Antitumor activity of sorafenib in human cancer cell lines with acquired resistance to EGFR and VEGFR tyrosine kinase inhibitors

Treatment of non small cell lung cancer (NSCLC) and colorectal cancer (CRC) have substantially changed in the last years with the introduction of epidermal growth factor receptor (EGFR) inhibitors in the clinical practice. The understanding of mechanisms which regulate cells sensitivity to these drugs is necessary for their optimal use.An in vitro model of acquired resistance to two tyrosine kinase inhibitors (TKI) targeting the EGFR, erlotinib and gefitinib, and to a TKI targeting EGFR and VEGFR, vandetanib, was developed by continuously treating the human NSCLC cell line CALU-3 and the human CRC cell line HCT116 with escalating doses of each drug. MTT, western blot analysis, migration, invasion and anchorage-independent colony forming assays were conducted in vitro and experiments with established xenografts in athymic nude mice were performed in vivo in sensitive, wild type (WT) and TKI-resistant CALU-3 and HCT116 cell lines.As compared to WT CALU-3 and HCT116 human cancer cells, TKI-resistant cell lines showed a significant increase in the levels of activated, phosphorylated AKT, MAPK, and of survivin. Considering the role of RAS and RAF as downstream signals of both the EGFR and VEGFR pathways, we treated resistant cells with sorafenib, an inhibitor of C-RAF, B-RAF, c-KIT, FLT-3, RET, VEGFR-2, VEGFR-3, and PDGFR-β. Sorafenib reduced the activation of MEK and MAPK and caused an inhibition of cell proliferation, invasion, migration, anchorage-independent growth in vitro and of tumor growth in vivo of all TKI-resistant CALU-3 and HCT116 cell lines.These data suggest that resistance to EGFR inhibitors is predominantly driven by the RAS/RAF/MAPK pathway and can be overcame by treatment with sorafenib.     

Adsorption of ethylenediaminetetraacetic dianhydride modified oxalate decarboxylase on calcium oxalate

We used ethylenediaminetetraacetic acid dianhydride (EDTAD) to modify oxalate decarboxylase (OXDC) to improve its adsorption on calcium oxalate stones. The modified sites were identified by Ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and the adsorption mechanism of the EDTAD-modified OXDC on calcium oxalate (CaOx) was investigated. We investigated adsorption time, initial enzyme concentration, temperature and solution pH on the adsorption process. Data were analyzed using kinetics, thermodynamics and isotherm adsorption models. UPLC-MS showed that EDTAD was attached to OXDC covalently and suggested that the chemical modification occurred at both the free amino of the side chain and the α-NH₂ of the peptide. The adsorption capacity of the EDTAD-OXDC on calcium oxalate was 53.37% greater than that of OXDC at the initial enzyme concentration of 5 mg/ml, pH = 7.0, at 37° C. The modified enzyme (EDTAD-OXDC) demonstrated improved oxalate degradation activity at pH 4.5?6.0. Kinetic data fitting analysis suggested a pseudo second order kinetic model. Estimates of the thermodynamic parameters including ΔG⁰, ΔH⁰ and ΔS⁰ of the adsorption process showed it to be feasible, spontaneous and endothermic. Isotherm data fitting analysis indicated that the adsorption process is reduced to monolayer adsorption at a low enzyme concentration and to multilayer adsorption at a high enzyme concentration. It may be possible to apply OXDC to degradation of calcium oxalate stones.     

Sustained Photosynthetic Performance of Coffea spp. under Long-Term Enhanced [CO2]

Coffee is one of the world’s most traded agricultural products. Modeling studies have predicted that climate change will have a strong impact on the suitability of current cultivation areas, but these studies have not anticipated possible mitigating effects of the elevated atmospheric [CO₂] because no information exists for the coffee plant. Potted plants from two genotypes of Coffea arabica and one of C. canephora were grown under controlled conditions of irradiance (800 μmol m^-2 s^-1), RH (75%) and 380 or 700 μL CO₂ L^-1 for 1 year, without water, nutrient or root development restrictions. In all genotypes, the high [CO₂] treatment promoted opposite trends for stomatal density and size, which decreased and increased, respectively. Regardless of the genotype or the growth [CO₂], the net rate of CO₂ assimilation increased (34-49%) when measured at 700 than at 380 μL CO₂ L^-1. This result, together with the almost unchanged stomatal conductance, led to an instantaneous water use efficiency increase. The results also showed a reinforcement of photosynthetic (and respiratory) components, namely thylakoid electron transport and the activities of RuBisCo, ribulose 5-phosphate kinase, malate dehydrogenase and pyruvate kinase, what may have contributed to the enhancements in the maximum rates of electron transport, carboxylation and photosynthetic capacity under elevated [CO₂], although these responses were genotype dependent. The photosystem II efficiency, energy driven to photochemical events, non-structural carbohydrates, photosynthetic pigment and membrane permeability did not respond to [CO₂] supply. Some alterations in total fatty acid content and the unsaturation level of the chloroplast membranes were noted but, apparently, did not affect photosynthetic functioning. Despite some differences among the genotypes, no clear species-dependent responses to elevated [CO₂] were observed. Overall, as no apparent sign of photosynthetic down-regulation was found, our data suggest that Coffea spp. plants may successfully cope with high [CO₂] under the present experimental conditions.     

Paradoxical effect of l-arginine: Acceleration of endothelial cell senescence

We have recently shown that inhibition of nitric oxide (NO) synthesis by asymmetrical dimethylarginine (ADMA) accelerated endothelial cell (EC) senescence which was prevented by coincubation with l-arginine; however the effect of long-term treatment of l-arginine alone on senescence of ECs have not been investigated. Human ECs were cultured in medium containing different concentrations of l-arginine until senescence. l-Arginine paradoxically accelerated senescence indicated by inhibiting telomerase activity. Moreover, l-arginine decreased NO metabolites, increased peroxynitrite, and 8-iso-prostaglandin F_2α formation. In old cells, the mRNA expression of human amino acid transporter (hCAT)2B, the activity and protein expression of arginase II were upregulated indicated by enhanced urea, l-ornithine, and l-arginine consumption. Inhibition of arginase activity, or transfection with arginase II siRNA prevented l-arginine-accelerated senescence. The most possible explanation for the paradoxical acceleration of senescence by l-arginine so far may be the translational and posttranslational activation of arginase II.     

Red wine decreases asymmetric dimethylarginine via SIRT1 induction in human endothelial cells

To investigate the effect of three red wines (RWs) from different growing areas and made from different grapes on asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, in young and senescent human endothelial cells (ECs). All RWs decreased ADMA levels, but 2-fold concentration of German RW was necessary to reach the same effect on ADMA compared to Italian RW and French RW without affecting the cell viability and morphology. The ADMA-lowering effect of RW was increased in senescent compared to young cells, accompanied by enhanced activity of the metabolizing enzyme: dimethylarginine dimethylaminohydrolase (DDAH) II, whereas the same amount in the upregulated protein expression of DDAH II and the downregulated protein expression of the synthesizing enzyme: protein arginine methyltransferase 1 was revealed. These effects were associated with decreased 8-iso-prostaglandin F_2α and peroxynitrite formation, enhanced protein expression of NAD⁺-dependent class III histone deacetylase sirtuin (SIRT) 1, and downregulated protein expression of histone senescence factor p53. Blockade of SIRT1 activity abolished the effect of red wine on ADMA. These data are the first demonstration that RW by activating SIRT1 impairs synthesis and increases metabolism of ADMA. This effect of RW is accentuated in senescent cells probably due to enhanced DDAH activity.     

SOS-red fluorescent protein (RFP) bioassay system for monitoring of antigenotoxic activity in plant extracts

An optical antigenotoxicity assay using genetically engineered red fluorescent bacteria is presented. Exposure of Escherichia coli RS4U to genotoxicants [mitomycin C (MMC), nalidixic acid (NA) and hydrogen peroxide (HP)] resulted in phenotypic red fluorescence proportional to the concentration of the inducer. Except for tannic acid (TA), co-treatment of the genotoxicant-activated bacteria with ascorbic acid (AA) and aqueous plant extracts (Mangifera indica, Psidium guajava and Syzygium cumini) afforded protection against all three genotoxicants. TA was effective in suppressing the genotoxic effect of MMC and HP. The antigenotoxic effect is seen as inhibition of the genotoxicant-triggered red fluorescence. The IC50 of the plant extracts and AA varied with the genotoxicant used. Rec assay verified the antigenotoxic activity of the plant extracts. Folin-Ciocalteu test, FeCl3 test and DPPH assay confirmed the presence of polyphenolic compounds and hydrolyzable tannins in the plant extracts and the antioxidant capacity of the plant samples.     

Chromosome studies in southern species of Mimosa (Fabaceae,Mimosoideae) and their taxonomic and evolutionary inferences

In this work, chromosome numbers and karyotype parameters of 36 taxa of the genus Mimosa were studied, especially from the southern South America center of diversification. Results support that x = 13 is the basic chromosome number in the genus. Polyploidy is very frequent, ca. 56 % of the total of the studied species here are polyploid, confirming that polyploids are more frequent at higher latitudes. The most common ploidy levels found are 2x and 4x, but some species studied exhibit 6x and 8x. In different groups, several ploidy levels were found. Parameters of chromosome size show statistically significant differences between close species, and asymmetry index A ₂ exhibited low variation between them. It is possible to infer variations of chromosome size between diploids and tetraploids and between basal and derived taxa. The present studies confirm or reveal polyploidy in several groups of South America which are highly diversified in the southernmost area of distribution of the genus, such as sect. Batocaulon ser. Stipellares and sect. Calothamnos. Our data are discussed in a taxonomic context, making inferences about the origin of some polyploid taxa. Polyploidy could be an important phenomenon that increases the morphologic diversity and specific richness in southern South America. On basis of our data, it is possible to hypothesize hybridization between same-ploidy level or different ploidy level taxa. As already shown in the literature, our results confirm the importance of the polyploidy in the speciation of the genus.     

The Protective Role of Fucosylated Chondroitin Sulfate,a Distinct Glycosaminoglycan,in a Murine Model of Streptozotocin-Induced Diabetic Nephropathy

Background

Heparanase-1 activation, albuminuria, and a decrease in glomerular heparan sulfate (HS) have been described in diabetic nephropathy (DN). Glycosaminoglycan (GAG)-based drugs have been shown to have renoprotective effects in this setting, although recent trials have questioned their clinical effectiveness. Here, we describe the effects of fucosylated chondroitin sulfate (FCS), a novel GAG extracted from a marine echinoderm, in experimentally induced DN compared to a widely used GAG, enoxaparin (ENX).

Methods

Diabetes mellitus (DM) was induced by streptozotocin in male Wistar rats divided into three groups: DM (without treatment), FCS (8 mg/kg), and ENX (4 mg/kg), administered subcutaneously. After 12 weeks, we measured blood glucose, blood pressure, albuminuria, and renal function. The kidneys were evaluated for mesangial expansion and collagen content. Immunohistochemical quantifications of macrophages, TGF-β, nestin and immunofluorescence analysis of heparanase-1 and glomerular basement membrane (GBM) HS content was also performed. Gene expression of proteoglycan core proteins and enzymes involved in GAG assembly/degradation were analyzed by TaqMan real-time PCR.

Results

Treatment with GAGs prevented albuminuria and did not affect the glucose level or other functional aspects. The DM group exhibited increased mesangial matrix deposition and tubulointerstitial expansion, and prevention was observed in both GAG groups. TGF-β expression and macrophage infiltration were prevented by the GAG treatments, and podocyte damage was halted. The diabetic milieu resulted in the down-regulation of agrin, perlecan and collagen XVIII mRNAs, along with the expression of enzymes involved in GAG biosynthesis. Treatment with FCS and ENX positively modulated such changes. Heparanase-1 expression was significantly reduced after GAG treatment without affecting the GBM HS content, which was uniformly reduced in all of the diabetic animals.

Conclusions

Our results demonstrate that the administration of FCS prevented several pathological features of ND in rats. This finding should stimulate further research on GAG treatment for this complication of diabetes.     

Evaluation of chemokine and cytokine profiles in osteoblast progenitors from umbilical cord blood stem cells by BIO-PLEX technology

We have used cytokine protein array to analyze the secretion of cytokines from an osteoblastic clone derived from human umbilical cord blood mesenchymal stem cells (MSCs) cultured in an osteogenic differentiation medium. The analysis demonstrated the unexpected ability of osteoblast committed cells and their early progenitors to produce significant amounts of a range of soluble immune mediators without in vitro exposure to clinically relevant bacterial pathogens. The cells were expanded and their osteogenic potential analyzed over 45 days of culture was revealed by the expression of osteoblast-specific markers (alkaline phosphatase and Runx2), and by matrix mineralization. Over this culture period, the cells secreted particularly high levels of IL-8, MCP-1 and VEGF, but did not express IL-2, IL-7, IL-17, eotaxin, G-CSF and IFN-gamma. These findings should encourage the use of human umbilical cord blood as a potential stem cells source for bone regeneration.