Nox4 mediates renal cell carcinoma cell invasion through hypoxia-induced interleukin 6- and 8- production

Background

Inflammatory cytokines are detected in the plasma of patients with renal cell carcinoma (RCC) and are associated with poor prognosis. However, the primary cell type involved in producing inflammatory cytokines and the biological significance in RCC remain unknown. Inflammation is associated with oxidative stress, upregulation of hypoxia inducible factor 1-alpha, and production of pro-inflammatory gene products. Solid tumors are often heterogeneous in oxygen tension together suggesting that hypoxia may play a role in inflammatory processes in RCC. Epithelial cells have been implicated in cytokine release, although the stimuli to release and molecular mechanisms by which they are released remain unclear. AMP-activated protein kinase (AMPK) is a highly conserved sensor of cellular energy status and a role for AMPK in the regulation of cell inflammatory processes has recently been demonstrated.

Methods and Principal Findings

We have identified for the first time that interleukin-6 and interleukin-8 (IL-6 and IL-8) are secreted solely from RCC cells exposed to hypoxia. Furthermore, we demonstrate that the NADPH oxidase isoform, Nox4, play a key role in hypoxia-induced IL-6 and IL-8 production in RCC. Finally, we have characterized that enhanced levels of IL-6 and IL-8 result in RCC cell invasion and that activation of AMPK reduces Nox4 expression, IL-6 and IL-8 production, and RCC cell invasion.

Conclusions/Significance

Together, our data identify novel mechanisms by which AMPK and Nox4 may be linked to inflammation-induced RCC metastasis and that pharmacological activation of AMPK and/or antioxidants targeting Nox4 may represent a relevant therapeutic intervention to reduce IL-6- and IL-8-induced inflammation and cell invasion in RCC.     

Separation and quantitation of azimilide and its putative metabolites by capillary electrophoresis

Reliable methods based on capillary electrophoresis (CE) have been developed for the separation and quantitation of azimilide, an antiarrhythmic drug under development at Procter & Gamble Pharmaceuticals (P&GP). Both capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) were employed in the separation of azimilide from its impurities, degradants and/or metabolites. Separation of azimilide from NE-11178, F-410, F-1054 and F-1292 was obtained by MECC at pH 9 with 50 mM sodium dodecyl sulfate (SDS). The separation of azimilide and NE-10171, a key metabolite of azimilide, was difficult because their structures differ by only a single methyl group. The best separation was achieved under acidic pH conditions with cetyltriethyl ammonium chloride (CTAC) additive in the buffer. All of the CE separations were completed within a substantially shorter time and with better resolution than the corresponding high-performance liquid chromatography (HPLC) separations. Quantitation was done with azimilide and NE-10171. Calibration curves ranging from 10 to 1000 μg/ml were obtained with R² greater than 0.997 for both azimilide and NE-10171. The back-calculated concentrations of the calibration standards and the recoveries of the quality control (QC) samples were within the acceptance range currently used for HPLC methods. These results demonstrated the viability of CE as an alternative technique for drug metabolism studies in support of pharmaceutical development.     

Comparing lipid remodeling of brown adipose tissue,white adipose tissue,and liver after one-week high fat diet intervention with quantitative Raman microscopy

Brown adipose tissue (BAT) consists of highly metabolically active adipocytes that catabolize nutrients to produce heat. Playing an active role in triacylglycerol (TAG) clearance, research has shown that dietary fatty acids can modulate the TAG chemistry deposition in BAT after weeks-long dietary intervention, similar to what has been shown in white adipose tissue (WAT). Our objective was to compare the influence of sustained, nonchronic dietary intervention (a 1-week interval) on WAT and interscapular BAT lipid metabolism and deposition in situ. We use quantitative, label-free chemical microscopy to show that 1 week of high fat diet (HFD) intervention results in dramatically larger lipid droplet (LD) growth in BAT (and liver) compared to LD growth in inguinal WAT (IWAT). Moreover, BAT showed lipid remodeling as increased unsaturated TAGs in LDs, resembling the dietary lipid composition, while WAT (and liver) did not show lipid remodeling on this time scale. Concurrently, expression of genes involved in lipid metabolism, particularly desaturases, was reduced in BAT and liver from HFD-fed mice after 1 week. Our data show that BAT lipid chemistry remodels exceptionally fast to dietary lipid intervention compared WAT, which further points towards a role in TAG clearance.     

Local Ca2+ influx through Ca2+ release-activated Ca2+ (CRAC) channels stimulates production of an intracellular messenger and an intercellular pro-inflammatory signal

Ca2+ entry through store-operated Ca2+ channels drives the production of the pro-inflammatory molecule leukotriene C4 (LTC4) from mast cells through a pathway involving Ca2+-dependent protein kinase C, mitogen-activated protein kinases ERK1/2, phospholipase A2, and 5-lipoxygenase. Here we examine whether local Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane stimulates this signaling pathway. Manipulating the amplitude and spatial extent of Ca2+ entry by altering chemical and electrical gradients for Ca2+ influx or changing the Ca2+ buffering of the cytoplasm all impacted on protein kinase C and ERK activation, generation of arachidonic acid and LTC4 secretion, with little change in the bulk cytoplasmic Ca2+ rise. Similar bulk cytoplasmic Ca2+ concentrations were achieved when CRAC channels were activated in 0.25 mm external Ca2+ versus 2 mm Ca2+ and 100 nm La3+, an inhibitor of CRAC channels. However, despite similar bulk cytoplasmic Ca2+, protein kinase C activation and LTC4 secretion were larger in 2 mm Ca2+ and La3+ than in 0.25 mm Ca2+, consistent with the central involvement of a subplasmalemmal Ca2+ rise. The nonreceptor tyrosine kinase Syk coupled CRAC channel opening to protein kinase C and ERK activation. Recombinant TRPC3 channels also activated protein kinase C, suggesting that subplasmalemmal Ca2+ rather than a microdomain exclusive to CRAC channels is the trigger. Hence a subplasmalemmal Ca2+ increase in mast cells is highly versatile in that it triggers cytoplasmic responses through generation of intracellular messengers as well as long distance changes through increased secretion of paracrine signals.     

Rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of clonidine in human plasma

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the estimation of clonidine in human plasma. Clonidine was extracted from human plasma by using solid-phase extraction technique. Nizatidine was used as the internal standard. A Hypurity C18 (50 mm x 4.6 mm i.d., 5 microm particle size) column provided chromatographic separation of analyte followed by detection with mass spectrometry. The method involves a rapid solid-phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables detection up to picogram levels with a total run time of 3.0 min only. The method was validated over the range of 50-2500 pg/mL. The absolute recoveries for clonidine (71.86%) and IS (69.44%) achieved from spiked plasma samples were consistent and reproducible.     

Root cause investigation of a viral contamination incident occurred during master cell bank (MCB) testing and characterization – A case study

An adventitious agent contamination occurred during a routine 9 CFR bovine viral screening test at BioReliance for an Eli Lilly Chinese Hamster Ovary (CHO) cell-derived Master Cell Bank (MCB) intended for biological production. Scientists from the sponsor (Eli Lilly and Company) and the testing service company (BioReliance) jointly conducted a systematic investigation in an attempt to determine the root cause of the contamination. Our investigation resulted in the identification of the viral nature of the contaminant. Subsequent experiments indicated that the viral contaminant was a non-enveloped and non-hemadsorbing virus. Transmission electron microscopy (TEM) revealed that the viral contaminant was 25–30 nm in size and morphologically resembled viruses of the family Picornaviridae. The contaminant virus was readily inactivated when exposed to acidic pH, suggesting that the viral contaminant was a member of rhinoviruses. Although incapable of infecting CHO cells, the viral contaminant replicated efficiently in Vero cell with a life cycle of 16 h. Our investigation provided compelling data demonstrating that the viral contaminant did not originate from the MCB. Instead, it was introduced into the process during cell passaging and a possible entry point was proposed. We identified the viral contaminant as an equine rhinitis A virus using molecular cloning and DNA sequencing. Finally, our investigation led us to conclude that the source of the viral contaminant was the equine serum added to the cell growth medium in the 9 CFR bovine virus test.     

A GRK5 polymorphism that inhibits beta-adrenergic receptor signaling is protective in heart failure

Beta-adrenergic receptor (betaAR) blockade is a standard therapy for cardiac failure and ischemia. G protein-coupled receptor kinases (GRKs) desensitize betaARs, suggesting that genetic GRK variants might modify outcomes in these syndromes. Re-sequencing of GRK2 and GRK5 revealed a nonsynonymous polymorphism of GRK5, common in African Americans, in which leucine is substituted for glutamine at position 41. GRK5-Leu41 uncoupled isoproterenol-stimulated responses more effectively than did GRK5-Gln41 in transfected cells and transgenic mice, and, like pharmacological betaAR blockade, GRK5-Leu41 protected against experimental catecholamine-induced cardiomyopathy. Human association studies showed a pharmacogenomic interaction between GRK5-Leu41 and beta-blocker treatment, in which the presence of the GRK5-Leu41 polymorphism was associated with decreased mortality in African Americans with heart failure or cardiac ischemia. In 375 prospectively followed African-American subjects with heart failure, GRK5-Leu41 protected against death or cardiac transplantation. Enhanced betaAR desensitization of excessive catecholamine signaling by GRK5-Leu41 provides a 'genetic beta-blockade' that improves survival in African Americans with heart failure, suggesting a reason for conflicting results of beta-blocker clinical trials in this population.     

Mitochondrial regulation of store-operated CRAC channels

In eukaryotic cells, one major route for Ca(2+) influx is through store-operated CRAC channels, which are activated following a fall in Ca(2+) content within the endoplasmic reticulum. Mitochondria are key regulators of this ubiquitous Ca(2+) influx pathway. Respiring mitochondria rapidly take up some of the Ca(2+) released from the stores, resulting in more extensive store depletion and thus robust activation of CRAC channels. As CRAC channels open, the ensuing rise in cytoplasmic Ca(2+) feeds back to inactivate the channels. By buffering some of the incoming Ca(2+) mitochondria reduce Ca(2+)-dependent inactivation of the CRAC channels, resulting in more prolonged Ca(2+) influx. However, mitochondria can release Ca(2+) close to the endoplasmic reticulum, accelerating store refilling and thus promoting deactivation of the CRAC channels. Mitochondria thus regulate all major transitions in CRAC channel gating, revealing remarkable versatility in how this organelle impacts upon Ca(2+) influx. Recent evidence suggests that mitochondria also control CRAC channels through mechanisms that are independent of their Ca(2+)-buffering actions and ability to generate ATP. Furthermore, pyruvic acid, a key intermediary metabolite and precursor substrate for the Krebs cycle, reduces the extent of Ca(2+)-dependent inactivation of CRAC channels. Hence mitochondrial metabolism impacts upon Ca(2+) influx through CRAC channels and thus on a range of key downstream cellular responses.