NaYF4-based upconverting nanoparticles with optimized phosphonate coatings for chemical stability and viability of human endothelial cells.

The increasing interest in upconverting nanoparticles (UCNPs) in biodiagnostics and therapy fuels the development of biocompatible UCNPs platforms. UCNPs are typically nanocrystallites of rare-earth fluorides codoped with Yb3+and Er3+or Tm3+. The most studied UCNPs are based on NaYF4but are not chemically stable in water. They dissolve significantly in the presence of phosphates. To prevent any adverse effects on the UCNPs induced by cellular phosphates, the surfaces of UCNPs must be made chemically inert and stable by suitable coatings. We studied the effect of various phosphonate coatings on chemical stability andin vitrocytotoxicity of the Yb3+,Er3+-codoped NaYF4UCNPs in human endothelial cells obtained from cellular line Ea.hy926. Cell viability of endothelial cells was determined using the resazurin-based assay after the short-term (15 min), and long-term (24 h and 48 h) incubations with UCNPs dispersed in cell-culture medium. The coatings were obtained from tertaphosphonic acid (EDTMP), sodium alendronate and poly(ethylene glycol)-neridronate. Regardless of the coating conditions, 1 - 2 nm-thick amorphous surface layers were observed on the UCNPs with transmission electron microscopy. The upconversion fluorescence was measured in the dispersions of all UCNPs. Surafce quenching in aqueous suspensions of the UCNPs was reduced by the coatings. The dissolution degree of the UCNPs was determined from the concentration of dissolved fluoride measured with ion-selective electrode after the ageing of UCNPs in water, physiological buffer (i.e., phosphate-buffered saline-PBS) and cell-culture medium. The phosphonate coatings prepared at 80 °C significantly suppressed the dissolution of UCNPs in PBS while only minor dissolution of bare and coated UCNPs was measured in water and cell-culture medium. The viability of human endothelial cells was significantly reduced when incubated with UCNPs, but it increased with the improved chemical stability of UCNPs by the phosphonate coatings with negligible cytotoxicity when coated with EDTMP at 80 °C.

Dietary vitamin D equilibrium in serum ameliorates direct bilirubin associated diabetes mellitus.

Diabetes mellitus (DM), a non-communicable endocrine disease that is marked by a differing degree of tolerance to insulin and dysfunction. The connection between diabetes and liver failure important to doctors in general practice diabetologists and hepatologists. DM is linked with an elevated risk of hepatic consequences and mortality of liver cirrhosis patients. DM may facilitate to insult the liver by inducing inflammation and fibrosis by elevating mitochondrial oxidative stress. The conventional liver function indices are bilirubin including Indirect Bilirubin (IBil), Direct Bilirubin (DBil), and Total Bilirubin (TBil). DBil, IBil, and TBil, have diverse clinical implications as the standard index of liver disorder. An elevated level of DBil may suggest damage to the hepatic cell whereas TBil is within the normal range. Thus, increased liver enzymes are correlated with hepatic insulin resistance in healthy subjects. Notably, a significant correlation between DBil levels and Insulin resistance risk could indicate a connection between liver dysfunction and diabetes mellitus risk. Thus, our primary goal via the current review to examine the impact of dietary vitamin D (VitD) in serum mediated risk reduction of insulin resistance and further incidence of DM through inflammatory liver associated high DBil. Therefore, modifying these inflammatory pathways may be a therapeutic alternative approach for diabetes treatment.

Assessment of toxicity and genotoxicity of low doses of 5-fluorouracil in zebrafish (Danio rerio) two-generation study.

Residues of anti-neoplastic drugs represent new and emerging pollutants in aquatic environments. Many of these drugs are genotoxic, and it has been postulated that they can cause adverse effects in aquatic ecosystems. 5-Fluorouracil (5-FU) is one of the most extensively used anti-neoplastic drugs in cancer therapy, and this article describes the results of the first investigation using a two-generation toxicity study design with zebrafish (Danio rerio). Exposure of zebrafish to 5-FU (0.01, 1.0 and 100 µg/L) was initiated with adult zebrafish (F0 generation) and continued through the hatchings and adults of the F1 generation, and the hatchings of the F2 generation, to day 33 post-fertilisation. The exposure did not affect survival, growth and reproduction of the zebrafish; however, histopathological changes were observed in the liver and kidney, along with genotoxic effects, at all 5-FU concentrations. Increases in DNA damage determined using the comet assay were significant in the liver and blood cells, but not in the gills and gonads. In erythrocytes, a significant, dose-dependent increase in frequency of micronuclei was observed at all 5-FU concentrations. Whole genome transcriptomic analysis of liver samples of F1 generation zebrafish exposed to 0.01 µg/L and 1 µg/L 5-FU revealed dose-dependent increases in the number of differentially expressed genes, including up-regulation of several DNA-damage-responsive genes and oncogenes (i.e., jun, myca). Although this chronic exposure to environmentally relevant concentrations of 5-FU did not affect the reproduction of the exposed zebrafish, it cannot be excluded that 5-FU can lead to degenerative changes, including cancers, which over long-term exposure of several generations might affect fish populations. The data from this study contribute to a better understanding of the potential consequences of chronic exposure of fish to low concentrations of anti-neoplastic drugs, and they demonstrate that further studies into multi-generation toxicity are needed.

Monocyclic 4-amino-6-(phenylamino)-1,3,5-triazines as inhibitors of human DNA topoisomerase IIα.

Human DNA topoisomerase IIα (htIIα) is a validated target for the development of anticancer agents. Starting from the available information about the binding of the purine-based htIIα inhibitors in the ATP binding site we designed a virtual screening campaign combining structure-based and ligand-based pharmacophores with a molecular docking calculation searching for compounds that would contain a monocycle mimetic of the purine moiety. We discovered novel 4-amino-6-(phenylamino)-1,3,5-triazines 6, 7 and 11 as monocyclic htIIα inhibitors targeting the ATP binding site. Compound 6 from the 1,3,5-triazine series also displayed cytotoxicity properties in hepatocellular carcinoma (HepG2) cell lines and selectivity against human umbilical vein endothelial (HUVEC) cell lines.

Arsenic trioxide (ATO) influences the gene expression of metallothioneins in human glioblastoma cells.

Arsenic trioxide (As(2)O(3); ATO, TRISENOX®) is used to treat patients with refractory or relapsed acute promyelocytic leukaemia while its application for treatment of solid cancers like glioblastoma is still under evaluation. In the present study, we investigated the interaction of arsenic trioxide with metallothionein (MT) isoforms as a possible (protective response) resistance of glioblastoma cells to arsenic-induced cytotoxicity. Special attention was focused on MT3, the isoform expressed mainly in the brain. MT3 has low metal inducibility, fast metal binding/releasing properties and outstanding neuronal inhibitory activity. The human astrocytoma (glioblastoma) cell line U87 MG was treated with 0.6, 2 and 6-7 µM arsenic (equivalent to 0.3, 1 and 3-3.5 µM As(2)O(3)) for 12, 24 or 48 h and gene expression for different MT isoforms, namely MT2A, MT1A, MT1F, MT1X, MT1E and MT3, was measured by real time qPCR using SYBR Green I and Taqman® gene expression assays. TfR, 18S rRNA, GAPDH and AB were tested as reference genes, and the last two evaluated to be appropriate in conditions of low (GAPDH) and high (AB) arsenic exposure. The gene expression of MT3 gene was additionally tested and confirmed by restriction enzyme analysis with PvuII. In the given conditions the mRNAs of six MT isoforms were identified in human glioblastoma cell line U87 MG. Depending on arsenic exposure conditions, an increase or decrease of MT gene expression was observed for each isoform, with the highest increase for isoforms MT1X, MT1F and MT2A mRNA (up to 13-fold) and more persistent decreases for MT1A, MT1E and MT3 mRNA. Despite the common assumption of the noninducibility of MT3, the evident MT3 mRNA increase was observed during high As exposure (up to 4-fold). In conclusion, our results clearly demonstrate the influence of As on MT isoform gene expression. The MT1X, MT1F and MT2A increase could represent brain tumour acquired resistance to As cytotoxicity while the MT3 increase is more enigmatic, with its possible involvement in arsenic-related induction of type II cell death.

CD133/prominin1 is prognostic for GBM patient's survival, but inversely correlated with cysteine cathepsins' expression in glioblastoma derived spheroids.

INTRODUCTION: CD133 is a marker for a population of glioblastoma (GBM) and normal neural stem cells (NNSC). We aimed to reveal whether the migratory potential and differentiation of these stem cells is associated with CD133 expression and with cathepsin proteases (Cats). MATERIALS AND METHODS.: The invasiveness of normal NNSC, GBM/CD133+ cell lines and GBM spheroids was evaluated in 3D collagen, as well as of U87-MG and normal astrocytes (NHA) grown in monolayers in 2D Matrigel. Expression of Cats B, L and S was measured at mRNA and activity levels and their relation to invasiveness, to CD133 mRNA in 26 gliomas, and to the survival of these patients. RESULTS: The average yield of CD133+ cells from GBM samples was 9.6 %. Survival of patients with higher CD133 mRNA expression was significantly shorter (p< 0.005). Invasion, associated with proteolytic degradation of matrix, was higher in normal stem cells and GBM spheroids and cells than in isolated GBM CD133+ cells. In glioma samples, there was no correlation between CD133 mRNA expression and Cat mRNAs, but there was an inverse correlation with Cat activities. CONCLUSIONS: The study confirms CD133 as a prognostic marker for the survival of GBM patients. We demonstrated that NNSC have higher invasion potential and invade the collagen matrix in a mode different from that of GBM, initiating stem cell spheres. This result could have implications for the design of new therapeutics, including protease inhibitors that specifically target invasive tumour stem cells. Increased activity of cathepsins in CD133- cells suggests their role in the invasive behaviour of GBM.

Prognostic impact of CD68 and kallikrein 6 in human glioma.

AIMS: To evaluate the expression of CD68 and kallikrein 6 in human gliomas, and investigate their prognostic significance for survival of brain cancer patients in comparison to some known prognostic markers. PATIENTS AND METHODS: Histological sections of 51 primary astrocytic tumours (11 benign, 40 malignant) were immunohistochemically stained for CD68, cathepsin B, kallikrein 6 and Ki-67. CD68 and kallikrein 6 expressions were also analyzed by real-time PCR in nine brain tumour biopsies. RESULTS: Both microglia and tumour cells expressed CD68. High CD68 and cathepsin B staining scores were significantly, more frequent in the malignant than in the benign tumours (p=0.036 and p=0.014, respectively). In contrast, the benign group presented a stronger immunoreactivity for kallikrein 6 compared with the malignant tumours (p=0.013). A CD68 staining score of tumour cells higher than 3 was a significant predictor of shorter overall survival (p<0.01) in all patients and of borderline significance in the malignant group (p=0.057). Strong CD68 staining was of greater predictive value in the subgroup of anaplastic astrocytomas (p=0.021). Furthermore, as expected on the basis of our previous studies, prognostic significance was confirmed for cathepsin B, but not for any of the other markers under evaluation. CONCLUSION: Kallikrein 6 was down-regulated in malignant glioma, but this differential expression did not have an impact on patient prognosis. In contrast, immunostaining of glioma tissue for CD68 and for cathepsin B may be used for prognosis of survival in these patients. This finding suggests that besides the known role of cathepsin B in invasion and angiogenesis, CD68 may be also associated with glioma progression.

Effects of model organophosphorous pesticides on DNA damage and proliferation of HepG2 cells.

Organophosphorous compounds (OPs) are commonly used pesticides. The primary mechanism of OP toxicity is the inhibition of acetylcholine esterase in the nervous system leading to a variety of acute and chronic effects. Recent studies have revealed several other targets of OPs that disturb noncholinergic biological systems. We investigated whether low concentrations of model OPs-methyl parathion (PT), methyl paraoxon (PO), and dimefox (DF)-induce DNA damage and/or affect cell proliferation in human hepatoma HepG2 cells. Genotoxicity of OPs was evaluated using the comet assay. The effect on cell proliferation was tested using the MTT assay and proliferation marker Ki-67 immunocytochemistry. The effects of OPs on mRNA expression of the DNA damage responsivegenes p53, p21, GADD45alpha, and MDM2 were determined using qRT-PCR. PT induced DNA damage at lower concentrations (1 microg/mL) than PO (100 microg/mL), whereas DF did not induce DNA damage. PT and PO caused a reduction of cell proliferation at their highest concentrations (100 microg/mL), while DF increased cell proliferation at all concentrations used (0.01-100 microg/mL). PT and PO upregulated expression of DNA damage responsive genes, while DF upregulated expression of p53, downregulated expression of p21, and had no effect on the expression of MDM2 and GADD45alpha. We conclude that PT and PO are genotoxic, while DF shows mitogenic activity. An important finding of this study is that PT had higher genotoxic potential than PO, which warrants for further investigations to correctly evaluate the hazards of exposure to these chemicals.

Patterns of microcystin-LR induced alteration of the expression of genes involved in response to DNA damage and apoptosis.

Microcystins (MCs) are hepatotoxic cyclic heptapeptides produced by freshwater cyanobacteria. They are inhibitors of serine/threonine protein phosphatases 1A and 2A and are involved in liver tumour promotion. Several recent studies indicated that MCs are genotoxic and may also act as tumour initiators. Based on our previous results showing that microcystin-LR (MCLR) induces DNA damage in HepG2 cells, we have now explored the effect of MCLR on the expression of selected genes known to be involved in the cell response to DNA damage and apoptosis. The HepG2 cells were exposed to non-cytotoxic concentrations (0.01, 0.1 and 1 microg/ml) of MCLR for various periods of time (2-16 h) and the mRNA expression was determined with the quantitative real-time polymerase chain reaction (QRT-PCR). We found a significantly elevated expression of tumour suppressor gene p53 and its downstream-regulated genes involved in DNA repair and cell cycle regulation (p21, gadd 45a, mdm2), as well as increased expression of the pro-apoptotic gene bax, but no alterations of the anti-apoptotic bcl-2. Up-regulation of the expression of mdm2, p21 and gadd45a provides strong support for our previous suggestion that MCLR is a genotoxic carcinogen. The increased ratio of expression of bax to that of bcl-2 induced by MCLR suggests that apoptosis in HepG2 cells proceeds via the mitochondrial pathway.

Cathepsin L affects apoptosis of glioblastoma cells: a potential implication in the design of cancer therapeutics.

BACKGROUND: Previous studies indicate that Cathepsin L (CatL) is involved in brain tumour progression. Here, CatL in tumour cell invasion and apoptosis has been studied. MATERIALS AND METHODS: Human glioblastoma cell line U87 was transfected with CatL cDNA in sense and antisense orientations. The in vitro invasiveness was tested in modified Boyden chambers. Apoptosis was determined by fluorescent staining, caspase activity, and by Bax and Bcl-2 mRNA levels. RESULTS: Surprisingly, the invasiveness of U87 cells was not impaired by genetic down-regulation of CatL expression. In the CatL antisense clones, the apoptotic rate induced by either intrinsic or extrinsic stimuli was increased, whereas CatL sense transfection seemed to protect the cells from apoptosis. CONCLUSION: Increased chemoresistance of tumour cells may be associated with increased levels of CatL and may have potential application in gene therapy, which would augment the apoptosis of glioblastoma cells induced by chemotherapy.

Invasiveness of transformed human breast epithelial cell lines is related to cathepsin B and inhibited by cysteine proteinase inhibitors.

The activities'of the lysosomal cysteine proteinases cathepsin B and L are regulated by their endogenous inhibitors, stefins A and B, and cystatin C, and their imbalance may be associated with increased invasiveness and development of the malignant cell phenotype. The aim of this study was to investigate mRNA, protein and activity levels of the above proteins in relation to in vitro invasiveness and to the reported in vivo tumorigenicity of four human breast tumor cell lines: the spontaneously immortalized cell line MCF10A, its c-Ha-ras transfectant MCF10AT, and two tumorigenic derivative cell lines, MCF10AT-Ca1a and MCF10AT-Ca1d. Invasiveness did not correlate with tumorigenicity, since the MCF10AT cell was the most invasive and the remaining three were at about half of its level. Cathepsin B expression paralleled the in vitro invasiveness through matrigel at all levels of expression, but cathepsin L did not. Stefin levels were elevated several-fold in the tumorigenic cell lines, but not in MCF10AT. The hypothesis that cathepsin B plays an active role in the invasion of breast cancer cell lines was confirmed by the fact that synthetic cysteine proteinase inhibitors, particularly those selective for cathepsin B, significantly reduced the invasion of the MCF10AT cells.

Expression of cysteine peptidase cathepsin L and its inhibitors stefins A and B in relation to tumorigenicity of breast cancer cell lines.

The in vitro invasiveness of human breast cancer cell lines was compared with their reported tumorigenicity in vivo, increasing from MCF7, MDA-MB468, MDA-MB231 to MDA-MB435 cells. The invasiveness roughly corresponded to the tumorigenicity of the cell lines. The levels of cathepsin L mRNA and protein correlated with the invasiveness of the cells. Stefin A protein decreased with the invasiveness and the reported tumorigenicity, whereas stefin B protein was significantly lower in all MDA-MB lines compared with the least invasive and tumorigenic MCF7 line. Our results suggest that the imbalance between cathepsin L and the stefins contributes to the development of a malignant cell phenotype.

Evolutionary relationships among Europan newts (genus Triturus) as inferred from two mtDNA fragments.

European newts (genus Triturus) are widely studied, but their phylogeny is not yet unambiguously resolved. Fragments of mitochondrial DNA experiencing different rates of evolution (the ATPase and 12S rDNA genes) were sequenced in order to test a phylogenetic hypothesis derived from biochemical and behavioural data. Well supported branches of the existing phylogeny also gained support in our study. Within the subgenus Palaeotriton (the group of small-bodied newts) the monophyletic origin of the hypothesized T. boscai - T. italicus clade remained ambiguous, whereas strong support was gained for the sister-taxon relationship of T. vulgaris and T. montandoni. The position of T. vittatus within the subgenus Triturus as a sister taxon to the clade of big-bodied newts (T. marmoratus and T. cristatus superspecies) was also supported. However, the phylogenetic position of the medium-sized newt, T. alpestris could not be clarified.

Cathepsin B and its inhibitor stefin A in brain tumors.

Cysteine protease cathepsin B (CatB) and its endogenous inhibitor stefin A (StA) play an important role in tumor progression. Increase of CatB expression and lower levels of its inhibitors were associated with tumor malignancy in brain tumors. In this study of 100 patients, CatB was localized by immunostaining to both, tumor and endothelial cells of primary brain tissue. Significant correlation with poor prognosis was found by univariate Cox's regression model. Intense overall immunostaining and immunostaining in endothelial cells alone were prognostic for survival (p=0.003 in both). When comparing CatB expression at mRNA level, we found considerable differences between center and periphery of a tumor as well as between different tumor samples. StA mRNA was only detected in benign, but not in malignant tissues. We suggest that screening of cysteine-protease genes expression can be applied in clinical prognosis of brain tumors.