Very long chain fatty acid metabolism is required in acute myeloid leukemia

Acute myeloid leukemia (AML) cells have an atypical metabolic phenotype characterized by increased mitochondrial mass, as well as a greater reliance on oxidative phosphorylation and fatty acid oxidation (FAO) for survival. To exploit this altered metabolism, we assessed publicly available databases to identify FAO enzyme overexpression. Very long chain acyl-CoA dehydrogenase (VLCAD; ACADVL) was found to be overexpressed and critical to leukemia cell mitochondrial metabolism. Genetic attenuation or pharmacological inhibition of VLCAD hindered mitochondrial respiration and FAO contribution to the tricarboxylic acid cycle, resulting in decreased viability, proliferation, clonogenic growth, and AML cell engraftment. Suppression of FAO at VLCAD triggered an increase in pyruvate dehydrogenase activity that was insufficient to increase glycolysis but resulted in adenosine triphosphate depletion and AML cell death, with no effect on normal hematopoietic cells. Together, these results demonstrate the importance of VLCAD in AML cell biology and highlight a novel metabolic vulnerability for this devastating disease

Thymoquinone from nutraceutical black cumin oil activates Neu4 sialidase in live macrophage, dendritic, and normal and type I sialidosis human fibroblast cells via GPCR G alpha i proteins and matrix metalloproteinase-9

Anti-inflammatory activities of thymoquinone (TQ) have been demonstrated in in vitro and in vivo studies. However, the precise mechanism(s) of TQ in these anti-inflammatory activities is not well understood. Using a newly developed assay to detect sialidase activity in live macrophage cells (Glycoconj J doi: 10.1007/s10719-009-9239-8), here we show that TQ has no inhibitory effect on endotoxin lipopolysaccharide (LPS) induced sialidase activity in live BMC-2 macrophage cells. In contrast, the parent black seed oil (BSO) and another constituent of BSO para-cymene (p-CY) completely block LPS induced sialidase activity. All of these compounds had no effect on cell viability. On the other hand, TQ induces a vigorous sialidase activity in live BMC-2 macrophage cells in a dose dependent manner as well in live DC-2.4 dendritic cells, HEK-TLR4/MD2, HEK293, SP1 mammary adenocarcinoma cells, human WT and 1140F01 and WG0544 type I sialidosis fibroblast cells. Tamiflu (oseltamivir phosphate) inhibits TQ-induced sialidase activity in live BMC-2 cells with an IC50 of 0.0194 mu M compared to an IC50 of 19.1 mu M for neuraminidase inhibitor DANA (2-deoxy-2,3-dehydro-N-acetylneuraminic acid). Anti-Neu1, -2 and -3 antibodies have no inhibition of TQ-induced sialidase activity in live BMC-2 and human THP-1 macrophage cells but anti-Neu4 antibodies completely block this activity. There is a vigorous sialidase activity associated with TQ treated live primary bone marrow (BM) macrophage cells derived from WT and hypomorphic cathepsin A mice with a secondary Neu1 deficiency (NeuI KD), but not from Neu4 knockout (Neu4 KO) mice. Pertussis toxin (PTX), a specific inhibitor of G alpha i proteins of G-protein coupled receptor (GPCR) and the broad range inhibitors of matrix metalloproteinase (MMP) galardin and piperazine applied to live BMC-2, THP-1 and primary BM macrophage cells completely block TQ-induced sialidase activity. These same inhibitory effects are not observed with the GM1 ganglioside specific cholera toxin subunit B (CTXB) as well as with CTX, tyrosine kinase inhibitor K252a, and the broad range GPCR inhibitor suramin. The specific inhibitor of MMP-9, anti-MMP-9 antibody and anti-Neu4 antibody, but not the specific inhibitor of MMP-3 completely block TQ-induced sialidase activity in live THP-1 cells, which express Neu4 and MMP-9 on the cell surface. Neu4 sialidase activity in cell lysates from TQ-treated live THP-1 cells desialylates natural gangliosides and mucin substrates. RT-PCR and western blot analyses reveal no correlation between mRNA and protein values for Neu3 and Neu4 in human monocytic THP-1 cells, suggesting for the first time a varied post-transcriptional mechanism for these two mammalian sialidases independent of TQ activation. Our findings establish an unprecedented activation of Neu4 sialidase on the cell surface by thymoquinone, which is derived from the nutraceutical black cumin oil. The potentiation of GPCR-signaling by TQ via membrane targeting of G alpha i subunit proteins and matrix metalloproteinase-9 activation may be involved in the activation process of Neu4 sialidase on the cell surface

Thymoquinone-induced Neu4 sialidase activates NFκB in macrophage cells and pro-inflammatory cytokines in vivo

Thymoquinone (TQ) derived from the nutraceutical black cumin oil has been reported to be a novel agonist of Neu4 sialidase activity in live cells (Glycoconj J DOI 10.1007/s10719-010-9281-6). The activation of Neu4 sialidase on the cell surface by TQ was found to involve GPCR-signaling via membrane targeting of G alpha i subunit proteins and matrix metalloproteinase-9 activation. Contrary to other reports, TQ had no anti-inflammatory effects in vitro. Here, we show that MyD88/TLR4 complex formation and subsequent NF kappa B activation are induced by the Neu4 activity associated with TQ-stimulated live primary bone marrow (BM) macrophage cells from WT and Neu1-deficient mice, HEK-TLR4/MD2 cells and BMC-2 macrophage cell line but not with primary macrophage cells from Neu4-knockout mice. Tamiflu (oseltamivir phosphate), pertussis toxin (PTX), a specific inhibitor of G alpha i proteins of G-protein coupled receptor (GPCR) and the broad range inhibitor of matrix metalloproteinase (MMP) galardin applied to live primary BM macrophage cells completely block TQ-induced MyD88/TLR4 complex formation. Using immunocytochemistry and western blot analyses, Tamiflu, galardin and PTX inhibit NF kappa B activation induced by Neu4 activity associated with TQ-stimulated BMC-2 cells, HEK-TLR4/MD2 cells and primary BM macrophages from WT mice. EMSA analyses on HEK-TLR4/MD2 nuclear cell extracts confirm the nuclear localization and DNA binding of TQ-induced NF kappa B activation in a biphasic manner within 30 min. Co-immunoprecipitation experiments reveal for the first time that MMP-9 may be an important intermediate link in the TQ-induced Neu4 activity circuitously targeting TLR4 receptors. Central to this process is that Neu4 forms a complex with MMP-9, which is already bound to TLR4 receptors. Fluorescence spectrophotometer analyses of live CD14-THP1 cells treated with TQ show Neu4 sialidase activity over 5 min. Using flow cytometry analyses, CD14-THP1 cells treated with TQ express stable protein levels of Neu4, TLR4 and MMP9 on the cell surface over 30 min except for a marked diminution of MMP9 at 15 min. Using cytokine array profiling analyses of serum, Neu4-knockout mice respond poorly to TQ in producing pro-inflammatory cytokines and chemokines after 5-h treatment compared to the wild-type or hypomorphic cathepsin A mice with a secondary 90% Neu1 deficient mice. Our findings establish an unprecedented signaling paradigm for TQ-induced Neu4 sialidase activity. It signifies that MMP-9 forms an important molecular signaling platform in complex with TLR4 receptors at the ectodomain and acts as the intermediate link for TQ-induced Neu4 sialidase in generating a functional receptor with subsequent NF kappa B activation and pro-inflammatory cytokine production in vivo

Dependence of pathogen molecule-induced Toll-like receptor activation and cell function on Neu1 sialidase

The signaling pathways of mammalian Toll-like receptors (TLR) are well characterized, but the initial molecular mechanisms activated following ligand interactions with the receptors remain poorly defined. Here, we show a membrane controlling mechanism that is initiated by ligand binding to TLR-2, -3 and-4 to induce Neu1 sialidase activity within minutes in live primary bone marrow (BM) macrophage cells and macrophage and dendritic cell lines. Central to this process is that Neu1 and not Neu2,-3 and-4 forms a complex with TLR-2,-3 and-4 on the cell surface of na < ve macrophage cells. Neuraminidase inhibitors BCX1827, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA), zanamivir and oseltamivir carboxylate have a limited significant inhibition of the LPS-induced sialidase activity in live BMC-2 macrophage cells but Tamiflu (oseltamivir phosphate) completely blocks this activity. Tamiflu inhibits LPS-induced sialidase activity in live BMC-2 cells with an IC50 of 1.2 mu M compared to an IC50 of 1015 mu M for its hydrolytic metabolite oseltamivir carboxylate. Tamiflu blockage of LPS-induced Neu1 sialidase activity is not affected in BMC-2 cells pretreated with anticarboxylesterase agent clopidogrel. Endotoxin LPS binding to TLR4 induces Neu1 with subsequent activation of NF kappa B and the production of nitric oxide and pro-inflammatory IL-6 and TNF alpha cytokines in primary and macrophage cell lines. Hypomorphic cathepsin A mice with a secondary Neu1 deficiency respond poorly to LPS-induced pro-inflammatory cytokines compared to the wild-type or hypomorphic cathepsin A with normal Neu1 mice. Our findings establish an unprecedented mechanism for pathogen molecule-induced TLR activation and cell function, which is critically dependent on Neu1 sialidase activity associated with TLR ligand treated live primary macrophage cells and macrophage and dendritic cell lines

Improvement in the design of MMTV LTR-based transfer vectors and their test in the <i>in vivo</i> packaging assay.

<p>(<b>A</b>) Schematic representation of the second set of MMTV transfer vectors in which the U3 region containing the promoter within the 5′ LTR was replaced with the promoter sequences of human cytomegalovirus (hCMV) generating a chimeric CMV-R/U5 LTR. In addition, the constitutive transport element (CTE) from the Mason-Pfizer monkey virus (MPMV) was inserted between the Δ<i>env</i> and the 3′ LTR to facilitate efficient nuclear export of viral genomic RNA in the absence of an intact MMTV Rem/RmRE export pathway. (<b>B</b>) Schematic representation of the three series of transfer vectors tested in the study containing chimeric 5′ MMTV LTR and therefore following transfection the RNA transcription was initiated by the hCMV promoter sequences. The first series of transfer vectors (DA19–24) tested the entire 5′ UTR in the presence of incremental amounts of <i>gag</i> sequences, the second series (FA21–26) tested only incremental amounts of 5′ UTR sequences in the absence of any <i>gag</i> sequences, while the third series (NS07–12) tested the same incremental amounts of 5′ UTR sequences in the presence of 400 nt of <i>gag</i>. (<b>C</b>) RT-PCR of cytoplasmic RNA fractions to ensure the integrity of the fractionation technique and conventional PCR on RNA samples to monitor the absence of any contaminating DNA in the RNA preparations. Panel I: Multiplex PCR amplification of cytoplasmic cDNAs with 18S and unspliced ß-actin primers; Panel II: PCR amplification of cytoplasmic cDNAs with spliced ß-actin primers; Panel III: PCR amplification of DNase-treated RNA with GAPDH primers. Lane numbers 1–18 correspond to the numbers of the three series of chimeric LTR transfer vectors (DA, FA, and NS) in sequential order, whereas lanes 19 and 20 correspond to cytoplasmic fraction from Mock (containing the packaging construct, JA10 + the VSV-G-Env expression plasmid, MD.G + luciferase-expression vector, pGL3) and no DNA transfected cultures. +C, positive control (cDNA from cellular mRNA); M, molecular weight markers.</p

Propagation efficiency of MMTV transfer vector RNAs containing chimeric 5′ LTRs.

*<p>JA10, MMTV gag/pol packaging expression vector; MD.G, vesicular stomatitis virus (VSV-G) envelope expression vector; pGL3, luciferase expression vector.</p>**<p>Propagation of the transfer vector RNA expressed as hygromycin resistance colony forming units (CFU)/ml of viral supernatant that was used to infect target cells. The data represents the mean of at least three independent transfection and infection experiments testing all mutants and was derived after normalization to the transfection efficiencies observed by luciferase expression from a co-transfected luciferase expression vector. SD, standard deviation.</p>***<p>Entire UTR refers to 160 bp excluding 17 bp of primer binding site (PBS).</p>****<p>The differences observed in the RNA propagation abilities of DA24 and NS12, both containing same amounts of 5′ UTR and gag sequences could be attributed to an artificially introduced <i>Spe</i>I site in NS12 at the junction of 5′ UTR and gag during cloning. This <i>Spe</i>I site may have destabilized some sequence/structural motifs important for MMTV transfer vector RNA packaging and propagation (compare DA24 and NS12 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047088#pone-0047088-g005" target="_blank">Figure 5C</a>).</p

Experimental design to determine the role of the 5′ untranslated region (UTR) and <i>gag</i> sequences in MMTV RNA packaging.

<p>(<b>A</b>) Schematic representations of the complete MMTV genome and the MMTV long terminal repeats (LTRs)-based transfer vectors used as RNA packaging substrates in the study. In these transfer vectors, the wild type (WT) MMTV LTRs were maintained and therefore following transfection the RNA transcription was initiated by the promoter sequences within the U3 region of the 5′ LTR. Region encompassing most of the structural and enzymatic genes (<i>gag</i>, <i>pro, pol</i>, part of <i>env</i> and <i>rem</i>) were replaced by the SV-40 <i>hygromycin</i> resistance gene cassette as a marker for successful transduction of the target cells by packaged transfer vector RNA. (<b>B</b>) Three series of deletion mutations at the 5′ end of MMTV transfer vector sequences were introduced to monitor their effect on RNA packaging and propagation. The first series of deletion mutants (DA07–12) contained the entire 5′ UTR in the presence of incremental amounts of <i>gag</i> sequences, the second series of mutants (FA07–12) contained deletions in the 5′ untranslated region (UTR) sequences in the absence of any <i>gag</i> sequences, while the third series of mutants (NS01–06) contained the same incremental amounts of 5′ UTR sequences in the presence of 400 nucleotides (nt) of <i>gag</i>. (<b>C</b>) Table showing viral titers observed post transduction of HeLaT4 target cells by transfer vector RNAs tested. None of the transfer vector RNAs containing deletions in the 5′ UTR in the absence (FA07–FA12) or the presence (NS01–NS05) of <i>gag</i> sequences could be propagated (<1 CFU/ml) except NS06. *JA10, MMTV packaging Gag/Pol expression construct; MD.G, vesicular stomatitis virus (VSV-G) Env expression plasmid; pGL3, luciferase expression plasmid. **Propagation of the transfer vector RNA expressed as hygromycin resistance colony forming units (CFU)/ml of viral supernatant that was used to infect target cells. ***The entire UTR refers to 160 nt excluding 17 nt of primer binding site (PBS). The data represents mean of at least three independent transfection and infection experiments testing all mutants and was derived after normalization to the transfection efficiencies observed by luciferase expression from a co-transfected luciferase expression vector. SD, standard deviation. (<b>D</b>) Reverse transcriptase (RT) PCR analysis of the MMTV LTR-based DA series of transfer vectors containing incremental amounts of <i>gag</i> sequences in the presence of the entire 5′ UTR followed by Southern blotting. The probe was prepared by PCR amplification of a 142 nt long R/UR/5′ UTR region (nt 1179–1321) common to all the transfer vectors, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047088#s4" target="_blank">Materials and Methods</a>. Amplification was carried out for either 25 (lower panel) or 30 cycles (upper panel) using transfer vector-specific primers OTR671 and OTR 672.</p