Red Kale (Brassica oleracea L. ssp. acephala L. var. sabellica) Induces Apoptosis in Human Colorectal Cancer Cells In Vitro.

This article presents the results of studies investigating the effect of red kale (Brassica oleracea L. ssp. acephala L. var. sabellica) extract on cancer cells (HT-29). The cytotoxicity of the red kale extract was assessed using MTT and LDH assays, while qRT-PCR was employed to analyze the expression of genes associated with the p53 signaling pathway to elucidate the effect of the extract on cancer cells. Furthermore, HPLC-ESI-QTOF-MS/MS was applied to identify bioactive compounds present in red kale. The obtained results indicated that red kale extract reduced the viability and suppressed the proliferation of HT-29 cells (the IC50 value of 60.8 µg/mL). Additionally, mRNA expression analysis revealed significant upregulation of several genes, i.e., casp9, mapk10, mapk11, fas, kat2 b, and ubd, suggesting the induction of cell apoptosis through the caspase-dependent pathway. Interestingly, the study revealed a decrease in the expression of genes including cdk2 and cdk4 encoding cell cycle-related proteins, which may lead to cell cycle arrest. Furthermore, the study identified certain bioactive compounds, such as sinigrin, spirostanol, hesperetin and usambarensine, which could potentially contribute to the apoptotic effect of red kale extracts. However, further investigations are necessary to elucidate the specific role of these individual compounds in the anti-cancer process.

Reliable chromatographic assay for measuring of indoleamine 2,3-dioxygenase 1 (IDO1) activity in human cancer cells.

The kynurenine pathway is the major tryptophan degradation routes generating bioactive compounds important in physiology and diseases. Depending on cell type it is initiated enzymatically by tryptophan-2,3-dioxygenase (TDO) or indoleamine-2,3-dioxygenase 1 and 2 (IDO1 and IDO2) to yield N-formylkynurenine as the precursor of further metabolites. Herein, we describe an accurate high-pressure liquid chromatography coupled with a diode array detector (HPLC-DAD) method to serve for IDO1 activity determination in human cancer cells cultured in vitro. Enzymatic activity was expressed as the rate of ʟ-kynurenine generation by 1 mg of proteins obtained from cancer cells. Our approach shows the limit of detection and limit of quantification at 12.9 and 43.0 nM Kyn, respectively. Applicability of this method was demonstrated in different cells (ovarian and breast cancer)exposed to various conditions and has successfully passed the validation process. This approach presents a useful model to study the role of kynurenine pathway in cancer biology.

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures.

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines. Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode. This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method generates reliable results and can be further applied to other in vitro cellular models.

Application of the optimized and validated LC-MS method for simultaneous quantification of tryptophan metabolites in culture medium from cancer cells.

Kynurenine pathway is the main route of tryptophan degradation generating a number of immunoregulatory compounds. Some conditions like oxidative stress, inflammatory factors might enhance tryptophan degradation. Process is active in several cells including fibroblasts, cancer cells, and immune cells, therefore it is intensively studied in context of cancer microenvironment. The validated and standardized methodology for kynurenine quantification is crucial for reliable comparison of results obtained in different studies. This paper concerns an approach for simultaneous quantification of four major tryptophan metabolites of the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in cell culture supernatants by liquid chromatography coupled with single quadrupole mass spectrometer. During development of the novel method, the principal component analysis was used to select the best mobile phase and to ensure the optimal conditions for simultaneous quantification of metabolites. The analysis involves simple protein precipitation with acidified methanol and 3-nitrotyrosine as an internal standard. The obtained limits of detection and quantification in cell culture medium were in the range of 3.31-10.80 nmol/L and 9.60-19.50 nmol/L, respectively. At the validation step, other method parameters (linearity, precision, accuracy, recovery, matrix effects) were also evaluated and satisfactory results were obtained for all target compounds. The method was applied to study tryptophan metabolites by determination of kynurenines in cell culture medium from two different human cancer cell lines (MDA-MD-231 and SK-OV-3) in context of exposure to glycation products.

Mutation in the pssA gene involved in exopolysaccharide synthesis leads to several physiological and symbiotic defects in Rhizobium leguminosarum bv. trifolii.

The symbiotic nitrogen-fixing bacterium Rhizobium leguminosarum bv. trifolii 24.2 secretes large amounts of acidic exopolysaccharide (EPS), which plays a crucial role in establishment of effective symbiosis with clover. The biosynthesis of this heteropolymer is conducted by a multi-enzymatic complex located in the bacterial inner membrane. PssA protein, responsible for the addition of glucose-1-phosphate to a polyprenyl phosphate carrier, is involved in the first step of EPS synthesis. In this work, we characterize R. leguminosarum bv. trifolii strain Rt270 containing a mini-Tn5 transposon insertion located in the 3'-end of the pssA gene. It has been established that a mutation in this gene causes a pleiotropic effect in rhizobial cells. This is confirmed by the phenotype of the mutant strain Rt270, which exhibits several physiological and symbiotic defects such as a deficiency in EPS synthesis, decreased motility and utilization of some nutrients, decreased sensitivity to several antibiotics, an altered extracellular protein profile, and failed host plant infection. The data of this study indicate that the protein product of the pssA gene is not only involved in EPS synthesis, but also required for proper functioning of Rhizobium leguminosarum bv. trifolii cells.