Ewing's sarcoma: Analysis of single nucleotide polymorphism in the EWS gene

AbstractWe aimed to investigate single nucleotide polymorphisms (SNPs) in the EWS gene breaking region in order to analyze Ewing's sarcoma susceptibility. The SNPs were investigated in a healthy subject population and in Ewing's sarcoma patients from Southern Brazil. Genotyping was performed by TaqMan® assay for allelic discrimination using Real-Time PCR. The analysis of incidence of SNPs or different SNP-arrangements revealed a higher presence of homozygote TT-rs4820804 in Ewing's sarcoma patients (p=0.02; Chi Square Test). About 300bp from the rs4820804 SNP lies a palindromic hexamer (5′-GCTAGC-3′) and three nucleotides (GTC), which were previously identified to be in close vicinity of the breakpoint junction in both EWS and FLI1 genes. This DNA segment surrounding the rs4820804 SNP is likely to indicate a breakpoint region. If the T-rs4820804 allele predisposes a DNA fragment to breakage, homozygotes (TT-rs4820804) would have double the chance of having a chromosome break, increasing the chances for a translocation to occur. In conclusion, the TT-rs4820804 EWS genotype can be associated with Ewing's sarcoma and the SNP rs4820804 can be a candidate marker to understand Ewing's sarcoma susceptibility

Conformational Characterization of Ipomotaosides and Their Recognition by COX-1 and 2

The aerial parts of Ipomoea batatas are described herein to produce four new resin glycosides, designated as ipomotaosides A, B, C, and D. Ipomotaoside A was found to present inhibitory activity on both cyclooxygenases. However, the conformational elucidation of these molecules may be difficult due to their high flexibility. In this context, the current work presents a conformational characterization of ipomotaosides A–D in aqueous and nonaqueous solvents. The employed protocol includes metadynamics evaluation and unrestrained molecular dynamics simulations (MD). The obtained data provided structural models for the ipomotaosides in good agreement with previous ROESY distances measured in pyridine. Accordingly, the most abundant conformation of ipomotaoside A in solution was employed in flexible docking studies, providing a structural basis for the compound’s inhibition of COX enzymes. The so-obtained complex supports resin glycosides’ role as original scaffolds for future studies, aiming at structural optimization and development of potential new anti-inflammatory agents

Discovery of a Series of Acridinones as Mechanism-Based Tubulin Assembly Inhibitors with Anticancer Activity

<div><p>Microtubules play critical roles in vital cell processes, including cell growth, division, and migration. Microtubule-targeting small molecules are chemotherapeutic agents that are widely used in the treatment of cancer. Many of these compounds are structurally complex natural products (e.g., paclitaxel, vinblastine, and vincristine) with multiple stereogenic centers. Because of the scarcity of their natural sources and the difficulty of their partial or total synthesis, as well as problems related to their bioavailability, toxicity, and resistance, there is an urgent need for novel microtubule binding agents that are effective for treating cancer but do not have these disadvantages. In the present work, our lead discovery effort toward less structurally complex synthetic compounds led to the discovery of a series of acridinones inspired by the structure of podophyllotoxin, a natural product with important microtubule assembly inhibitory activity, as novel mechanism-based tubulin assembly inhibitors with potent anticancer properties and low toxicity. The compounds were evaluated <i>in vitro</i> by wound healing assays employing the metastatic and triple negative breast cancer cell line MDA-MB-231. Four compounds with IC₅₀ values between 0.294 and 1.7 μM were identified. These compounds showed selective cytotoxicity against MDA-MB-231 and DU-145 cancer cell lines and promoted cell cycle arrest in G₂/M phase and apoptosis. Consistent with molecular modeling results, the acridinones inhibited tubulin assembly in <i>in vitro</i> polymerization assays with IC₅₀ values between 0.9 and 13 μM. Their binding to the colchicine-binding site of tubulin was confirmed through competitive assays.</p></div

Synthesis of Limonene β-Amino Alcohol Derivatives in Support of New Antileishmanial Therapies

A series of seven limonene β-amino alcohol derivatives has been regioselectively synthesised in moderate to good yields. Two of these compounds were found to be significantly effective against in vitro cultures of the Leishmania (Viannia) braziliensis promastigote form in the micromolar range. The activities found for 3b and 3f were about 100-fold more potent than the standard drug, Pentamidine, in the same test, while limonene did not display any activity. This is the first report of antileishmanial activity by limonene β-amino alcohol derivatives

Flow cytometry analyses.

<p>(A), the left panel presents the fluorescence histogram with the DNA profile of the cell-cycle phases (Sub-G₁, G₁, S and G₂) of MDA-MB-231 cells in the presence of the control (DMSO 0.1%), colchicine and <b>10</b>. The right panel shows the distribution of cells in different phases of the cell cycle for the control and after treatment with colchicine, <b>6</b>, <b>7</b>, <b>9</b> and <b>10</b> (B), the left panel displays the dot plot for the Annexin-V FITC/PI double stain of MDA-MB-231 cells for the control and after treatment with colchicine and <b>10</b>. In the FITC- PI- quadrant are the alive cells, in the FITC+ quadrant are the apoptotic cells, and in the FITC+ PI+ and PI+ quadrants are the dead cells. The right panel shows the percentage of alive, apoptotic and dead cells of the control and after treatment with colchicine, <b>6</b>, <b>7</b>, <b>9</b> and <b>10</b>. Statistical significance relative to control * <i>P</i> <0.05, ** <i>P</i> <0.01 and *** <i>P</i> <0.001.</p

<i>In vitro</i> tubulin assembly inhibition.

<p>(A), results for the fluorescence-based assay; blue lines and circles represent the negative control (DMSO 1%), orange lines and squares represent the stabilizing control (paclitaxel) and green lines and triangles represent inhibition by the control (colchicine). <b>6</b> results are presented as purple lines and inverted triangles, <b>7</b> are dark red lines and hexagons and the results for <b>10</b> are presented as light red lines and circles (B), results for the light-scattering assay negative control, paclitaxel, colchicine, <b>6</b>, <b>7</b> and <b>10</b> are represented by the same colors and shapes as in (A), <b>9</b> is presented as dark blue lines and hexagons (C), competitive colchicine binding assay; black lines and circles represent the fluorescence of the negative control (DMSO 1%) as competitor, and the results for positive control colchicine as competitor are presented as red lines and diamonds; fluorescence in the presence of <b>10</b> as competitor is presented as green lines and triangles. Data are representative of two experiments. *To monitor a non-competitive colchicine-binding behavior, experiments using an inactive compound from our <i>in house</i> collection of compounds were conducted (data not shown).</p

Molecular modeling studies of the acridinones series.

<p>(A), steric complementarity between the colchicine site and <b><i>S</i>-7</b> (B), binding mode of <b><i>S</i>-11</b> showing a hydrogen bond between the NH groups of acridinones and Thr α179 (C), binding mode of <b><i>S</i>-10</b> where the trimethoxybenzyl group occupies the hydrophobic pocket of the colchicine site (D), docking pose for <b><i>S</i>-7</b> with a hydrogen bond between the NO₂ group and Leu β252 (E), docking pose for <b><i>S</i>-6</b> with a hydrogen bond between oxygen from dioxalane and Cys β241 (F), Comparison of predicted interactions between the docking pose of <b><i>S</i>-7</b> and the crystal structure of tubulin (PDB ID: 1SA0) and the predicted interactions between crystallographic DAMA-colchicine (PDB ID: CN2) and tubulin (PDB ID: 1SA0). These interactions were calculated using the online software PoseView [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160842#pone.0160842.ref038" target="_blank">38</a>]. The green lines represent hydrophobic interactions and the black dotted lines represent hydrogen bonds.</p

Effects of the acridinones on MDA-MB-231 cell migration.

<p>(A), inhibition of wound closure (concentration of 10 μM) (B), pictures of the wound healing assay at 0 h and 22 h for the negative control and <b>10</b> (C), representative pictures of migrated cells in a Boyden chamber assay for the negative control and <b>10</b> in different concentrations (D), a representative IC₅₀ curve for the antimigratory response against <b>10</b>.</p