Overexpression of Prolidase Induces Autophagic Death in MCF-7 Breast Cancer Cells.

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.

LC-QTOF-MS and 1H NMR Metabolomics Verifies Potential Use of Greater Omentum for Klebsiella pneumoniae Biofilm Eradication in Rats.

Bacterial wound infections are a common problem associated with surgical interventions. In particular, biofilm-forming bacteria are hard to eradicate, and alternative methods of treatment based on covering wounds with vascularized flaps of tissue are being developed. The greater omentum is a complex organ covering the intestines in the abdomen, which support wound recovery following surgical procedures and exhibit natural antimicrobial activity that could improve biofilm eradication. We investigated changes in rats' metabolome following Klebsiella pneumoniae infections, as well as the greater omentum's ability for Klebsiella pneumoniae biofilm eradication. Rats received either sterile implants or implants covered with Klebsiella pneumoniae biofilm (placed in the peritoneum or greater omentum). Metabolic profiles were monitored at days 0, 2, and 5 after surgery using combined proton nuclear magnetic resonance (1H NMR) and high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (LC-QTOF­MS) measurements of urine samples followed by chemometric analysis. Obtained results indicated that grafting of the sterile implant to the greater omentum did not cause major disturbances in rats' metabolism, whereas the sterile implant located in the peritoneum triggered metabolic perturbations related to tricarboxylic acid (TCA) cycle, as well as choline, tryptophan, and hippurate metabolism. Presence of implants colonized with Klebsiella pneumoniae biofilm resulted in similar levels of metabolic perturbations in both locations. Our findings confirmed that surgical procedures utilizing the greater omentum may have a practical use in wound healing and tissue regeneration in the future.

Maternal Plasma Metabolomic Profiles in Spontaneous Preterm Birth: Preliminary Results.

OBJECTIVE: To profile maternal plasma metabolome in spontaneous preterm birth. METHOD: In this retrospective case-control study, we have examined plasma of patient with preterm birth (between 22 and 36 weeks of pregnancy (n = 57)), with threatened preterm labor (between 23 and 36 weeks of pregnancy (n = 49)), and with term delivery (n = 25). Plasma samples were analysed using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) in positive and negative polarity modes. RESULTS: We found 168 differentially expressed metabolites that were significantly distinct between study groups. We determined 51 metabolites using publicly available databases that could be subdivided into one of the five groups: amino acids, fatty acids, lipids, hormones, and bile acids. PLS-DA models, verified by SVM classification accuracy, differentiated preterm birth and term delivery groups. CONCLUSIONS: Maternal plasma metabolites are different between term and preterm parturitions. Part of them may be related with preterm labor, while others may be affected by gestational age or the beginning of labor. Metabolite profile can classify preterm or term delivery groups raising the potential of metabolome as a biomarker to identify high-risk pregnancies. Metabolomic studies are also a tool to detect individual compounds that may be further tested in targeted researches.

Constituents of Propolis: Chrysin, Caffeic Acid, p-Coumaric Acid, and Ferulic Acid Induce PRODH/POX-Dependent Apoptosis in Human Tongue Squamous Cell Carcinoma Cell (CAL-27).

Propolis evokes several therapeutic properties, including anticancer activity. These activities are attributed to the action of polyphenols. Previously it has been demonstrated, that one of the most abundant polyphenolic compounds in ethanolic extracts of propolis are chrysin, caffeic acid, p-coumaric acid, and ferulic acid. Although their pro-apoptotic activity on human tongue squamous cell carcinoma cells (CAL-27) was established previously, the detailed mechanism of this process remains unclear. Considering the crucial role of proline metabolism and proline dehydrogenase/proline oxidase (PRODH/POX) in the regulation of cancer cell survival/apoptosis, we studied these processes in polyphenol-treated CAL-27 cells. All studied polyphenols evoked anti-proliferative activity, accompanied by increased PRODH/POX, P53, active caspases-3 and -9 expressions and decreased collagen biosynthesis, prolidase activity and proline concentration in CAL-27 cells. These data suggest that polyphenols of propolis induce PRODH/POX-dependent apoptosis through up-regulation of mitochondrial proline degradation and down-regulation of proline utilization for collagen biosynthesis.

New potential biomarkers of acetaminophen-induced hepatotoxicity.

Acetaminophen (APAP) is one of the most common antipyretic and analgesic drugs. Despite various precautions patients use APAP in amounts exceeding acceptable daily doses. APAP overdosing contributes to APAP intoxication, which leads to acute liver injury or necessity of exigent liver transplantation. Biomarkers that can be helpful in early diagnosis of liver injury during APAP overdosing are studied worldwide. This review presents recent reports on new potential biomarkers and their prospective application in clinical practice.

Verification of chemical composition of commercially available propolis extracts by gas chromatography-mass spectrometry analysis.

Propolis is a resin that is collected by honeybees from various plant sources. Due to its pharmacological properties, it is used in commercial production of nutritional supplements in pharmaceutical industry. In this study, gas chromatography-mass spectrometry was applied for quality control analysis of the three commercial specimens containing aqueous-alcoholic extracts of bee propolis. More than 230 constituents were detected in analyzed products, including flavonoids, chalcones, cinnamic acids and their esters, phenylpropenoid glycerides, and phenylpropenoid sesquiterpenoids. An allergenic benzyl cinnamate ester was also identified in all tested samples. This analytical method allows to evaluate biological activity and potential allergenic components of bee glue simultaneously. Studies on chemical composition of propolis samples may provide new approach to quality and safety control analysis in production of propolis supplementary specimens.

Metabolomic study of plasma of patients with abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is an important health problem, both because of AAA rupture and death and because of increased cardiovascular mortality. Identification of new biomarkers of AAA may suggest novel pathological mechanisms and targets for new medical treatments to slow AAA progression. Metabolic changes in AAA patients were mainly related to carbohydrate and lipid metabolism and many of these changes can be associated with a situation of insulin resistance (which can be related to metabolic syndrome) together with altered amino acid metabolism. For the first time, metabolites that can be associated with differential metabolism by the gut microflora of AAA patients have also been found. Moreover, aminomalonic acid in plasma has been shown to be the metabolite with the biggest difference between patients suffering from large aneurysm (>5 cm) and controls.

Metabolomics with LC-QTOF-MS permits the prediction of disease stage in aortic abdominal aneurysm based on plasma metabolic fingerprint.

Abdominal aortic aneurysm (AAA) is a permanent and localized aortic dilation, defined as aortic diameter ≥3 cm. It is an asymptomatic but potentially fatal condition because progressive enlargement of the abdominal aorta is spontaneously evolving towards rupture.Biomarkers may help to explain pathological processes of AAA expansion, and allow us to find novel therapeutic strategies or to determine the efficiency of current therapies. Metabolomics seems to be a good approach to find biomarkers of AAA. In this study, plasma samples of patients with large AAA, small AAA, and controls were fingerprinted with LC-QTOF-MS. Statistical analysis was used to compare metabolic fingerprints and select metabolites that showed a significant change. Results presented here reveal that LC-QTOF-MS based fingerprinting of plasma from AAA patients is a very good technique to distinguish small AAA, large AAA, and controls. With the use of validated PLS-DA models it was possible to classify patients according to the disease stage and predict properly the stage of additional AAA patients. Identified metabolites indicate a role for sphingolipids, lysophospholipids, cholesterol metabolites, and acylcarnitines in the development and progression of AAA. Moreover, guanidinosuccinic acid, which mimics nitric oxide in terms of its vasodilatory action, was found as a strong marker of large AAA.

Targeted and non-targeted metabolic time trajectory in plasma of patients after acute coronary syndrome.

Metabolite fingerprinting (metabolomics/metabonomics) is perfectly suited for assessing the biological response following acute coronary syndrome (ACS) as relevant information can be identified in both the change and the absence of change in metabolite concentrations as time progresses post syndrome. During this study the metabolic pattern of plasma from patients at time points 0, four days, two months and six months after the onset of ACS were compared to controls using a non-targeted approach with gas chromatography mass spectrometry (GC-MS). Fatty acid profiles of the sample set were also analysed in a targeted way. The methods were employed with the aim to identify specific biomarkers, which vary with time. Using the non-targeted approach 27 statistically significant metabolites of interest were found: glucose, fructose, myoinositol, pyruvate, lactate, oxalate, citrate, isocitrate, succinate, malate, valine, alanine, serine, glycine, cysteine, threonine, aspartate, tryptophan, tyrosine, 4-hydroxyproline, 2-hydroxybutyrate, 2-aminobutyrate, 2,3,4-trihydroxybutyrate, 3-hydroxybutyrate, creatinine and aminomalonate. In addition, the targeted analysis of 21 fatty acids revealed patients within the group ACS at day 0 had the highest values for all 21. After 4 days, values decreased and were maintained at a lower level during the 6 months. Whereas the overall fatty acid profile did not change, different patterns of concentration trajectories over time were identified, which can reflect the underlying metabolic alterations as a result of the initial ACS, interestingly these levels had not fully reverted six months later.

Improving metabolite knowledge in stable atherosclerosis patients by association and correlation of GC-MS and 1H NMR fingerprints.

The plasma of patients with stable carotid atherosclerosis (n = 9), and healthy subjects (n = 10) have been fingerprinted with both GC-MS and (1)H NMR. Principal component analysis (PCA), partial least-squares-discriminant analysis (PLS-DA) and orthogonal partial least-squares-discriminant analysis (OPLS-DA) have been applied to the profiles from each technique both separately and in combination. These techniques complement each other and enable a clearer picture of the biological samples to be interpreted not only for classification purposes, but also more importantly to define the metabolic state of patients with carotid atherosclerosis. The results showed at least 24 metabolites that were significantly modified in the group of atherosclerotic patients by this nontargeted procedure. Most of the changes can be associated to alterations of the metabolism characteristics of insulin resistance that can be strongly related to the metabolic syndrome. In addition, correlations among variables accounting for the classification show amino acids as variables whose changes showed a high degree of correlation. GC-MS and (1)H NMR fingerprints can provide complementary information in the identification of altered metabolic pathways in patients with carotid atherosclerosis. Moreover, correlations among the results with both techniques, instead of a single study, can provide a deeper insight into the patient state.