Biological Effect of Silver-modified Nanostructured Titanium Dioxide in Cancer.

BACKGROUND/AIM: Nanomedicine is a promising scientific field that exploits the unique properties of innovative nanomaterials, providing alternative solutions in diagnostics, prevention and therapeutics. Titanium dioxide nanoparticles (TiO2 NPs) have a great spectrum of photocatalytic antibacterial and anticancer applications. The chemical modification of TiO2 optimizes its bioactive performance. The aim of this study was the development of silver modified NPs (Ag/TiO2 NPs) with anticancer potential. MATERIALS AND METHODS: Ag/TiO2 NPs were prepared through the sol-gel method, were fully characterized and were tested on cultured breast cancer epithelial cells (MCF-7 and MDA-MB-231). The MTT colorimetric assay was used to estimate cellular viability. Western blot analysis of protein expression along with a DNA-laddering assay were employed for apoptosis detection. RESULTS AND CONCLUSION: We show that photo-activated Ag/TiO2 NPs exhibited significant cytotoxicity on the highly malignant MDA-MB-231 cancer cells, inducing apoptosis, while MCF-7 cells that are characterized by low invasive properties were unaffected under the same conditions.

Liraglutide, a human glucagon-like peptide-1 analogue, stimulates AKT-dependent survival signalling and inhibits pancreatic ß-cell apoptosis.

Liraglutide, a human long-lasting GLP-1 analogue, is currently regarded as a powerful treatment option for type 2 diabetes. Apart from glucoregulatory and insulinotropic actions, liraglutide increases ß-cell mass through stimulation of ß-cell proliferation and islet neogenesis, as well as inhibition of ß-cell apoptosis. However, the underline molecular mechanisms have not been fully characterized. In this study, we investigated the mechanism by which liraglutide preserves islet ß-cells in an animal model of overt diabetes, the obese db/db mice, and protects a mouse pancreatic ß-cell line (ßTC-6 cells) against apoptosis. Treatment of 12-week-old diabetic mice with liraglutide for 2 weeks had no appreciable effects on blood non-fasting glucose concentration, islet insulin content and body weight. However, morphological and biochemical examination of diabetic mouse pancreatic islets demonstrated that liraglutide restores islet size, reduces islet ß-cell apoptosis and improves nephrin expression, a protein involved in ß-cell survival signalling. Our results indicated that liraglutide protects ßTC-6 cells from serum withdrawal-induced apoptosis through inhibition of caspase-3 activation. The molecular mechanism of the anti-apoptotic action of liraglutide in ßTC-6-cells comprises stimulation of PI3-kinase-dependent AKT phosphorylation leading to the phosphorylation, hence inactivation of the pro-apoptotic protein BAD and inhibition of FoxO1 transcription factor. In conclusion, we provided evidence that the GLP-1 analogue liraglutide exerts important beneficial effects on pancreatic islet architecture and ß-cell survival by protecting cells against apoptosis. These findings extend our understanding of the actions of liraglutide and further support the use of GLP-1R agonists in the treatment of patients with type 2 diabetes.

Effects of Dietary Supplementation with Sea Buckthorn (Hippophae rhamnoides L.) Seed Oil on an Experimental Model of Hypertensive Retinopathy in Wistar Rats.

BACKGROUND/AIMS: Sea buckthorn (Hippophae rhamnoides L.) oil is a rich source of phytosterols, flavonoids, unsaturated fatty acids, and carotenoids, known for their antioxidant and neuroprotective activity. In this study, we investigated the neuroprotective and antioxidant effect of sea buckthorn oil on rat retina in hypertensive retinopathy. METHODS: Twenty-eight male 6-month-old Wistar rats were separated into 3 groups: (1) controls, (2) unilateral nephrectomized rats receiving drinking water with 1% NaCl, (3) unilateral nephrectomized rats receiving 0.5 mL sea buckthorn oil and drinking water with 1% NaCl. Systemic pressures were being measured with the tail-cuff method. The antiapoptotic effect of sea buckthorn was determined by measuring glial fibrillary acidic protein (GFAP), cleaved caspase-3, and glutamine synthetase levels with immunohistochemistry and Western blot. RESULTS: Nephrectomy and salt intake caused increases in both systolic and diastolic pressures. Both types of analysis showed that group 2 had statistically significant increases in the expression of GFAP and cleaved caspase-3, while group 3 showed no significant differences compared with the control group. The expression of glutamine synthetase showed no significant differences between the 3 groups. CONCLUSIONS: Our findings suggest that sea buckthorn could notably protect the retina from damage induced by hypertensive retinopathy.

Nephrin, a transmembrane protein, is involved in pancreatic beta-cell survival signaling.

Nephrin, a cell surface signaling receptor, regulates podocyte function in health and disease. We study the role of nephrin in ß-cell survival signaling. We report that in mouse islet ß-cells and the mouse pancreatic beta-cell line (ßTC-6 cells) nephrin is associated and partly co-localized with PI3-kinase. Incubation of cells with functional anti-nephrin antibodies induced nephrin clustering at the plasma membrane, nephrin phosphorylation and recruitment of PI3-kinase to nephrin thus resulting in increased PI3K-dependent Akt phosphorylation and augmented phosphorylation/inhibition of pro-apoptotic Bad and FoxO. Nephrin silencing abolished Akt activation and increased susceptibility of cells to apoptosis. High glucose impaired nephrin signaling, increased nephrin internalization and up-regulated PKCα expression. Interestingly, a marked decrease in nephrin expression and phosphorylated Akt was observed in pancreatic islets of db/db lepr-/- diabetic mice. Our findings revealed that nephrin is involved in ß-cell survival and suggest that glucose-induced changes in nephrin signaling may contribute to gradual pancreatic ß-cell loss in type 2 diabetes.

Effect of nanostructured TiO2 crystal phase on photoinduced apoptosis of breast cancer epithelial cells.

PURPOSE: The use of nanoparticles has seen exponential growth in the area of health care, due to the unique physicochemical properties of nanomaterials that make them desirable for medical applications. The aim of this study was to examine the effects of crystal phase-nanostructured titanium dioxide particles on bioactivity/cytotoxicity in breast cancer epithelial cells. MATERIALS AND METHODS: Cultured Michigan Cancer Foundation (MCF)-7 and human breast adenocarcinoma (MDA-MB-468) breast cancer epithelial cells were exposed to ultraviolet A light (wavelength 350 nm) for 20 minutes in the presence of aqueous dispersions of two different nanostructured titanium dioxide (TiO2) crystal phases: anatase and an anatase-rutile mixture. Detailed characterization of each titanium dispersion was performed by dynamic light scattering. A 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) colorimetric assay was employed to estimate the percentage of viable cells after each treatment. Western blot analysis of protein expression and characterization, as well as a deoxyribonucleic acid (DNA)-laddering assay, were used to detect cell apoptosis. RESULTS: Our results documented that 100% anatase TiO2 nanoparticles (110-130 nm) exhibited significantly higher cytotoxicity in the highly malignant MDA-MB-468 cancer cells than anatase- rutile mixtures (75%/25%) with the same size. On the contrary, MCF-7 cells (characterized by low invasive properties) were not considerably affected. Exposure of MDA-MB-468 cells to pure anatase nanoparticles or anatase-rutile mixtures for 48 hours resulted in increased proapoptotic Bax expression, caspase-mediated poly(adenosine diphosphate ribose) polymerase (PARP) cleavage, DNA fragmentation, and programmed cell death/apoptosis. CONCLUSION: The obtained results indicated that pure anatase TiO2 nanoparticles exhibit superior cytotoxic effects compared to anatase-rutile mixtures of the same size. The molecular mechanism of TiO2 nanoparticle cytotoxicity involved increased Bax expression and caspase-mediated PARP inactivation, thus resulting in DNA fragmentation and cell apoptosis.

High glucose induces suppression of insulin signalling and apoptosis via upregulation of endogenous IL-1beta and suppressor of cytokine signalling-1 in mouse pancreatic beta cells.

Chronic hyperglycemia and inflammatory cytokines disrupt and/or attenuate signal transduction pathways that promote normal beta-cell survival, leading to the destruction of endocrine pancreas in type 2 diabetes. There is convincing evidence that autocrine insulin signalling exerts protective anti-apoptotic effects on beta cells. Suppressors of cytokine signalling (SOCS) were induced by several cytokines and inhibit insulin-initiated signal transduction. The aim of this study was to investigate whether high glucose can influence endogenous interleukin-1beta (IL-1beta) and SOCS expression thus affecting insulin signalling and survival in insulin-producing mouse pancreatic beta cells (betaTC-6). Results showed that prolonged exposure of betaTC-6 cells to increased glucose concentrations resulted in significant inhibition of insulin-induced tyrosine phosphorylation of the insulin receptor (IR), and insulin receptor substrate-2 (IRS-2) as well as PI3-kinase activation. These changes were accompanied by impaired activation of the anti-apoptotic signalling protein Akt and annulment of Akt-mediated suppression of the Forkhead family of transcription factors (FoxO) activation. Glucose-induced attenuation of IRS-2/Akt-mediated signalling was associated with increased IL-1beta expression. Enhanced endogenous IL-1beta specifically induced mRNA and protein expression of SOCS-1 in betaTC-6 cells. Inhibition of SOCS-1 expression by SOCS-1-specific small interfering RNA restored IRS-2/PI3K-mediated Akt phosphorylation suppressed by high glucose. The upregulation of endogenous cytokine signalling and FoxO activation were accompanied by enhanced caspase-3 activation and increased susceptibility of cells to apoptosis. These results indicated that glucose-induced endogenous IL-1beta expression increased betaTC-6 cells apoptosis by inhibiting, at least in part, IRS-2/Akt-mediated signalling through SOCS-1 upregulation.

Selective binding of integrins from different renal cell types to the NC1 domain of alpha3 and alpha1 chains of type IV collagen.

BACKGROUND: Cells interact with type IV collagen (Col IV) via integrins through the triple-helical and NC1 domains. We examined interactions of human glomerular and proximal tubular epithelial cells with recombinant alpha1 and alpha3 NC1 chains of Col IV, to explore the ability of different cell types to interact with Col IV of different trimer composition. METHODS: Interactions of TSV-40-immortalized human glomerular epithelial cells (HGECs), HPV-16-immortalized human proximal tubular epithelial (HK-2) cells and primary human mesangial cells (MES) with recombinant alpha1 and alpha3 NC1 chains of Col IV were examined by affinity chromatography and solid-phase binding assays. The expression of integrin-regulated metalloproteinases was examined by zymography. RESULTS: HGECs bound to both alpha3 and alpha1(IV)NC1, albeit there was preferential binding to alpha3(IV)NC1, through the alpha3beta1 and alpha2beta1 integrin receptors; HK-2 cells and MES bound almost exclusively to alpha1(IV)NC1 via the alpha3beta1/alphavbeta3 and alpha1beta1/alpha2beta1 receptors, respectively. It was demonstrated that the expression of MMP-2 and MMP-9 by HGECs was down-regulated in the presence of alpha3(IV)NC1. CONCLUSIONS: The observed data indicate that the isoform NC1 chains of Col IV serve for selective, integrin-mediated cell binding which probably triggers different signaling mechanisms, resulting in the activation of specific transcription factors and the modulation of gene expression.

Chronic exposure of human glomerular epithelial cells to high glucose concentration results in modulation of high-affinity glucose transporters expression.

INTRODUCTION: GLUTs are specific membrane proteins that transport glucose down a concentration gradient. There have been few studies on their expression in the kidney. The aim of this study was to identify the expression of GLUTs 1, 3, and 4 in HGEC and their regulation under diabetic milieu. MATERIAL AND METHODS: An immortalized cell line of HGEC was used. Cells were cultured in medium containing 5 or 25 mM D-glucose. Western blotting and flow cytometry were used to examine the presence of GLUTs (1, 3, 4) and alterations in expression. RESULTS: Western blotting analysis revealed that GLUT-1 levels were increased by 53% in HGEC cultured under experimental diabetes compared to cells grown in 5mM glucose. GLUT-3 levels were also increased by 15% under diabetic conditions. GLUT-4 levels were decreased by 20% in diabetes. Fluorescence Activated Cell Sorting (FACS) analysis demonstrated that cell surface expression of GLUT-1 was increased by 28% in cells grown in 25mM glucose. High glucose concentration did not affect cell surface expression of GLUT-3 and GLUT-4. DISCUSSION: These findings suggest that depressed GLUT4 expression in glomerulus and overexpression of GLUT-1 and in a lesser extent of GLUT-3 may alter the glucose uptake in these cells. It has been suggested that the overexpression of GLUT-1 in glomerulus, being the major isoform, may lead to the initial pathologic hallmarks of diabetic nephropathy.

Enhanced podocalyxin expression alters the structure of podocyte basal surface.

Glomerular basement membrane (GBM) and podocalyxin are essential for podocyte morphology. We provide evidence of functional interconnections between basement membrane components (collagen IV and laminin), the expression of podocalyxin and the morphology of human glomerular epithelial cells (podocytes). We demonstrated that GBM and laminin, but not collagen IV, up-regulated the expression of podocalyxin. Scanning electron microscopy revealed that laminin induced a modified morphology of podocytes with process formation, which was more extensive in the presence of GBM. Under high magnification, podocytes appeared ruffled. Using transmission electron microscopy we observed that raised areas occurred in the basal cell surface. Furthermore, the presence of anti-podocalyxin antibody increased the extent of adhesion and spreading of podocytes to both collagen IV and laminin, thus podocalyxin apparently inhibits cell-matrix interactions. We also performed adhesion and spreading assays on podocytes grown under increased glucose concentration (25 mM). Under these conditions, the expression of podocalyxin was almost totally suppressed. The cells adhered and spread to basement membrane components but there was no increase in the extent of adhesion and spreading in the presence of anti-podocalyxin antibody, or ruffling of the cell edges. Additionally, in podocytes expressing podocalyxin, the presence of anti-podocalyxin antibody partially reversed the inhibition of adhesion to collagen IV provoked by anti-beta1 integrin antibody, thus podocalyxin should compete with beta1-related cell adhesion. We suggest that the observed podocalyxin-mediated inhibition of binding to the matrix could be in part responsible for the specialized conformation of the basal surface of podocytes.

Glucose-induced changes in integrins and matrix-related functions in cultured human glomerular epithelial cells.

In cultured human glomerular epithelial cells (HGEC), 25 mM glucose resulted in decreased expression of alpha(3)-, alpha(2)-, and beta(1)-integrins and increased expression of alpha(5)- and alpha(v)beta(3)-integrins. This change was accompanied by decreased binding of HGEC to type IV collagen. In the presence of normal (5 mM) glucose concentration, cell binding to type IV collagen was primarily mediated by alpha(2)beta(1)- and alpha(5)beta(1)-integrins, as indicated by experiments in which cell adhesion to type IV collagen was competed by specific anti-integrin monoclonal antibodies. In the presence of high (25 mM) glucose, the upregulated alpha(5)- and alpha(v)beta(3)-integrins were mainly involved in cell binding to type IV collagen. Furthermore, high glucose decreased expression of matrix metalloproteinase-2 (MMP-2), a collagenase regulated in part by alpha(3)beta(1)-integrin, as suggested by the use of ligand-mimicking antibodies against these integrins, which resulted in release of increased amounts of MMP-2 in the culture medium. Finally, tissue inhibitor of metalloproteinase-2, the specific inhibitor of MMP-2, was upregulated in high glucose and could contribute to matrix accumulation. These changes could help explain basement membrane thickening in diabetes.

Proximal tubular epithelial cell integrins respond to high glucose by altered cell-matrix interactions and differentially regulate matrixin expression.

Thickening of the tubular basement membrane (TBM) occurs in diabetic nephropathy, but the effects of high glucose on the functional aspects of proximal tubular epithelial cells are not clearly understood. In the present study, we examined the effects of elevated glucose concentrations on (a) integrin expression by human proximal tubular epithelial cells (HK-2) and integrin-mediated interactions with type IV collagen (colIV) and laminin, major components of TBM; (b) the expression of matrixins/matrix metalloproteinases (MMPs), which is regulated by integrins; and (c) the expression of tissue inhibitors of metalloproteinases (TIMPs). HK-2 cells cultured in 25 mM glucose underwent a reduction of the expression of alpha3, beta1, alpha(v)beta3, and alpha5 integrin subunits, with a concomitant increase of the alpha2 subunit, compared with cells grown in 5 mM glucose. Adhesion experiments demonstrated that high glucose led to increased cell adhesion on either colIV or laminin. Experiments of competition of adhesion using anti-integrin antibodies indicated that HK-2 cells in 5 mM glucose used mainly alpha(v)beta3 and alpha5beta1 integrins to adhere to colIV, whereas in 25 mM glucose they additionally used alpha2beta1. In the case of laminin, a beta1-mediated adhesion was observed when HK-2 cells were in 5 mM glucose, whereas in 25 mM glucose, alpha2beta1 and alpha(v)beta3 were also involved. Elevated glucose concentrations resulted in decreased expression of MMP-9 and MMP-2, whereas an increase in TIMP-1 and a decrease in TIMP-2 expression were observed. We also examined which integrins mediated the expression and secretion of matrixins MMP-2 and MMP-9. Ligation of alpha3beta1 with mAbs resulted in induction of MMP-2 expression and secretion, whereas antibody ligation of alpha(v)beta3 led to down-regulation of MMP-9. The above data implicate integrins of proximal tubular epithelial cells in the regulation of MMPs and in the development of TBM thickening in diabetic nephropathy.

Effects of collagen IV on neuroblastoma cell matrix-related functions.

Integrin-mediated interactions with collagen IV and its domains were examined in a human neuroblastoma cell line (SK-N-SH). By adhesion assays we demonstrated that neuroblastoma cells bound to solid-phase intact collagen IV and synthetic cell-binding peptide HEP-III, derived from the collagenous part of the molecule, but not to the main noncollagenous NC1 domain or to the synthetic cell-binding peptide HEP-I, derived from this domain. Monoclonal antibodies against beta1, alpha3, and alpha(v)beta3 integrins resulted in inhibition of cell binding to collagenous substrates by 95, 30, and 35%, respectively. By flow cytometry and immunoblotting it was shown that culture of SK-N-SH cells on collagen IV resulted in alteration in the expression of major neuroblastoma cell integrins. Binding to collagen IV induced the expression and activation of matrix metalloproteinases A and B (MMP-2, MMP-9), with a concomitant increase at the protein level of tissue-specific inhibitors of metalloproteinases (TIMP-1, TIMP-2). Finally, the expression of MMP-2 was significantly up-regulated by anti-alpha3beta1 antibodies, whereas ligation of anti-alpha(v)beta3 antibodies resulted in a modest down-regulation of MMP-2. Our results indicate that the presence of collagen IV modulates the expression of integrins, which are used for binding to this glycoprotein, and MMP-2 secreted by SK-N-SH cells.