Abstract: Parathyroid hormone (PTH) strongly stimulates hyaluronan (HA) synthesis and secretion of both normal and carcinogenic cells of the osteoblastic lineage and improves skeletal microarchitecture. HA, a glycosaminoglycan component of the extracellular matrix (ECM), is capable of transmitting ECM-derived signals to regulate cellular function. In this study, we investigated whether the changes of HA metabolism induced by PTH (1-34) and PTH (7-84) peptides in moderately MG-63 and well-differentiated Saos 2 osteosarcoma cell lines, are correlated to their migration capabilities. Our results demonstrate that intermittent PTH (1-34) treatment significantly (P < or = 0.01) supported the migration of MG-63 cells, increased their HA-synthase-2 (HAS2) expression (P < or = 0.001), and enhanced their high-molecular size HA deposition in the pericellular matrix. Both increased endogenous HA production (P < or = 0.01) and treatment with exogenous high-molecular weight HA (P < or = 0.05) correlated to a significant increase of MG-63 cell migration capacity. Transfection with siHAS2 showed that PTH (1-34), mainly through HAS2, enhanced HA and regulated MG-63 cell motility. Interestingly, continuous PTH (1-34) treatment stimulated both Saos 2 cell HAS2 (P < or = 0.001) and HAS1 (P < or = 0.001) isoform expression inhibited their HYAL2 expression (P < or = 0.001) and modestly (P < or = 0.05) enhanced their migration. Therefore, the PTH (1-34) administration mode appears to distinctly modulate the migratory responses of the MG-63 moderately and Saos 2 well-differentiated osteosarcoma cell lines. Conclusively, the obtained data suggest that there is a regulatory effect of PTH (1-34), in an administration mode-dependent manner, on HA metabolism that is essential for osteosarcoma cell migration.
Abstract: BACKGROUND: Although oxidative stress plays an important role in the pathophysiology of restenosis, its role following the implantation of sirolimus-eluting stents (SES) is unknown. METHODS: We examined the relation between total peroxides (TP), a marker of oxidative stress, and in-stent late luminal loss over a 6-month follow-up in patients with stable coronary artery disease and compared the results from SES with those from bare metal stents (BMS). We enrolled 75 consecutive patients, who underwent successful PCI and were randomly allocated to SES (n=37) or BMS (n=38). Blood samples were taken 24 h before, at 24 h, 48 h and 1 month after angioplasty; levels of TP were determined on each occasion. Follow-up coronary angiography was performed 6-8 months later. RESULTS: TP levels in the BMS group were significantly higher at 24 h and 48 h compared to baseline (p=0.006 for both). At one month there was a significant decline from the 48 h levels (p=0.029) to levels slightly, but not significantly higher than baseline. In contrast, in SES TP levels showed no significant changes during the first 48 h, while they declined to levels somewhat lower than baseline at 30 days. A significant correlation was found between TP changes and in-stent late luminal loss at 6 months in both groups. CONCLUSION: Our study showed that patients with stable coronary artery disease who received SES have a different behavior of oxidative stress after stenting compared with BMS, and this could contribute to the difference in restenosis rate between these 2 types of stents.
Abstract: OBJECTIVES: Heparin acts as an extracellular stimulus capable of activating major cell signalling pathways. Thus, we examined the putative mechanisms utilized by heparin to stimulate HT29, SW1116 and HCT116 colon cancer cell growth. MATERIALS AND METHODS: Possible participation of the mitogen-activated protein kinase (MAPK) cascade on heparin-induced HT29, SW1116 and HCT116 colon cancer cell growth was evaluated using specific MAPK cascade inhibitors, Western blot analysis, real-time quantitative PCR and FACS apoptosis analysis. RESULTS: Treatment with a highly specific p38 kinase inhibitor, SB203580, significantly (50-70%) inhibited heparin-induced colon cancer cell growth, demonstrating that p38 MAPK signalling is involved in their heparin-induced proliferative response. This was shown to be correlated with increased (up to 3-fold) phosphorylation of 181/182 threonine/tyrosine residues on p38 MAP kinase. Furthermore, heparin inhibited cyclin-dependent kinase inhibitor p21(WAF1/CIP1) and p53 tumour suppressor gene and protein expression up to 2-fold or 1.8-fold, respectively, and stimulated cyclin D1 expression up to 1.8-fold, in these cell lines through a p38-mediated mechanism. On the other hand, treatment with heparin did not appear to affect HT29, SW1116 and HCT116 cell levels of apoptosis. CONCLUSIONS: This study demonstrates that an extracellular glycosaminoglycan, heparin, finely modulates expression of genes crucial to cell cycle regulation through specific activation of p38 MAP kinase to stimulate colon cancer cell growth.
Abstract: BACKGROUND: Hyaluronan (HA) a glycosaminoglycan, is capable of transmitting extracellular matrix derived signals to regulate cellular functions. In this study, we investigated whether the changes in HT1080 and B6FS fibrosarcoma cell lines HA metabolism induced by basic fibroblast growth factor (bFGF) are correlated to their migration. METHODS: Real-time PCR, in vitro wound healing assay, siRNA transfection, enzyme digestions, western blotting and immunofluorescence were utilized. RESULTS: bFGF inhibited the degradation of HA by decreasing hyaluronidase-2 expression in HT1080 cells (p=0.0028), increased HA-synthase-1 and -2 expression as we previously found and enhanced high molecular weight HA deposition in the pericellular matrix. Increased endogenous HA production (p=0.0022) and treatment with exogenous high molecular weight HA (p=0.0268) correlated with a significant decrease of HT1080 cell migration capacity. Transfection with siHAS2 and siHAS1 showed that mainly HAS1 synthesized high molecular weight HA regulates HT1080 cell motility. Induced degradation of the HA content by hyaluronidase treatment and addition of low molecular weight HA, resulted in a significant stimulation of HT1080 cells' motility (p<0.01). In contrast, no effects on B6FS fibrosarcoma cell motility were observed. CONCLUSIONS: bFGF regulates, in a cell-specific manner the migration capability of fibrosarcoma cells by modulating their HA metabolism. HA metabolism is suggested to be a potential therapeutic target in fibrosarcoma.
Abstract: Fibroblast growth factor-2 (FGF-2), the most abundant growth factor produced by melanoma cells but not by normal melanocytes, is an important regulator of cell proliferation, migration and differentiation. In this study we show that M5 human metastatic melanoma cells' ability to migrate is significantly enhanced by exogenously added FGF-2 while, neutralization of endogenous FGF-2 stimulates their adhesion. Previously, we have demonstrated that FGF-2 distinctly modulates the synthesis of individual glycosaminoglycans/proteoglycans (GAGs/PGs) subclasses, changing both their amounts and distribution in M5 cells. Here, treatment with FGF-2 strongly reduces the expression levels of the heparan sulfate-containing proteoglycan, syndecan-4. Syndecan-4 is a focal adhesion component in a range of cell types, adherent to several different matrix molecules, including fibronectin (FN). The reduction in syndecan-4 expression by utilizing specific siRNA discriminately increased melanoma cell motility and decreased their attachment on FN, demonstrating a regulatory role of syndecan-4 on these cell functions. Syndecan-4 has previously been demonstrated to regulate focal adhesion kinase (FAK) phosphorylation. In this study FGF-2 was shown to downregulate FAK Y397-phosphorylation during FN-mediated M5 cell adhesion, promoting their migration. The observed decrease in FAK Y397 activation was correlated to syndecan-4 expression levels. Thus, a balance in syndecan-4 expression perpetrated by FGF-2 may be required for optimal M5 cell migration. These results suggest that essential in melanoma progression FGF-2, specifically regulates melanoma cell ability to migrate through a syndecan-4-dependent mechanism.
Abstract: 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.
Abstract: Basic fibroblast growth factor (FGF-2) and its respective tyrosine kinase receptors, form an autocrine loop that affects human melanoma growth and metastasis. The aim of the present study was to examine the possible participation of various glycosaminoglycans, i.e. chondroitin sulfate, dermatan sulfate and heparin on basal and FGF-2-induced growth of WM9 and M5 human metastatic melanoma cells. Exogenous glycosaminoglycans mildly inhibited WM9 cell's proliferation, which was abolished by FGF-2. Treatment with the specific inhibitor of the glycosaminoglycan sulfation, sodium chlorate, demonstrated that endogenous glycosaminoglycan/proteoglycan production is required for both basal and stimulated by FGF-2 proliferation of these cells. Heparin capably restored their growth, and unexpectedly exogenous chondroitin sulfate to WM9 and both chondroitin sulfate and dermatan sulfate to M5 cells allowed FGF-2 mitogenic stimulation. Furthermore, in WM9 cells the degradation of membrane-bound chondroitin/dermatan sulfate stimulates basal growth and even enhances FGF-2 stimulation. The specific tyrosine kinase inhibitor, genistein completely blocked the effects of FGF-2 and glycosaminoglycans on melanoma proliferation whereas the use of the neutralizing antibody for FGF-2 showed that the mitogenic effect of chondroitin sulfate involves the interaction of FGF-2 with its receptors. Both the amounts of chondroitin/dermatan/heparan sulfate and their sulfation levels differed between the cell lines and were distinctly modulated by FGF-2. In this study, we show that chondroitin/dermatan sulfate-containing proteoglycans, likely in cooperation with heparan sulfate, participate in metastatic melanoma cell FGF-2-induced mitogenic response, which represents a novel finding and establishes the central role of sulfated glycosaminoglycans on melanoma growth.
Abstract: The cancer microenvironment and the interactions between cancer and surrounding tissue cells are thought to play a pivotal role in tumor development and progression. Glycosaminoglycans (GAGs)/proteoglycans (PGs) are major constituents of the extracellular matrix, the composition of which may affect various cellular functions. In the present study, the effects of GAGs on the proliferation of HT29, SW1116, and HCT116 human colon cancer cell lines were examined using exogenously added GAGs, an inhibitor of endogenous GAG sulfation and specific glycosidase digestions. Our results demonstrate that colon cancer cell growth was exclusively stimulated by exogenously added heparin and insensitive to endogenous GAGs/PGs production, in a sulfation pattern-related manner. Treatment of the tested cell lines with the FGF-2 neutralizing antibody showed that the stimulatory effect of heparin on the cells' growth was not FGF-2-dependent. Responsiveness of colon cancer cell lines to exogenous heparin/heparan sulfate may play a role in their growth and metastasis.
Abstract: 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-beta2-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.
Abstract: The small leucine-rich repeat proteoglycans (SLRPs) constitute a group of structurally and functionally related molecules that participate in the organization of the extracellular matrix ECM and have important effects on cell behavior. Osteosarcomas are heterogeneous bone tumors whose common characteristic is the production of an abundant nonmineralized (ECM)-osteoid. The scope of this minireview is to briefly present the current state of knowledge on the role of the SLRPs in osteosarcoma pathogenesis, with special emphasis on the recently described in osteosarcoma, proteoglycan lumican.
Abstract: Lumican belongs to the family of small leucine-rich repeat proteoglycans. Recent studies have shown that lumican participates in the maintenance of tissue homeostasis and modulates cellular functions including cell proliferation, migration, and differentiation. The expression of lumican has been correlated to the growth and metastasis of various malignancies; however, its exact role in tumorogenesis remains elusive. This review focuses upon the role of lumican in cell biology, providing insights into molecular mechanisms that lumican likely utilizes to control processes relevant to tumorogenesis.
Abstract: N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) are the dominant sialic acids (Sia) in mammals usually found in the non-reducing terminal of oligosaccharide side chains in glycoproteins and glycolipids. Their expression and distribution pattern have been correlated both with the malignant phenotype and tumor grade of human cancers. The aim of the present study was to determine by reversed-phase HPLC method the amounts of Neu5Ac and Neu5Gc as well as their distribution among the culture media and cell surface of MG-63 and Saos-2 human osteosarcoma cell lines of high and low metastatic potential. It was determined that MG-63 cells produce up to 5-fold more total sialic acid as compared with the Saos 2 cells. Neu5Ac accounts for ca 60% of the total sialic acids secreted by MG-63 cells, whereas Neu5Gc is the predominant sialic acid present on the MG-63 cell membrane. Saos 2 cells secrete considerable amounts of Neu5Ac to culture media. The obtained data indicate that the human osteosarcoma cells express both forms of Sia-containing glycoconjugates; the differences in the amounts of each of the two major Sia types and their distribution may be related to their differences in morphology and/or metastatic potentials.
Abstract: Peripheral artery stiffness is altered in diabetic patients with end-stage renal disease (ESRD), whereas few data exist to confirm this trend for proximal aortic stiffness. The pulse wave velocity of the proximal aorta (PWVr) and of the carotid-to-femoral aortic segment (PWVcf) were determined by ultrasound imaging in 160 patients with ESRD (70 diabetic) and in 160 matched control subjects. Also, plasma levels of endothelin, homocysteine, and high-sensitivity C-reactive protein were determined in both groups. Patients with ESRD had increased pulse pressure, left ventricular (LV) end-diastolic diameter, LV mass index, PWVr, and PWVcf compared with control subjects (p < 0.05). Diabetic patients had increased LV mass index, PWVr, and PWVcf compared with nondiabetic patients with ESRD (p < 0.05). Endothelin levels exhibited a strong relation with PWVr (r = 0.32, p < 0.001) and PWVcf (r = 0.33, p < 0.001) measurements in ESRD patients. Multivariate linear regression analysis revealed that age, diabetes, and plasma levels of endothelin were major determinants of increased PWVr measurements in the total ESRD population. After adjustment for age, body surface area, time on dialysis, systolic blood pressure, history of hypertension, and plasma endothelin levels, diabetes was an independent factor associated with PWVr in ESRD subjects. Diabetic patients with ESRD had significantly increased proximal aortic stiffness and significantly altered plasma levels of endothelin as compared with the nondiabetic.
Abstract: The soy isoflavone genistein can affect cell metabolism by specifically inhibiting protein tyrosine kinase (PTK) and/or interacting with the estrogen receptors (ERs). Glycosaminoglycans (GAG)/proteoglycans (PG) may participate in tumor development and progression. The synthesis of GAG by two human colon cancer cell lines, HT-29 and SW-1116, and the effects of genistein on their production and distribution between culture medium and cell membrane were studied. The mitogenic activity of genistein on both cell lines growth was also examined. Metabolic labeling, sensitive high pressure liquid chromatography (HPLC) techniques and fluorometric cell proliferation assays were utilized. The results demonstrate that both estrogen receptor beta-positive (ERbeta+) cancer cell lines produced hyaluronan (HA), both extracellular and membrane-associated galactosaminoglycans (GalAG) and heparan sulfate (HS), with the HT-29 cells producing all GAG fractions at significantly higher rates. The observed dose-dependent inhibitory effect of genistein on the synthesis of both secreted and cell-associated GAG/PG by the SW-1116 cells, as well as on their growth, was suggestive of a PTK mechanism. On the other hand, the synthesis of GAGs/PGs by HT-29 cells in the presence of genistein was dependent on their type and localization which implies the active participation of the ERs, which was further supported by the observed growth stimulation at low concentrations of genistein.
Abstract: BACKGROUND: The incidence of subclinical cardiotoxicity following anthracycline treatment for childhood cancer varies according to the method used for its detection. The aim of the study was to document the prevalence of left ventricular myocardial mass (LVM) reduction and its possible association with plasma N-terminal pro-brain natriuretic peptide (NT-proBNP) levels in asymptomatic children treated with anthracyclines. PATIENTS AND METHODS: Nineteen asymptomatic children who had received anthracyclines during their treatment for cancer were evaluated. They had received an equivalent of doxorubicin dose 240 mg/m2 (22-1200 mg/m2) on average 3.9 years (0.6-8.3) before (median age at diagnosis 3.8 years). The LVM was determined by M-Mode echocardiography and compared to the expected value, obtained from the regression equation of LVM on height of a group of 160 healthy children. Additionally the patients' plasma NT-pro BNP levels were determined. RESULTS: A high prevalence of reduced LVM associated with increased NT-proBNP levels was found. The average LVM value was -14.4% (+/-4.9) lower than expected whereas fourteen patients (73%) had a lower LVM than predicted. The NT-pro BNP levels in patients with reduced LVM were significantly higher than those measured in patients without LVM reduction (0.316+/-0.02 versus 0.17+/-0.01 pmol/ml respectively, p=0.009). A cut off NT-pro BNP level of 0.2 pmol/ml could differentiate patients with LVM reduction from those with normal or greater than expected LVM. CONCLUSION: The association of higher NT-proBNP levels with reduced LVM in asymptomatic children after anthracycline administration could be an early indication of subclinical cardiotoxicity.
Abstract: The authors investigated the time-dependent action of atorvastatin and simvastatin on oxidative stress and cytokine levels immediately after the start of treatment. These factors play a role in endothelial dysfunction. Hyperlipidemic patients (n = 132) were assigned to treatment with 40 mg atorvastatin, 40 mg simvastatin, or placebo. Blood samples were taken before, 2 hours, 24 hours, 7 days, and 3 weeks after the administration of the statin or placebo to evaluate serum concentrations of total peroxides (TP), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and soluble intercellular vascular adhesion molecule 1 (sICAM 1). In the atorvastatin group the TP changes were significantly different at 2 hours and 24 hours (p = 0.005), whereas in the simvastatin group there was a gradual, more or less linear decline in TP until 7 days (p = 0.006) and then a plateau. Simvastatin exhibited a faster statistically significant decrease over time in IL-6 and sICAM 1 levels (at 7 days, p = 0.014 and p = 0.001, respectively). TNF-alpha demonstrated a faster linear trend in the simvastatin group, but the significant effect appeared late (p = 0.006). Both simvastatin and atorvastatin exerted early beneficial effects on oxidative stress, proinflammatory cytokines, and endothelial activation in hyperlipidemic subjects. These effects became significant 2 hours following the initiation of therapy.
Abstract: Melanoma is a frequent and therapy-resistant human disease. Malignant melanocytes modulate their microenvironment in order to penetrate the dermal/epidermal junction and eventually invade the dermis. The small leucine-rich proteoglycans (SLRPs) constitute important constituents of the dermis extracellular matrix (ECM), participating in both the structural and the functional organization of the skin. The role of a keratan sulphate SLRP lumican, has recently been investigated in the growth and metastasis of several cancers. In this study, the expression of lumican was studied in two human melanoma cell lines (WM9, M5) as well as in normal neonatal human melanocytes (HEMN) using real time PCR, western blotting with antibodies against the protein core and keratan sulfate, and treatments with specific enzymes. Both human metastatic melanoma cell lines were found to express lumican mRNA and effectively secrete lumican in a proteoglycan form, characterized to be substituted mostly with keratan sulfate chains. Lumican mRNA was not detected in normal melanocytes. This is the first time that the synthesis and secretion of lumican in human melanoma cell lines is reported. The role of this proteoglycan in the development and progression of malignant melanoma has to be further investigated.
Abstract: Versican, a large sized chondroitin-sulphate proteoglycan (PG), and its binding partner, hyaluronan (HA), are extracellular matrix (ECM) components that play an essential role in transformed cell behavior. Expression of certain versican isoforms has been implicated in cell migration and proliferation of cancer cells and, on the other hand, disruption of HA synthesis by inhibiting hyaluronan synthase-2 (HAS2) expression in osteosarcoma cells by suppressing cell proliferation, invasiveness and motility. Considering that growth factors, such as TGF-beta, bFGF and PDGF-BB, are important regulators for the expression of the ECM macromolecules, in this study we examined the effect of these growth factors on the expression of the various versican isoforms, HA synthases as well as HA synthesis by MG-63 osteosarcoma cells and normal human osteoblastic periodontal ligament cells (hPDL). Real-time PCR and metabolic labelling followed by fine HPLC analysis coupled to radiochemical detection were the methods utilized. It was found that, contrary to normal hPDL cells, osteosarcoma MG-63 cells do not constitutively express the versican isoforms V0 and V1. Exogenous addition of TGF-beta2 stimulated the versican transcript levels mainly by forcing osteosarcoma cells to express V1 and V0 isoforms. PDGF-BB and bFGF had only minor effects in these cells. In hPDL cells a strong stimulation of the V3 transcript by all growth factors was observed. TGF-beta2 was also the major stimulator of HAS2 isoform expression as well as hyaluronan synthesis in osteosarcoma cells, while PDGF-BB exerted dominant influence on HAS2 isoform expression and hyaluronan biosynthesis by osteoblasts. The obtained results show for the first time that TGF-beta2 triggers the malignant phenotype pattern of versican and hyaluronan expression in human osteosarcoma cells and indicate that this growth factor may account for the metastatic potential of these cells.
Abstract: Osteoblastic cells produce a complex extracellular matrix (ECM) composed of a mixture of proteoglycans (PGs), collagens and non-collagenous proteins. The interaction of proteoglycans with matrix effector macromolecules via either their glycosaminoglycan (GAG) chains or their protein core is critical in regulating a variety of cellular events. Alterations in the structural composition of the GAG/PG component of the ECM may have important consequences on cell proliferation and/or differentiation. Human osteoblasts and two osteosarcoma cell lines, able to produce galactosaminoglycan (GalAGs) and heparan sulphate (HS)-containing proteoglycans, were treated with their main GAG chain types, and the effects on cell growth were examined. Chondroitin sulphate (CSA) and dermatan sulphate (DS) inhibited cell proliferation of all osteoblastic cell lines at high concentration (100 microg/ml). DS showed the stronger inhibitory effect, probably due to the presence of flexible IdoA residues that provide a greater variety in conformation to these macromolecules. Heparin strongly inhibited the proliferation rates of both normal osteoblasts and transformed osteoblastic cells at concentrations > or = 1 microg/ml. The presence of large amounts of IdoA-derived trisulphated disaccharides, responsible for the overall negative charge of heparin, should be considered as a critical factor for the inhibition of cell proliferation. The obtained results suggest that matrix GAGs are factors which affect cell growth of both malignant and normal cells of the osteoblastic lineage in a concentration-dependent manner. This effect is closely related to the fine chemical structure of GAGs, i.e. the presence of L-iduronic acid and the degree of sulphation.
Abstract: The insulin-like growth factor I (IGF-I) has been implicated in breast cancer development acting through insulin-like growth factor I receptor (IGF-IR), but also through estrogen receptor (ER). The effect of IGF on proteoglycan (PG) synthesis by two human breast cancer epithelial cell lines, the ER-positive MCF-7 and the ER-negative BT-20, was studied alone and in combination with genistein. Both cell lines synthesise hyaluronan (HA), matrix secreted and cell membrane-associated galactosaminoglycan containing proteoglycans (GalAGPGs) and heparan sulphate proteoglycans (HSPGs) in variable amounts. IGF-I affects the synthesis of PGs by BT-20 cells by decreasing the amounts of HA and secreted GalAGPGs and HSPGs and upregulates the expression of cell membrane-associated GalAGPGs and HSPGs. IGF-I exerts this effect on BT-20 cells acting mainly through receptors with protein tyrosine kinase activity (PTK). In contrast, IGF-I stimulates the synthesis of secreted GalAGPGs and HSPGs by MCF-7 cells, exhibiting only a slight suppression on synthesis of cell-associated GalAGPGs and HSPGs. The regulatory effect of IGF-I on PGs distribution in MCF-7 cells is mediated through a mix of pathways, which involves both receptors with PTK activity and PTK-independent signalling. It is suggested that the effects of IGF-I on the synthesis and distribution of PGs by epithelial breast cancer cells also depend on the presence or the absence of ER. The result of the IGF-I action is the balanced biosynthesis between the matrix and cell-associated PGs in both cell lines, approaching a common biosynthetic phenotype.
Abstract: OBJECTIVES: Although heart failure (HF) is characterized by increased proinflammatory cytokines, natriuretic peptide levels and impaired exercise capacity, the effect of concomitant diastolic dysfunction on those parameters has not been adequately studied. METHODS: We analyzed circulating levels of IL-1, IL-6, TNF-alpha and its soluble receptors, sTNFRI and sTNFRII, Nt-ANP and Nt-BNP natriuretic peptides in 81 patients, aged 56+/-12 years, with non-ischemic dilated cardiomyopathy (NIDC), LVEF 29.7+/-7.75% and functional NYHA class II-III. An echocardiographic study and cardiopulmonary exercise test (CPE) were performed in all patients. RESULTS: Patients were divided into restrictive (24 patients, group I) and non-restrictive (57 patients, group II) groups, according to their transmitral-filling pattern. No differences in LV dimensions or LVEF were found between the two groups. Group I showed increased levels of IL-6 (P=0.006), TNF-alpha (P=0.05), sTNFRII (P=0.02), Nt-ANP (P<0.001) and Nt-BNP (P<0.001) and decreased exercise duration (P<0.001) and PVO(2) (P<0.001) compared to group II. The strongest independent predictors for restrictive filling pattern were Nt-ANP and IL-6 levels, while Nt-BNP levels were the strongest PVO(2) predictor. CONCLUSIONS: Restrictive filling pattern implying greater diastolic dysfunction may contribute to increased cytokine production in the heart failure syndrome, as well as greater increases in natriuretic peptides and decreased exercise tolerance.
Abstract: The frequency and severity of crop protection product (pesticide) contamination of peaches grown conventionally were compared with those of peaches grown by integrated crop management (ICM). The peach samples (n = 150) were collected preharvest (June-August 2001) from both conventional (n = 55) and ICM (n = 95) cultivations from the Pella and Imathia districts of Macedonia, Northern Greece. The residue levels of selected insecticides, fungicides and acaricides in peach samples were determined by gas chromatography-mass spectrometry following solid-phase extraction. The concentrations of all detected pesticides were lower than the maximum residue limits (MRLs) in all peach samples grown with the ICM system (p<0.001). However, chlorpyrifos residues at levels higher than the MRLs were detected in four peach samples (i.e. 7% of the total samples) grown by the conventional system. Comparing the results for both cultivation methods with the reported average percentage (3.6%) of fruit samples with pesticide residues above the MRLs (European Union report for Greece in 2001), it was concluded that the initial implementation of the ICM in Greece was successful. The present study indicates that ICM cultivation has a higher efficiency in terms of product safety and quality. Furthermore, the results suggest that the application of conventional cultivation requires continuous monitoring of various crop protection product levels.
Abstract: The soy isoflavone genistein is a phytochemical which can affect the proliferation of both normal and cancer cells. The specific inhibition of protein tyrosine kinase (PTK) by which it effects the proliferation of cancer cells is a well-known mechanism of its action. Glycosaminoglycans (GAGs)/proteoglycans (PGs) are considered to be of great importance in the development and progression of cancer. The synthesis of GAGs by two osteosarcoma cell lines, MG-63 and SAOS-2, which differ in the density of estrogen receptors (ER) they express, and the effects of genistein on their synthesis and distribution among culture medium and cell membrane were studied. The obtained results showed that both cell lines synthesized extracellular hyaluronan (HA) and both extracellular and cell-associated galactosaminoglycans (GalAGs) and heparan sulfate (HS). Even though both cell lines synthesized considerable amounts of PGs, the SAOS-2 cells produced HA, GalAGs and HS at considerably lower rates than the MG-63 cells. The inhibitory effect of genistein on the synthesis of both extracellularly secreted and cell-associated GAGs/PGs in SAOS-2 2 cells was found to be dose-dependent and mediated most probably by a PTK mechanism. The synthesis of GAGs/PGs by the MG-63 cells in the presence of genistein was dependent on their type and localization, suggesting that a more complex mechanism regulates the PG synthesis. This may well involve the effect of genistein via the estrogen receptors, which are present in much higher density in MG-63 cells as compared to SAOS-2 cells.
Abstract: Experimental studies have shown that cytokine production by the heart may be regulated by sympathetic nervous system stimulation of cardiac beta-adrenergic receptors. Proinflammatory cytokine levels are increased in heart failure, whereas cardiac fixation of 123-I-metaiodobenzylguanidine (MIBG) has been used to study myocardial adrenergic innervation. This study was designed to assess the relation between cardiac MIBG uptake and circulating levels of proinflammatory cytokines in patients with idiopathic dilated cardiomyopathy (IDC). Forty-seven patients (12 women; mean age 56.5 +/- 9 years) with angiographically proved IDC, in New York Heart Association functional classes II to III, and who had left ventricular ejection fraction 30.6 +/- 9.5%, and 20 healthy controls were studied with planar MIBG. The early (10 minutes) and late (4 hours) heart to mediastinum uptake ratio and washout were calculated. Circulating plasma levels of interleukins (IL)-1 and IL-6, tumor necrosis factor-alpha, and solube receptors of TNF (sTNFR) I and II were measured. The patient group had significantly lower values of MIBG uptake at 10 minutes (p <0.001) and 4 hours (p <0.001) and higher washout (p <0.001) than the controls. Late MIBG uptake was significantly correlated with New York Heart Association class (r = -0.42, p = 0.02), left ventricular ejection fraction (r = 0.34, p = 0.01), left ventricular systolic wall stress (r = -0.40, p = 0.05), oxygen consumption at peak exercise (r = 0.32, p = 0.03), IL-1 (r = -0.55, p <0.001), TNF (r = -0.33, p = 0.02), and sTNFRII (r = -0.44, p = 0.001). Multivariate linear regression analysis revealed that MIBG at 4 hours was independently associated with IL-1 levels (p <0.001). Thus, reduced cardiac sympathetic innervation in heart failure is associated with elevated levels of inflammatory cytokines, suggesting that it has a potential inflammatory effect via modulation of the cardiac production of these cytokines.
Abstract: Genistein, a soy isoflavone, affects the proliferation of both estrogen receptor (ER)-positive and ER-negative cancer cells. Glycosaminoglycans (GAGs)/proteoglycans (PGs) are considered to be of great importance in the treatment of cancer. The synthesis of GAGs by two human breast cancer epithelial cell lines, the ER-positive MCF-7 and the ER-negative BT-20, was studied under the effects of genistein, and their distribution in the culture medium and the cell membranes was determined. The results obtained show that both cell lines synthesize extracellular hyaluronic acid (HA) and both extracellular and cell-associated galactosaminoglycans (GalAGs) and heparan sulphate (HS). The MCF-7 cell line synthesizes HA, GalAGs and HS at considerably lower rates than the BT-20 cell line. The effect of genistein on the synthesis of extracellularly secreted GAGs/PGs by ER-positive MCF-7 cells is dose-dependent and follows two mechanisms; one at low concentrations (< or = 35 microM) mediated via the estrogen receptor and the other at higher concentrations via protein tyrosine kinase (PTK). The synthesis of cell-associated GAGs/PGs by ER-positive MCF-7 cells and of both secreted and associated with the cell membrane GAGs/PGs by ER-negative BT-20 cells is mediated by a PTK mechanism. It is concluded that genistein affects the synthesis of GAGs/PGs, by breast cancer epithelial cells depending on the presence or absence of estrogen receptor and the localisation of PGs.
Abstract: INTRODUCTION: Patients with persistent atrial fibrillation (AF) have hemodynamic changes, which impair endothelial cell function resulting in decreased nitric oxide (NO) production. The aim of this work was to assess endothelial function in AF patients before and at various time points after cardioversion. METHODS: Forty-two patients with AF and 21 normal and age-adjusted healthy controls were studied. Nitrites and nitrates (NO(x)) and von Willebrand factor (vWf) concentrations were measured on blood samples taken just before cardioversion and over a 30 day period after the procedure. RESULTS: Plasma levels of NO(x) in AF were significantly lower compared to healthy controls (p < 0.001), but after cardioversion gradually increased to approach to those of the healthy controls by the end of the first month of sustained sinus rhythm (p = 0.004). Interestingly plasma levels of NO(x) were negatively correlated to left atrial volume measured by ultrasonography (r = -0.34, p < 0.05). Plasma levels of vWf in AF patients were significantly higher compared to the healthy controls (p < 0.01) but with sustained sinus rhythm decreased (p = 0.02). CONCLUSION: The parallel normalization of the NO(x) titers and vWf levels suggests that vascular endothelial function improves after 30 days of normal sinus rhythm.
Abstract: Nitric oxide (NO), which has diverse biological effects, can modulate AP-1 activity. Since DNA binding of Jun-Jun and Jun-Fos dimers is regulated in vitro by redox control involving conserved cysteines, we hypothesized that the action of NO is mediated via these residues. We performed electrophoretic mobility-shift analyses using Jun and Fos recombinant proteins and NO solutions. Cysteine-to-serine mutants showed that the inhibition of AP-1 activity following NO treatment was dependent on the presence of Cys7272 and Cys154 in the DNA binding domain of Jun and Fos, respectively. The inhibitory effect of NO was reversed by DTT and the thioredoxin system. Our results demonstrate that NO mediates its inhibitory effect by reacting specifically with the conserved cysteine residues in Jun and Fos.
Abstract: In activated human neutrophils a burst of nitric oxide (NO) converts intracellular GSH to S-nitrosoglutathione (GSNO) which is subsequently cleaved to restore GSH by an unknown mechanism. We discovered that GSNO is an NADPH oxidizing substrate for human or calf thymus thioredoxin reductase (TR) with an apparent Km value of 60 microM and a Kcat of 0.6 x s-1. Addition of human thioredoxin (Trx) stimulated the initial NADPH oxidation rate severalfold but was accompanied by progressive inactivation of TR. Escherichia coli TR lacked activity with GSNO, but with E. coli Trx present, GSNO was reduced without inhibition of the enzyme. Chemically reduced E. coli Trx-(SH)2 was oxidized to Trx-S2 by GSNO with a rate constant of 760 M-1s-1 (7-fold faster than by GSSG) as measured by tryptophan fluorescence. Analysis of this reaction in the presence of oxymyoglobin revealed quantitative formation of metmyoglobin indicative of NO. release. Analysis of GSNO reduction demonstrated that oxidation of NADPH produced a stoichiometric amount of free GSH. These results demonstrate a homolytic cleavage mechanism of GSNO, giving rise to GSH and NO.. GSNO efficiently inhibited the protein disulfide reductase activity of the complete human or calf thymus thioredoxin systems. Our results demonstrate enzymatic cleavage of GSNO by TR or Trx and suggest novel mechanisms for redox signaling.
