Ultrahigh resolution MS1/MS2-based reconstruction of metabolic networks in mammalian cells reveals changes for selenite and arsenite action

Metabolic networks are complex, intersecting, and composed of numerous enzyme-catalyzed biochemical reactions that transfer various molecular moieties among metabolites. Thus, robust reconstruction of metabolic networks requires metabolite moieties to be tracked, which cannot be readily achieved with mass spectrometry (MS) alone. We previously developed an Ion Chromatography-ultrahigh resolution-MS¹/data independent-MS² method to track the simultaneous incorporation of the heavy isotopes ¹³C and ¹⁵N into the moieties of purine/pyrimidine nucleotides in mammalian cells. Ultrahigh resolution-MS¹ resolves and counts multiple tracer atoms in intact metabolites, while data independent-tandem MS (MS²) determines isotopic enrichment in their moieties without concern for the numerous mass isotopologue source ions to be fragmented. Together, they enabled rigorous MS-based reconstruction of metabolic networks at specific enzyme levels. We have expanded this approach to trace the labeled atom fate of [¹³C₆]-glucose in 3D A549 spheroids in response to the anticancer agent selenite and that of [¹³C₅,¹⁵N₂]-glutamine in 2D BEAS-2B cells in response to arsenite transformation. We deduced altered activities of specific enzymes in the Krebs cycle, pentose phosphate pathway, gluconeogenesis, and UDP-GlcNAc synthesis pathways elicited by the stressors. These metabolic details help elucidate the resistance mechanism of 3D versus 2D A549 cultures to selenite and metabolic reprogramming that can mediate the transformation of BEAS-2B cells by arsenite.     

Inhibition of Anaplerotic Glutaminolysis Underlies Selenite Toxicity in Human Lung Cancer

Large clinical trials and model systems studies suggest that the chemical form of selenium dictates chemopreventive and chemotherapeutic efficacy. Selenite induces excess ROS production, which mediates autophagy and eventual cell death in non‐small cell lung cancer adenocarcinoma A549 cells. As the mechanisms underlying these phenotypic effects are unclear, the clinical relevance of selenite for cancer therapy remains to be determined. The authors' previous stable isotope‐resolved metabolomics and gene expression analysis showed that selenite disrupts glycolysis, the Krebs cycle, and polyamine metabolism in A549 cells, potentially through perturbed glutaminolysis, a vital anaplerotic process for proliferation of many cancer cells. Herein, the role of the glutaminolytic enzyme glutaminase 1 (GLS1) in selenite's toxicity in A549 cells and in patient‐derived lung cancer tissues is investigated. Using [¹³C₆]‐glucose and [¹³C₅,¹⁵N₂]‐glutamine tracers, selenite's action on metabolic networks is determined. Selenite inhibits glutaminolysis and glutathione synthesis by suppressing GLS1 expression, and blocks the Krebs cycle, but transiently activates pyruvate carboxylase activity. Glutamate supplementation partially rescues these anti‐proliferative and oxidative stress activities. Similar metabolic perturbations and necrosis are observed in selenite‐treated human patients' cancerous lung tissues ex vivo. The results support the hypothesis that GLS1 suppression mediates part of the anti‐cancer activity of selenite both in vitro and ex vivo.     

The anti-cancer effect of amygdalin on human cancer cell lines

Derived from rosaceous plant seed, amygdalin belongs to aromatic cyanogenic glycoside group, and its anticancer effects have been supported by mounting evidence. In this study, we objected to investigate amygdalin effect on two antiapoptotic genes (Survivin, XIAP) and two lncRNAs (GAS5, MALAT1) in human cancer cells (A549, MCF7, AGS). Employing RT-qPCR analysis, we compared the mRNA levels of the genes related to apoptosis in A549, MCF7, and AGS cancer cells between amygdalin-treated (24, 48 and 72 h) and un-treated groups. RNA was extracted from both cell groups and then cDNAs were synthesized. The changes in the gene expression levels were specified using ΔΔCt method. RT-qPCR analysis has revealed that the expression of Survivin, XIAP, GAS5 and MALAT1 in amygdala-treated cancer cells were significantly different, compared to the un-treated cells. However, these expressions were different depending on the treatment time. According to the results, amygdalin significantly inhibited the expression level of Survivin, and XIAP genes in treated via untreated group. Our findings suggest that amygdalin might have an anticancer effect due to the various gene expressions in A549, MCF7, and AGS human cancer cells, showing it’s potential as a natural therapeutic anticancer drug.

     

Markers of squamous cell carcinoma in sarco/endoplasmic reticulum Ca ATPase 2 heterozygote mice keratinocytes

A mutation of Atp2a2 gene encoding the sarco/endoplasmic reticulum Ca²⁺-ATPase 2 (SERCA2) causes Darier's disease in human and null mutation in one copy of Atp2a2 leads to a high incidence of squamous cell tumor in a mouse model. In SERCA2 heterozygote (SERCA2^+/−) mice keratinocytes, mechanisms involved in partial depletion of SERCA2 gene and its related tumor induction have not been studied. In this study, we investigated Ca²⁺ signaling and differential gene expression in primary cultured keratinocytes from SERCA2^+/− mice. SERCA2^+/− keratinocytes showed reduced initial increases in intracellular concentration of calcium in response to ATP, a G-protein coupled receptor agonist, and higher store-operated Ca²⁺ entry with the treatment of thapsigargin, an inhibitor of SERCA, compared to wild type kerationcytes. Protein expressions of plasma membrane Ca²⁺ ATPases, NFATc1, phosphorylated ERK, JNK, and phospholipase γ1 were increased in SERCA2^+/− keratinocytes. Using the gene fishing system, we first found in SERCA2^+/− keratinocytes that gene level of tumor-associated calcium signal transducer 1, crystalline αB, procollagen XVIII α1, and nuclear factor I-B were increased. Expression of involucrin, a marker of keratinocyte differentiation, was decreased in SERCA2^+/− keratinocytes. These results suggest that the alterations of Ca²⁺ signaling by SERCA2 haploinsufficiency alternate the gene expression of tumor induction and differentiation in keratinocytes.     

Long-term exposure of the freshwater shrimp Macrobrachium olfersii to elevated salinity: Effects on gill (Na,K)-ATPase α-subunit expression and K-phosphatase activity

The kinetic properties of a microsomal gill (Na⁺,K⁺)-ATPase from the freshwater shrimp, Macrobrachium olfersii, acclimated to 21‰ salinity for 10 days were investigated using the substrate p-nitrophenylphosphate. The enzyme hydrolyzed this substrate obeying cooperative kinetics at a rate of 123.6 ± 4.9 U mg^− 1 and K_0.5 = 1.31 ± 0.05 mmol L^− 1. Stimulation of K⁺-phosphatase activity by magnesium (V_max = 125.3 ± 7.5 U mg^− 1; K_0.5 = 2.09 ± 0.06 mmol L^− 1), potassium (V_max = 134.2 ± 6.7 U mg^− 1; K_0.5 = 1.33 ± 0.06 mmol L^− 1) and ammonium ions (V_max = 130.1 ± 5.9 U mg^− 1; K_0.5 = 11.4 ± 0.5 mmol L^− 1) was also cooperative. While orthovanadate abolished p-nitrophenylphosphatase activity, ouabain inhibition reached 80% (K_I = 304.9 ± 18.3 μmol L^− 1). The kinetic parameters estimated differ significantly from those for freshwater-acclimated shrimps, suggesting expression of different isoenzymes during salinity adaptation. Despite the ≈2-fold reduction in K⁺-phosphatase specific activity, Western blotting analysis revealed similar α-subunit expression in gill tissue from shrimps acclimated to 21‰ salinity or fresh water, although expression of phosphate-hydrolyzing enzymes other than (Na⁺,K⁺)-ATPase was stimulated by high salinity acclimation.     

Leishmania amazonensis META2 protein confers protection against heat shock and oxidative stress

The META cluster of Leishmania amazonensis contains both META1 and META2 genes, which are upregulated in metacyclic promastigotes and encode proteins containing the META domain. Previous studies defined META2 as a 48.0-kDa protein, which is conserved in other Leishmania species and in Trypanosoma brucei. In this work, we demonstrate that META2 protein expression is regulated during the Leishmania life cycle but constitutive in T. brucei. META2 protein is present in the cytoplasm and flagellum of L. amazonensis promastigotes. Leishmania META2-null replacement mutants are more sensitive to oxidative stress and, upon heat shock, assume rounded morphology with shortened flagella. The increased susceptibility of null parasites to heat shock is reversed by extra-chromosomal expression of the META2 gene. Defective Leishmania promastigotes exhibit decreased ability to survive in macrophages. By contrast, META2 expression is decreased by 80% in RNAi-induced T. brucei bloodstream forms with no measurable effect on survival or resistance to heat shock.     

Fluorine-18 labeled galactosyl-neoglycoalbumin for imaging the hepatic asialoglycoprotein receptor

Asialoglycoprotein receptors (ASGP-R) are well known to exist on the mammalian liver, situate on the surface of hepatocyte membrane. Quantitative imaging of asialoglycoprotein receptors could estimate the function of the liver. ^99mTc labeled galactosyl-neoglycoalbumin (NGA) and diethylenetriaminepentaacetic acid galactosyl human serum albumin (GSA) have been developed for SPECT imaging and clinical used in Japan. In this study, we labeled the NGA with ¹⁸F to get a novel PET tracer [¹⁸F]FNGA and evaluated its hepatic-targeting efficacy and pharmacokinetics. Methods: NGA was labeled with ¹⁸F by conjugation with N-succinimidyl-4-¹⁸F-fluorobenzoate ([¹⁸F]SFB) under a slightly basic condition. The in vivo metabolic stability of [¹⁸F]FNGA was determined. Ex vivo biodistribution of [¹⁸F]FNGA and blocking experiment was investigated in normal mice. MicroPET images were acquired in rat with and without block at 5 min and 15 min after injection of the radiotracer (3.7 MBq/rat), respectively. Results: Starting with ¹⁸F⁻ Kryptofix 2.2.2./K₂CO₃ solution, the total reaction time for [¹⁸F]FNGA is about 150 min. Typical decay-corrected radiochemical yield is about 8–10%. After rapid purified with HiTrap desalting column, the radiochemical purity of [¹⁸F]FNGA was more than 99% determined by radio-HPLC. [¹⁸F]FNGA was metabolized to produce [¹⁸F]FB-Lys in urine at 30 min. Ex vivo biodistribution in mice showed that the liver accumulated 79.18 ± 7.17% and 13.85 ± 3.10% of the injected dose per gram at 5 and 30 min after injection, respectively. In addition, the hepatic uptake of [¹⁸F]FNGA was blocked by pre-injecting free NGA as blocking agent (18.55 ± 2.63%ID/g at 5 min pi), indicating the specific binding to ASGP receptor. MicroPET study obtained quality images of rat at 5 and 15 min post-injection. Conclusion: The novel ASGP receptor tracer [¹⁸F]FNGA was synthesized with high radiochemical yield. The promising biological properties of [¹⁸F]FNGA afford potential applications for assessment of hepatocyte function in the future. It may provide quantitative information and better resolution which particularly help to the liver surgery.     

Metabolic phenotyping of genetically modified mice: An NMR metabonomic approach

Monocyte chemoattractant protein-1 (MCP-1) plays a relevant role in macrophage migration but recent findings suggest an additional role in lipid and glucose metabolism. We report the use of ¹H NMR spectroscopy as a useful complementary method to assess the metabolic function of this gene in a comparative strategy. This metabonomic analysis was rapid, simple, quantitative and reproducible, and revealed a suggestive relationship between the expression of the MCP-1 gene and hepatic glucose and taurine concentrations. This approach should be considered in genetically modified mice when a metabolic alteration is suspected, or in routine assessment of metabolic phenotype.     

Contribution of organic anion transporting polypeptide OATP2B1 to amiodarone accumulation in lung epithelial cells

The accumulation mechanisms of amiodarone (AMD) involving transporters in lung alveolar epithelial type II cells were studied. The uptake of AMD was examined using human alveolar epithelial-derived cell line A549 as a model. AMD was transported by the carrier-mediated system, and the apparent K_m and V_max values were 66.8 ± 30.3 μM and 49.7 ± 9.7 nmol/mg protein/5 min, respectively. The uptake of AMD by A549 cells was Na⁺-independent and was inhibited by substrates of human organic anion transporting polypeptide (OATP). The inhibition profiles were similar to the inhibitory effects of several compounds on OATP2B1-mediated E-3-S transport, and RT-PCR analysis showed mRNA expression of OATP2B1 and 1B3 in A549 cells. SiRNAs targeted to the OATP2B1 gene decreased the OATP2B1 mRNA expression level in A549 cells up to about 50% and reduced the uptake of AMD up to about 40%. These results indicate that AMD uptake mediated by carriers, including OATP2B1, might lead to accumulation of AMD in the lung and AMD-induced pulmonary toxicity (AIPT).     

Effects of selenium on altered mechanical and electrical cardiac activities of diabetic rat

Since selenium compounds can restore some metabolic parameters and structural alterations of diabetic rat heart, we were tempted to investigate whether these beneficial effects extend to the diabetic rat cardiac dysfunctions. Diabetes was induced by streptozotocin (50mg/kg body weight) and rats were then treated with sodium selenite (5 micromol/kg body weight/day) for four weeks. Electrically stimulated isometric contraction and intracellular action potential in isolated papillary muscle strips and transient (I(to)) and steady state (I(ss)) outward K(+) currents in isolated cardiomyocytes were recorded. Sodium selenite treatment could reverse the prolongation in both action potential duration and twitch duration of the diabetic rats, and also cause significant increases in the diminished amplitudes of the two K(+) currents. Treatment of rats with sodium selenite also markedly increased the depressed acid-soluble sulfhydryl levels of the hearts. Our data suggest that the beneficial effects of sodium selenite treatment on the mechanical and electrical activities of the diabetic rat heart appear to be due to the restoration of the diminished K(+) currents, partially, related to the restoration of the cell glutathione redox cycle.     

Ouabain activates signaling pathways associated with cell death in human neuroblastoma

Cardiotonic steroids (CTS) like ouabain are not only specific inhibitors of the sodium pump (Na⁺,K⁺-ATPase), they also can influence various cytosolic signaling events in a hormone-like manner. In the neuroblastoma cell line SH-SY5Y ouabain triggers multiple signaling pathways. Within 30 min of incubation with 1 or 10 μM ouabain, SH-SY5Y cells generate reactive oxygen species to a level approximately 50% above control and show a modest but significant elevation in cytosolic [Ca²⁺] of about 25%. After 6 h of exposure, ouabain stimulates a series of anti-apoptotic actions in SH-SY5Y cells, including concentration-dependent phosphorylation of Erk1/2, Akt, and Bad. Nevertheless, at the same time this CTS also induces a series of events that inhibit retinoic acid-induced neuritogenesis and promote cell death. Both of these latter phenomena are possibly associated with the observed ouabain-induced reduction in the abundance of the anti-apoptotic proteins Bcl-XL and Bcl-2. In addition, ouabain treatment results in cytochrome c release into the cytosol and induces activation of caspase 3, events that point towards the stimulation of apoptotic pathways that are probably enhanced by the stimulation of p53 phosphorylation at Ser15 also observed in this study. These pathways may eventually lead to cell death: treatment with 10 nM ouabain results in a 20% decrease in cell number after 4 days of incubation and treatment with 1 μM ouabain decreases cells number by about 75%. The results obtained here emphasize the importance of further research in order to elucidate the various signalling cascades triggered by ouabain and possibly other CTS that are used in the treatment of heart failure and to identify their primary receptor(s).     

High-level expression of the HIV-1 Pr55gag polyprotein in transgenic tobacco chloroplasts

Plants have been recognized as a promising production platform for recombinant pharmaceutical proteins. The human immunodeficiency virus Gag (Pr55^gag) structural polyprotein precursor is a prime candidate for developing a HIV-1 vaccine, but, so far, has been expressed at very low level in plants. The aim of this study was to investigate factors potentially involved in Pr55^gag expression and increase protein yield in plant cells. In transient expression experiments in various subcellular compartments, the native Pr55^gag sequence could be expressed only in the chloroplast. Experiments with truncated subunits suggested a negative role of the 5′-end on the expression of the full gene in the cytosol. Stable transgenic plants were produced in tobacco by Agrobacterium-mediated nuclear transformation with protein targeted to plastids, and biolistic-mediated plastid transformation. Compared to the nuclear genome, the integration and expression of the gag transgene in the plastome resulted in significantly higher protein accumulation levels (up to 7–8% TSP, equivalent to 312–363 mg/kg FW). In transplastomic plants, a 25-fold higher protein accumulation was obtained by translationally fusing the Pr55^gag polyprotein to the N-terminus of the plastid photosynthetic RbcL protein. In chloroplasts, the Pr55^gag polyprotein was processed in a pattern similar to that achieved by the viral protease, the processing being more extended in older leaves of mature plants. The Gag proteins produced in transgenic plastids were able to assemble into particles resembling VLPs produced in baculovirus/insect cells and E. coli systems. These results indicate that plastid transformation is a promising tool for HIV antigen manufacturing in plant cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. IGV publication no. 330