Computational selection of inhibitors of Abeta aggregation and neuronal toxicity.

Alzheimer\u27s disease (AD) is characterized by the cerebral accumulation of misfolded and aggregated amyloid-beta protein (Abeta). Disease symptoms can be alleviated, in vitro and in vivo, by \u27beta-sheet breaker\u27 pentapeptides that reduce plaque load. However the peptide nature of these compounds, made them biologically unstable and unable to penetrate membranes with high efficiency. The main goal of this study was to use computational methods to identify small molecule mimetics with better drug-like properties. For this purpose, the docked conformations of the active peptides were used to identify compounds with similar activities. A series of related beta-sheet breaker peptides were docked to solid state NMR structures of a fibrillar form of Abeta. The lowest energy conformations of the active peptides were used to design three dimensional (3D)-pharmacophores, suitable for screening the NCI database with Unity. Small molecular weight compounds with physicochemical features and a conformation similar to the active peptides were selected, ranked by docking and biochemical parameters. Of 16 diverse compounds selected for experimental screening, 2 prevented and reversed Abeta aggregation at 2-3microM concentration, as measured by Thioflavin T (ThT) fluorescence and ELISA assays. They also prevented the toxic effects of aggregated Abeta on neuroblastoma cells. Their low molecular weight and aqueous solubility makes them promising lead compounds for treating AD

Combination therapy with vemurafenib (PLX4032/RG7204) and metformin in melanoma cell lines with distinct driver mutations

<p>Abstract</p> <p>Background</p> <p>A molecular linkage between the MAPK and the LKB1-AMPK energy sensor pathways suggests that combined MAPK oncogene inhibition and metabolic modulation of AMPK would be more effective than either manipulation alone in melanoma cell lines.</p> <p>Materials and methods</p> <p>The combination of the BRAF inhibitor vemurafenib (formerly PLX4032) and metformin were tested against a panel of human melanoma cell lines with defined BRAF and NRAS mutations for effects on viability, cell cycle and apoptosis. Signaling molecules in the MAPK, PI3K-AKT and LKB1-AMPK pathways were studied by Western blot.</p> <p>Results</p> <p>Single agent metformin inhibited proliferation in 12 out of 19 cell lines irrespective of the BRAF mutation status, but in one NRAS^Q61Kmutant cell line it powerfully stimulated cell growth. Synergistic anti-proliferative effects of the combination of metformin with vemurafenib were observed in 6 out of 11 BRAF^V600Emutants, including highly synergistic effects in two BRAF^V600Emutant melanoma cell lines. Antagonistic effects were noted in some cell lines, in particular in BRAF^V600Emutant cell lines resistant to single agent vemurafenib. Seven out of 8 BRAF wild type cell lines showed marginally synergistic anti-proliferative effects with the combination, and one cell line had highly antagonistic effects with the combination. The differential effects were not dependent on the sensitivity to each drug alone, effects on cell cycle or signaling pathways.</p> <p>Conclusions</p> <p>The combination of vemurafenib and metformin tended to have stronger anti-proliferative effects on BRAF^V600Emutant cell lines. However, determinants of vemurafenib and metformin synergism or antagonism need to be understood with greater detail before any potential clinical utility of this combination.</p

The interplay of growth differentiation factor 15 (GDF15) expression and M2 macrophages during prostate carcinogenesis

M2 (tumor-supportive) macrophages may upregulate growth differentiation factor 15 (GDF15), which is highly expressed in prostate tumors, but the combined utility of these markers as prognostic biomarkers are unclear. We retrospectively studied 90 prostate cancer cases that underwent radical prostatectomy as their primary treatment and were followed for biochemical recurrence (BCR). These cases also had a benign prostate biopsy at least 1 year or more before their prostate cancer surgery. Using computer algorithms to analyze digitalized immunohistochemically stained slides, GDF15 expression and the presence of M2 macrophages based on the relative density of CD204- and CD68-positive macrophages were measured in prostate: (i) benign biopsy, (ii) cancer and (iii) tumor-adjacent benign (TAB) tissue. Both M2 macrophages (P = 0.0004) and GDF15 (P \u3c 0.0001) showed significant inter-region expression differences. Based on a Cox proportional hazards model, GDF15 expression was not associated with BCR but, in men where GDF15 expression differences between cancer and TAB were highest, the risk of BCR was significantly reduced (hazard ratio = 0.26; 95% confidence interval = 0.09-0.94). In addition, cases with high levels of M2 macrophages in prostate cancer had almost a 5-fold increased risk of BCR (P = 0.01). Expression of GDF15 in prostate TAB was associated with M2 macrophage levels in both prostate cancer and TAB and appeared to moderate M2-macrophage-associated BCR risk. In summary, the relationship of GDF15 expression and CD204-positive M2 macrophage levels is different in a prostate tumor environment compared with an earlier benign biopsy and, collectively, these markers may predict aggressive disease

Racial differences in the systemic inflammatory response to prostate cancer

Systemic inflammation may increase risk for prostate cancer progression, but the role it plays in prostate cancer susceptibility is unknown. From a cohort of over 10,000 men who had either a prostate biopsy or transurethral resection that yielded a benign finding, we analyzed 517 incident prostate cancer cases identified during follow-up and 373 controls with one or more white blood cell tests during a follow-up period between one and 18 years. Multilevel, multivariable longitudinal models were fit to two measures of systemic inflammation, neutrophil-to-lymphocyte ratio (NLR) and monocyte-to-lymphocyte ratio (MLR), to determine NLR and MLR trajectories associated with increased risk for prostate cancer. For both measures, we found no significant differences in the trajectories by case/control status, however in modeling NLR trajectories there was a significant interaction between race (white or Black and case-control status. In race specific models, NLR and MLR values were consistently higher over time among white controls than white cases while case-control differences in NLR and MLR trajectories were not apparent among Black men. When cases were classified as aggressive as compared to non-aggressive, the case-control differences in NLR and MLR values over time among white men were most apparent for non-aggressive cases. For NLR among white men, significant case-control differences were observed for the entire duration of observation for men who had inflammation in their initial prostate specimen. It is possible that, among white men, monitoring of NLR and MLR trajectories after an initial negative biopsy may be useful in monitoring prostate cancer risk

Early-life cockroach allergen and polycyclic aromatic hydrocarbon exposures predict cockroach sensitization among inner-city children

Background: Sensitization to cockroach is one of the strongest identified risk factors for greater asthma morbidity in low-income urban communities; however, the timing of exposures relevant to the development of sensitization has not been elucidated fully. Furthermore, exposure to combustion byproducts, including polycyclic aromatic hydrocarbons (PAHs), can augment the development of allergic sensitization. Objective: We sought to test the hypotheses that domestic cockroach allergen measured prenatally would predict cockroach sensitization in early childhood and that this association would be greater for children exposed to higher PAH concentrations. Methods: Dominican and African American pregnant women living in New York City were enrolled. In the third trimester expectant mothers wore personal air samplers for measurement of 8 nonvolatile PAHs and the semivolatile PAH pyrene, and dust was collected from homes for allergen measurement. Glutathione-S-transferase μ 1 (GSTM1) gene polymorphisms were measured in children. Allergen-specific IgE levels were measured from the children at ages 2, 3, 5, and 7 years. Results: Bla g 2 in prenatal kitchen dust predicted cockroach sensitization at the ages of 5 to 7 years (adjusted relative risk [RR], 1.15; P = .001; n = 349). The association was observed only among children with greater than (RR, 1.22; P = .001) but not less than (RR, 1.07; P = .24) the median sum of 8 nonvolatile PAH levels. The association was most pronounced among children with higher PAH levels and null for the GSTM1 gene (RR, 1.54; P = .001). Conclusions: Prenatal exposure to cockroach allergen was associated with a greater risk of allergic sensitization. This risk was increased by exposure to nonvolatile PAHs, with children null for the GSTM1 mutation particularly vulnerable

Growth and differentiation factor 15 and NF-κB expression in benign prostatic biopsies and risk of subsequent prostate cancer detection

Growth and differentiation factor 15 (GDF-15), also known as macrophage inhibitory cytokine 1 (MIC-1), may act as both a tumor suppressor and promotor and, by regulating NF-κB and macrophage signaling, promote early prostate carcinogenesis. To determine whether expression of these two inflammation-related proteins affect prostate cancer susceptibility, dual immunostaining of benign prostate biopsies for GDF-15 and NF-κB was done in a study of 503 case-control pairs matched on date, age, and race, nested within a historical cohort of 10,478 men. GDF-15 and NF-κB expression levels were positively correlated (r = 0.39; p \u3c 0.0001), and both were significantly lower in African American (AA) compared with White men. In adjusted models that included both markers, the odds ratio (OR) for NF-κB expression was statistically significant, OR =0.87; p = 0.03; 95% confidence interval (CI) =0.77-0.99, while GDF-15 expression was associated with a nominally increased risk, OR =1.06; p = 0.27; 95% CI =0.96-1.17. When modeling expression levels by quartiles, the highest quartile of NF-κB expression was associated with almost a fifty percent reduction in prostate cancer risk (OR =0.51; p = 0.03; 95% CI =0.29-0.92). In stratified models, NF-κB had the strongest negative association with prostate cancer in non-aggressive cases (p = 0.03), older men (p = 0.03), and in case-control pairs with longer follow-up (p = 0.02). Risk associated with GDF-15 expression was best fit using nonlinear regression modeling where both first (p = 0.02) and second (p = 0.03) order GDF-15 risk terms were associated with significantly increased risk. This modeling approach also revealed significantly increased risk associated with GDF-15 expression for subsamples defined by AA race, aggressive disease, younger age, and in case-control pairs with longer follow-up. Therefore, although positively correlated in benign prostatic biopsies, NF-κB and GDF-15 expression appear to exert opposite effects on risk of prostate tumor development

Differential sensitivity of melanoma cell lines with BRAFV600E mutation to the specific Raf inhibitor PLX4032

Blocking oncogenic signaling induced by the BRAFV600E mutation is a promising approach for melanoma treatment. We tested the anti-tumor effects of a specific inhibitor of Raf protein kinases, PLX4032/RG7204, in melanoma cell lines. PLX4032 decreased signaling through the MAPK pathway only in cell lines with the BRAFV600E mutation. Seven out of 10 BRAFV600E mutant cell lines displayed sensitivity based on cell viability assays and three were resistant at concentrations up to 10 μM. Among the sensitive cell lines, four were highly sensitive with IC50 values below 1 μM, and three were moderately sensitive with IC50 values between 1 and 10 μM. There was evidence of MAPK pathway inhibition and cell cycle arrest in both sensitive and resistant cell lines. Genomic analysis by sequencing, genotyping of close to 400 oncogeninc mutations by mass spectrometry, and SNP arrays demonstrated no major differences in BRAF locus amplification or in other oncogenic events between sensitive and resistant cell lines. However, metabolic tracer uptake studies demonstrated that sensitive cell lines had a more profound inhibition of FDG uptake upon exposure to PLX4032 than resistant cell lines. In conclusion, BRAFV600E mutant melanoma cell lines displayed a range of sensitivities to PLX4032 and metabolic imaging using PET probes can be used to assess sensitivity

Single-cell proteomic chip for profiling intracellular signaling pathways in single tumor cells

We describe a microchip designed to quantify the levels of a dozen cytoplasmic and membrane proteins from single cells. We use the platform to assess protein–protein interactions associated with the EGF-receptor-mediated PI3K signaling pathway. Single-cell sensitivity is achieved by isolating a defined number of cells (n = 0–5) in 2 nL volume chambers, each of which is patterned with two copies of a miniature antibody array. The cells are lysed on-chip, and the levels of released proteins are assayed using the antibody arrays. We investigate three isogenic cell lines representing the cancer glioblastoma multiforme, at the basal level, under EGF stimulation, and under erlotinib inhibition plus EGF stimulation. The measured protein abundances are consistent with previous work, and single-cell analysis uniquely reveals single-cell heterogeneity, and different types and strengths of protein–protein interactions. This platform helps provide a comprehensive picture of altered signal transduction networks in tumor cells and provides insight into the effect of targeted therapies on protein signaling networks