Cancer cells can undergo a metabolic reprogramming from oxidative phosphorylation to glycolysis that allows them to adapt to nutrient-poor microenvironments, thereby imposing a selection for aggressive variants. However, the mechanisms underlying this reprogramming are not fully understood. Using complementary approaches in validated cell lines and freshly obtained human specimens, we report here that mitochondrial respiration and oxidative phosphorylation are slowed in metastatic melanomas, even under normoxic conditions due to the persistence of a high nuclear expression of hypoxia-inducible factor-1a (HIF-1a). Pharmacologic or genetic blockades of the HIF-1a pathway decreased glycolysis and promoted mitochondrial respiration via specific reduction in the expression of pyruvate dehydrogenase kinase-3 (PDK3). Inhibiting PDK3 activity by dichloroacetate (DCA) or siRNA-mediated attenuation was sufficient to increase pyruvate dehydrogenase activity, oxidative phosphorylation, and mitochondrial reactive oxygen species generation. Notably, DCA potentiated the antitumor effects of elesclomol, a pro-oxidative drug currently in clinical development, both by limiting cell proliferation and promoting cell death. Interestingly, this combination was also effective against BRAF V600E-mutant melanoma cells that were resistant to the BRAF inhibitor vemurafenib. Cotreatment of melanomas with DCA and elesclomol in vivo achieved a more durable response than single agent alone. Our findings offer a preclinical validation of the HIF-1/PDK3 bioenergetic pathway as a new target for therapeutic intervention in metastatic melanoma, opening the door to innovative combinations that might eradicate this disease. Cancer Res; 72(19); 5035-47. Ó2012 AACR.

A low dose (0.5 mg/kg) of lipopolysaccharide (LPS), administered 72 hours before 60-minute middle cerebral artery occlusion, induced a delayed neuroprotection proven by the significant decrease (-35%) of brain infarct volume in comparison with control, whereas infarct volumes remained unchanged in rats treated 12, 24, or 168 hours before ischemia. This delayed neuroprotective effect of LPS was induced only with low doses (0.25 to 1 mg/kg), whereas this effect disappeared with a higher dose (2 mg/kg). The delayed neuroprotection of LPS was induced in the cortical part of the infarcted zone, not in the subcortical part. The beneficial effect of LPS on consequences of middle cerebral artery occlusion was suppressed by dexamethasone (3 mg/kg) and indomethacin (3 mg/ kg) administered 1 hour before LPS, whereas both drugs had no direct effect on infarct volume by themselves, suggesting that activation of inflammatory pathway is involved in the development of LPS-induced brain ischemic tolerance. Preadministration of cycloheximide, an inhibitor of protein synthesis, also blocked LPS-induced brain ischemic tolerance suggesting that a protein synthesis is also necessary as a mediating mechanism. Superoxide dismutase (SOD) could be one of the synthesized proteins because lipopolysaccharide increased SOD brain activity 72 hours, but not 12 hours, after its administration, which paralleled the development of brain ischemic tolerance. In contrast, catalase brain activity remained unchanged after LPS administration. The LPS-induced delayed increase in SOD brain content was suppressed by a previous administration of indomethacin. These data suggest that the delayed neuroprotective effect of low doses of LPS is mediated by an increased synthesis of brain SOD that could be triggered by activation of inflammatory pathway.

Targeted therapies as BRAF and MEK inhibitor combination have been approved as first-line treatment for BRAF-mutant melanoma. However, disease progression occurs in most of the patients within few months of therapy. Metabolic adaptations have been described in the context of acquired resistance to BRAF inhibitors (BRAFi). BRAFi-resistant melanomas are characterized by an increase of mitochondrial oxidative phosphorylation and are more prone to cell death induced by mitochondrial-targeting drugs. BRAFi-resistant melanomas also exhibit an enhancement of oxidative stress due to mitochondrial oxygen consumption increase. To understand the mechanisms responsible for survival of BRAFi-resistant melanoma cells in the context of oxidative stress, we have established a preclinical murine model that accurately recapitulates in vivo the acquisition of resistance to MAPK inhibitors including several BRAF or MEK inhibitors alone and in combination. Using mice model and melanoma cell lines generated from mice tumors, we have confirmed that the acquisition of resistance is associated with an increase in mitochondrial oxidative phosphorylation as well as the importance of glutamine metabolism. Moreover, we have demonstrated that BRAFi-resistant melanoma can adapt mitochondrial metabolism to support glucose-derived glutamate synthesis leading to increase in glutathione content. Besides, BRAFi-resistant melanoma exhibits a strong activation of NRF-2 pathway leading to increase in the pentose phosphate pathway, which is involved in the regeneration of reduced glutathione, and to increase in xCT expression, a component of the xc—amino acid transporter essential for the uptake of cystine required for intracellular glutathione synthesis. All these metabolic modifications sustain glutathione level and contribute to the intracellular redox balance to allow survival of BRAFi-resistant melanoma cells.

Renal proximal tubular epithelial cells play a central role in renal physiology and are among the cell types most sensitive to ischemia and xenobiotic nephrotoxicity. In order to investigate the molecular and cellular mechanisms underlying the pathophysiology of kidney injuries, a stable and well-characterized primary culture model of proximal tubular cells is required. An existing model of proximal tubular cells is hampered by the cellular heterogeneity of kidney; a method based on cell sorting for specific markers must therefore be developed. In this study, we present a primary culture model based on the mechanical and enzymatic dissociation of healthy tissue obtained from nephrectomy specimens. Renal epithelial cells were sorted using co-labeling for CD10 and CD13, two renal proximal tubular epithelial markers, by flow cytometry. Their purity, phenotypic stability and functional properties were evaluated over several passages. Our results demonstrate that CD10/CD13 double-positive cells constitute a pure, functional and stable proximal tubular epithelial cell population that displays proximal tubule markers and epithelial characteristics over the long term, whereas cells positive for either CD10 or CD13 alone appear to be heterogeneous. In conclusion, this study describes a method for establishing a robust renal proximal tubular epithelial cell model suitable for further experimentation.

Challenges today concern chronic myeloid leukemia (CML) patients resistant to imatinib. There is growing evidence that imatinib-resistant leukemic cells present abnormal glucose metabolism but the impact on mitochondria has been neglected. Our work aimed to better understand and exploit the metabolic alterations of imatinib-resistant leukemic cells. Imatinib-resistant cells presented high glycolysis as compared to sensitive cells. Consistently, expression of key glycolytic enzymes, at least partly mediated by HIF-1α, was modified in imatinib-resistant cells suggesting that imatinib-resistant cells uncouple glycolytic flux from pyruvate oxidation. Interestingly, mitochondria of imatinib-resistant cells exhibited accumulation of TCA cycle intermediates, increased NADH and low oxygen consumption. These mitochondrial alterations due to the partial failure of ETC were further confirmed in leukemic cells isolated from some imatinib-resistant CML patients. As a consequence, mitochondria generated more ROS than those of imatinib-sensitive cells. This, in turn, resulted in increased death of imatinib-resistant leukemic cells following in vitro or in vivo treatment with the pro-oxidants, PEITC and Trisenox, in a syngeneic mouse tumor model. Conversely, inhibition of glycolysis caused derepression of respiration leading to lower cellular ROS. In conclusion, these findings indicate that imatinib-resistant leukemic cells have an unexpected mitochondrial dysfunction that could be exploited for selective therapeutic intervention.

ObjectivesAmylase concentration in respiratory secretions was reported to be a potentially useful marker for aspiration and pneumonia. The aim of this study was to determine accuracy of α-amylase in diagnosing microaspiration in critically ill patients.MethodsRetrospective analysis of prospectively collected data collected in a medical ICU. All patients requiring mechanical ventilation for at least 48 h, and included in a previous randomized controlled trial were eligible for this study, provided that at least one tracheal aspirate was available for α-amylase measurement. As part of the initial trial, pepsin was quantitatively measured in all tracheal aspirates during a 48-h period. All tracheal aspirates were frozen, allowing subsequent measurement of α-amylase for the purpose of the current study. Microaspiration was defined as the presence of at least one positive tracheal aspirate for pepsin (>200 ng.mL−1). Abundant microaspiration was defined as the presence of pepsin at significant level in >74% of tracheal aspirates.ResultsAmylase was measured in 1055 tracheal aspirates, collected from 109 patients. Using mean α-amylase level per patient, accuracy of α-amylase in diagnosing microaspiration was moderate (area under the receiver operator curve 0.72±0.05 [95%CI 0.61–0.83], for an α-amylase value of 1685 UI.L−1). However, when α-amylase levels, coming from all samples, were taken into account, area under the receiver operator curve was 0.56±0.05 [0.53–0.60]. Mean α-amylase level, and percentage of tracheal aspirates positive for α-amylase were significantly higher in patients with microaspiration, and in patients with abundant microaspiration compared with those with no microaspiration; and similar in patients with microaspiration compared with those with abundant microaspiration. α-amylase and pepsin were significantly correlated (r2 = 0.305, p = 0.001).ConclusionAccuracy of mean α-amylase in diagnosing microaspiration is moderate. Further, when all α-amylase levels were taken into account, α-amylase was inaccurate in diagnosing microaspiration, compared with pepsin.

No abstract

IntroductionBased on the potential interest in sodium lactate as an energy substrate and resuscitative fluid, we investigated the effects of hypertonic sodium lactate in a porcine endotoxic shock.MethodsFifteen anesthetized, mechanically ventilated pigs were challenged with intravenous infusion of E. coli endotoxin. Three groups of five animals were randomly assigned to receive 5 mL/kg/h of different fluids: a treatment group received hypertonic sodium lactate 11.2% (HSL group); an isotonic control group receiving 0.9% NaCl (NC group); a hypertonic control group with the same amount of osmoles and sodium than HSL group receiving hypertonic sodium bicarbonate 8.4% (HSB group). Hemodynamic and oxygenation variables, urine output and fluid balance were measured at baseline and at 30, 60, 120, 210 and 300 min. Skin microvascular blood flow at rest and during reactive hyperemia was obtained using a laser Doppler flowmetry technique. Results were given as median with interquartile ranges.ResultsEndotoxin infusion resulted in hypodynamic shock. At 300 min, hemodynamics and oxygenation were significantly enhanced in HSL group: mean arterial pressure (103 [81–120] mmHg vs. 49 [41–62] in NC group vs. 71 [60–78] in HSB group), cardiac index (1.6 [1.2–1.8] L/min/m2 vs. 0.9 [0.5–1.1] in NC group vs. 1.3 [0.9–1.6] in HSB group) and partial pressure of oxygen (366 [308–392] mmHg vs. 166 [130–206] in NC group vs. 277 [189–303] in HSB group). At the same time, microvascular reactivity was significantly better in HSL group with a lower venoarterial CO2 tension difference (5.5 [4–10] mmHg vs. 17 [14–25] in NC group vs. 14 [12–15] in HSB group). The cumulative fluid balance was lower in HSL group (-325 [-655; -150] mL) compared to NC (+560 [+230; +900] mL, p = 0.008) and HSB (+185 [-110; +645] mL, p = 0.03) groups.ConclusionsIn our hypodynamic model of endotoxic shock, infusion of hypertonic sodium lactate improves hemodynamic and microvascular reactivity with a negative fluid balance and a better oxygenation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13054-014-0467-3) contains supplementary material, which is available to authorized users.