Chemical, Physical and Biological Triggers of Evolutionary Conserved Bcl-xL-Mediated Apoptosis

Background: The evidence that pan-Bcl-2 or Bcl-xL-specific inhibitors prematurely kill virus-infected or RNA/DNA-transfected cells provides rationale for investigating these apoptotic inducers further. We hypothesized that not only invasive RNA or DNA (biological factors) but also DNA/RNA-damaging chemical or physical factors could trigger apoptosis that have been sensitized with pan-Bcl-2 or Bcl-xL-specific agents; Methods: We tested chemical and physical factors plus Bcl-xL-specific inhibitor A-1155463 in cells of various origins and the small roundworms (C. elegans); Results: We show that combination of a A-1155463 along with a DNA-damaging agent, 4-nitroquinoline-1-oxide (4NQO), prematurely kills cells of various origins as well as C. elegans. The synergistic effect is p53-dependent and associated with the release of Bad and Bax from Bcl-xL, which trigger mitochondrial outer membrane permeabilization. Furthermore, we found that combining Bcl-xL-specific inhibitors with various chemical compounds or physical insults also induced cell death; Conclusions: Thus, we were able to identify several biological, chemical and physical triggers of the evolutionarily conserved Bcl-xL-mediated apoptotic pathway, shedding light on strategies and targets for novel drug development

Bisbenzimidazole derivatives as potent inhibitors of the trypsin-like sites of the immunoproteasome core particle

International audienceIn this study, a monomeric (MB) and a dimeric (DB) bisbenzimidazoles were identified as novel proteasome inhibitors of the trypsin-like activity located on beta 2c sites of the constitutive 20S proteasome (IC50 values at 2-4 mu M range). Remarkably, they were further shown to be 100- and 200-fold more potent inhibitors of the immunoproteasome trypsin-like activity (beta 2i sites, IC50 = 24 nM) than of the homologous constitutive activity. Molecular models of inhibitor/enzyme complexes in the two types of trypsin-like sites and corresponding computed binding energy values corroborated kinetic data. Different binding modes were suggested for MB and DB to the beta 2c and beta 2i trypsic sites. Each pointed to better contacts of the ligand inside the beta 2i active site than for beta 2c site. MB and DB represent the first selective inhibitors of the immunoproteasome trypsin-like activity described to date and can be considered as prototypes for inhibiting this activity

Une âme de jeune fille [ : Gabrielle***] / Jean Vaudon,...

<p>A. Distribution of CpG-motifs within rDNA (transcribed region of human ribosomal repeat). The digits indicate the nucleotide order number, the vertical bar shows the motif location. Red color is used to mark region A and region B, that were analyzed for the presence of methylated CCGG sites. B. Determination of methylation index of three genes in DNA from cells treated with 20 μM DBP(1–4), 72 h (description is given in Methods).</p

Chemical, Physical and Biological Triggers of Evolutionary Sonserved Bcl-xL-Mediated Apoptosis

Background: The evidence that pan-Bcl-2 or Bcl-xL-specific inhibitors prematurely kill virus-infected or RNA/DNA-transfected cells provides rationale for investigating these apoptotic inducers further. We hypothesized that not only invasive RNA or DNA (biological factors) but also DNA/RNA-damaging chemical or physical factors could trigger apoptosis that have been sensitized with pan-Bcl-2 or Bcl-xL-specific agents; Methods: We tested chemical and physical factors plus Bcl-xL-specific inhibitor A-1155463 in cells of various origins and the small roundworms (C. elegans); Results: We show that combination of a A-1155463 along with a DNA-damaging agent, 4-nitroquinoline-1-oxide (4NQO), prematurely kills cells of various origins as well as C. elegans. The synergistic effect is p53-dependent and associated with the release of Bad and Bax from Bcl-xL, which trigger mitochondrial outer membrane permeabilization. Furthermore, we found that combining Bcl-xL-specific inhibitors with various chemical compounds or physical insults also induced cell death; Conclusions: Thus, we were able to identify several biological, chemical and physical triggers of the evolutionarily conserved Bcl-xL-mediated apoptotic pathway, shedding light on strategies and targets for novel drug development

Chemical, Physical and Biological Triggers of Evolutionary Sonserved Bcl-xL-Mediated Apoptosis

Background: The evidence that pan-Bcl-2 or Bcl-xL-specific inhibitors prematurely kill virus-infected or RNA/DNA-transfected cells provides rationale for investigating these apoptotic inducers further. We hypothesized that not only invasive RNA or DNA (biological factors) but also DNA/RNA-damaging chemical or physical factors could trigger apoptosis that have been sensitized with pan-Bcl-2 or Bcl-xL-specific agents; Methods: We tested chemical and physical factors plus Bcl-xL-specific inhibitor A-1155463 in cells of various origins and the small roundworms (C. elegans); Results: We show that combination of a A-1155463 along with a DNA-damaging agent, 4-nitroquinoline-1-oxide (4NQO), prematurely kills cells of various origins as well as C. elegans. The synergistic effect is p53-dependent and associated with the release of Bad and Bax from Bcl-xL, which trigger mitochondrial outer membrane permeabilization. Furthermore, we found that combining Bcl-xL-specific inhibitors with various chemical compounds or physical insults also induced cell death; Conclusions: Thus, we were able to identify several biological, chemical and physical triggers of the evolutionarily conserved Bcl-xL-mediated apoptotic pathway, shedding light on strategies and targets for novel drug development

DNA methylation in nuclei of the control MCF-7 cells.

<p>A. Cells were processed for immunofluorescence staining with anti 5-mC antibody. (1)—Magnification X60; (2)—For analysis of the nuclei the photo was enlarged by an order using computer processing. The arrow points to the methylated DNA block close to the nucleolus. B. (1)—DNA methylation in nuclei of the control MCF-7 cells and the cells treated with 10 μM AzaC or 20μM DBP(1–4), 72 h. Background: Control cells were only treated with secondary antibodies conjugated with FITC. (2) The photo was enlarged considerably for the analysis of nuclei size. The nucleus of the cell cultivated with DBP(4) is shown for an example. C. An example of the different DNA methylation level in cells’ nuclei that were cultivated in the presence of 20μM DBP(4), 72 h. A,B(1) and C—Cell nuclei were additionally colored with DAPI.</p

Incorporation of DBP(1–4) in MCF-7 cells.

<p>Excitation of fluorescence at 380 nm. Magnification X 20. MCF-7 were incubated with DBP(1–4) (7, 10 or 20 μM) for 24 h and the fluorescence was analyzed in unfixed cells. Staining of the cells was heterogeneous and practically did not depend on the concentrations of DBP(1–4). The 20 μM concentrations of DBP(1–4) were used for an example. The photos of cells were taken with identical exposure. For example the most common nuclei staining types are shown with arrows and numbers: 1 –nucleus is stained only, 2 –nucleus and the cytoplasm are stained. 3- cells show strong blue fluorescence signal while nuclei do not contrast. Background: the photo of control cells in visible light (VIS) and at λex = 380 nm (FL), the exposure is increased two times.</p

A (FL-reader).

<p>(1)–Example of reaction rate constant determination for DCF formation when DCFH reacts with ROS. Cells were cultivated for 24 hours in the presence of 40 μM DBP(1). The cultivation environment was replaced by 5 μm H2DCFH-DA in PBS solution and a relative fluorescence intensity increase was detected. I_t,I0 –sample’s signal at time t and immediately after H2DCFH-DA addition respectively. The slope of the line– <a href="http://universal_ru_en.academic.ru/378785/reaction_rate_constant" target="_blank">reaction rate constant</a> for DCF formation (k). ROS index Δk/k₀ = (k_DBP−k₀)×100/k₀ (%). (2)—Relationship between ROS index Δk/k₀ and DBP(1–4) concentration. Time of cultivation is shown on the figure. B (FCA). Proliferation of MCF-7 cells exposed to DBP(1–4) at final concentration 20 μM for 24 hours. (1)—Distribution of fluorescence of fixed cells stained with anti-Ki-67 antibodies (control—dark green color, DBP(2) as example—blue color). Background fluorescence was quantified using FITC-conjugated secondary antibodies (green color). (2)—The median signal intensity of FL1 (Ki-67+) (gate R). Each experiment was repeated at least three times. Results represent the average of the medians of the FL parameter in independent experiments. C (FCA). (1)—Distribution of fluorescence of fixed cells stained with propidium iodide. The fractions of (1–4) cells with different DNA amounts are shown. (2)–Proportion MCF-7 culture cells with DNA amount corresponding to the G1-, S—and G2/M phases of the cell cycle. The data shown is an average value from three independent experiments. (3) The total number of cells in culture. (4) The number hypodiploid cells (fraction SubG0/G1).D. The proportion of cells in culture with signs of apoptotic nuclei (condensed chromatin, the irregular nucleus shape, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189826#pone.0189826.g004" target="_blank">Fig 4</a>). B, C, D–cells were cultivated in the presence of 20 μM DBP(1–4) for 24 hours.</p

Overall 5-methylcytosine level in DNA of cells treated with 20 μM DBP(1–4) for 72 hours.

<p>A (ELISA). (1, 2)–example of calibration dependence between methylated DNA in samples and integral stain density (I). (3) example of DNA samples analysis from cells treated with DBP(1–4). 10 ng of DNA was applied. pBR322 plasmid was used as a negative control. (4) Relative signal change reflecting difference in DNA methylation level. B (<i>UPLC/MS/MS</i>). Average 5mC content in DNA samples.</p