Ketogenic diet induces p53-dependent cellular senescence in multiple organs.

A ketogenic diet (KD) is a high-fat, low-carbohydrate diet that leads to the generation of ketones. While KDs improve certain health conditions and are popular for weight loss, detrimental effects have also been reported. Here, we show mice on two different KDs and, at different ages, induce cellular senescence in multiple organs, including the heart and kidney. This effect is mediated through adenosine monophosphate-activated protein kinase (AMPK) and inactivation of mouse double minute 2 (MDM2) by caspase-2, leading to p53 accumulation and p21 induction. This was established using p53 and caspase-2 knockout mice and inhibitors to AMPK, p21, and caspase-2. In addition, senescence-associated secretory phenotype biomarkers were elevated in serum from mice on a KD and in plasma samples from patients on a KD clinical trial. Cellular senescence was eliminated by a senolytic and prevented by an intermittent KD. These results have important clinical implications, suggesting that the effects of a KD are contextual and likely require individual optimization.

Development of cell-based high throughput luminescence assay for drug discovery in inhibiting OCT4/DNA-PKcs and OCT4-MK2 interactions.

Amplification-independent c-MYC overexpression is suggested in multiple cancers. Targeting c-MYC activity has therapeutic potential, but efforts thus far have been mostly unsuccessful. To find a druggable target to modulate c-MYC activity in cancer, we identified two kinases, MAPKAPK2 (MK2) and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which phosphorylate the Ser111 and the Ser93 residues of OCT4, respectively, to transcriptionally activate c-MYC. Using these observations, we present here a novel cell-based luminescence assay to identify compounds that inhibit the interaction between these kinases and OCT4. After screening approximately 80,000 compounds, we identified 56 compounds ("hits") that inhibited the luminescence reaction between DNA-PKcs and OCT4, and 65 hits inhibiting the MK2-OCT4 interaction. Using custom antibodies specific for pOCT4S93 and pOCT4S111 , the "hits" were validated for their effect on OCT4 phosphorylation and activation. Using a two-step method for validation, we identified two candidate compounds from the DNA-PKcs assay and three from the MK2 assay. All five compounds demonstrate a significant ability to kill cancer cells in the nanomolar range. In conclusion, we developed a cell-based luminescence assay to identify novel inhibitors targeting c-MYC transcriptional activation, and have found five compounds that may function as lead compounds for further development.

MYC transcription activation mediated by OCT4 as a mechanism of resistance to 13-cisRA-mediated differentiation in neuroblastoma.

Despite the improvement in clinical outcome with 13-cis-retinoic acid (13-cisRA) + anti-GD2 antibody + cytokine immunotherapy given in first response ~40% of high-risk neuroblastoma patients die of recurrent disease. MYCN genomic amplification is a biomarker of aggressive tumors in the childhood cancer neuroblastoma. MYCN expression is downregulated by 13-cisRA, a differentiating agent that is a component of neuroblastoma therapy. Although MYC amplification is rare in neuroblastoma at diagnosis, we report transcriptional activation of MYC medicated by the transcription factor OCT4, functionally replacing MYCN in 13-cisRA-resistant progressive disease neuroblastoma in large panels of patient-derived cell lines and xenograft models. We identified novel OCT4-binding sites in the MYC promoter/enhancer region that regulated MYC expression via phosphorylation by MAPKAPK2 (MK2). OCT4 phosphorylation at the S111 residue by MK2 was upstream of MYC transcriptional activation. Expression of OCT4, MK2, and c-MYC was higher in progressive disease relative to pre-therapy neuroblastomas and was associated with inferior patient survival. OCT4 or MK2 knockdown decreased c-MYC expression and restored the sensitivity to 13-cisRA. In conclusion, we demonstrated that high c-MYC expression independent of genomic amplification is associated with disease progression in neuroblastoma. MK2-mediated OCT4 transcriptional activation is a novel mechanism for activating the MYC oncogene in progressive disease neuroblastoma that provides a therapeutic target.

Índices de oxigenación como predictores de ventilación mecánica en neumonía a 2600 metros de altitud / Oxygen indexes as predictors of mechanical ventilation in pneumonia at 2600 meters above sea level

Resumen Antecedentes: los valores de la diferencia alveolo arterial de oxígeno D(A-a)O2 y de la relación presión alveolar de oxígeno y fracción inspirada de oxígeno (PaO2/FiO2), son pobremente conocidos a gran altitud para predecir ventilación mecánica (VM) en pacientes con neumonía adquirida en comunidad (NAC) mayores de 65 años. Objetivo: conocer los valores de D(A-a)O2 y PaO2/FiO2 en pacientes con NAC que requirieron soporte ventilatorio. Métodos: estudio de cohorte prospectivo donde se obtuvo la D(A-a)O2 y PaO2/FiO2 de los gases arteriales de ingreso a urgencias, con cálculo de sensibilidad (S), especificidad (E), valor predictivo positivo (VPP), valor predictivo negativo VPN) y área bajo la curva ROC para el requerimiento de VM en las primeras 72 horas. Resultados: se siguieron 247 pacientes, 37 (15%) requirieron VM, no se encontraron diferencias en edad, género, y comorbilidades entre los grupos de VM y no VM. El área bajo la curva ROC para D(A-a) O2 como predictor de VM fue de 0.84 (IC95%:0.77-0.92), para la PaO2/FiO2 de 0.85 (IC 5%: 0.78-0.92) (p<0.0001). Para una D(A-a)O2 en 55 se obtuvo una sensibilidad para predecir VM en 70.27%, especificidad 86.19%, VPP: 47%, VPN: 94%, razón de verosimilitud positiva (LR+): 5.1, razón de verosimilitud negativa (LR-): 0.3. Una PaO2/FiO2 de 180 tiene una sensibilidad para predecir VM de: 86.65%, especificidad: 70.27%, VPP: 34%, VPN: 97%, LR+: 2.9, LR-: 0.2. La mortalidad global fue 3.2%. Conclusión: los valores de D(A-a)O2 y PaO2/FiO2 se relacionan con el requerimiento de VM en pacientes mayores de 65 años con NAC. (Acta Med Colomb 2016; 41: 169-175).

Abstract Background: the values of the difference of alveolar arterial oxygen D(A-a)O2 and ratio of the alveolar oxygen pressure and fraction of inspired oxygen (PaO2/FiO2) are poorly known at high altitude to predict mechanical ventilation (MV) in patients over 65 years with community-acquired pneumonia (CAP). Objective: to know the values of D(A-a)O2 and PaO2/FiO2 in CAP patients requiring ventilatory support. Methods: prospective cohort study where D(A-a)O2 y PaO2/FiO2 were obtained from arterial blood gases at entrance to the emergency room, with calculation of sensitivity (S), specificity (E), positive predictive value (PPV), negative predictive value (NPP) and area under the ROC curve for MV requirement within the first 72 hours. Results: 247 patients were followed; 37 (15%) required MV. No differences were found in age, gender and comorbidities between the groups of MV and no MV. The area under the ROC curve for D(A-a) O2 as a predictor of MV was 0.84 (95% CI: 0.77 to 0.92), for the la PaO2/FiO2 of 0.85 (95% CI: 0.78 to 0.92) (p <0.0001). For a D(A-a)O2 in 55 patients was obtained a sensibility to predict MV in 70.27%, specificity 86.19%, PPV 47%, NPV 94%, positive likelihood ratio (LR +): 5.1, negative likelihood ratio (LR -): 0.3. A PaO2/FiO2 of 180 has a sensitivity to predict MV of 86.65%, specificity: 70.27%, PPV 34%, NPV 97%, LR +: 2.9, LR: 0.2. Overall mortality was 3.2%. Conclusion: the values of D(A-a)O2 and PaO2/FiO2 relate to the requirement of MV in patients older than 65 with CAP. (Acta Med Colomb 2016; 41: 169-175).

Plasmalemmal vacuolar H+-ATPase is decreased in microvascular endothelial cells from a diabetic model.

Angiogenesis requires invasion of extracellular matrix (ECM) proteins by endothelial cells and occurs in hypoxic and acidic environments that are not conducive for cell growth and survival. We hypothesize that angiogenic cells must exhibit a unique system to regulate their cytosolic pH in order to cope with these harsh conditions. The plasmalemmal vacuolar type H(+)-ATPase (pmV-ATPase) is used by cells exhibiting an invasive phenotype. Because angiogenesis is impaired in diabetes, we hypothesized that pmV-ATPase is decreased in microvascular endothelial cells from diabetic rats. The in vitro angiogenesis assays demonstrated that endothelial cells were unable to form capillary-like structures in diabetes. The proton fluxes were slower in cells from diabetic than normal model, regardless of the presence or absence of Na(+) and HCO(3) (-) and were suppressed by V-H(+)-ATPase inhibitors. Immunocytochemical data revealed that pmV-ATPases were inconspicuous at the plasma membrane of cells from diabetic whereas in normal cells were prominent. The pmV-ATPase activity was lower in cells from diabetic than normal models. Inhibition of V-H(+)-ATPase suppresses invasion/migration of normal cells, but have minor effects in cells from diabetic models. These novel observations suggest that the angiogenic abnormalities in diabetes involve a decrease in pmV-ATPase in microvascular endothelial cells.

Plasmalemmal V-H(+)-ATPases regulate intracellular pH in human lung microvascular endothelial cells.

The lung endothelium layer is exposed to continuous CO(2) transit which exposes the endothelium to a substantial acid load that could be detrimental to cell function. The Na(+)/H(+) exchanger and HCO(3)(-)-dependent H(+)-transporting mechanisms regulate intracellular pH (pH(cyt)) in most cells. Cells that cope with high acid loads might require additional primary energy-dependent mechanisms. V-H(+)-ATPases localized at the plasma membranes (pmV-ATPases) have emerged as a novel pH regulatory system. We hypothesized that human lung microvascular endothelial (HLMVE) cells use pmV-ATPases, in addition to Na(+)/H(+) exchanger and HCO(3)(-)-based H(+)-transporting mechanisms, to maintain pH(cyt) homeostasis. Immunocytochemical studies revealed V-H(+)-ATPase at the plasma membrane, in addition to the predicted distribution in vacuolar compartments. Acid-loaded HLMVE cells exhibited proton fluxes in the absence of Na(+) and HCO(3)(-) that were similar to those observed in the presence of either Na(+), or Na(+) and HCO(3)(-). The Na(+)- and HCO(3)(-)-independent pH(cyt) recovery was inhibited by bafilomycin A(1), a V-H(+)-ATPase inhibitor. These studies show a Na(+)- and HCO(3)(-)-independent pH(cyt) regulatory mechanism in HLMVE cells that is mediated by pmV-ATPases.

Vacuolar H+-ATPase in human breast cancer cells with distinct metastatic potential: distribution and functional activity.

Tumor cells thrive in a hypoxic microenvironment with an acidic extracellular pH. To survive in this harsh environment, tumor cells must exhibit a dynamic cytosolic pH regulatory system. We hypothesize that vacuolar H(+)-ATPases (V-ATPases) that normally reside in acidic organelles are also located at the cell surface, thus regulating cytosolic pH and exacerbating the migratory ability of metastatic cells. Immunocytochemical data revealed for the first time that V-ATPase is located at the plasma membrane of human breast cancer cells: prominent in the highly metastatic and inconspicuous in the lowly metastatic cells. The V-ATPase activities in isolated plasma membranes were greater in highly than in lowly metastatic cells. The proton fluxes via V-ATPase evaluated by fluorescence spectroscopy in living cells were greater in highly than in lowly metastatic cells. Interestingly, lowly metastatic cells preferentially used the ubiquitous Na(+)/H(+) exchanger and HCO(3)(-)-based H(+)-transporting mechanisms, whereas highly metastatic cells used plasma membrane V-ATPases. The highly metastatic cells were more invasive and migratory than the lowly metastatic cells. V-ATPase inhibitors decreased the invasion and migration in the highly metastatic cells. Altogether, these data indicate that V-ATPases located at the plasma membrane are involved in the acquisition of a more metastatic phenotype.