Marilena Kampa

Antiproliferative and apoptotic effects of selective phenolic acids on T47D human breast cancer cells: potential mechanisms of action

IntroductionThe oncoprotective role of food-derived polyphenol antioxidants has been described but the implicated mechanisms are not yet clear. In addition to polyphenols, phenolic acids, found at high concentrations in a number of plants, possess antioxidant action. The main phenolic acids found in foods are derivatives of 4-hydroxybenzoic acid and 4-hydroxycinnamic acid.MethodsThis work concentrates on the antiproliferative action of caffeic acid, syringic acid, sinapic acid, protocatechuic acid, ferulic acid and 3,4-dihydroxy-phenylacetic acid (PAA) on T47D human breast cancer cells, testing their antioxidant activity and a number of possible mechanisms involved (interaction with membrane and intracellular receptors, nitric oxide production).ResultsThe tested compounds showed a time-dependent and dose-dependent inhibitory effect on cell growth with the following potency: caffeic acid > ferulic acid = protocatechuic acid = PAA > sinapic acid = syringic acid. Caffeic acid and PAA were chosen for further analysis. The antioxidative activity of these phenolic acids in T47D cells does not coincide with their inhibitory effect on tumoral proliferation. No interaction was found with steroid and adrenergic receptors. PAA induced an inhibition of nitric oxide synthase, while caffeic acid competes for binding and results in an inhibition of aryl hydrocarbon receptor-induced CYP1A1 enzyme. Both agents induce apoptosis via the Fas/FasL system.ConclusionsPhenolic acids exert a direct antiproliferative action, evident at low concentrations, comparable with those found in biological fluids after ingestion of foods rich in phenolic acids. Furthermore, the direct interaction with the aryl hydrocarbon receptor, the nitric oxide synthase inhibition and their pro-apoptotic effect provide some insights into their biological mode of action.

The human prostate cancer cell line LNCaP bears functional membrane testosterone receptors that increase PSA secretion and modify actin cytoskeleton

Recent findings have shown that, in addition to the genomic action of steroids, through intracellular receptors, short‐time effects could be mediated through binding to membrane sites. In the present study of prostate cancer LNCaP cells, we report that dihydrotestosterone and the non‐internalizable analog testosterone‐BSA increase rapidly the release of prostate‐specific antigen (PSA) in the culture medium. Membrane testosterone binding sites were identified through ligand binding on membrane preparations, flow cytometry, and confocal laser microscopy of the non‐internalizable fluorescent analog testosterone‐BSA‐FITC, on whole cells. Binding on these sites is time‐ and concentration‐dependent and specific for testosterone, presenting a Kd of 10.9 nM and a number of 144 sites/mg protein (~13000 sites/cell). Membrane sites differ immunologically for intracellular androgen receptors. The secretion of PSA after membrane testosterone receptor stimulation was inhibited after pretreatment with the actin cytoskeleton disrupting agent cytochalasin B. In addition, membrane testosterone binding modifies the intracellular dynamic equilibrium of monomeric to filamentous actin and remodels profoundly the actin cytoskeleton organization. These results are discussed in the context of a possible involvement of these sites in cancer chemotherapy.

Polyphenols and cancer cell growth

Polyphenols constitute an important group of phytochemicals that gained increased research attention since it was found that they could affect cancer cell growth. Initial evidence came from epidemiologic studies suggesting that a diet that includes regular consumption of fruits and vegetables (rich in polyphenols) significantly reduces the risk of many cancers. In the present work we briefly review the effects of polyphenols on cancer cell fate, leading towards growth, differentiation and apoptosis. Their action can be attributed not only to their ability to act as antioxidants but also to their ability to interact with basic cellular mechanisms. Such interactions include interference with membrane and intracellular receptors, modulation of signaling cascades, interaction with the basic enzymes involved in tumor promotion and metastasis, interaction with oncogenes and oncoproteins, and, finally, direct or indirect interactions with nucleic acids and nucleoproteins. These actions involve almost the whole spectrum of basic cellular machinery--from the cell membrane to signaling cytoplasmic molecules and to the major nuclear components--and provide insights into their beneficial health effects. In addition, the actions justify the scientific interest in this class of compounds, and provide clues about their possible pharmaceutical exploitation in the field of oncology.

A new automated method for the determination of the Total Antioxidant Capacity (TAC) of human plasma, based on the crocin bleaching assay

BackgroundAntioxidant molecules, which scavenge free radical species to prevent or delay oxidative damage of important macromolecules, membrane lipids and lipoproteins, are prevalent in plasma and other biological fluids. Among them, bilirubin, uric acid and protein thiols are the major endogenous antioxidants, while vitamins C and E, as well as a number of food-derived (poly)aromatic substances, belonging to stilbens, flavonoids and phenolic acids, are the main classes of nutritional antioxidants. Assays for total antioxidant capacity in plasma differ in their type of oxidation source, target and measurement used to detect the oxidized product.MethodsIn the present work we present an automated assay for the estimation of blood total antioxidant capacity (TAC assay), based on the crocin bleaching (oxidation) method. This method was adapted on a modern autoanalyzer, was linear over a wide range of values (0–3 mmol/L), and performed using an end point measurement.ResultsThe TAC method presented a linear correlation with another automated commercial Total Antioxidant Status (TAS) test. Detection of the interference of different metabolites revealed a significant participation of TAC from uric acid, bilirubin, albumin, a minor interference from ascorbic acid, and no interference from hemoglobin. TAC was not modified by two freeze/thawing cycles, and was stable in samples stored at room temperature for 4 hours. K-EDTA and heparin were the best anticoagulants, while citrate decreased TAC by 20%. Reference values derived from samples of normal blood donors was 1.175 ± 0.007 mmol/L (mean ± SEM), while a diet rich in antioxidants more than doubled this value.ConclusionsThe proposed TAC assay, is fully automated, stable and reliable, and could be of value in the estimation of the AC of plasma. It is further proposed to calculate the antioxidant capacity of plasma after a subtraction of all interference deriving from endogenous and/or exogenous metabolites. The antioxidant capacity of plasma thus calculated can be used as a useful indicator of the antioxidant value of foods and beverages in the daily diet.

Adipocytes as Immune Cells: Differential Expression of TWEAK, BAFF, and APRIL and Their Receptors (Fn14, BAFF-R, TACI, and BCMA) at Different Stages of Normal and Pathological Adipose Tissue Development

Adipose tissue represents a rich source of multipotent stem cells. Mesenchymal cells, isolated from this source, can differentiate to other cell types in vitro and therefore can be used for a number of regenerative therapies. Our view of adipose tissue has recently changed, establishing adipocytes as new members of the immune system, as they produce a number of proinflammatory cytokines (such as IL-6 and TNFα and chemokines, in addition to adipokines (leptin, adiponectin, resistin) and molecules associated with the innate immune system. In this paper, we report the differential expression of TNF-superfamily members B cell activating factor of the TNF Family (BAFF), a proliferation inducing ligand (APRIL), and TNF-like weak inducer of apoptosis (TWEAK) in immature-appearing and mature adipocytes and in benign and malignant adipose tissue-derived tumors. These ligands act through their cognitive receptors, BAFF receptor, transmembrane activator and calcium signal-modulating cyclophilic ligand (TACI), B cell maturation Ag (BCMA), and fibroblast growth factor-inducible 14 (Fn14), which are also expressed in these cells. We further report the existence of functional BCMA, TACI, and Fn14 receptors and their ligands BAFF, APRIL, and TWEAK on adipose tissue-derived mesenchymal cells, their interaction modifying the rate of adipogenesis. Our data integrate BAFF, APRIL, and TWEAK and their receptors BCMA, TACI, and Fn14 as novel potential mediators of adipogenesis, in addition to their specific role in immunity, and define immature and mature adipocytes as source of immune mediators.

Estrogen exerts neuroprotective effects via membrane estrogen receptors and rapid Akt/NOS activation

The neuroprotective role of estrogen (E2) is supported by a multitude of experimental and epidemiological data, although its mode of action is not fully understood. The present work was conducted to study the underlying mechanisms of its neuroprotective action, using the rat cell line PC12, an established model for neuronal cell apoptosis and survival. Our results show that E2 (but not androgens or progestins) prevent growth inhibition and apoptosis of PC12 cells, induced by serum deprivation. Several mechanisms of action were investigated: 1) intracellular estrogen receptors (ERs) have been identified but do not appear to mediate the protective effect of E2. 2) The antioxidant properties of E2 cannot explain their protective actions at the concentrations used (10−12‐10−6 M). 3) Finally, membrane sites for E2 have been identified, and the underlying initial signaling cascade (2‐30 min after E2) has been tested, showing Ca2+ mobilization→PI3K activation→Akt phosporylation→NOS activation. Inhibition of PI3K or NOS completely reversed the anti‐apoptotic effect of E2. These results suggest a new mechanism of neuroprotective action of estrogen.

Alterations in gut hormones after laparoscopic sleeve gastrectomy: a prospective clinical and laboratory investigational study.

Objective:To evaluate the effect of laparoscopic sleeve gastrectomy (LSG) on fasting and meal-stimulated release of the gut hormones ghrelin, pancreatic polypeptide (PP), peptide-YY (PYY), glucagon-like peptide-1 (GLP-1), and amylin and of the adipocytokine leptin. Background:Mounting evidence suggests that the mechanisms of weight loss and the improvement in glucose metabolism seen after LSG are related not only to gastric restriction but also to neurohormonal changes. Methods:Fasting and postprandial levels at 60 and 120 minutes after a standard test meal of the above peptides and glucose metabolism indices were evaluated in 15 consecutive morbidly obese (MO) subjects before and 6 and 12 months after LSG. As study controls, 15 lean subjects matched for age and sex were also assessed. Results:Body mass index values notably decreased at 6 and 12 months (P < 0.01), postoperatively. In addition, an overall improvement of the glycemic profile of MO patients was noted. After LSG, markedly decreased fasting and postprandial levels of ghrelin, amylin, and leptin were observed. A significant postprandial increase of PYY and GLP-1 levels was also noted postoperatively. Interestingly, significantly increased levels of PP were noted only at 60 minutes postprandially after LSG. Conclusions:LSG markedly improved glucose homeostasis and generated significant changes in ghrelin, PP, PYY, GLP-1, amylin, and leptin levels. These multiple hormonal actions may have several beneficial effects on the underlying mechanism of weight loss, demonstrating that LSG could be more than just a restrictive bariatric operation.

Membrane-initiated steroid action in breast and prostate cancer

The mode of action of steroid hormones has been extended in recent years. In addition to their classical nuclear action (acting as transcription factors), they can also regulate cell-signaling phosphorylation cascades and exert actions that are initiated at the membrane and which, in most cases, are rapid. Even though research in this field was intensified during the last decade the nature of the up-stream receptor targets that mediates these rapid non-genomic actions remains to be better established. However, it became obvious that steroid signaling is not uniform, with a variety of modes of rapid action being described. There are several studies speculating a classical steroid receptor involvement in the rapid effects of steroids, localized at the cytoplasmic membrane and mediating effects directly or indirectly, via interactions with specific membrane structures (estrogen receptor (ER) isoforms have been shown to localize in caveolae) and/or other membrane receptors (like growth factor receptor). In addition, there are reports that suggest the existence of a distinct receptor, associated to the plasma membrane, being different from the classical, intracellular one. Non-genomic/extranuclear actions of steroids have been described in a number of different normal or cancer tissues independently of the presence of classical nuclear steroid receptors. In the present work, we review briefly the identification and signaling events of membrane-initiated steroid (androgen and estrogen) action in breast and prostate cancer cell lines and clinical specimens. Furthermore, we discuss the interaction of cytokine/growth factor receptors with membrane-acting steroids and their potential clinical implications.

Activation of membrane estrogen receptors induce pro-survival kinases.

Experimental and epidemiological data suggest a neuroprotective role for estrogen (E(2)). We have recently shown that, in PC12 cells, non-permeable estradiol conjugated to bovine serum albumin (BSA) prevent serum-deprivation induced apoptosis through activation of specific membrane estrogen receptors (mER). In the present study, we explored in detail the early signaling events involved in this anti-apoptotic action, downstream to activation of mER. Our findings suggest that mER is associated to G-proteins, and its activation with non-permeable E(2)-BSA results in the activation of the following downstream pro-survival kinases pathways: (1) the PKB/Akt pathway, (2) the Src-->MEK-->ERK kinases and finally (3) the MAPK-->ERK kinases. Activation of these pro-survival signals leads to CREB phosphorylation and NFkappaB nuclear translocation, two transcription factors controlling the expression of anti-apoptotic Bcl-2 proteins. These data suggest that major pro-survival kinases are involved in the mER-mediated anti-apoptotic effects of estrogen. This is further supported by experiments with specific kinases inhibitors, which partially but significantly reversed the mER-mediated anti-apoptotic effect of E(2)-BSA. Our findings suggest that estrogen act via mER as potent cytoprotective factors, downstream activating pro-survival kinases, assuring thus an efficient and multipotent activation of the anti-apoptotic machinery.

Expression of TNF-superfamily members BAFF and APRIL in breast cancer: Immunohistochemical study in 52 invasive ductal breast carcinomas

BackgroundRecent studies suggest an association between chronic inflammation, modulating the tissue microenvironment, and tumor biology. Tumor environment consists of tumor, stromal and endothelial cells and infiltrating macrophages, T lymphocytes, and dendritic cells, producing an array of cytokines, chemokines and growth factors, accounting for a complex cell interaction and regulation of differentiation, activation, function and survival of tumor and surrounding cells, responsible for tumor progression and spreading or induction of antitumor immune responses and rejection. Tumor Necrosis Factor (TNF) family members (19 ligands and 29 receptors) represent a pleiotropic family of agents, related to a plethora of cellular events from proliferation and differentiation to apoptosis and tumor reduction. Among these members, BAFF and APRIL (CD257 and CD256 respectively) gained an increased interest, in view of their role in cell protection, differentiation and growth, in a number of lymphocyte, epithelial and mesenchymal structures.MethodsWe have assayed by immunohistochemistry 52 human breast cancer biopsies for the expression of BAFF and APRIL and correlated our findings with clinicopathological data and the evolution of the disease.ResultsBAFF was ubiquitely expressed in breast carcinoma cells, DCIS, normal-appearing glands and ducts and peritumoral adipocytes. In contrast, APRIL immunoreactive expression was higher in non-malignant as compared to malignant breast structures. APRIL but not BAFF immunoreactivity was higher in N+ tumors, and was inversely related with the grade of the tumors. Neither parameter was related to DFS or the OS of patients.ConclusionOur data show, for the first time, an autocrine secretion of BAFF and APRIL from breast cancer cells, offering new perspectives for their role in neoplastic and normal breast cell biology and offering new perspectives for possible selective intervention in breast cancer.