Alexandros Zafiropoulos

Estradiol-estrogen receptor: a key interplay of the expression of syndecan-2 and metalloproteinase-9 in breast cancer cells.

Estrogens are related with the growth and development of target tissues and play a critical role in breast cancer progression. The effects of estrogens are mediated by the estrogen receptors ERα and ERβ, which are members of the nuclear steroid receptor superfamily. To date, it is not known how these hormones elicit many of their effects on extracellular matrix molecules and how these effects can be connected with ER expression. For this purpose, the effect of estradiol on ER expression as well as on proteoglycan and metalloproteinase expression was studied. The effect of E2 on extracellular matrix molecule expression has been studied using ERα suppression in breast cancer cells. Our studies using ERα‐positive MCF‐7 cells show that estradiol affects the expression of syndecan‐2, but not of syndecan‐4, through ERα. Furthermore, the ability of estradiol to affect MMP‐9 and TIMP‐1 expression is connected with ERα status. Together, these data demonstrate the significant role of ERα on mediating the effect of estradiol on extracellular matrix molecules.

Lumican expression is positively correlated with the differentiation and negatively with the growth of human osteosarcoma cells

Osteosarcoma is the most common primary bone tumour associated with childhood and adolescence. The possible role of the small leucine‐rich proteoglycan, lumican, in the growth and metastasis of various cancer types has recently been investigated. In this study, the expression of lumican was examined in moderately differentiated (MG‐63) and well‐differentiated (Saos 2) human osteosarcoma cell lines of high and low metastatic capability, respectively. Real‐time PCR, western blotting with antibodies against the protein core and keratan sulfate, and specific enzymatic digestions were the methods employed. The two human osteosarcoma cell lines were found to express and secrete lumican partly substituted with keratan sulfate glycosaminoglycans. Importantly, the non‐metastatic, well‐differentiated Saos 2 cells produced lumican at rates that were up to sevenfold higher than those of highly metastatic MG‐63 cells. The utilization of short interfering RNA specific for the lumican gene resulted in efficient down‐regulation of its mRNA levels in both cell lines. The growth of Saos 2 cells was inhibited by lumican, whereas their migration and chemotactic response to fibronectin were found to be promoted. Lumican expression was negatively correlated with the basal level of Smad 2 activation in these cells, suggesting that lumican may affect the bioavailability of Smad 2 activators. By contrast, these cellular functions of highly aggressive MG‐63 cells were demonstrated not to be sensitive to a decrease in their low endogenous lumican levels. These results suggest that lumican expression may be positively correlated with the differentiation and negatively correlated with the progression of osteosarcoma.

Induction of antigen-specific isotype switching by in vitro immunization of human naive B lymphocytes

Abstract The use of in vitro immunization technology for the generation of human antigen-specific antibodies has essentially resulted in low affinity IgM antibodies, resembling an in vivo primary immune response. We now describe a detailed reproducible protocol for a two-step in vitro immunization, which yields isotype switched, antigen-specific human antibodies. The immunizing antigen was a 30aa synthetic peptide, containing both a B (15aa V3 peptide of the HIV-1) and a T helper cell epitope (15aa peptide from tetanus toxin). The immunization protocol includes: (i) a selection procedure of donors with a memory T cell response against tetatus toxoid; (ii) immunization of mature naive peripheral B lymphocytes in two distinct phases, involving a primary and a secondary step. None of the donors which were examined after primary 7immunization showed at any time an IgG anti-V3 specific antibody response, while all the donors showed an IgM response. After the secondary immunization step, anti-V3 antibodies of both IgM and IgG isotypes were detected. The switch frequency event was high among the tested donors (5/8).

Decorin-induced growth inhibition is overcome through protracted expression and activation of epidermal growth factor receptors in osteosarcoma cells.

Decorin is an established natural oncosuppressive factor whose action is being studied in detail. Recently, decorin gene therapy formulations using adenoviral vectors have been shown in several animal models with very promising results. The present study describes the first exception to the established oncosuppression model using human osteosarcoma cells. MG-63 osteosarcoma cells were found to constitutively produce decorin, and furthermore, to be resistant to decorin-induced growth arrest. On the contrary, decorin seemed to be beneficial to osteosarcoma cells because it was necessary for MG-63 cell migration and acted as a mediator, counteracting the transforming growth factor-β2–induced cytostatic function. Efforts to determine how MG-63 cells could overcome the decorin-induced cytostatic effect established that decorin in MG-63 cells does not induce p21 expression nor does it cause protracted retraction and inactivation of the epidermal growth factor receptor. Conversely, epidermal growth factor receptor seemed to be overexpressed and continuously phosphorylated. In view of the proposed design of decorin-based anticancer therapeutic strategies, our study provides new data on pathways that cancer cells might employ to overcome the established decorin-induced growth suppression. (Mol Cancer Res 2008;6(5):785–94)

Peripheral monocytes from diabetic patients with coronary artery disease display increased bFGF and VEGF mRNA expression

BackgroundMacrophages can produce vascular endothelial growth factor (VEGF) in response to hypoxia, transforming growth factor β1 (TGF-β1), angiotensin II, basic fibroblast growth factor (bFGF), and interleukin-1. These factors have been found in the serum of coronary artery disease (CAD) patients as well as in atherosclerotic lesions. The aim of the present study was to test the hypothesis that the expression of VEGF, TGF-β1 and bFGF in peripheral monocytes and lymphocytes is related to CAD.MethodsHuman Mononuclear cells and lymphocytes from peripheral blood were isolated from 53 donors undergoing angiography. Seventeen were found to be healthy and 36 were diagnosed with CAD. The respective mRNAs were extracted and quantified.ResultsThe statistical analysis revealed a significant increase of the basal level expression for macrophage VEGF and bFGF in the CAD SA (stable angina) patient group compared to the noCAD (control) (p = 0.041 and p = 0.022 respectively) and CAD UA (unstable angina) (p = 0.024 and p = 0.005 respectively) groups, which was highly dependent on the diabetic status of the population. Furthermore, we demonstrated with an in vitro cell culture model that the levels of VEGF and bFGF in monocytes of healthy donors are not affected by short term exposure to increased glucose levels (usually observed in the diabetic patients) and/or statin.ConclusionOur findings display a statistically significant association of the increased VEGF and bFGF levels in peripheral monocytes, with stable angina and diabetes in coronary artery disease. The results give new insight to CAD and the impaired collateral vessel formation in diabetics.

Glycosaminoglycans and PDGF Signaling in Mesenchymal Cells

Platelet derived growth factor (PDGF) is involved in the autocrine growth stimulation of normal and malignant cells, the stimulation of angiogenesis, and the recruitment and regulation of tumor fibroblasts. PDGF has been shown to physically interact with glycosaminoglycans which are abundant in the extracellular microenvironment. The present review discusses the effects of glycosaminoglycans on the functions mediated by the PDGF on cells of mesenchymal origin. Recent studies have demonstrated that both soluble and surface bound glycosaminoglycan chains can modulate PDGF-BB isoform signaling depending on the cell type. These data demonstrated that the microenvironment rich in GAGs/PGs is able to significantly modify the cellular response to PDGF-BB signaling in a critical way for cell growth and differentiation.

Associations of xenobiotic-metabolizing enzyme genotypes PON1Q192R, PON1L55M and CYP1A1*2A MspI with pathological symptoms of a rural population in south Greece

Paraoxonases and cytochromes P450 constitute two major classes of xenobiotic-metabolizing enzymes involved in the detoxification of pesticide chemicals. In this study, we examined the distribution of two common genetic polymorphisms of the paraoxonase 1 gene and one common polymorphism of the CYP1A1 gene, in relation to pathological diseases occurring in a rural population. Blood and hair samples were collected from 220 participants of an agricultural cohort in the south of Greece for genotype and pesticide analysis. Demographic information and disease status of the participants was obtained by questionnaire, medical examination and medical record. Organochlorine pesticides and metabolites (DDTs, HCHs) were extracted from hair and analyzed using gas chromatography combined with mass spectrometry techniques. Our results indicate exposure of the rural population of Amaliada to organophosphate and past exposure to organochlorine pesticides. Genotypic analysis of PON1Q192R, PON1L55M and CYP1A1*2A MspI polymorphisms was performed using PCR-RFLP. The PON1 192R and 55M alleles absence was significantly associated with hypertension (OR: 2.59; 95% CI: 1.10–6.09) and hepatitis (OR: 21.43; 95% CI: 2.53–181.50), respectively, as indicated from backward logistic regression. Although the presence of PON1 192R allele significantly affected the occurrence of prostate hyperplasia (OR: 0.35; 95% CI: 0.03–0.40), no associations were obtained between the paraoxonase serum activity or the CYP1A1 genotype and the disease status.

Decorin-Mediated Effects in Cancer Cell Biology

Decorin is a multifunctional molecule of the extracellular matrix. Among the multitude of assigned functions the most intriguing is the ability to inhibit the growth and the metastasis of a wide range of cancer cells in vitro. Decorin was established to directly interact with EGFR and erb2, inducing protracted receptor internalization, which results in attenuation of the receptor-mediated intacellular signaling and induction of apoptosis. Studies by our group of osteosarcoma cells described the first exception to the established decorin-mediated growth suppression model. Osteosarcoma cells constitutively produced decorin and they were not sensitive to decorin-induced growth arrest. On the contrary, decorin seemed to be beneficial to osteosarcoma cells, since it was necessary for cell migration and acted as mediator, counteracting the TGFβ2-induced cytostatic function. Importantly, decorin did not induce p21 expression whereas EGFR appeared to be overexpressed and continuously phosphorylated in our osteosarcoma model. These data provide new insight on pathways that cancer cells might employ to overcome the established decorin-induced growth suppression.

Paraoxonase 1 R/Q alleles are associated with differential accumulation of saturated versus 20:5n3 fatty acid in human adipose tissue

Serum paraoxonase 1 (PON1) function has been associated with human cardiovascular disease. The projected mechanism postulates interaction of PON1 with lipoproteins and insulin signaling resulting in alterations in lipid homeostasis. Recently, PON2 was shown to directly regulate triglyceride accumulation in macrophages and PON1 was detected in the interstitial space of adipocytes. The aims of the present study were a) to examine the relationship of the PON1 function with serum parameters related to lipid homeostasis, and b) to examine a possible role of PON1 in the regulation of lipid composition in the human adipose tissue. Two important genetic variations with functional impact on PON1 activity in humans are the Q192R and the L55M. The present study evaluated the impact of the Q192R and the L55M polymorphisms in a cross-section of the population on the island of Crete, as regards to PON1 activity, plasma lipids/lipoproteins, parameters of the metabolic syndrome, and the fatty acid composition of the adipose tissue. We detected a significant association of the polymorphisms with blood pressure, fasting blood glucose, triglycerides, apolipoprotein B, serum iron, and homocysteine. Furthermore, a novel function is suggested for PON1 on the fatty acid composition in the adipose tissue through the positive association of the R allele with saturated fatty acid and of the Q allele with 20:5n3 fatty acid deposition.

The HIV-1 gp120/V3 modifies the response of uninfected CD4 T cells to antigen presentation: mapping of the specific transcriptional signature

BackgroundThe asymptomatic phase of HIV-1 infection is characterized by a progressive depletion of uninfected peripheral effector/memory CD4+ T cells that subsequently leads to immune dysfunction and AIDS symptoms. We have previously demonstrated that the presence of specific gp120/V3 peptides during antigen presentation can modify the activation of normal T-cells leading to altered immune function. The aim of the present study was to map the specific transcriptional profile invoked by an HIV-1/V3 epitope in uninfected T cells during antigen presentation.MethodsWe exposed primary human peripheral blood monocytes to V3 lipopeptides using a liposome delivery system followed by a superantigen-mediated antigen presentation system. We then evaluated the changes in the T-cell transcriptional profile using oligonucleotide microarrays and performed Ingenuity Pathway Analysis (IPA) and DAVID analysis. The results were validated using realtime PCR, FACS, Western blotting and immunofluorescence.ResultsOur results revealed that the most highly modulated transcripts could almost entirely be categorized as related to the cell cycle or transcriptional regulation. The most statistically significant enriched categories and networks identified by IPA were associated with cell cycle, gene expression, immune response, infection mechanisms, cellular growth, proliferation and antigen presentation. Canonical pathways involved in energy and cell cycle regulation, and in the co-activation of T cells were also enriched.ConclusionsTaken together, these results document a distinct transcriptional profile invoked by the HIV-1/V3 epitope. These data could be invaluable to determine the underlying mechanism by which HIV-1 epitopes interfere with uninfected CD4+ T-cell function causing hyper proliferation and AICD.