cancer markers. [ABSTRACT FROM AUTHOR]

Objectives Data from studies performed in Japanese and Korean populations suggest that free amino acid profiles have the potential to aid in non-small cell lung cancer (NSCLC) detection. However, there is still no data regarding abnormalities of free amino acids and their usefulness in NSCLC detection in European populations. The aim of the study was an evaluation of utility of amino acid profiles in NSCLC detection in Polish patients. Materials and methods Levels of 31 free amino acids were determined in 153 serum samples applying a liquid chromatography-tandem mass spectrometry-based methodology. Patients with I stage lung cancer represented a significant part of the studied group (46.7%). The obtained metabolite profiles along with clinical data were subjected to multivariate statistical tests. Results The presented study indicated that the increased serum level of phenylalanine and decreased level of citrulline are among the most robust cancer signatures in blood of NSCLC group. In addition, increased levels of aspartic acid and β-alanine were also recognized as important features of NSCLC. Amino acid selected based on studies of Asian patients were found to have insufficient specificity in NSCLC detection in the studied population. Therefore, we proposed a new set of 6 amino acids (aspartic acid, β-alanine, histidine, asparagine, phenylalanine and serine), which ensured higher accuracy in sample classification (from 90.3% to 77.1% depending of histological type). Conclusion We indicated that some of the free amino acid alterations occur in serum of NSCLC patients in early stage of disease and thus they can be valuable components of a blood multi-marker panel for NSCLC detection. [ABSTRACT FROM AUTHOR]

Due to high mortality rates of lung cancer, there is a need for identification of new, clinically useful markers, which improve detection of this tumor in early stage of disease. In the current study, serum peptide profiling was evaluated as a diagnostic tool for non-small cell lung cancer patients. The combination of the ZipTip technology with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for the analysis of peptide pattern of cancer patients (n = 153) and control subjects (n = 63) was presented for the first time. Based on the observed significant differences between cancer patients and control subjects, the classification model was created, which allowed for accurate group discrimination. The model turned out to be robust enough to discriminate a new validation set of samples with satisfactory sensitivity and specificity. Two peptides from the diagnostic pattern for non-small cell lung cancer (NSCLC) were identified as fragments of C3 and fibrinogen chain. Since ELISA test did not confirm significant differences in the expression of complement component C3, further study will involve a quantitative approach to prove clinical utility of the other proteins from the proposed multi-peptide cancer signature. [ABSTRACT FROM AUTHOR]

The aim of this pilot study was to apply a novel combined metabolomic and proteomic approach in analysis of gestational diabetes mellitus. The investigation was performed with plasma samples derived from pregnant women with diagnosed gestational diabetes mellitus (n = 18) and a matched control group (n = 13). The mass spectrometry-based analyses allowed to determine 42 free amino acids and low molecular-weight peptide profiles. Different expressions of several peptides and altered amino acid profiles were observed in the analyzed groups. The combination of proteomic and metabolomic data allowed obtaining the model with a high discriminatory power, where amino acids ethanolamine, L-citrulline, L-asparagine, and peptide ions with m/z 1488.59; 4111.89 and 2913.15 had the highest contribution to the model. The sensitivity (94.44%) and specificity (84.62%), as well as the total group membership classification value (90.32%) calculated from the post hoc classification matrix of a joint model were the highest when compared with a single analysis of either amino acid levels or peptide ion intensities. The obtained results indicated a high potential of integration of proteomic and metabolomics analysis regardless the sample size. This promising approach together with clinical evaluation of the subjects can also be used in the study of other diseases. [ABSTRACT FROM AUTHOR]

Beehive products possess nutritional value and health-promoting properties and are recommended as so-called "superfoods". However, because of their natural origin, they may contain relevant elemental contaminants. Therefore, to assess the quality of bee products, we examined concentrations of a broad range of 24 selected elements in propolis, bee pollen, and royal jelly. The quantitative analyses were performed with inductively coupled plasma-mass spectrometry (ICP-MS) and inductively coupled plasma optical emission spectrometry (ICP-OES) techniques. The results of our research indicate that bee products contain essential macronutrients (i.e., K, P, and S) and micronutrients (i.e., Zn and Fe) in concentrations depending on the products' type. However, the presence of toxic heavy metals makes it necessary to test the quality of bee products before using them as dietary supplements. Bearing in mind that bee products are highly heterogenous and, depending on the environmental factors, differ in their elemental content, it is necessary to develop standards regulating the acceptable levels of inorganic pollutants. Furthermore, since bees and their products are considered to be an effective biomonitoring tool, our results may reflect the environment's condition in west-central Poland, affecting the health and well-being of both humans and bees. [ABSTRACT FROM AUTHOR]

A growing interest in metabolomics studies of cultured cells requires development not only untargeted methods capable of fingerprinting the complete metabolite profile but also targeted methods enabling the precise and accurate determination of a selected group of metabolites. Proline metabolism affects many crucial processes at the cellular level, including collagen biosynthesis, redox balance, energetic processes as well as intracellular signaling. The study aimed to develop a robust and easy-to-use targeted metabolomics method for the determination of the intracellular level of proline and the other two amino acids closely related to proline metabolism: glutamic acid and arginine. The method employs hydrophilic interaction liquid chromatography followed by high-resolution, accurate-mass mass spectrometry for reliable detection and quantification of the target metabolites in cell lysates. The sample preparation consisted of quenching by the addition of ice-cold methanol and subsequent cell scraping into a quenching solution. The method validation showed acceptable linearity (r > 0.995), precision (%RSD < 15%), and accuracy (88.5–108.5%). Pilot research using HaCaT spontaneously immortalized human keratinocytes in a model for wound healing was performed, indicating the usefulness of the method in studies of disturbances in proline metabolism. The developed method addresses the need to determine the intracellular concentration of three key amino acids and can be used routinely in targeted mammalian cell culture metabolomics research. [ABSTRACT FROM AUTHOR]

Organic acids are important active small molecules present in venoms and toxins, which have not been fully explored yet. The aim of the study was the determination of organic acids in honeybee venom (HBV) samples by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two protocols for sample preparation were employed. A solid-phase extraction was used for the determination of malonic acid, fumaric acid, glutaric acid, and kynurenic acid. A dilute-and-shoot method was optimal for: citric acid, malic acid, and succinic acid. Chromatographic separation was performed using a Synergi Hydro-RP column. Detection was performed on a triple-quadrupole mass spectrometer operating in multiple reaction monitoring mode. Among the analytes, glutaric acid and kynurenic acid were present in HBV samples in the lowest concentrations, whereas citric acid was the most abundant acid in each sample, and accounted for an average of 86 mg/g (8.6%) of the venom dry weight. Organic acids were discussed in terms of function. This is the first study in the available literature that provides specific data on the content of organic acids in HBV using a validated quantitative method. [ABSTRACT FROM AUTHOR]

Both venoms and poisonous secretions are complex mixtures that assist in defense, predation, communication, and competition in the animal world. They consist of variable bioactive molecules, such as proteins, peptides, salts and also metabolites. Metabolomics opens up new perspectives for the study of venoms and poisons as it gives an opportunity to investigate their previously unexplored low molecular-weight components. The aim of this article is to summarize the available literature where metabolomic technologies were used for examining the composition of animal venoms and poisons. The paper discusses only the low molecular-weight components of venoms and poisons collected from snakes, spiders, scorpions, toads, frogs, and ants. An overview is given of the analytical strategies used in the analysis of the metabolic content of the samples. We paid special attention to the classes of compounds identified in various venoms and poisons and potential applications of the small molecules (especially bufadienolides) discovered. The issues that should be more effectively addressed in the studies of animal venoms and poisons include challenges related to sample collection and preparation, species-related chemical diversity of compounds building the metabolome and a need of an online database that would enhance identification of small molecule components of these secretions. [ABSTRACT FROM AUTHOR]

Highlights • Amino acid profile of RA samples was made using targeted metabolomics methodology. • Tryptophan and threonine were found as potential markers of RA therapy monitoring. • Arginine and glycine were selected as potential indicators due Steinbrocker scale. • Proposed amino acids are involved in the inflammatory or immune response. Abstract Background In presented study the amino acid analysis was performed in serum derived from rheumatoid arthritis patients (RA) according to undertaken therapy and classification of physical disability. The results were compared with previously published data. Methods The levels of 31 free amino acids were determined in 50 serum samples derived from RA subjects and 51 controls. The RA patients were divided into two groups according to the therapy (methotrexate/leflunomide, infliximab/adalimumab/etanercept/tocilizumab, prednisolone/NSAID) and classification of physical disability of the patients. Levels of amino acids were measured by LC-MS/MS. The obtained results were subjected to multivariate statistical tests. Results According to the therapy that was being used, threonine differentiated RA patients treated with methotrexate/leflunomide - infliximab/adalimumab/etanercept/tocilizumab (p = 0.00954) and infliximab/adalimumab/etanercept/tocilizumab - prednisolone/NSAID (p = 0.03109), while tryptophan differentiated RA patients treated with methotrexate/leflunomide - infliximab/adalimumab/etanercept/tocilizumab (p = 0.01723). In the functional classification, arginine differentiated RA samples between class III and IV (p = 0.02332), while glycine differentiated them between class I+II and III of the Steinbrocker functional classification (p = 0.03366). Conclusions An analysis of the metabolome profile requires the use of validated bioanalytical methods that are strictly dedicated for this purpose. The obtained results are not accidental (p value less than 0.05), and all of the selected amino acids play an important role in inflammation and immune response. It is suggested that studied amino acids can be considered as a markers for diagnosis of RA and monitoring pharmacotherapy of the disease. [ABSTRACT FROM AUTHOR]

Organic acids are important active small molecules present in venoms and toxins, which have not been fully explored yet. The aim of the study was the determination of organic acids in honeybee venom (HBV) samples by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two protocols for sample preparation were employed. A solid-phase extraction was used for the determination of malonic acid, fumaric acid, glutaric acid, and kynurenic acid. A dilute-and-shoot method was optimal for: citric acid, malic acid, and succinic acid. Chromatographic separation was performed using a Synergi Hydro-RP column. Detection was performed on a triple-quadrupole mass spectrometer operating in multiple reaction monitoring mode. Among the analytes, glutaric acid and kynurenic acid were present in HBV samples in the lowest concentrations, whereas citric acid was the most abundant acid in each sample, and accounted for an average of 86 mg/g (8.6%) of the venom dry weight. Organic acids were discussed in terms of function. This is the first study in the available literature that provides specific data on the content of organic acids in HBV using a validated quantitative method.

There is a great interest in searching for diagnostic biomarkers in prostate cancer patients. The aim of the pilot study was to evaluate free amino acid profiles in their serum and urine. The presented paper shows the first comprehensive analysis of a wide panel of amino acids in two different physiological fluids obtained from the same groups of prostate cancer patients (n = 49) and healthy men (n = 40). The potential of free amino acids, both proteinogenic and non-proteinogenic, as prostate cancer biomarkers and their utility in classification of study participants have been assessed. Several metabolites, which deserve special attention in the further metabolomic investigations on searching for prostate cancer markers, were indicated. Moreover, free amino acid profiles enabled to classify samples to one of the studied groups with high sensitivity and specificity. The presented research provides a strong evidence that ethanolamine, arginine and branched-chain amino acids metabolic pathways can be a valuable source of markers for prostate cancer. The altered concentrations of the above-mentioned metabolites suggest their role in pathogenesis of prostate cancer and they should be further evaluated as clinically useful markers of prostate cancer., Competing Interests: The authors have declared that no competing interest exists.