Chemical Components of Oxytropis pseudoglandulosa Induce Apoptotic-Type Cell Death of Caco-2 Cells.

Oxytropis pseudoglandulosa plant is used in traditional Mongolian medicine. However, its chemical composition and biological properties are poorly explored. In this study, the total content of polyphenols and flavonoids as well as antioxidant activity were verified in plant extract. The total phenolic and flavonoid contents were determined by spectrometric (6.62 mg GAE/g and 10.32 mg QE/g) and chromatographic (17,598 mg/kg and 17,467 mg/kg) assays. The antioxidant potential was investigated by DPPH assay and yielded IC50 at 18.76 µg/mL. Twelve phenolic compounds were identified as components of O. pseudoglandulosa extract. Kaempferol-3-O-robinosyl-7-O-rhamnoside and kaempferol-3-(p-coumaroyl)-rutinosyl-7-rhamnoside made up 80% of determined components and were found to be the major polyphenolic compounds. The biological properties of O. pseudoglandulosa extracts were determined in vitro using human epithelial adenocarcinoma Caco-2 cell line. Low concentrations of extract (0-30 µg/mL) exhibited protective effects against cell damage caused by chemically induced oxidative stress. Elevated concentrations, on the other hand, resulted in apoptotic-type cell death induction. Metabolic failure, ROS elevation and membrane permeabilization observed in cells upon incubation with extract dosages above 50 µg/mL allowed us to conclude on O. pseudoglandulosa being predominantly a necrosis inducer.

Extracellular Nucleotides Affect the Proangiogenic Behavior of Fibroblasts, Keratinocytes, and Endothelial Cells.

Chronic wound healing is currently a severe problem due to its incidence and associated complications. Intensive research is underway on substances that retain their biological activity in the wound microenvironment and stimulate the formation of new blood vessels critical for tissue regeneration. This group includes synthetic compounds with proangiogenic activity. Previously, we identified phosphorothioate analogs of nucleoside 5'-O-monophosphates as multifunctional ligands of P2Y6 and P2Y14 receptors. The effects of a series of unmodified and phosphorothioate nucleotide analogs on the secretion of VEGF from keratinocytes and fibroblasts, as well as their influence on the viability and proliferation of keratinocytes, fibroblasts, and endothelial cells were analyzed. In addition, the expression profiles of genes encoding nucleotide receptors in tested cell models were also investigated. In this study, we defined thymidine 5'-O-monophosphorothioate (TMPS) as a positive regulator of angiogenesis. Preliminary analyses confirmed the proangiogenic potency of TMPS in vivo.

Phenolics-Rich Extracts of Dietary Plants as Regulators of Fructose Uptake in Caco-2 Cells via GLUT5 Involvement.

The latest data link the chronic consumption of large amounts of fructose present in food with the generation of hypertension and disturbances in carbohydrate and lipid metabolism, which promote the development of obesity, non-alcoholic fatty liver disease, insulin resistance, and type 2 diabetes. This effect is possible after fructose is absorbed by the small intestine cells and, to a lesser extent, by hepatocytes. Fructose transport is dependent on proteins from the family of glucose transporters (GLUTs), among which GLUT5 selectively absorbs fructose from the intestine. In this study, we examined the effect of four phenolic-rich extracts obtained from A. graveolens, B. juncea, and M. chamomilla on fructose uptake by Caco-2 cells. Extracts from B. juncea and M. chamomilla most effectively reduced fluorescent fructose analogue (NBDF) accumulation in Caco-2, as well as downregulated GLUT5 protein levels. These preparations were able to decrease the mRNA level of genes encoding transcription factors regulating GLUT5 expression-thioredoxin-interacting protein (TXNIP) and carbohydrate-responsive element-binding protein (ChREBP). Active extracts contained large amounts of apigenin and flavonols. The molecular docking simulation suggested that some of identified phenolic constituents can play an important role in the inhibition of GLUT5-mediated fructose transport.

Comparison of Chemical Composition and Antioxidant Capacity of Fruit, Flower and Bark of Viburnum opulus.

In this work, the profiles of phenolics, fiber, pectins, sugars, organic acids and carotenoids, vitamin C, ash, protein and fat contents, as well as antioxidant capacity were compared in fruits, flowers, and bark of Viburnum opulus (VO). Antioxidant capacity was evaluated against ABTS, hydroxyl, peroxyl and superoxide free radicals, and as a reducing power by using in vitro test. The results showed great quantitative differences in the composition of the VO morphological parts tested. Fruits contained the highest concentrations of fat, organic acids, sugars, soluble dietary fiber (10.57 ± 0.54; 7.34 ± 0.06; 32.27 ± 1.25; 6.82 ± 0.38 g/100 g DW, respectively) and carotenoids (2.70 ± 0.07 mg/100 g DW). Whereas, the bark exceeded the remaining parts of the VO in terms of antioxidant capacity, ash (9.32 ± 0.17 g/100 g DW), total (59.34 ± 0.75 g/100 g DW) and insoluble dietary fiber (58.20 ± 0.73 g/100 g DW) contents as well as phenolic compounds (3.98 ± 0.04 g/100 g DW). Among the phenolic compounds quantified in this study, chlorogenic acid and (+)-catechin had the highest concentrations (> 1 g/100 g DW) in the flowers and bark, respectively.

2-OMe-lysophosphatidylcholine analogues are GPR119 ligands and activate insulin secretion from ßTC-3 pancreatic cells: Evaluation of structure-dependent biological activity.

GPR119 receptor has been proposed as a metabolic regulator playing a pivotal role in the modulation of glucose homeostasis in type 2 diabetes. GPR119 was identified on pancreatic ß cells and its ligands have the ability to enhance glucose-stimulated insulin secretion (GSIS). Lysophosphatidylcholine (LPC) was shown to potentiate GSIS and our present studies indicate that 2-methoxy-lysophosphatidylcholine (2-OMe-LPC) analogues, unable to undergo 1→2 acyl migration, stimulate GSIS from murine ßTC-3 pancreatic cells even more efficiently. Moreover, biological assays in engineered Tango™ GPR119-bla U2OS cells were carried out to ascertain the agonist activity of 2-OMe-LPC at GPR119. 2-OMe-LPC possessing in sn-1 position the residues of myristic, palmitic, stearic and oleic acid were also evaluated as factors regulating [Ca2+]i mobilization and cAMP levels. Extension of these studies to R- and S-enantiomers of 14:0 2-OMe-LPC revealed that the overall impact on GSIS does not depend on chirality, however, the intracellular calcium mobilization data show that the R enantiomer is significantly more active than S one. Taking into account differences in chemical structure between various native LPCs and their 2-methoxy counterparts the possible binding mode of 2-OMe-LPC to the GPR119 receptor was determined using molecular modeling approach.

Lysophosphatidylcholine elicits intracellular calcium signaling in a GPR55-dependent manner.

The GPR55 signaling is fertile ground for drug discovery, however despite considerable research progress during the past 10 years, many open questions remain. The GPR55 pharmacology remains controversial, as many ligands have been reported with inconsistent results. Here, we show that various molecular species of lysophosphatidylcholine (LPC) elicit intracellular Ca2+ mobilization in GPR55-expressing PC-3 human prostate carcinoma cells. The response was even stronger than [Ca2+]i flux evoked by endogenous (OEA) and synthetic (Abn-CBD) agonists. Treatment with GPR55 antagonists CID16020046 and ML193 as well as the lipid raft disrupter methyl-ß-cyclodextrin strongly blunted LPC-induced calcium signal. Additionally, molecular modeling analysis revealed that LPC 16:0 and LPC 18:1 interact stronger with the receptor than to OEA. Identified electrostatic interactions between GPR55 residues and the ligands overlap with the binding site identified previously for lysophosphatidylinositol. Therefore, we prove that LPC is another GPR55-sensitive ligand. This finding is relevant in understanding lysophospolipids-mediated signaling and opens new avenues to develop therapeutic approach based on GPR55 targeting.

The chemical synthesis and preliminary biological studies of phosphodiester and phosphorothioate analogues of 2-methoxy-lysophosphatidylethanolamine.

The chemical synthesis of phosphorothioate/phosphodiester analogues of 2-methoxy-lysophosphatidylethanolamine has been described. For the preparation of phosphorothioate derivatives oxathiaphospholane approach has been employed. The phosphodiester compounds were prepared by OXONE® oxidation of corresponding phosphorothioates. Each lysophospholipid analogue was synthesized as a series of four compounds, bearing different fatty acid residues both saturated (14:0, 16:0, 18:0) and unsaturated (18:1). The methylation of glycerol 2-hydroxyl function was applied in order to increase the stability of prepared analogues by preventing 1→2 acyl migration. The cytotoxicity of newly synthesized 2-methoxy-lysophosphatidylethanolamine derivatives was evaluated with resazurin-based method in prostate cancer PC3 cell line. The highest reduction of cell viability was noted for LPE analogues containing myristoyl acyl chain.

Influence of polyphenol extract from evening primrose (Oenothera paradoxa) seeds on human prostate and breast cancer cell lines.

There is growing interest in plant polyphenols which exhibit pleiotropic biological activities, including anti-inflammatory, antioxidant, and anticancer effects. The objective of our study was to evaluate the influence of an evening primrose extract (EPE) from defatted seeds on viability and invasiveness of three human cell lines: PNT1A (normal prostate cells), DU145 (prostate cancer cells) and MDA-MB-231 (breast cancer cells). The results revealed that after 72 h of incubation the tested extract reduced the viability of DU 145 and MDA-MB-231 with IC50 equal to 14.5 µg/mL for both cell lines. In contrast, EPE did not inhibit the viability of normal prostate cells. Furthermore, EPE reduced PNT1A and MDA-MB-231 cell invasiveness; at the concentration of 21.75 µg/mL the suppression of invasion reached 92% and 47%, respectively (versus control). Additionally, zymographic analysis revealed that after 48 h of incubation EPE inhibited metalloproteinase-2 (MMP-2) and metalloproteinase-9 (MMP-9) activities in a dose-dependent manner. For PNT1A the activities of MMP-2 and MMP-9 decreased 4- and 2-fold, respectively, at EPE concentration of 29 µg/mL. In the case of MDA-MB-231 and DU 145 the decrease in MMP-9 activity at EPE concentration of 29 µg/mL was 5.5-fold and almost 1.9-fold, respectively. In conclusion, this study suggests that EPE may exhibit antimigratory, anti-invasive and antimetastatic potential towards prostate and breast cancer cell lines.

Lysophosphatidylcholine and lysophosphatidylinosiol--novel promissing signaling molecules and their possible therapeutic activity.

For many years the role of lysophospholipids (LPLs) was associated only with structural and storage components of the cell without any informational function. Today, based on many research projects performed during the last decades, it is clear that some of the LPLs act as hormone-like signaling molecules and thus are very important inter- and intracellular lipid mediators. They can activate specific membrane receptors and/or nuclear receptors regulating many crucial physiological and pathophysiological processes. The LPLs were iden- tified as involved in a majority of cellular processes, including modulation of disease-related mechanisms observed, for instance, in case of diabetes, obesity, atherosclerosis and cancer. Among LPLs, lysophosphatidylcholine (LPC) and lysophosphatidylinositol (LPI) are becoming attractive research topics. Their recently revealed activities as novel ligands of orphan G protein-coupled receptors (i.e., GPR55 and GPR119) involved in modulation of tumor physiology and insulin secretion seem to be one of the most interesting aspects of these compounds. Moreover, the most recent scientific reports emphasize the significance of the acyl chain structure bound to the glycerol basis of LPL, as it entails different biological properties and activities of the compounds.

Lysophosphatidylcholines Enriched with cis and trans Palmitoleic Acid Regulate Insulin Secretion via GPR119 Receptor.

Among lipids, lysophosphatidylcholines (LPCs) with various fatty acyl chains have been identified as potential agonists of G protein-coupled receptors (GPCRs). Recently, targeting GPCRs has been switched to diabetes and obesity. Concomitantly, our last findings indicate the insulin secretagogue properties of cis and trans palmitoleic acid (16:1, n-7) resulting from GPCR activation, however, associated with different signaling pathways. We here report the synthesis of LPCs bearing two geometrical isomers of palmitoleic acids and investigation of their impact on human pancreatic ß cells viability, insulin secretion, and activation of four GPCRs previously demonstrated to be targeted by free fatty acids and LPCs. Moreover, molecular modeling was exploited to investigate the probable binding sites of tested ligands and calculate their affinity toward GPR40, GPR55, GPR119, and GPR120 receptors.

Flavanols from Japanese quince (Chaenomeles japonica) fruit inhibit human prostate and breast cancer cell line invasiveness and cause favorable changes in Bax/Bcl-2 mRNA ratio.

Polyphenols are natural compounds of high structural diversity which translates into a very wide spectrum of biological activities, including chemoprevention. Here we report that a Japanese quince fruit flavanol preparation (JQFFP) caused favorable changes in Bax/Bcl-2 mRNA ratio, which rendered normal and cancer cells more resistant and more sensitive, respectively, to apoptosis. DU145 human prostate cancer cells were characterized by the most advantageous Bax/Bcl-2 ratio. The growth and invasiveness of MDA-MB-231 human breast cancer cells were strongly suppressed by JQFFP, which was accompanied with a decrease in MMP-9 activity and stimulation of TIMP-1 expression. Importantly, JQFFP did not decrease normal human prostate PNT1A cell number, whereas Bax/Bcl-2 ratio decreased which implies increased resistance to apoptosis. In conclusion, JQFFP exhibited a potent antiproliferative effect against cancer cells, inhibited their invasiveness, and decreased expression level of several genes involved in apoptosis, angiogenesis, and metastasis.

Trichostatin A inhibits expression of the human SLC2A5 gene via SNAI1/SNAI2 transcription factors and sensitizes colon cancer cells to platinum compounds.

GLUT5, a key protein encoded by the SLC2A5 gene, is involved in the uptake of fructose from the intestine. Currently, with the increased consumption of this sugar and the associated increased incidence of obesity, diabetes and cancer, GLUT5 may represent an important molecular target in the prevention and treatment of these diseases. Here, we demonstrate that overexpression of the SNAI1 and SNAI2 transcription factors in cells expressing high levels of SLC2A5 mRNA reduced SLC2A5 gene expression. Furthermore, a histone deacetylase inhibitor, trichostatin A, which induces SNAI1 and SNAI2 expression, inhibits SLC2A5/GLUT5 expression and sensitizes colon cancer cells to cisplatin and oxaliplatin. This finding might have potential relevance for the development of therapeutic treatments aimed at modulating fructose transport or genes involved in this process for use with certain cancers.

Trans-palmitoleic acid, a dairy fat biomarker, stimulates insulin secretion and activates G protein-coupled receptors with a different mechanism from the cis isomer.

Dietary trans-palmitoleic acid (trans 16:1n-7, tPOA), a biomarker for high-fat dairy product intake, has been associated with a lower risk of type 2 diabetes mellitus (T2DM) in some cross-sectional and prospective epidemiological studies. Here, we investigated the insulin secretion-promoting activity of tPOA and compared them with the effects evoked by the cis-POA isomer (cPOA), an endogenous lipokine biosynthesized in the liver and adipose tissue, and found in some natural food sources. The debate about the positive and negative relationships of those two POA isomers with metabolic risk factors and the underlying mechanisms is still going on. Therefore, we examined the potency of both POA isomers to potentiate insulin secretion in murine and human pancreatic ß cell lines. We also investigated whether POA isomers activate G protein-coupled receptors proposed as potential targets for T2DM treatment. We show that tPOA and cPOA augment glucose-stimulated insulin secretion (GSIS) to a similar extent; however, their insulin secretagogue activity is associated with different signaling pathways. We also performed ligand docking and molecular dynamics simulations to predict the preferred orientation of POA isomers and the strength of association between those two fatty acids and GPR40, GPR55, GPR119, and GPR120 receptors. Overall, this study provides insight into the bioactivity of tPOA and cPOA toward selected GPCR functions, indicating them as targets responsible for the insulin secretagogue action of POA isomers. It reveals that both tPOA and cPOA may promote insulin secretion and subsequently regulate glucose homeostasis.

[Cyclic phosphatidic acids and their analogues--unique lipid mediators]. / Cykliczne kwasy fosfatydowe i ich analogi--mediatory lipidowe o unikalnych wlasciwosciach.

Lysophosphatidic acid (1-acyl-2-sn-glycerol-3-phosphate; LPA) and its naturally occurring analog, cyclic phosphatidic acid (1-acyl-sn-glycerol-2,3-cyclic phosphate; cPA) belong to a group of bioactive glycerophospholipids, which attract attention of many scientists because of their biological functions. Among these two compounds LPA is known better; information about unique biological properties of cPA appeared for the first time in the 90's. The synthesis of various, chemically modified analogues of cPA was performed to highlight mechanisms of the compound actions. Both native cPA and its derivatives emerge into the limelight because of their anti-cancer activities. Knowledge about pathways of biosynthesis and biodegradation of LPA and cPA as well as understanding of mechanisms of their action are increasing gradually. Previous studies have shown that both the metabolism and signaling cascades of these compounds have numerous common points. What is even more interesting, LPA and cPA seem to induce opposite biological activities.

Advanced Glycation End-Products (AGEs): Formation, Chemistry, Classification, Receptors, and Diseases Related to AGEs.

Advanced glycation end-products (AGEs) constitute a non-homogenous, chemically diverse group of compounds formed either exogeneously or endogeneously on the course of various pathways in the human body. In general, they are formed non-enzymatically by condensation between carbonyl groups of reducing sugars and free amine groups of nucleic acids, proteins, or lipids, followed by further rearrangements yielding stable, irreversible end-products. In the last decades, AGEs have aroused the interest of the scientific community due to the increasing evidence of their involvement in many pathophysiological processes and diseases, such as diabetes, cancer, cardiovascular, neurodegenerative diseases, and even infection with the SARS-CoV-2 virus. They are recognized by several cellular receptors and trigger many signaling pathways related to inflammation and oxidative stress. Despite many experimental research outcomes published recently, the complexity of their engagement in human physiology and pathophysiological states requires further elucidation. This review focuses on the receptors of AGEs, especially on the structural aspects of receptor-ligand interaction, and the diseases in which AGEs are involved. It also aims to present AGE classification in subgroups and to describe the basic processes leading to both exogeneous and endogeneous AGE formation.

Procyanidins from Japanese quince (Chaenomeles japonica) fruit induce apoptosis in human colon cancer Caco-2 cells in a degree of polymerization-dependent manner.

Plant proanthocyanidins, including procyanidins, display various biological activities. Here we report an inhibition of human colon cancer Caco-2 cell growth by the extract from Japanese quince fruit and the procyanidin-rich fractions of the extract. We observed that the amount of apoptotic Caco-2 cells increased by 52.1% vs. control after 72-h incubation with 50 µg extract/mL, as assessed by flow cytometry and image cytometry. Under the same experimental conditions the corresponding values for human colon cancer HT-29 cells and for rat normal intestinal IEC-6 cells were 5.0% and 8.1%, respectively. The extract fractions enriched with higher oligomers exhibited the highest proapoptotic activity. In conclusion, the Japanese quince procyanidins exhibited proapoptotic activity in Caco-2 cells within a submilimolar concentration range.

Formation of ßTC3 and MIN6 Pseudoislets Changes the Expression Pattern of Gpr40, Gpr55, and Gpr119 Receptors and Improves Lysophosphatidylcholines-Potentiated Glucose-Stimulated Insulin Secretion.

The impaired spatial arrangement and connections between cells creating islets of Langerhans as well as altered expression of G protein-coupled receptors (GPCRs) often lead to dysfunction of insulin-secreting pancreatic ß cells and can significantly contribute to the development of diabetes. Differences in glucose-stimulated insulin secretion (GSIS) are noticeable not only in diabetic individuals but also in model pancreatic ß cells, e.g., ßTC3 and MIN6 ß cell lines with impaired and normal insulin secretion, respectively. Now, we compare the ability of GPCR agonists (lysophosphatidylcholines bearing fatty acid chains of different lengths) to potentiate GSIS in ßTC3 and MIN6 ß cell models, cultured as adherent monolayers and in a form of pseudoislets (PIs) with pancreatic MS1 endothelial cells. Our aim was also to investigate differences in expression of the GPCRs responsive to LPCs in these experimental systems. Aggregation of ß cells into islet-like structures greatly enhanced the expression of Gpr40, Gpr55, and Gpr119 receptors. In contrast, the co-culture of ßTC3 cells with endothelial cells converted the GPCR expression pattern closer to the pattern observed in MIN6 cells. Additionally, the efficiencies of various LPC species in ßTC3-MS1 PIs also shifted toward the MIN6 cell model.

Targeting GPCRs Activated by Fatty Acid-Derived Lipids in Type 2 Diabetes.

G protein-coupled receptors (GPCRs) are the most intensively studied drug targets, because of their diversity, cell-specific expression, and druggable sites accessible at the cell surface. Preclinical and clinical studies suggest that targeting GPCRs activated by fatty acid-derived lipids may have potential to improve glucose homeostasis and reduce complications in patients with type 2 diabetes (T2D). Despite the discontinued development of fasiglifam (TAK-875), the first FFA1 agonist to reach late-stage clinical trials, lipid-sensing receptors remain a viable target, albeit with a need for further characterization of their binding mode, intracellular signaling, and toxicity. Herein, we analyze general discovery trends, various signaling pathways, as well as possible challenges following activation of GPCRs that have been validated clinically to control blood glucose levels.

Viburnum opulus Fruit Phenolic Compounds as Cytoprotective Agents Able to Decrease Free Fatty Acids and Glucose Uptake by Caco-2 Cells.

In recent years, there has been increasing interest in studying food-originated phytocompounds with beneficial influences for humans. Amongst the most active natural substances are polyphenols, for which high content has been identified in the Viburnum opulus berry, and which are unused in Western Europe. Due to its strong antioxidant activity we explored the potential of V. opulus as a preventive agent against diet-related chronic diseases, such as obesity and type 2 diabetes. Among the causes of these ailments is oxidative stress, as well as impaired glucose and free fatty acids (FFA) uptake. Thus, the purpose of this study was to determine biological activity of V. opulus phenolic extracts as cytoprotective agents able to decrease induced oxidative stress, lower lipid accumulation and attenuate glucose and FFA uptake by Caco-2 cells via GLUT2 and CD36/FAT transporters. To determine the source of the most biologically active phenolic compounds, we obtained four phenolic compounds extracts as crude juice, phenolics isolated from juice and two preparations of phenolics obtained with different extraction agents from fruit pomace. Among the studied extracts, the phenolic rich fraction obtained from fruit juice revealed the strongest activity to decrease uptake of glucose, FFA and accumulation of lipid droplets in Caco-2 cells without affecting their viability (IC0 50 µg/mL). Observed uptake attenuation was followed by decrease of the CD36/FAT gene expression, without influence on the GLUT2 and PPARα levels. We suspect that V. opulus phenolics were able to modulate the cellular membrane dynamic, although that hypothesis requires further, more detailed studies. Extracts revealed strong chemo-preventive activity against oxidative stress induced chemically by tert-butylhydroperoxide (t-BOOH), as well as against DNA damage through the induction of DNA repair after cell exposition to methylnitronitrosoguanidine (MNNG) and H2O2. Our findings suggest Viburnum opulus fruit as a dietary source of phytocompounds, which could be considered as a tailored design food supplement components for the prevention and treatment of postprandial elevation of glucose and fatty acids through delaying the rate of glucose and fatty acid absorption by intestinal cells.

Lysophosphatidylcholine and its phosphorothioate analogues potentiate insulin secretion via GPR40 (FFAR1), GPR55 and GPR119 receptors in a different manner.

Lysophosphatidylcholine (LPC) is an endogenous ligand for GPR119 receptor, mediating glucose-stimulated insulin secretion (GSIS). We demonstrate that LPC facilitates GSIS in MIN6 pancreatic ß-cell line and murine islets of Langerhans by recognizing not only GPR119 but also GPR40 (free fatty acid receptor 1) and GPR55 activated by lysophosphatidylinositol. Natural LPCs are unstable when administered in vivo limiting their therapeutic value and therefore, we present phosphorothioate LPC analogues with increased stability. All the modified LPCs under study (12:0, 14:0, 16:0, 18:0, and 18:1) significantly enhanced GSIS. The 16:0 sulfur analogue was the most potent, evoking 2-fold accentuated GSIS compared to the native counterpart. Interestingly, LPC analogues evoked GPR40-, GPR55-and GPR119-dependent [Ca2+]i signaling, but did not stimulate cAMP accumulation as in the case of unmodified molecules. Thus, introduction of a phosphorothioate function not only increases LPC stability but also modulates affinity towards receptor targets and evokes different signaling pathways.