Raffaella Comitato

A novel mechanism of natural vitamin E tocotrienol activity: involvement of ERβ signal transduction

Vitamin E is a generic term used to indicate all tocopherol (TOC) and tocotrienol (TT) derivates. In the last few years, several papers have shown that a TT-rich fraction (TTRF) extracted from palm oil inhibits proliferation and induces apoptosis in a large number of cancer cells. However, the molecular mechanism(s) involved in TT action is still unclear. In the present study, we proposed for the first time a novel mechanism for TT activity that involves estrogen receptor (ER) signaling. In silico simulations and in vitro binding analyses indicated a high affinity of TTs for ERbeta but not for ERalpha. In addition, in ERbeta-containing MDA-MB-231 breast cancer cells, we demonstrated that TTs increase the ERbeta translocation into the nucleus, which in turn activates estrogen-responsive genes (MIC-1, EGR-1 and cathepsin D), as demonstrated by cell preincubation with the ER inhibitor ICI-182,780. Finally, we observed that TT treatment is associated with alteration of cell morphology, DNA fragmentation, and caspase-3 activation. Altogether, these experiments elucidated the molecular mechanism underling gamma- and delta-TT effects.

Gender Differences in Food Choice and Dietary Intake in Modern Western Societies

A significant “male oriented” bias in science is a matter of fact (Marino et al., 2011), even though the number of women majoring in science has increased dramatically (Yokoo, 1996). Considering people graduated in mathematics, science and technology per 1,000 of population aged 20-29, since 1993 up to 2009 (EUROSTAT, 2011), proportion of women is 4.4% vs. 3.4% of men. Interestingly, 4 out of 5 authors of this paper are women. It is justifiable to ask the reason for this gender bias. We can advocate two “reasonable” reasons: the first one has mainly sociologic concern in that different aspects of the human society still present an odd distribution. Research is not an exception and even though the proportion of women within professionals involved in public and private research has reached and possibly overtaken the other gender, the target of scientific investigations is still to be actually considered somehow unbalanced in favour of one gender. The second one has an exquisitely pragmatic origin: in the majority of cases, and unless your research is not to be focused on events strictly connected to females (pregnancy, lactation, few organ specific disease), males are a simpler and cheaper experimental model than females. No needs to carefully evaluate risk factors bound to pregnancy and lactation, to consider hormonal cycle, no sharp changes of tissue functionality associated with ageing.

The anti-inflammatory pharmacology of Pycnogenol® in humans involves COX-2 and 5-LOX mRNA expression in leukocytes.

We investigated the effects of Pycnogenol supplementation on the arachidonic acid pathway in human polymorphonuclear leukocytes (PMNL) in response to an inflammatory stimulus. Pycnogenol is a standardised extract of French maritime pine bark consisting of procyanidins and polyphenolic monomers. Healthy volunteers aged 35 to 50 years were supplemented with 150 mg Pycnogenol a day for five days. Before and after the final day of supplementation, blood was drawn and PMNL were isolated. PMNL were primed with lipopolysaccharide (LPS) and stimulated with the receptor-mediated agonist formyl-methionyl-leucyl-phenylalanine (fMLP) to activate the arachidonic acid pathway and the biosynthesis of leukotrienes, thromboxane and prostaglandins. Pycnogenol supplementation inhibited 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) gene expression and phospholipase A2 (PLA2) activity. This effect was associated with a compensatory up-regulation of COX-1 gene expression. Interestingly, Pycnogenol suspended the interdependency between 5-LOX and 5-lipoxygenase activating protein (FLAP) expression. Pycnogenol supplementation reduced leukotriene production but did not leave prostaglandins unaltered, which we attribute to a decline of COX-2 activity in favour of COX-1. Here we show for the first time that Pycnogenol supplementation simultaneously inhibits COX-2 and 5-LOX gene expression and reduces leukotriene biosynthesis in human PMNL upon pro-inflammatory stimulation ex vivo.

Tocotrienols activity in MCF‐7 breast cancer cells: Involvement of ERβ signal transduction

The term Vitamin E is utilized to describe eight molecules, subdivided into two groups, tocopherols and tocotrienols (TTs). It has been shown that specific TTs affect the growth of several lines of tumour cells, and that this activity is not shared by tocopherols. In agreement with these observations, a TTs-rich fraction from palm oil (PTRF) was reported to inhibit proliferation and induce apoptosis in several cancer cells. However, the molecular mechanism involved in TTs activity is still unclear. We have recently proposed that TTs pro-apoptotic activity involves estrogen receptor beta (ERbeta) signalling. In this study, we report that, in MCF-7 breast cancer cell, expressing both ERalpha and ERbeta, PTRF treatment increases ERbeta nuclear translocation, as demonstrated by immunofluorescence experiments and significantly inhibits ERalpha expression (-458.91-fold of change) and complete disappearing of the protein from the nucleus. Moreover, PTRF treatment induces ER-dependent genes expression (macrophage inhibitory cytokine-1, early growth response-1 and Cathepsin D) which is inhibited by the ER inhibitor, ICI 182.780, and induces DNA fragmentation. Finally, cDNA-array experiments suggest that the activation of specific pathways in cells treated with gamma-TT with respect to alpha-TT. Our data suggest a novel potential molecular mechanism for TTs activity.

Red wine metabolites modulate NF-κB, activator protein-1 and cAMP response element-binding proteins in human endothelial cells

We have studied the effect of human serum, collected after red wine consumption (RWS), on TNF-alpha-dependent activation of transcription factors (NF-kappaB, activator protein-1 (AP-1) and cAMP response element-binding proteins) and on the expression of selected genes involved in cell adhesion or fibrinolysis processes in human primary endothelial cells (human umbilical vein endothelial cells (HUVEC)). Our data indicate that RWS containing RW metabolites, isolated after 40 min from an acute consume of wine (5 ml/kg body weight), induces nuclear translocation of NF-kappaB and AP-1 in the absence of any further stimulus. On the other hand, TNF-alpha treatment in the presence of RWS is associated with a delay in transcription factor activation and to a negative modulation on the expression of specific genes. Moreover, RWS stimulates c-jun binding to the tissue-type plasminogen activator cAMP responsive element consensus site modulating the expression of the specific gene downstream. These results confirm that RW metabolites affect the activity of different transcription factors playing an important preconditioning role in the modulation of the inflammatory pathway in endothelial cells. This is the first report on the effects of a complex food matrix, on the molecular mechanisms associated with inflammatory response in HUVEC cultured in condition that reproduces the physiological environment occurring in vivo.

Absorption, metabolism, and effects at transcriptome level of a standardized French oak wood extract, Robuvit, in healthy volunteers: pilot study.

The consumption of wine and spirits, traditionally aged in oak barrels, exposes humans to roburin ingestion. These molecules belong to a class of ellagitannins (ETs), and their only known source is oak wood. Very little is currently known about roburin bioavailability and biological activity. We reported for the first time human absorption of roburins from a French oak wood (Quercus robur) water extract (Robuvit) by measuring the increase of total phenols (from 0.63 ± 0.06 to 1.26 ± 0.18 μg GAE equiv/mL plasma) and the appearance of roburin metabolites (three different glucoronidate urolithins and ellagic acid), in plasma, after 5 days of supplementation. Robuvit supplementation induced also the increase of plasma antioxidant capacity from 1.8 ± 0.05 to 1.9 ± 0.01 nmol Trolox equiv/mL plasma. Moreover, utilizing a combined ex vivo cell culture approach, we assessed the effect of Q. robur metabolites (present in human serum after supplementation) on gene expression modulation, utilizing an Affymetrix array matrix, in endothelial, neuronal, and keratinocyte cell lines. The functional analysis reveals that Robuvit metabolites affect ribosome, cell cycle, and spliceosome pathways.

Sex Hormones and Macronutrient Metabolism

The biological differences between males and females are determined by a different set of genes and by a different reactivity to environmental stimuli, including the diet, in general. These differences are further emphasized and driven by the exposure to a different hormone flux throughout the life. These differences have not been taken into appropriate consideration by the scientific community. Nutritional sciences are not immune from this “bias” and when nutritional needs are concerned, females are considered only when pregnant, lactating or when their hormonal profile is returning back to “normal,” i.e., to the male-like profile. The authors highlight some of the most evident differences in aspects of biology that are associated with nutrition. This review presents and describes available data addressing differences and similarities of the “reference man” vs. the “reference woman” in term of metabolic activity and nutritional needs. According to this assumption, available evidences of sex-associated differences of specific biochemical pathways involved in substrate metabolism are reported and discussed. The modulation by sexual hormones affecting glucose, amino acid and protein metabolism and the metabolization of nutritional fats and the distribution of fat depots, is considered targeting a tentative starting up background for a gender concerned nutritional science.

Nanomolar Caffeic Acid Decreases Glucose Uptake and the Effects of High Glucose in Endothelial Cells

Epidemiological studies suggest that moderate and prolonged consumption of coffee is associated with a reduced risk of developing type 2 diabetes but the molecular mechanisms underlying this effect are not known. In this study, we report the effects of physiological concentrations of caffeic acid, easily achievable by normal dietary habits, in endothelial cells cultured in 25 mM of glucose (high glucose, HG). In HG, the presence of 10 nM caffeic acid was associated with a decrease of glucose uptake but not to changes of GLUT-1 membrane localization or mRNA levels. Moreover, caffeic acid countered HG-induced loss of barrier integrity, reducing actin rearrangement and FITC-dextran passage. The decreased flux of glucose associated to caffeic acid affected HG induced apoptosis by down-regulating the expression of initiator (caspase 8 and 9) and effector caspases (caspase 7 and 3) and by increasing the levels of phosphorylated Bcl-2. We also observed that caffeic acid in HG condition was associated to a reduction of p65 subunit nuclear levels with respect to HG alone. NF-κB activation has been shown to lead to apoptosis in HG treated cells and the analysis of the expression of a panel of about 90 genes related to NF-κB signaling pathway revealed that caffeic acid significantly influenced gene expression changes induced by HG. In conclusion, our results suggest that caffeic acid, decreasing the metabolic stress induced by HG, allows the activation of survival mechanisms mediated by a different modulation of NF-κB-related signaling pathways and to the activation of anti-apoptotic proteins.

Tocotrienols: A Family of Molecules with Specific Biological Activities

Vitamin E is a generic term frequently used to group together eight different molecules, namely: α-, β-, γ- and δ-tocopherol and the corresponding tocotrienols. The term tocopherol and eventually Vitamin E and its related activity was originally based on the capacity of countering foetal re-absorption in deficient rodents or the development of encephalomalacia in chickens. In humans, Vitamin E activity is generally considered to be solely related to the antioxidant properties of the tocolic chemical structure. In recent years, several reports have shown that specific activities exist for each different tocotrienol form. In this short review, tocotrienol ability to inhibit cancer cell growth and induce apoptosis thanks to specific mechanisms, not shared by tocopherols, such as the binding to Estrogen Receptor-β (ERβ) and the triggering of endoplasmic reticulum (EndoR) stress will be described. The neuroprotective activity will also be presented and discussed. We propose that available studies strongly indicate that specific forms of tocotrienols have a distinct mechanism and biological activity, significantly different from tocopherol and more specifically from α-tocopherol. We therefore suggest not pooling them together within the broad term “Vitamin E” on solely the basis of their putative antioxidant properties. This option implies obvious consequences in the assessment of dietary Vitamin E adequacy and, probably more importantly, on the possibility of evaluating a separate biological variable, determinant in the relationship between diet and health.