Identification of a new bisindolinone arresting IGROV1 cells proliferation.

In an initial screening, a series of novel Knoevenagel adducts were submitted to the National Cancer Institute for evaluation of antitumor activity in human cell lines. In particular, compound 5f showed remarkable selectivity against IGROV1, an ovarian cancer cell line, without affecting healthy human fibroblast cells. Analyses of cytotoxicity, cell proliferation, cell migration, epigenetic changes, gene expression, and DNA damage were performed to obtain detailed information about its antitumor properties. Our results show that 5f causes proliferation arrest, decrease in motility, histone hyperacetylation, downregulation of cyclin D1 and α5 subunit of integrin ß1 gene transcription. In addition, 5f treatment reduces transcript and protein levels of cyclin D1, which increases sensitivity to ionizing radiation and results in DNA damage comparable to cyclin D1 gene silencing.

Naxitamab Activity in Neuroblastoma Cells Is Enhanced by Nanofenretinide and Nanospermidine.

Neuroblastoma cells highly express the disialoganglioside GD2, a tumor-associated carbohydrate antigen, which is also expressed in neurons, skin melanocytes, and peripheral nerve fibers. Immunotherapy with monoclonal anti-GD2 antibodies has a proven efficacy in clinical trials and is included in the standard treatment for children with high-risk neuroblastoma. However, the strong neuro-toxicity associated with anti-GD2 antibodies administration has hindered, until now, the possibility for dose-escalation and protracted use, thus restraining their therapeutic potential. Strategies to increase the efficacy of anti-GD2 antibodies are actively sought, with the aim to enable chronic treatments that could eradicate minimal residual disease and subsequent relapses, often occurring after treatment. Here, we report that Nanofenretinide and Nanospermidine improved the expression of GD2 in neuroblastoma cells (CHP-134) and provided different effects in combination with the anti-GD2 antibody naxitamab. In particular, Nanofenretinide significantly increased the cytotoxic effect of naxitamab while Nanospermidine inhibited cell motility at extents proportional to naxitamab concentration. In neuroblastoma cells characterized by a low and heterogeneous basal expression of GD2, such as SH-SY5Y, which may represent the cell heterogeneity in tumors after chemotherapy, both Nanofenretinide and Nanospermidine increased GD2 expression in approximately 50% of cells, thus shifting the tumor population towards improved sensitivity to anti-GD2 antibodies.

ERα-LBD, an isoform of estrogen receptor alpha, promotes breast cancer proliferation and endocrine resistance.

Estrogen receptor alpha (ERα) drives mammary gland development and breast cancer (BC) growth through an evolutionarily conserved linkage of DNA binding and hormone activation functions. Therapeutic targeting of the hormone binding pocket is a widely utilized and successful strategy for breast cancer prevention and treatment. However, resistance to this endocrine therapy is frequently encountered and may occur through bypass or reactivation of ER-regulated transcriptional programs. We now identify the induction of an ERα isoform, ERα-LBD, that is encoded by an alternative ESR1 transcript and lacks the activation function and DNA binding domains. Despite lacking the transcriptional activity, ERα-LBD is found to promote breast cancer growth and resistance to the ERα antagonist fulvestrant. ERα-LBD is predominantly localized to the cytoplasm and mitochondria of BC cells and leads to enhanced glycolysis, respiration and stem-like features. Intriguingly, ERα-LBD expression and function does not appear to be restricted to cancers that express full length ERα but also promotes growth of triple-negative breast cancers and ERα-LBD transcript (ESR1-LBD) is also present in BC samples from both ERα(+) and ERα(-) human tumors. These findings point to ERα-LBD as a potential mediator of breast cancer progression and therapy resistance.

Structural and electrochemical characterization of lawsone-dependent production of tellurium-metal nanoprecipitates by photosynthetic cells of Rhodobacter capsulatus.

Cells of the facultative photosynthetic bacterium Rhodobacter capsulatus exploit the simultaneous presence in the cultural medium of the toxic oxyanion tellurite (TeO32-) and the redox mediator lawsone (2-hydroxy-1,4-naphthoquinone) by reducing tellurite to metal Te0 nanoprecipitates (TeNPs) outside the cells. Here we have studied the mechanism by which lawsone interacts with metabolically active cells and analysed both structure and composition of the TeNPs collected from the growth medium of phototrophycally grown R. capsulatus. High Resolution Transmission Electron Microscopy (HR-TEM) images and Energy-Dispersive X-ray (EDX) microanalysis of TeNPs showed a central core of polycrystalline tellurium interspersed in an organic matrix with a predominant protein-based composition. The main proteins from Te0 nanostructures were identified by Liquid Chromatography tandem-Mass Spectrometry and were all correlated with the cell outer membrane composition. The interaction of reduced lawsone with tellurite and with the bacterial cells was probed by Cyclic Voltammetry and Scanning ElectroChemical Microscopy (SECM). We concluded that lawsone is required for the reduction of tellurite to metal Te0 in a reaction mechanism dependent on reducing equivalents deriving from the cell photosynthetic metabolism. SECM experiments demonstrate that lawsone, by diffusing inside the bacterial cells, is effectively available at the membrane site of the photosynthetic electron transport chain.

Manganese is a Deinococcus radiodurans growth limiting factor in rich culture medium.

To understand the effects triggered by Mn2+ on Deinococcus radiodurans, the proteome patterns associated with different growth phases were investigated. In particular, under physiological conditions we tested the growth rate and the biomass yield of D. radiodurans cultured in rich medium supplemented or not with MnCl2. The addition of 2.5-5.0 µM MnCl2 to the medium neither altered the growth rate nor the lag phase, but significantly increased the biomass yield. When higher MnCl2 concentrations were used (10-250 µM), biomass was again found to be positively affected, although we did observe a concentration-dependent lag phase increase. The in vivo concentration of Mn2+ was determined in cells grown in rich medium supplemented or not with 5 µM MnCl2. By atomic absorption spectroscopy, we estimated 0.2 and 0.75 mM Mn2+ concentrations in cells grown in control and enriched medium, respectively. We qualitatively confirmed this observation using a fluorescent turn-on sensor designed to selectively detect Mn2+in vivo. Finally, we investigated the proteome composition of cells grown for 15 or 19 h in medium to which 5 µM MnCl2 was added, and we compared these proteomes with those of cells grown in the control medium. The presence of 5 µM MnCl2 in the culture medium was found to alter the pI of some proteins, suggesting that manganese affects post-translational modifications. Further, we observed that Mn2+ represses enzymes linked to nucleotide recycling, and triggers overexpression of proteases and enzymes linked to the metabolism of amino acids.

Cyclooxygenase-2 silencing for the treatment of colitis: a combined in vivo strategy based on RNA interference and engineered Escherichia coli.

Nonpathogenic-invasive Escherichia coli (InvColi) bacteria are suitable for genetic transfer into mammalian cells and may act as a vehicle for RNA Interference (RNAi) in vivo. Cyclooxygenase-2 (COX-2) is overexpressed in ulcerative colitis (UC) and Crohn's disease (CD), two inflammatory conditions of the colon and small intestine grouped as inflammatory bowel disease (IBD). We engineered InvColi strains for anti-COX-2 RNAi (InvColi(shCOX2)), aiming to investigate the in vivo feasibility of a novel COX-2 silencing strategy in a murine model of colitis induced by dextran sulfate sodium (DSS). Enema administrations of InvColi(shCOX2) in DSS-treated mice led to COX-2 downregulation, colonic mucosa preservation, reduced colitis disease activity index (DAI) and increased mice survival. Moreover, DSS/InvColi(shCOX2)-treated mice showed lower levels of circulating pro-inflammatory cytokines and a reduced colitis-associated shift of gut microbiota. Considering its effectiveness and safety, we propose our InvColi(shCOX2) strategy as a promising tool for molecular therapy in intestinal inflammatory diseases.

Identification of TLR4 as the receptor that recognizes Shiga toxins in human neutrophils.

Hemolytic uremic syndrome (HUS) caused by intestinal Shiga toxin-producing Escherichia coli infections is a worldwide health problem, as dramatically exemplified by the German outbreak occurred in summer 2011 and by a constant burden of cases in children. Shiga toxins (Stx) play a pivotal role in HUS by triggering endothelial damage in kidney and brain through globotriaosylceramide (Gb3Cer) receptor targeting. Moreover, Stx interact with human neutrophils, as experimentally demonstrated in vitro and as observed in patients with HUS. A neutrophil-protective role on endothelial damage (sequestration of circulating toxins) and a causative role in toxin delivery from the gut to the kidney (piggyback transport) have been suggested in different studies. However, the receptor that recognizes Stx in human neutrophils, which do not express Gb3Cer, has not been identified. In this study, by competition and functional experiments with appropriate agonists and antagonists (LPS, anti-TLR4 Abs, respectively), we have identified TLR4 as the receptor that specifically recognizes Stx1 and Stx2 in human neutrophils. Accordingly, these treatments displaced both toxin variants from neutrophils and, upon challenge with Stx1 or Stx2, neutrophils displayed the same pattern of cytokine expression as in response to LPS (assessed by quantitative RT-PCR, ELISA, or multiplexed Luminex-based immunoassays). Moreover, data were supported by adequate controls excluding any potential interference of contaminating LPS in Stx-binding and activation of neutrophils. The identification of the Stx-receptor on neutrophils provides additional elements to foster the understanding of the pathophysiology of HUS and could have an important effect on the development of therapeutic strategies.

The bacterial response to the chalcogen metalloids Se and Te.

Microbial metabolism of inorganics has been the subject of interest since the 1970s when it was recognized that bacteria are involved in the transformation of metal compounds in the environment. This area of research is generally referred to as bioinorganic chemistry or microbial biogeochemistry. Here, we overview the way the chalcogen metalloids Se and Te interact with bacteria. As a topic of considerable interest for basic and applied research, bacterial processing of tellurium and selenium oxyanions has been reviewed a few times over the past 15 years. Oddly, this is the first time these compounds have been considered together and their similarities and differences highlighted. Another aspect touched on for the first time by this review is the bacterial response in cell-cell or cell-surface aggregates (biofilms) against the metalloid oxyanions. Finally, in this review we have attempted to rationalize the considerable amount of literature available on bacterial resistance to the toxic metalloids tellurite and selenite.

The thiol:disulfide oxidoreductase DsbB mediates the oxidizing effects of the toxic metalloid tellurite (TeO32-) on the plasma membrane redox system of the facultative phototroph Rhodobacter capsulatus.

The highly toxic oxyanion tellurite (TeO3(2-)) is a well known pro-oxidant in mammalian and bacterial cells. This work examines the effects of tellurite on the redox state of the electron transport chain of the facultative phototroph Rhodobacter capsulatus, in relation to the role of the thiol:disulfide oxidoreductase DsbB. Under steady-state respiration, the addition of tellurite (2.5 mM) to membrane fragments generated an extrareduction of the cytochrome pool (c- and b-type hemes); further, in plasma membranes exposed to tellurite (0.25 to 2.5 mM) and subjected to a series of flashes of light, the rate of the QH2:cytochrome c (Cyt c) oxidoreductase activity was enhanced. The effect of tellurite was blocked by the antibiotics antimycin A and/or myxothiazol, specific inhibitors of the QH2:Cyt c oxidoreductase, and, most interestingly, the membrane-associated thiol:disulfide oxidoreductase DsbB was required to mediate the redox unbalance produced by the oxyanion. Indeed, this phenomenon was absent from R. capsulatus MD22, a DsbB-deficient mutant, whereas the tellurite effect was present in membranes from MD22/pDsbB(WT), in which the mutant gene was complemented to regain the wild-type DsbB phenotype. These findings were taken as evidence that the membrane-bound thiol:disulfide oxidoreductase DsbB acts as an "electron conduit" between the hydrophilic metalloid and the lipid-embedded Q pool, so that in habitats contaminated with subinhibitory amounts of Te(IV), the metalloid is likely to function as a disposal for the excess reducing power at the Q-pool level of facultative phototrophic bacteria.

Tellurite effects on Rhodobacter capsulatus cell viability and superoxide dismutase activity under oxidative stress conditions.

Cells of the facultative photosynthetic bacterium Rhodobacter capsulatus (MT1131 strain) incubated with 10 microg ml-1 of the toxic oxyanion tellurite (TeO2-(3)) exhibited an increase in superoxide dismutase activity. The latter effect was also seen upon incubation with sublethal amounts of paraquat, a cytosolic generator of superoxide anions (O2-), in parallel with a strong increase in tellurite resistance (TeR). A mutant strain (CW10) deficient in SenC, a protein with similarities to peroxiredoxin/thiol:disulfide oxidoreductases and a homologue of mitochondrial Sco proteins, was constructed by interposon mutagenesis via the gene transfer agent system. Notably, the absence of SenC affected R. capsulatus resistance to periplasmic O2- generated by xanthine/xanthine oxidase but not to cytosolic O2- produced by paraquat. Further, the absence of SenC did not affect R. capsulatus tellurite resistance. We conclude that: (1) cytosolic-generated O2- enhances TeR of this bacterial species; (2) small amounts of tellurite increase SOD activity so as to mimic the early cell response to oxidative stress; (3) SenC protein is required in protection of R. capsulatus against periplasmic oxidative stress; and finally, (4) SenC protein is not involved in TeR, possibly because tellurite does not generate O-2 at the periplasmic space level.

Effects of the metalloid oxyanion tellurite (TeO32-) on growth characteristics of the phototrophic bacterium Rhodobacter capsulatus.

This work examines the effects of potassium tellurite (K2TeO3) on the cell viability of the facultative phototroph Rhodobacter capsulatus. There was a growth mode-dependent response in which cultures anaerobically grown in the light tolerate the presence of up to 250 to 300 microg of tellurite (TeO3(2-)) per ml, while dark-grown aerobic cells were inhibited at tellurite levels as low as 2 microg/ml. The tellurite sensitivity of aerobic cultures was evident only for growth on minimal salt medium, whereas it was not seen during growth on complex medium. Notably, through the use of flow cytometry, we show that the cell membrane integrity was strongly affected by tellurite during the early growth phase (< or =50% viable cells); however, at the end of the growth period and in parallel with massive tellurite intracellular accumulation as elemental Te0 crystallites, recovery of cytoplasmic membrane integrity was apparent (> or =90% viable cells), which was supported by the development of a significant membrane potential (Deltapsi = 120 mV). These data are taken as evidence that in anaerobic aquatic habitats, the facultative phototroph R. capsulatus might act as a natural scavenger of the highly soluble and toxic oxyanion tellurite.

Tellurite uptake by cells of the facultative phototroph Rhodobacter capsulatus is a Delta pH-dependent process.

The uptake by light-grown cells of Rhodobacter capsulatus of the highly toxic metalloid oxyanion tellurite (TeO(3)(2-)) was examined. We show that tellurite is rapidly taken up by illuminated cells in a process which is inhibited by the protonophore carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP) and by the K(+)/H(+) exchanger nigericin. Notably, the light-driven membrane potential (Delta psi) is enhanced by K(2)TeO(3)> or =200 microM. Further, tellurite uptake is largely insensitive to valinomycin, strongly repressed by the sulfhydryl reagent N-ethylethylmaleimide (NEM) and competitively inhibited by phosphate. We conclude that tellurite is transported into cells by a Delta pH-dependent, non-electrogenic process which is likely to involve the phosphate transporter (PiT family).