Targeting breast cancer metabolism with a novel inhibitor of mitochondrial ATP synthesis.

Inhibitors of mitochondrial respiration and ATP synthesis may promote the selective killing of respiration-competent cancer cells that are critical for tumor progression. We previously reported that CADD522, a small molecule inhibitor of the RUNX2 transcription factor, has potential for breast cancer treatment. In the current study, we show that CADD522 inhibits mitochondrial oxidative phosphorylation by decreasing the mitochondrial oxygen consumption rate (OCR) and ATP production in human breast cancer cells in a RUNX2-independent manner. The enzyme activity of mitochondrial ATP synthase was inhibited by CADD522 treatment. Importantly, results from cellular thermal shift assays that detect drug-induced protein stabilization revealed that CADD522 interacts with both α and β subunits of the F1-ATP synthase complex. Differential scanning fluorimetry also demonstrated interaction of α subunits of the F1-ATP synthase to CADD522. These results suggest that CADD522 might target the enzymatic F1 subunits in the ATP synthase complex. CADD522 increased the levels of intracellular reactive oxygen species (ROS), which was prevented by MitoQ, a mitochondria-targeted antioxidant, suggesting that cancer cells exposed to CADD522 may elevate ROS from mitochondria. CADD522-increased mitochondrial ROS levels were enhanced by exogenously added pro-oxidants such as hydrogen peroxide or tert-butyl hydroperoxide. Conversely, CADD522-mediated cell growth inhibition was blocked by N-acetyl-l-cysteine, a general ROS scavenger. Therefore, CADD522 may exert its antitumor activity by increasing mitochondrial driven cellular ROS levels. Collectively, our data suggest in vitro proof-of-concept that supports inhibition of mitochondrial ATP synthase and ROS generation as contributors to the effectiveness of CADD522 in suppression of tumor growth

Investigation of Mitochondrial Dysfunction by Sequential Microplate-Based Respiration Measurements from Intact and Permeabilized Neurons

Mitochondrial dysfunction is a component of many neurodegenerative conditions. Measurement of oxygen consumption from intact neurons enables evaluation of mitochondrial bioenergetics under conditions that are more physiologically realistic compared to isolated mitochondria. However, mechanistic analysis of mitochondrial function in cells is complicated by changing energy demands and lack of substrate control. Here we describe a technique for sequentially measuring respiration from intact and saponin-permeabilized cortical neurons on single microplates. This technique allows control of substrates to individual electron transport chain complexes following permeabilization, as well as side-by-side comparisons to intact cells. To illustrate the utility of the technique, we demonstrate that inhibition of respiration by the drug KB-R7943 in intact neurons is relieved by delivery of the complex II substrate succinate, but not by complex I substrates, via acute saponin permeabilization. In contrast, methyl succinate, a putative cell permeable complex II substrate, failed to rescue respiration in intact neurons and was a poor complex II substrate in permeabilized cells. Sequential measurements of intact and permeabilized cell respiration should be particularly useful for evaluating indirect mitochondrial toxicity due to drugs or cellular signaling events which cannot be readily studied using isolated mitochondria

Важливе історико-географічне дослідження

Рец. на кн. Темушева В.Н. "Гомельская земля в конце XV первой половине XVI в. Территориальные трансформации в пограничном регионе". — М.: "Квадрига", 2009. — 190 с.Review of the book: Temushev V.N. "Gomel Land in the Late 15th — the 1st half of the 16th Centuries. Territorial Transformations in the Frontier Area". — Moscow: "Kvadriga", 2009. — 190 p

Improved Mitochondrial Function with Diet-Induced Increase in Either Docosahexaenoic Acid or Arachidonic Acid in Membrane Phospholipids

Mitochondria can depolarize and trigger cell death through the opening of the mitochondrial permeability transition pore (MPTP). We recently showed that an increase in the long chain n3 polyunsaturated fatty acids (PUFA) docosahexaenoic acid (DHA; 22:6n3) and depletion of the n6 PUFA arachidonic acid (ARA; 20:4n6) in mitochondrial membranes is associated with a greater Ca2+ load required to induce MPTP opening. Here we manipulated mitochondrial phospholipid composition by supplementing the diet with DHA, ARA or combined DHA+ARA in rats for 10 weeks. There were no effects on cardiac function, or respiration of isolated mitochondria. Analysis of mitochondrial phospholipids showed DHA supplementation increased DHA and displaced ARA in mitochondrial membranes, while supplementation with ARA or DHA+ARA increased ARA and depleted linoleic acid (18:2n6). Phospholipid analysis revealed a similar pattern, particularly in cardiolipin. Tetralinoleoyl cardiolipin was depleted by 80% with ARA or DHA+ARA supplementation, with linoleic acid side chains replaced by ARA. Both the DHA and ARA groups had delayed Ca2+-induced MPTP opening, but the DHA+ARA group was similar to the control diet. In conclusion, alterations in mitochondria membrane phospholipid fatty acid composition caused by dietary DHA or ARA was associated with a greater cumulative Ca2+ load required to induced MPTP opening. Further, high levels of tetralinoleoyl cardiolipin were not essential for normal mitochondrial function if replaced with very-long chain n3 or n6 PUFAs

The Science Performance of JWST as Characterized in Commissioning

This paper characterizes the actual science performance of the James Webb Space Telescope (JWST), as determined from the six month commissioning period. We summarize the performance of the spacecraft, telescope, science instruments, and ground system, with an emphasis on differences from pre-launch expectations. Commissioning has made clear that JWST is fully capable of achieving the discoveries for which it was built. Moreover, almost across the board, the science performance of JWST is better than expected; in most cases, JWST will go deeper faster than expected. The telescope and instrument suite have demonstrated the sensitivity, stability, image quality, and spectral range that are necessary to transform our understanding of the cosmos through observations spanning from near-earth asteroids to the most distant galaxies.Comment: 5th version as accepted to PASP; 31 pages, 18 figures; https://iopscience.iop.org/article/10.1088/1538-3873/acb29

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure

Effect of the permeabilizing agent and time on FCCP-stimulated respiration.

<p>(<b>A</b>) Primary rat cortical neurons were permeabilized by saponin (sap, 25 µg/ml, filled squares) or digitonin (25 µg/ml, open squares, 50 µg/ml, open triangles, 100 µg/ml, open circles) plus EGTA (5 mM) in aCSF medium after three baseline O₂ consumption rate (OCR) measurements (first arrow). Pyruvate and malate (P/M, 5 mM each), ADP (1 mM), and excess K₂PHO₄ (3.6 mM for 4 mM final) were co-injected with saponin to measure complex I-dependent ADP-stimulated respiration. Oligomycin (oligo, 0.3 µg/ml), FCCP, (F, 2 µM) and antimycin A (AA, 1 µM) were added as indicated (arrows). (<b>B</b>) OCRs were measured and at the injection marked <i>a</i>, neurons were control-treated in the absence (filled squares) or presence of FCCP plus pyruvate (2 µM and 10 mM, respectively, open squares) or permeabilized using saponin (triangles). Complex I-linked respiration (P/M) in permeabilized neurons was stimulated by ADP (1 mM, filled triangles) or FCCP (F, 2 µM, open triangles). Intact (open squares) and permeabilized (open triangles) cells treated with FCCP received a second FCCP injection (1 µM) at <i>b</i> to insure respiration was maximally uncoupled, followed by a control injection at <i>c</i> and finally antimycin A (AA, 1 µM) at <i>d</i>. Control-treated intact cells (filled squares) and ADP-treated permeabilized cells (filled triangles) received injections of oligo, FCCP, (F, 2 µM) and AA in ports <i>b</i>, <i>c</i>, and <i>d</i>, respectively, with pyruvate (10 mM) included with FCCP for intact cells. OCRs in (<b>A</b>) and (<b>B</b>) are mean ± SD in quadruplicate, normalized to the third measurement point and expressed as % baseline OCR.</p

Methyl succinate fails to rescue KB-R7943-inhibited respiration in intact neurons.

<p>(<b>A</b>) Primary rat cortical neurons were control-treated (con, filled squares) or treated with KB-7943 (K-BR, 10 µM, open circles, 20 µM, filled triangles, or 30 µM, open triangles) after three baseline O₂ consumption rate (OCR) measurements (first arrow). Subsequently, oligomycin (oligo, 0.3 µg/ml), FCCP plus pyruvate (F, 2 µM and 10 mM, respectively), and antimycin A (AA, 1 µM) were added as indicated (arrows). (<b>B</b>) Neurons were control-treated (con, filled squares) or treated with KB-7943 (30 µM, all other groups) as in (<b>A</b>). Other additions were as (A) except the FCCP+pyruvate injection after KB-R7943 addition also contained methyl succinate (MeS, 10 mM, open triangles), methyl succinate plus rotenone (10 mM/0.5 µM, MeS/R, open circles), or no additional substrate (open squares). OCRs in (<b>A</b>) and (<b>B</b>) are mean ± SD in triplicate, normalized to the third measurement point and expressed as % baseline OCR.</p

Respiration rates and respiratory control ratios for permeabilized neurons oxidizing various substrates.

<p>Initial, state 3, state 4, and uncoupled O₂ consumption rates (OCRs) are expressed in pmol O₂/min/80,000 plated cells. The initial OCRs are the rates measured just prior to permeabilization or control injection (intact). The state 4 rates were measured in the presence of oligomycin. The respiratory control ratio (RCR) is the ratio of the state 3 rate to the state 4 rate, calculated on an individual well basis prior to averaging. Data are mean ± SE, n = 4 experiments of 3–5 wells per treatment. One well (P/M) was excluded from analysis because the antimycin OCR was higher than the oligomycin OCR.</p>*<p>indicates a significant difference compared to intact cells (p<0.05). # indicates a significant difference compared to pyruvate/malate (p<0.05).</p

Methyl succinate is a poor substrate for complex II.

<p>(<b>A</b>) Primary rat cortical neurons were incubated in aCSF in the presence of glucose (15 mM, filled squares), no substrate (open circles), pyruvate (10 mM, filled triangles), or methyl succinate (10 mM, open squares). Cells were control-treated (con, filled squares) or treated with 2-deoxyglucose (2-DG, 2 mM, all other groups) after three baseline O₂ consumption rate (OCR) measurements (first arrow). Rotenone (0.5 µM) was subsequently added (second arrow) to inhibit complex I. (<b>B</b>) Neurons were control-treated (con, filled squares) or permeabilized by saponin (sap, 25 µg/ml) plus EGTA (5 mM) in aCSF medium after three baseline O₂ consumption rate (OCR) measurements (first arrow). Succinate/rotenone (S/R, 5 mM and 0.5 mM respectively) or methyl succinate/rotenone (MeS/R, 5 mM and 0.5 mM respectively) as substrate (sub), ADP (1 mM), and excess K₂PHO₄ (3.6 mM for 4 mM final) were co-injected with saponin to measure complex II-dependent ADP-stimulated respiration. Subsequently, oligomycin (oligo, 0.3 µg/ml), FCCP, (F, 2 µM) and antimycin A (AA, 1 µM) were added as indicated (arrows). Pyruvate (10 mM) was included with FCCP for intact cells (filled squares) to insure that substrate supply was not rate-limiting for uncoupled respiration. OCRs in (<b>A</b>) and (<b>B</b>) are mean ± SD in triplicate, normalized to the third measurement point and expressed as % baseline OCR.</p