Eliana Spilioti

Phenolic acid composition, antiatherogenic and anticancer potential of honeys derived from various regions in Greece.

The phenolic acid profile of honey depends greatly on its botanical and geographical origin. In this study, we carried out a quantitative analysis of phenolic acids in the ethyl acetate extract of 12 honeys collected from various regions in Greece. Our findings indicate that protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, caffeic acid and p-coumaric acid are the major phenolic acids of the honeys examined. Conifer tree honey (from pine and fir) contained significantly higher concentrations of protocatechuic and caffeic acid (mean: 6640 and 397 µg/kg honey respectively) than thyme and citrus honey (mean of protocatechuic and caffeic acid: 437.6 and 116 µg/kg honey respectively). p-Hydroxybenzoic acid was the dominant compound in thyme honeys (mean: 1252.5 µg/kg honey). We further examined the antioxidant potential (ORAC assay) of the extracts, their ability to influence viability of prostate cancer (PC-3) and breast cancer (MCF-7) cells as well as their lowering effect on TNF- α-induced adhesion molecule expression in endothelial cells (HAEC). ORAC values of Greek honeys ranged from 415 to 2129 µmol Trolox equivalent/kg honey and correlated significantly with their content in protocatechuic acid (p<0.001), p-hydroxybenzoic acid (p<0.01), vanillic acid (p<0.05), caffeic acid (p<0.01), p-coumaric acid (p<0.001) and their total phenolic content (p<0.001). Honey extracts reduced significantly the viability of PC-3 and MCF-7 cells as well as the expression of adhesion molecules in HAEC. Importantly, vanillic acid content correlated significantly with anticancer activity in PC-3 and MCF-7 cells (p<0.01, p<0.05 respectively). Protocatechuic acid, vanillic acid and total phenolic content correlated significantly with the inhibition of VCAM-1 expression (p<0.05, p<0.05 and p<0.01 respectively). In conclusion, Greek honeys are rich in phenolic acids, in particular protocatechuic and p-hydroxybenzoic acid and exhibit significant antioxidant, anticancer and antiatherogenic activities which may be attributed, at least in part, to their phenolic acid content.

Advanced glycation end-products induce endoplasmic reticulum stress in human aortic endothelial cells.

Abstract Background: Advanced glycation end products (AGEs), the final products of the Maillard reaction, have been shown to impair endothelial proliferation and function, thus contributing to endothelial cell injury present in diabetes, inflammatory and cardiovascular diseases. Endoplasmic reticulum (ER) stress triggered under hyperglycemic, hypoxic and oxidative conditions has been implicated in endothelial dysfunction through activation of the unfolded protein response (UPR). The present study investigates the role of AGEs in ER stress induction in human aortic endothelial cells exposed to variable AGE treatments. Methods: Human aortic endothelial cells (HAEC) were treated with increasing concentrations (100, 200 μg/mL) of AGE-bovine serum albumin (AGE-BSA) at different time-points (24, 48, 72 h). The induction of ER stress and the involved UPR components were investigated on mRNA and protein levels. Apoptosis was quantitatively determined by flow cytometry detecting propidium iodide expression and annexin V binding simultaneously. Results: AGEs administration significantly reduced HAEC proliferation in a time- and dose-dependent manner. An immediate induction of the ER chaperones GRP78, GRP94 and the transcriptional activator, XBP-1 was observed at 24 h and 48 h. A later induction of the phospho-elF2α and proapoptotic transcription factor CHOP was observed at 48 h and 72 h, being correlated with elevated early apoptotic cell numbers at the same time-points. Conclusions: The present study demonstrates that AGEs directly induce ER stress in human aortic endothelial cells, playing an important role in endothelial cell apoptosis. Targeting AGEs signaling pathways in order to alleviate ER stress may prove of therapeutic potential to endothelial dysfunction-related disorders.

Advanced glycation end products upregulate lysyl oxidase and endothelin-1 in human aortic endothelial cells via parallel activation of ERK1/2–NF-κB and JNK–AP-1 signaling pathways

Endothelial dysfunction involves deregulation of the key extracellular matrix (ECM) enzyme lysyl oxidase (LOX) and the vasoconstrictor protein, endothelin-1 (ET-1), whose gene expression can be modulated by the transcriptional activators nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1). Advanced glycation end products (AGEs) present an aggravating factor of endothelial dysfunction which upon engagement to their receptor RAGE induce upregulation of mitogen-activated protein kinases (MAPKs), leading to NF-κB and AP-1 potentiation. We hypothesized that AGEs could induce NF-κΒ- and AP-1-dependent regulation of LOX and ET-1 expression via the AGE/RAGE/MAPK signaling axis. Western blot, real-time qRT-PCR, FACS analysis and electrophoretic mobility-shift assays were employed in human aortic endothelial cells (HAECs) following treatment with AGE-bovine serum albumin (AGE-BSA) to investigate the signaling pathway towards this hypothesis. Furthermore, immunohistochemical analysis of AGEs, RAGE, LOX and ET-1 expression was conducted in aortic endothelium of a rat experimental model exposed to high- or low-AGE content diet. HAECs exposed to AGE-BSA for various time points exhibited upregulation of LOX and ET-1 mRNA levels in a dose- and time-dependent manner. Exposure of HAECs to AGE-BSA also showed specific elevation of phospho(p)-ERK1/2 and p-JNK levels in a dose- and time-dependent fashion. AGE administration significantly increased NF-κΒ- and AP-1-binding activity to both LOX and ET-1 cognate promoter regions. Moreover, LOX and ET-1 overexpression in rat aortic endothelium upon high-AGE content diet confirmed the functional interrelation of these molecules. Our findings demonstrate that AGEs trigger NF-κΒ- and AP-1-mediated upregulation of LOX and ET-1 via the AGE/RAGE/MAPK signaling cascade in human endothelial cells, thus contributing to distorted endothelial homeostasis by impairing endothelial barrier function, altering ECM biomechanical properties and cell proliferation.

Lignans 7‐hydroxymatairesinol and 7‐hydroxymatairesinol 2 exhibit anti‐inflammatory activity in human aortic endothelial cells

SCOPE In the present study, we evaluated the anti-inflammatory properties of several plant lignans most commonly distributed in foods. 7-Hydroxymatairesinol (HMR) and its major isomer 7-hydroxymatairesinol 2 (HMR2), lariciresinol, secoisolariciresinol, and pinoresinol, isolated from Norway spruce knots were examined. METHODS AND RESULTS We investigated the anti-inflammatory effects of lignans on tumor necrosis factor-α-treated human aortic endothelial cells by measuring the expression of intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 by cell ELISA and the adhesion of U937 monocytes to activated endothelial cells using a cell adhesion assay. Among the lignans studied, HMR and HMR2 significantly reduced intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 levels as well as the adhesion of U937 to endothelial cells. To further characterize the molecular mechanisms involved in this regulation, the effect of HMR and HMR2 on nuclear factor-κB, SAPK/c-Jun NH2-terminal kinase and extracellular signal regulated kinase phosphorylation was assessed. CONCLUSION Our results demonstrated that the lignans HMR and HMR2, dominant in cereals such as in wheat, triticale, oat, barley, millet, corn bran, and in amaranth whole grain, exhibit strong anti-inflammatory properties in endothelial cells, at least in part, through attenuation of nuclear factor-κB and extracellular signal regulated kinase phosphorylation.

A monoterpene, unique component of thyme honeys, induces apoptosis in prostate cancer cells via inhibition of NF-κB activity and IL-6 secretion

We have previously demonstrated that Greek thyme honey inhibits significantly the cell viability of human prostate cancer cells. Herein, 15 thyme honey samples from several regions of Greece were submitted to phytochemical analysis for the isolation, identification and determination (through modern spectral means) of the unique thyme honey monoterpene, the compound trihydroxy ketone E-4-(1,2,4-trihydroxy-2,6,6-trimethylcyclohexyl)-but-3-en-2-one. We investigated the anti-growth and apoptotic effects of the trihydroxy ketone on PC-3 human androgen independent prostate cancer cells using MTT assay and Annexin V-FITC respectively. The molecular pathways involved to such effects were further examined by evaluating its ability to inhibit (a) the NF-κB phosphorylation (S536), (b) JNK and Akt phosphorylation (Thr183/Tyr185 and S473 respectively) and (c) IL-6 production, using ELISA method. The anti-microbial effects of the trihydroxy ketone against a panel of nine pathogenic bacteria and three fungi were also assessed. The trihydroxy ketone exerted significant apoptotic activity in PC-3 prostate cancer cells at 100 μM, while it inhibited NF-κB phosphorylation and IL-6 secretion at a concentration range 10(-6)-10(-4)M. Akt and JNK signaling were not found to participate in this process. The trihydroxy ketone exerted significant anti-microbial profile against many human pathogenic bacteria and fungi (MIC values ranged from 0.04 to 0.57 mg/ml). Conclusively, the Greek thyme honey-derived monoterpene exerted significant apoptotic activity in PC-3 cells, mediated, at least in part, through reduction of NF-κB activity and IL-6 secretion and may play a key role in the anti-growth effect of thyme honey on prostate cancer cells.

Biological properties of mud extracts derived from various spa resorts

Spa resorts are known for thousands of years for their healing properties and have been empirically used for the treatment of many inflammatory conditions. Mud is one of the most often used natural materials for preventive, healing and cosmetic reasons and although it has been used since the antiquity, little light has been shed on its physical, chemical and biological properties. In this study we examined the effect of mud extracts on the expression of adhesion molecules (CAMs) by endothelial cells as well as their effects on monocyte adhesion to activated endothelial cells. Most of mud extracts inhibited the expression of VCAM-1 by endothelial cells and reduced monocyte adhesion to activated endothelial cells, indicating a potent anti-inflammatory activity. Furthermore, the mud extracts were tested for their antimicrobial activity; however, most of them appeared inactive against S. aureus and S. epidermidis. One of the mud extracts (showing the best stabilization features) increased significantly the expression of genes involved in cell protection, longevity and hydration of human keratinocytes, such as, collagen 6A1, forkhead box O3, sirtuin-1, superoxide dismutase 1 and aquaporin-3. The present study reveals that mud exerts important beneficial effects including anti-inflammatory and anti-aging activity as well as moisturizing effects, implicating important cosmeceutical applications.

Sideritis euboea extract lowers total cholesterol but not LDL cholesterol in humans: a randomized controlled trial

Abstract Aim: Sideritis euboea is used to prepare a widely consumed beverage. We evaluated the biological activity of S. euboea in healthy human subjects, focusing on serum cardiovascular factors, including lipids, inflammation and glucose homeostasis markers. Patients & methods: In a double-blind study, 54 participants were randomly assigned to consume S. euboea aqueous extract food (n = 27, intervention group) or a placebo food (n = 27, control group) for a 1-month period. A total of 47 participants were included in the final ana lysis. Serum lipids (total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, lipoprotein lipase(a)), Homeostasis Model Assessment and inflammatory markers were determined. Results: Total cholesterol was reduced significantly in the intervention group, while no beneficial effects on other lipid parameters and inflammatory markers were observed. In females, S. euboea significantly ameliorated the Homeostasis Model Assessment index. Conclusion: The consumption of S. euboea induces only a significant total cholesterol lowering effect while it exerts insulin-sensitizing actions in females. Larger studies with a longer intervention period should be performed.

Altered expression of circadian clock genes in polyglandular autoimmune syndrome type III

PurposeCircadian timing system is a highly conserved, ubiquitous molecular “clock” which creates internal circadian rhythmicity. Dysregulation of clock genes expression is associated with various diseases including immune dysregulation. In this study we investigated the circadian pattern of Clock-related genes in patients with polyglandular autoimmune syndrome type III (PAS type III).MethodsNineteen patients diagnosed with PAS type III and 12 healthy controls were enrolled. mRNA and protein expression of Clock-related genes (CLOCK, BMAL1, ROR and Per-1,-2,-3), as well as the GR-a and the GILZ genes were determined by real-time quantitative PCR and western blot analysis from blood samples drawn at 8 pm and 8am. Serum cortisol and TSH, as well as plasma ACTH, were measured by chemiluminescence.ResultsThere were no statistical significant differences in the metabolic profile, cortisol, ACTH and TSH levels between patients and controls. Patients with PAS type III expressed higher transcript levels of CLOCK, BMAL1 and Per-1 in the evening than in the morning (p = 0.03, p = 0.029, p = 0.013, respectively), while the ratios (Rpm/am) of GR-a, CLOCK, BMAL1, and Per-3 mRNA levels were statistically different between patients and controls. Cortisol circadian variation (Fpm/am) was positively correlated with GILZ mRNA circadian pattern (Rpm/am) in the patient group and with the GR-a mRNA (Rpm/am) in the control group.ConclucionsOur findings suggest that there is an aberrant circadian rhythm of Clock-related genes in patients with PAS type III. The disruption of the expression of 4 circadian Clock-related genes could indicate a possible association with the pathogenesis of the disease.