Biomedical subjects

Agapios Sachinidis

Publications and source records attributed to Agapios Sachinidis.

At least 19 recordsLinked to original sources

PubMed2007

Identification of differentially expressed genes involved in the formation of multicellular tumor spheroids by HT-29 colon carcinoma cells.

The multicellular tumor spheroid (MCTS) model represents a suitable in vitro model recreating in vivo tumor formation. The aim of this study was to identify differentially expressed genes that could potentially serve as predictive gene markers for MCTS and be involved in the formation of MCTS. Using the suppression subtractive hybridization (SSH) method, we identified ERBB2/HER2-interacting protein (Erbin), Tumor rejection gp96 (Tr-gp96), 12S ribosomal RNA (12S rRNA), ATP synthase, Kruppel-like transcription factor 5 (KLF5), transcription factor-like 5 (TCFL5), and the dual-specificity phosphatase 11 (DUSP11) to be overexpressed in 3-day-old HT-29 colon carcinoma MCTSs compared to HT-29 colon carcinoma cells grown in monolayer. We could also confirm overexpression of these genes in HT-29 MCTSs and in MCTSs formed by the human glioblastoma tumor cell lines U343 MG, U373 MG, and DBTRG 05 MG. Knockdown of KLF5, Erbin, DUSP11, and TCFL5 was effectively achieved after transfection of HT-29 cells with the appropriate short-interfering RNAs (siRNAs), and correlated with a significant inhibition of MCTS formation in the case of KLF5, Erbin, and TCFL5 siRNAs. We suggest that KLF5, Erbin, and TCFL5 are essential for MCTS formation and play a key role in the development of tumor diseases.

Adaptor Proteins, Signal Transducing↗

PubMed2006

The FunGenES consortium: functional genomics in engineered embryonic stem cells.

Although the complete sequence of a mammalian genome defines the information content of each cell, understanding the selective usage of this information during the development of specific cell types is limited. The fundamental questions that remain to be answered includes, which are the gene subsets that define the pluripotential self-renewing state of embryonic stem (ES) cells, partially and terminally differentiated developmental states, and how are transitions between these states regulated (lineage commitment)? The FunGenES consortium has been formed to address this challenge by mapping the gene subsets involved in pluripotent, lineage committed, and selected differentiated cell types using gene expression profiling and functional screens. They create an atlas of mammalian genome participating in early and late developmental processes. To fulfil the aim, mouse ES cells were used as an in vitro developmental model system that is very close to the human as they are pluripotent. They can be differentiated through the three major developmental pathways ecto-, meso-, and endoderm into many committed cell types and can be genetically engineered with relative ease. Knowledge of genetic pathways in mouse ES cell differentiation and development might be translated to human ES cells and the potential development of stem cell-based therapies.

Animals↗

PubMed2006

Cardiovascular genomics: a current overview of in vivo and in vitro studies.

The cardiovascular system is the first system that is developed in the embryo. The cardiovascular development is a complex process involving the coordination, differentiation, and interaction of distinct cell lineages to form the heart and the diverse array of arteries, veins, and capillaries required to supply oxygen and nutrients to all tissues. Embryonic stem cells have been proposed as an interesting model system to investigate molecular and cellular mechanisms involved in mammalian development. The present review is focused on extrinsic soluble factors, intrinsic transcription factors, receptors, signal transduction pathways, and genes regulating the development of cardiovascular system in vivo and in vitro. Special emphasis has been given to cardiovascular genomics including gene expression studies on the cardiovascular system under developmental and pathophysiological conditions.

Animals↗

PubMed2006

Identification of small signalling molecules promoting cardiac-specific differentiation of mouse embryonic stem cells.

Identification of signalling cascades involved in cardiomyogenesis is crucial for optimising the generation of cardiomyocytes from embryonic stem cells (ES cells) (in vitro). We used a transgenic ES cell lineage expressing enhanced green fluorescent protein (EGFP) under the control of the alpha-myosin heavy chain (alpha-MHC) promoter (palphaMHC-EGFP) to investigate the effects of 33 small molecules interfering with several signalling cascades on cardiomyogenesis. Interestingly, the L-Type Ca2+ channel blocker Verapamil as well as Cyclosporin, an inhibitor of the protein phosphatase 2B, exerted the most striking pro-cardiomyogenic effect. Forskolin (adenylate cyclase stimulator) exerted the most striking anti-cardiomyogenic effect. The cardiomyogenic effect of Cyclosporin and Verapamil correlated with an expression of early cardiac markers Nkx2.5 and GATA4. Compared to the effects on late developmental stage embryoid bodies (EBs) stimulation of early developmental stage EBs (1-day old) with Verapamil or Cyclosporin for 48 h resulted in a potent cardiomyogenic effect. Accordingly, enhanced expression of alpha-MHC mRNA and EGFP mRNA was observed after stimulation of the early developmental stage EBs for 48 h. No expression of alpha-smooth muscle actin or platelet endothelial cell adhesion molecule-1 (PECM-1) as well as of neuronal genes (Nestin, Neurofilament H) has been observed demonstrating a preferentially pro-cardiomyogenic effect by both molecules.

Adenylyl Cyclases↗

PubMed2006

Generation and characterization of cardiomyocytes under serum-free conditions.

In vitro culturing of mammalian cells provides an elegant platform to study cell signaling, interactions, and metabolism as well as proliferation and differentiation processes. Often, these cells are cultured and maintained in sera obtained from animals such as horses, cows, and rabbits. The sera used for this purpose fluctuates in composition from individual animals and, hence, influences the cellular growth and differentiation at different magnitudes. This poses a need to use a substitute for sera in cell culture systems to overcome the observed variations. Here, we present and compare protocols for culturing of embryonic stem (ES) cells in serum-free conditions, derivation of germ layers, and cardiac differentiation of ES cells in both serum-free and serum-containing culture conditions. Differentiated embryoid bodies by serum-free protocols produce significantly increased frequencies of clusters of cardiac cells beating stronger than found in serum-containing media. Therefore, we conclude that the use of serum replacement media (SRM) in our experiments led to more specific differentiation but reduced proliferation because these SRMs contained reduced essential substances like growth factors and hormones. Unlike serum media, SRMs have a well-defined composition and are highly reproducible. Hence, SRM will be the ideal substitute for serum-containing media.

Animals↗

PubMed2006

Dietary small molecules and large-scale gene expression studies: an experimental approach for understanding their beneficial effects on the development of malignant and non-malignant proliferative diseases.

Epidemiological studies have repeatedly demonstrated a correlation between nutrition, development and the severity of malignant and non-malignant proliferative diseases such as cancer and atherosclerosis. Therefore, the prevention of chronic proliferative diseases through dietary intervention is currently receiving considerable attention. Until now, much of the research is being focused on the cellular and molecular action mechanisms of dietary small molecules explaining their beneficial effects. Dietary chemicals may affect gene expression in several human diseases. However, significant progress has been made and several molecular action mechanisms have been proposed. Alteration of genetical pathways by nutrition, also called "Nutrigenomics", may offer a new approach for understanding the beneficial effects of dietary compounds on the development of severe polygenic diseases, such as cardiovascular disease, diabetes and hypertension. This review focuses on the nutritional genomics of dietary chemicals with a special emphasis on catechins. Catechins belong to the flavonoid family, which are polyphenolic compounds available in foods of plant origin. Several epidemiological studies have reported that consumption of flavonoids, and especially catechins might function as chemopreventive agents against cancer and cardiovascular diseases.

Animals↗

PubMed2005

Trapping of growth factors by catechins: a possible therapeutical target for prevention of proliferative diseases.

The prevention of cancer through dietary intervention is currently receiving considerable attention. Several epidemiological studies substantiate that green tea has a protective effect against a variety of malignant proliferative disorders such as lung cancer, breast cancer and prostate cancer. This preventive potential of green tea against cancer is attributed to the biologically active flavonoids called catechins. Epigallocatechin 3-o-gallate, the major catechin found in green tea, mediates diverse physiological and pharmacological actions in bringing about the regression of the tumors and also lowers the risk of nonmalignant cardiovascular proliferative diseases. Much of the current research is being focused on how these catechins specifically bring about the regression of the experimentally induced tumors both in vitro and in vivo. These catechins exert diverse physiological effects against proliferative diseases by several mechanisms, most of which are not completely characterized. This review summarizes the mechanisms by which these catechins play an essential role in regulating the process of carcinogenesis, with a special emphasis on how these catechins antagonize the growth factor-induced proliferative disorders.

Animals↗

PubMed2005

An optimized embryonic stem cell model for consistent gene expression and developmental studies: a fundamental study.

In vitro differentiation of embryonic stem (ES) cells results in generation of tissue-specific somatic cells and may represent a powerful tool for general understanding of cellular differentiation and development in vivo. Culturing of most ES cell lines requires murine embryonic fibroblasts (MEF), which may influence adventitiously the genetic differentiation program of ES cells. We compared the expression profile of key developmental genes in the MEF-independent CGR8 ES cell line and in the MEF-dependent D3 ES cell line. Using neomycin-resistant MEFs we demonstrated that MEFs are able to contaminate the D3 ES cells even after removing the MEFs. Subsequently, optimal differentiation conditions were established for the differentiation of CGR8 ES cells into various germ layer cells. Detailed gene expression studies in differentiating CGR8 cells were done by RT-PCR analysis and by microarray analysis demonstrating a general trend of the assessed genes to be expressed either in 3 days- or 10-days old embryoid bodies (EBs) when compared to undifferentiated ES cells. Subsets within the various functional gene classes were defined that are specifically up- or down-regulated in concert. Interestingly, the present results demonstrate that developmental processes toward germ layer formation are irreversible and mostly independent of the culture conditions. Notably, apoptotic and mitochondrial ribosomal genes were down- and up-regulated in 10-days old EBs, respectively, whereas compared to the 3-days old EBs whereas the activity of the extracellular signal-regulated kinase (ERK) 1/2 decreased with progressive development. This article defines a platform for ES cell differentiation and gene expression studies.

Animals↗

PubMed2005

A novel component of the ubiquitin pathway, ubiquitin carboxyl extension protein 1 is overexpressed in prostate cancer.

Prostate cancer is among the most common tumors in industrialized nations. However, little is known about the molecular events underlying its development. In the present study we used suppressive subtraction hybridization (SSH) in combination with laser-assisted microdissection in order to compare gene expression between prostate carcinoma and the normal prostate proper. Both are mixed tissues which consist of an epithelial and a stromal compartment. We first compared mRNA (cDNA) expression by SSH and then used real-time quantitative RT-PCR analysis of microdissected tissue probes in order to verify differential expression of subtracted cDNA clones. We also used differentially expressed cDNAs for the synthesis of radiolabelled riboprobes in order to attribute differential expression to specific cell types in tissue sections by in situ hybridization. Using this approach we found an up-regulation of ubiquitin carboxyl extension protein 1 (UBCEP-1) mRNA in prostate carcinoma cells compared to the normal glandular epithelium of the prostate proper. UBCEP-1 mediated ubiquitin chain elongation may promote prostate carcinoma development by increasing via the proteasome pathway the degradation of proteins which are involved in growth inhibition or apoptosis.

Aged↗

PubMed2004

Embryonic stem cells for basic research and potential clinical applications in cardiology.

Embryonic stem (ES) cells are pluripotent, possessing the unique property to differentiate into any somatic cell type while retaining the ability to proliferate indefinitely. Due to their ability to recapitulate embryonic differentiation, ES cells are an ideal tool to study the process of early embryogenesis in vitro. Signalling cascades and genes involved in differentiation can be easily studied, and functional genomics approaches aim to identify the regulatory networks underlying lineage commitment. Their unique ability to differentiate into any cell type make ES cells a prime candidate for cell replacement therapy (CRT) of various degenerative disorders. Results from various disease models are promising and have demonstrated their principal suitability as a therapeutic agent in diseases such as myocardial infarctions, diabetes mellitus and Parkinson's disease. Prior to clinical trials in humans, two issues remain to be solved: due to their high proliferative potential, ES cells can form teratocarcinomas in the recipient, and depending on the source of the cells, ES cell grafts may be rejected by the host organism. This review discusses the current state of basic ES cell research with a focus on cardiac differentiation and gives an overview of their use in CRT approaches.

Animals↗

PubMed2004

Inhibition of the vascular-endothelial growth factor-induced intracellular signaling and mitogenesis of human endothelial cells by epigallocatechin-3 gallate.

Galloyl group-containing catechins, such as epigallocatechin-3 gallate, inhibit receptor tyrosine kinase activity of several growth factor receptors. This study investigated the effects of epigallocatechin-3 gallate, as compared to epicatechin, on vascular endothelial growth factor-induced intracellular signaling and mitogenesis of human umbilical endothelial cells. Epigallocatechin-3 gallate concentration-dependently inhibited vascular endothelial growth factor-induced DNA synthesis, cell proliferation, autophosphorylation of vascular endothelial growth factor receptors-1 and -2, phosphorylation of extracellular signal-regulated kinases-1 and -2, and mRNA expression of the early growth response factor-1. In contrast, epicatechin was not effective. Thus, epigallocatechin-3 gallate may be an attractive candidate drug to inhibit tumour angiogenesis.

Catechin↗

PubMed2004

Local delivery of green tea catechins inhibits neointimal formation in the rat carotid artery injury model.

It has been shown that green tea catechins (GTC) suppress proliferation of vascular smooth muscle cells (VSMCs) and that epigallocatechin-3-gallate (EGCG), which is a major constituent of GTC, selectively inhibits the platelet-derived growth factor-BB (PDGF-BB)-induced intracellular signaling transduction pathway. Vascular smooth muscle cell proliferation is one of major mechanisms of restenosis following percutaneous coronary intervention. This study tested whether GTC can inhibit VSMC proliferation and prevent neointimal formation in a rat carotid artery injury model. Vascular smooth muscle cell proliferation inhibition was analyzed with [3H]thymidine incorporation. Green tea catechins were applied to the endothelium-denuded carotid arteries of rats for 20 min. Angiography and morphometric analysis was performed after 2 weeks. Green tea catechins decreased [3H]thymidine incorporation stimulated with PDGF-BB dose dependently. In the absence of PDGF-BB, the decrement of [3H]thymidine incorporation was evident above a concentration of 10 micro g/ml of GTC. Carotid arteriographic evaluation showed that the minimum luminal diameter in the GTC-treated group (n=12) was 5.9 +/- 1.6 arbitrary units (a.u.) and was significantly larger than in the control group (4.3 +/- 1.4 a.u., n=10) ( P <0.05). The GTC-treated group also showed a significant reduction in neointimal formation compared with the control group (0.29 +/- 0.11 vs 0.42 +/- 0.10 mm2, P < 0.05). To identify the active ingredients, we performed a similar experiment using EGCG. The effects of EGCG were similar to those of GTC. Green tea catechins effectively inhibited VSMC proliferation. Neointimal formation was prevented in the rat carotid artery injury model by local delivery of GTC. As EGCG showed similar effects, it may be one of the major constituents of GTC having these effects.

Angiography↗

PubMed2004

Inhibitory effect of epigallocatechin 3-O-gallate on vascular smooth muscle cell hypertrophy induced by angiotensin II.

Recent evidence indicates that epigallocatechin 3-O-gallate (EGCG), the major catechin derived from green tea leaves, lowers the risk of cardiovascular diseases such as atherosclerosis and hypertension. However, a precise mechanism for this biologic function has not yet been clearly delineated. Angiotensin II (Ang II) stimulates vascular smooth muscle cell (VSMC) hypertrophy, which is a critical event in the development of atherosclerosis, hypertension, and angioplasty-induced restenosis. In the present study, we show that EGCG inhibits Ang II-stimulated VSMC hypertrophy, as determined by [3H]leucine incorporation into VSMC. Since mitogen-activated protein kinase (MAPK) families are involved in cell growth, we determined whether EGCG affects them. EGCG pretreatment did not exert any significant changes in Ang II-stimulated activation of extracellular signal-regulated kinase (ERK) and p38 MAPK. EGCG only inhibited Ang II-stimulated activation of c-Jun N-terminal kinase (JNK). Moreover, EGCG suppressed Ang II-induced c-jun mRNA expression. In contrast, EGC, a structural analogue of EGCG, did not inhibit the JNK activity or c-jun mRNA expression. In addition, a specific JNK inhibitor, SP600125, dose-dependently suppressed Ang II-stimulated VSMC hypertrophy. These results suggest that the effect of EGCG on Ang II-induced VSMC hypertrophy is due to specific inhibition of the JNK signaling pathway at both transcriptional and posttranslational levels, which may underlie its beneficial effect on the cardiovascular diseases.

Angiotensin II↗

PubMed2004

Transcriptional responses to epigallocatechin-3 gallate in HT 29 colon carcinoma spheroids.

Catechins have been reported to possess anti-cancer activity in vitro and in vivo. To identify target genes that may be involved in the anti-tumorigenic effect of catechins, gene expression profiles in adherent human HT 29 colon carcinoma cells, in HT 29 spheroids and in epigallocatechin-3 gallate (EGCG)-treated HT 29 cells have been analysed by high-density oligonucleotide microarrays. Treatment of HT 29 cells with EGCG (2.5-50 microm) resulted in a dose-dependent inhibition of spheroid formation of HT 29 cells. Forty transcripts were induced at least twofold in 3-day-old spheroids relative to normal adherent cells using three replicates. Oncogenes like c-fos and c-jun are significantly up-regulated in spheroids. We identified several signal transduction and proliferation genes which are down-regulated in response to EGCG treatment. Increase in the mRNA expression profile of c-Fos correlated well with protein levels in HT 29 spheroids whereas EGCG did not affect protein formation. In agreement with the DNA chip data, IQGAP2 protein was not increased in spheroids but protein formation was totally blocked in EGCG-treated cells. Interestingly, no change in expression of cytotoxic or apoptotic related genes has been observed in EGCG-treated cells. Our findings suggest that EGCG may exert its anti-cancer activity through modulation of expression of a number of genes that are involved in cell proliferation, cell-cell contacts and cell-matrix interactions.

Blotting, Western↗

PubMed2004

Regulation of mitogen-activated protein kinase cascades by low density lipoprotein and lysophosphatidic acid.

Aim of the present study was to elucidate the regulation of the mitogen activated protein (MAP) kinase cascades as well as the cross-talk between the various MAP kinase isoforms (ERK1/2, SAPK and p38) after stimulation of vascular smooth muscle cells (VSMC) with low density lipoprotein (LDL), 100 microg/ml or lysophosphatidic acid (LPA), 5 microg/ml. Furthermore, the role of the intracellular free Ca(2+) concentration ([Ca(2+)](i)) on the activation of the MAP kinase isoforms as well as on the protein expression of MAP kinase phosphatase (MKP)-1 was investigated. The methods used were Western blot analysis, immunoprecipitation and LDL isolation. Involvement of G(i)-proteins on LDL- and LPA-induced activation of the MAP kinase isoforms was examined by treatment of VSMC with pertussis toxin (PTX),100 ng/ml. LDL as well as LPA induced an activation of SAPK and p38 MAP kinase in a PTX-sensitive manner. The ERK1/2, SAPK and p38 MAP kinase activation was a Ca(2+)-dependent process, most likely regulated through modulation of MKP-1 protein expression. Inhibition of ERK1/2 by PD 98059 completely abolished LDL- and LPA-induced activation of SAPK, whereas the activation of p38 MAP kinase was not affected. We conclude, that [Ca(2+)](i) regulates the PTX-sensitive LDL- and LPA-induced stimulation of the MAP kinase cascades, probably via inhibition of the MKP-1 protein expression.

Animals↗

PubMed2004

Possible non-classic intracellular and molecular mechanisms of LDL cholesterol action contributing to the development and progression of atherosclerosis.

Elevated low density lipoprotein (LDL) cholesterol (LDL-C) levels represent one of the most important risk factors for atherosclerosis and therefore cardiovascular morbidity and mortality. LDL-C operates at different levels and through various classic and non-classic mechanisms. For example, it has been recently shown that both native and oxidized LDL are potent growth factors for several cell types such as vascular smooth muscle cells (VSMC) participating in the development and progression of atherosclerosis. Moreover, LDL-C modulates the expression of various growth factors and growth factor receptors that are involved in the process of atherosclerosis. More specifically, LDL-C can phosphorylate and therefore activate the epidermal growth factor (EGF) receptor and enhance the production of platelet derived growth factor (PDGF)-AA and of the PDGF receptors. LDL as well as oxidized LDL (oxLDL) signal transduction pathways involve trimeric G-proteins and cAMP, protein kinase C and ceramide, diacylglycerol and inositol-1,4,5-triphosphate, Ca(+2), Na(+)/H(+) exchange, c-fos and egr-1, phospholipases C, A2 and D, Raf-1, MEK1/2, the ERK1/2 (p42/44), SAP/JNK and p38 isoforms of the mitogen activated protein kinases (MAPK) as well as the signal transuding element gp 130. Furthermore, the mitogenic effects of oxLDL may be mediated by its oxidation products such as lysophosphatidylcholine (LPC), and lysophosphatidic acid (LPA), through LDL-induced lactosylceramide (LacCer) synthesis, and, as our group has recently shown, through LDL-adherent factors such as sphingosine-1-phosphate (S1P) and sphingosylphosphorylcholine (SPC). We review the various LDL-mediated signal transduction pathways implicated with the development and progression of atherosclerosis.

Animals↗

PubMed2003

Mechanisms of the inhibitory effects of epigallocatechin-3 gallate on platelet-derived growth factor-BB-induced cell signaling and mitogenesis.

An enhanced activity of receptor tyrosine kinases (RTKs), such as the platelet-derived growth factor (PDGF) alpha-receptor (PDGF-Ralpha) or the PDGF beta-receptor (PDGF-Rbeta), is involved in the development of proliferative diseases. We have previously demonstrated that green tea catechins containing a galloyl group in the third position of the catechin structure interfere with PDGF-BB-induced mitogenic signaling pathways by inhibiting tyrosine phosphorylation of the PDGF-Rbeta. However, the underlying cellular and molecular mechanisms are unknown. Using human vascular smooth muscle cells (VSMC) and porcine endothelial cells (AEC) stably transfected with PDGF-Ralpha and -beta, respectively, we demonstrate that EGCG preferably inhibited PDGF-BB isoform-mediated signal transduction pathways and cell proliferation. To elucidate cellular and molecular mechanisms of the inhibitory effects of EGCG, we studied the distribution of incorporated EGCG into cellular compartments after subcellular fractionation. Interestingly, most (85%) of the EGCG was found in the cytoplasmic fraction, whereas only ~2% was found within the cell plasma membranes. However, no alteration of membrane fluidity has been observed after treatment of VSMC with 50 microM EGCG. Binding studies with [125I]-PDGF-BB on EGCG-treated VSMC demonstrated that the specific binding of PDGF-BB was completely abolished. Moreover, when [125I]-PDGF-BB was incubated with VSMC in the presence of EGCG, a 50% reduction of cellular [125I]-PDGF-BB binding was observed. Our findings suggest that plasma membrane incorporated EGCG or soluble EGCG directly interacts with PDGF-BB, thereby preventing specific receptor binding.

Becaplermin↗

PubMed2003

Regulation of intrinsic prion protein by growth factors and TNF-alpha: the role of intracellular reactive oxygen species.

Function and regulation of the intrinsic prion protein (PrPc) are largely unknown. In the present study the regulation of PrPc expression by growth factors and cytokines that increase intracellular reactive oxygen species (ROS) levels was studied in glioma and neuroblastoma cells grown as multicellular tumor spheroids. PrPc protein was significantly increased when glioma spheroids were treated with either ATP, nerve growth factor (NGF), epidermal growth factor (EGF), or tumor necrosis factor alpha (TNF-alpha), whereas mRNA levels as evaluated by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) remained unchanged. ATP, NGF, EGF, and TNF-alpha raised intracellular ROS levels as evaluated using the redox-sensitive fluorescence dye 2'7'-dichlorodihydrofluorescein diacetate (H2DCFDA). The observed elevation in PrPc was completely abolished in the presence of the free radical scavengers vitamin E and ebselen, as well as following pretreatment with the NADPH-oxidase inhibitor diphenylen iodonium chloride (DPI), indicating that PrPc levels are regulated by intracellular ROS. The correlation of PrPc expression to the intracellular ROS levels was investigated by the use of neuroblastoma cells overexpressing either mutant V210I PrP, or wild-type PrPc. It was observed that the intracellular redox state was significantly reduced in PrPc as well as V210I PrP overexpressing cells as compared to non-transfected cells. Consequently, the observed elevation of ROS following treatment with ATP was completely abolished in PrP overexpressing cells. Our data are in line with the assumption that PrPc plays a role as free radical scavenger and/or sensor molecule for oxidative stress.

Adenosine Triphosphate↗