Vassilia-Ismini Alexaki

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.

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.

G protein-associated, specific membrane binding sites mediate the neuroprotective effect of dehydroepiandrosterone

The neurosteroid dehydroepiandrosterone (DHEA) at 1 nM protects NMDA‐/GABAA‐receptor negative neural crest‐derived PC12 cells from apoptosis. We now report that membrane‐impermeable DHEA‐BSA conjugate replaces unconjugated DHEA in protecting serum‐deprived PC12 cells from apoptosis (IC50=1.5 nM). Protection involves phosphorylation of the prosurvival factor Src and induction of the anti‐apoptotic protein Bcl‐2 and is sensitive to pertussis toxin. Binding assays of [3H]DHEA on isolated PC12 cell membranes revealed saturation within 30 min and binding of DHEA with a Kd of 0.9 nM. A similar binding activity was detectable in isolated membranes from rat hippocampus and from normal human adrenal chromaffin cells. The presence of DHEA‐specific membrane binding sites was confirmed by flow cytometry and confocal laser microscopy of DHEA‐BSA‐FITC stained cells. In contrast to estrogens and progestins, glucocorticoids and androgens displaced DHEA from its membrane binding sites but with a 10‐fold lower affinity than DHEA (IC50=9.3 and 13.6 nM, respectively). These agents acted as pure antagonists, blocking the antiapoptotic effect of DHEA as well as the induction of Bcl‐2 proteins and Src kinase activation. In conclusion, our findings suggest that neural crest‐derived cells possess specific DHEA membrane binding sites coupled to G proteins. Binding to these sites confers neuroprotection.

Neurosteroid Dehydroepiandrosterone Interacts with Nerve Growth Factor (NGF) Receptors, Preventing Neuronal Apoptosis

The neurosteroid dehydroepiandrosterone (DHEA), produced by neurons and glia, affects multiple processes in the brain, including neuronal survival and neurogenesis during development and in aging. We provide evidence that DHEA interacts with pro-survival TrkA and pro-death p75NTR membrane receptors of neurotrophin nerve growth factor (NGF), acting as a neurotrophic factor: (1) the anti-apoptotic effects of DHEA were reversed by siRNA against TrkA or by a specific TrkA inhibitor; (2) [3H]-DHEA binding assays showed that it bound to membranes isolated from HEK293 cells transfected with the cDNAs of TrkA and p75NTR receptors (KD: 7.4±1.75 nM and 5.6±0.55 nM, respectively); (3) immobilized DHEA pulled down recombinant and naturally expressed TrkA and p75NTR receptors; (4) DHEA induced TrkA phosphorylation and NGF receptor-mediated signaling; Shc, Akt, and ERK1/2 kinases down-stream to TrkA receptors and TRAF6, RIP2, and RhoGDI interactors of p75NTR receptors; and (5) DHEA rescued from apoptosis TrkA receptor positive sensory neurons of dorsal root ganglia in NGF null embryos and compensated NGF in rescuing from apoptosis NGF receptor positive sympathetic neurons of embryonic superior cervical ganglia. Phylogenetic findings on the evolution of neurotrophins, their receptors, and CYP17, the enzyme responsible for DHEA biosynthesis, combined with our data support the hypothesis that DHEA served as a phylogenetically ancient neurotrophic factor.

Topical application of autologous adipose-derived mesenchymal stem cells (MSCs) for persistent sterile corneal epithelial defect

Different causes of corneal persistent epithelial defects (PED) have been described including dry eye, corneal epithelial stem cell deficiency, diabetes mellitus, and neurotrophic keratopathy (after herpetic keratitis or corneal surgery). Corneal PEDs could lead to a cascade of events throughout the cornea, resulting in stromal degradation, thinning and in advanced cases, corneal perforation. Several treatment options have been proposed for corneal PEDs (based on the etiology) such as eye patching, unpreserved artificial tears, bandage contact lens, punctual plugging, tarsorrhaphy, autologous serum eyedrops, limbal stem cells transplantation, and amniotic membrane grafting. Mesenchymal stem cells (MSCs) are multipotent stem cells that can differentiate into a variety of cell types. Clinical uses of these cells have been proposed, such as in patients with degenerative joint disease (using percutaneously implanted autologous MSCs) [1], while adipose-derived MSCs have been used as a source for cell therapy of the corneal stroma in experimental animals [2]. In this case report we present, for the first time, a patient with post-traumatic persistent sterile corneal epithelial defect treated with topical application of autologous adipose-derived mesenchymal stem cells (MSCs).

TWEAK affects keratinocyte G2/M growth arrest and induces apoptosis through the translocation of the AIF protein to the nucleus.

The soluble TNF-like weak inducer of apoptosis (TWEAK, TNFSF12) binds to the fibroblast growth factor-inducible 14 receptor (FN14, TNFRSF12A) on the cell membrane and induces multiple biological responses, such as proliferation, migration, differentiation, angiogenesis and apoptosis. Previous reports show that TWEAK, which does not contain a death domain in its cytoplasmic tail, induces the apoptosis of tumor cell lines through the induction of TNFα secretion. TWEAK induces apoptosis in human keratinocytes. Our experiments clearly demonstrate that TWEAK does not induce the secretion of TNFα or TRAIL proteins. The use of specific inhibitors and the absence of procaspase-3 cleavage suggest that the apoptosis of keratinocytes follows a caspase- and cathepsin B-independent pathway. Further investigation showed that TWEAK induces a decrease in the mitochondrial membrane potential of keratinocytes. Confocal microscopy showed that TWEAK induces the cleavage and the translocation of apoptosis inducing factor (AIF) from the mitochondria to the nucleus, thus initiating caspase-independent apoptosis. Moreover, TWEAK induces FOXO3 and GADD45 expression, cdc2 phosphorylation and cdc2 and cyclinB1 degradation, resulting in the arrest of cell growth at the G2/M phase. Finally, we report that TWEAK and FN14 are normally expressed in the basal layer of the physiological epidermis and are greatly enhanced in benign (psoriasis) and malignant (squamous cell carcinoma) skin pathologies that are characterized by an inflammatory component. TWEAK might play an essential role in skin homeostasis and pathology.

APRIL Binding to BCMA Activates a JNK2–FOXO3–GADD45 Pathway and Induces a G2/M Cell Growth Arrest in Liver Cells

The TNF superfamily ligands APRIL and BAFF bind with different affinity to two receptors, BCMA and TACI, and induce cell survival and/or proliferation, whereas BAFF also binds specifically to BAFFR. These molecules were considered specific for the immune system. Recently, however, they were also found in epithelial and mesenchymal noncancerous and cancerous tissues and cell lines. In this article, we report that hepatocellular carcinoma (HCC) cell lines HepG2 and Hep3B and HCC specimens express APRIL and BAFF and their receptors BCMA and BAFFR, but not TACI; APRIL/BCMA is enhanced in HCC, compared with normal liver tissue. In contrast to previous reports, APRIL binding to BCMA decreases cell proliferation by inducing G2/M cell cycle arrest, whereas BAFF has no effect on cell growth. HCC cells therefore represent a rare system in which these two ligands (APRIL and BAFF) exert a differential effect and may serve as a model for specific APRIL/BCMA actions. We show that the effect of APRIL is mediated via BCMA, which does not activate the classical NF-κB pathway, whereas it induces a novel signaling pathway, which involves JNK2 phosphorylation, FOXO3A activation, and GADD45 transcription. In addition, JNK2 mediates the phosphorylation of Akt, which is activated but does not participate in the antiproliferative effect of APRIL. Furthermore, transcriptome analysis revealed that APRIL modifies genes specifically related to cell cycle modulation, including MCM2/4/5/6, CDC6, PCNA, and POLE2. Our data, therefore, identify a novel APRIL/BCMA signaling pathway in HCC and suggest that APRIL could have a pleiotropic role in tumor biology.

Polyphenol interaction with the T47D human breast cancer cell line

Experimental and epidemiological studies indicate that antioxidant food polyphenols could have antimitotic activities, interfering with cancer initiation, progression or mortality. Circulating polyphenols are far lower than the nominal value in foods. In the rare studies dealing with polyphenol bioavailability, it was noted that their active concentrations in the blood are <1% of their food concentration. In the present study we investigated the effect of four polyphenols (resveratrol, and the flavonoids quercetin, catechin and epicatechin, major constituents of wine) in the hormone-sensitive human cancer cell line T47D, at concentrations compatible with their calculated plasma concentrations after ingestion of a moderate quantity of wine (nM or pM). Our results indicate that cell growth was decreased, with cells being arrested at the S phase of the cycle. In addition, we provide evidence of a bimodal modulation of the NO/NOS system, affecting its activity and transcription. We show that modulation of this system is sufficient to explain polyphenol action on this cell line. This result suggests a potential importance of wine ingestion and possibly the consumption of other polyphenol-rich dietary foods and drinks in the control of breast cancer cell growth.

Adipose Tissue-Derived Mesenchymal Cells Support Skin Reepithelialization Through Secretion of KGF-1 and PDGF-BB: Comparison With Dermal Fibroblasts

Epidermal organization and homeostasis are regulated by mesenchymal influences through paracrine actions. Until today, dermal fibroblasts (DFs) are used in the “dermal” layer to support keratinocyte growth in vitro in dermal and skin substitutes. In the present work, we used human adipose tissue-derived mesenchymal cells (ADMCs) as a support of keratinocyte growth in vitro (in monolayer culture and in 3D skin cell culture models) and in vivo (mouse wound healing models) and compared our findings with those obtained using dermal fibroblasts. ADMCs induce reepithelialization during wound healing more efficiently than DFs, by enhancing keratinocyte proliferation through cell cycle progression, and migration. This effect is mediated (at least partially) by a paracrine action of KGF-1 and PDGF-BB, which are more prominently expressed in ADMCs than in DFs. Furthermore, replacement of DFs by ADMCs in the dermal compartment of organotypic skin cultures leads to an artificial epidermis resembling to that of normal skin, concerning the general histology, although with a higher expression of cytokeratins 5 and 19. In Rag1 knockout mice, ADMCs induced a more rapid reepithelialization and a more effective wound healing, compared to dermal fibroblasts. In conclusion, we provide evidence that ADMCs can serve as supportive cells for primary keratinocyte cultures. In addition, because of their abundance and the great cell yield achieved during ADMC isolation, they represent an interesting cell source, with potential aspects for clinical use.

Dehydroepiandrosterone protects human keratinocytes against apoptosis through membrane binding sites.

Although the epidermis is importantly affected by steroid hormones, little is known about the effects of dehydroepiandrosterone (DHEA) on human keratinocytes, in spite of its abundance in human serum. Here, we demonstrate for the first time a protective role of DHEA against apoptosis in keratinocytes, using non-cancerous immortalized human HaCaT cells. We show that DHEA transmits its signal via specific G protein-coupled, membrane binding sites and inhibits apoptosis, through prevention of mitochondrial disruption and altered balance of Bcl-2 proteins. DHEA conjugated to the membrane impermeable molecule BSA, as well as DHEA-S, the most abundant form of DHEA in human serum exhibit similar anti-apoptotic effect. Our data provide new insights in the treatment of the epidermis with steroid hormones in apoptosis-related conditions.