39 research outputs found
Lactate metabolic pathway regulates tumor cell metastasis and its use as a new therapeutic target
Abnormal energy metabolism is one of the ten hallmarks of tumors, and tumor cell metabolism provides energy and a suitable microenvironment for tumorigenesis and metastasis. Tumor cells can consume large amounts of glucose and produce large amounts of lactate through glycolysis even in the presence of oxygen, a process called aerobic glycolysis, also known as the Warburg effect. Lactate is the end product of the aerobic glycolysis. Lactate dehydrogenase A (LDHA), which is highly expressed in cancer cells, promotes lactate production and transports lactate to the tumor microenvironment and is taken up by surrounding stromal cells under the action of monocarboxylate transporter 1/4 (MCT1/4), which in turn influences the immune response and enhances the invasion and metastasis of cancer cells. Therapeutic strategies targeting lactate metabolism have been intensively investigated, focusing on its metastasis-promoting properties and various target inhibitors; AZD3965, an MCT1 inhibitor, has entered phase I clinical trials, and the LDHA inhibitor N-hydroxyindole (NHI) has shown cancer therapeutic activity in pre-clinical studies. Interventions targeting lactate metabolism are emerging as a promising option for cancer therapy, with chemotherapy or radiotherapy combined with lactate-metabolism-targeted drugs adding to the effectiveness of cancer treatment. Based on current research, this article outlines the role of lactate metabolism in tumor metastasis and the potential value of inhibitors targeting lactate metabolism in cancer therapy
Chlorella Virus PBCV-1 Encodes a Functional Homospermidine Synthase
Sequence analysis of the 330-kb genome of chlorella virus Paramecium bursaria chlorella virus 1 (PBCV-1) revealed an open reading frame, A237R, that encodes a protein with 34% amino acid identity to homospermidine synthase from Rhodopseudomonas viridis. Expression of the a237r gene product in Escherichia coli established that the recombinant enzyme catalyzes the NAD+-dependent formation of homospermidine from two molecules of putrescine. The a237r gene is expressed late in PBCV-1 infection. Both uninfected and PBCV-1-infected chlorella, as well as PBCV-1 virions, contain homospermidine, along with the more common polyamines putrescine, spermidine, and cadaverine. The total number of polyamine molecules per virion (~539) is too small to significantly neutralize the virus double-stranded DNA (\u3e660,000 nucleotides). Consequently, the biological significance of the homospermidine synthase gene is unknown. However, the gene is widespread among the chlorella viruses. To our knowledge, this is the first report of a virus encoding an enzyme involved in polyamine biosynthesis
Molecular Determinants of GS-9620-Dependent TLR7 Activation
<div><p>GS-9620 is an orally administered agonist of Toll-like receptor (TLR)7 currently being evaluated in clinical studies for the treatment of chronic HBV and HIV patients. GS-9620 has shown antiviral efficacy in preclinical models of chronic hepadnavirus infection in woodchuck as well as chimpanzee. However, the molecular determinants of GS-9620-dependent activation of TLR7 are not well defined. The studies presented here elucidate GS-9620 subcellular distribution and characterize its molecular interactions with human TLR7 using structure-guided mutational analysis. Based on our results we present a molecular model of TLR7 bound to GS-9620. We also determine that several coding SNPs had no effect on GS-9620-dependent TLR7 activation. In addition, our studies provide evidence that TLR7 exists in a ligand-independent oligomeric state and that, TLR7 activation by GS-9620 is likely associated with compound-induced conformational changes. Finally, we demonstrate that activation of NF-κB and Akt pathways in primary plasmacytoid dendritic cells occur as immediate downstream cellular responses to GS-9620 stimulation. The data presented here further our understanding of the molecular parameters governing TLR7 activation by GS-9620, and more generally by nucleos/tide-related ligands.</p></div
TLR7 dimers exist independent of GS-9620 binding.
<p>Immunoblot analysis of whole cell lysates of Huh7 cells transfected with V5-tagged TLR7 (TLR7-V5) or HA-tagged TLR7 (TLR7-HA) or HA-tagged TLR9 (TLR9-HA) untreated or treated with increasing amounts of GS-9620 (0.1μM, 1μM and 5μM) for 1 hour before preparation of cell lysates and immunoprecipitation (IP) analysis. Lysates were immunoprecipitated with anti-V5 agarose (panel C,D) and immunoblot probed with anti-V5 mAb (panel A,C) or anti-HA mAb (panel B,D). Total cell lysates used in panels A and B were probed with anti-V5 mAb and anti-HA mAb respectively, to control for protein expression and loading.</p
GS-9620 induces phosphorylation of NF-κB and Akt in pDCs.
<p>PBMC isolated from healthy donors were cultured with GS-9620, resiquimod, or DMSO control. Phosphorylation of NF-κB and Akt was assessed in gated pDC and mDC subsets. (a) Flow cytometric histogram of p-NF-κB (using a phosho NF-κB (S529) specific antibody) following 30min stimulation (left panel) or p-Akt (using a phospho Akt (S473) specific antibody) following 60min stimulation (right panel) with 1μM GS-9620 (blue histogram) or DMSO control (grey histogram). (b) Fold increase in mean fluorescence intensity (MFI) of pDCs for pNF-κB (left panel) or p-Akt (right panel) after stimulation with 1μM GS-9620 normalized to DMSO control treatment. Statistically significant differences relative to DMSO control (p<0.05) are observed with GS-9620 stimulation for all assessed time points (p-NF-κB), and for 10, 15, 30, and 60min time points (p-Akt). (c) Fold increase in MFI upon stimulation with 1μM GS-9620 or resiquimod (R848) normalized to DMSO control treatment in mDCs or pDCs. As expected, phospho-responses to GS-9620 and R848 for both readouts, p-NF-κB and p-Akt, were significantly stronger in pDC compared to donor-matched mDC (p<0.01 for all comparisons). Graphs show data obtained from 6 (p-NF-κB) and 4 (p-Akt) independent healthy donors with mean ±SEM (bars).</p
GS-9620 rapidly distributes to and signals through the endo-lysosomal compartments.
<p>(a-b) Chemical structure of (a) GS-9620 and (b) Resiquimod. (c) Kinetics of intracellular accumulation of <sup>3</sup>H-GS-9620 in Daudi cells. (d) Sub-cellular distribution of <sup>3</sup>H-GS-9620 in relation to major organelle markers obtained by isopycnic density gradient centrifugation. (e) IFN-α2 secretion by enriched pDCs upon stimulation with GS-9620 or resiquimod (control) in the presence of bafilomycin A1 (BAF) or PBS (mock). Data shown in (c), (d), (e) are representative of 3 independent experiments. (e) Error bars represent mean of triplicate assay conditions and SEM. Abbreviations: Early endosome (Early Endo), endoplasmic reticulum (ER), mitochondria (Mt), lysosome (Lyso).</p
Structure-based mutational analysis identifies residues in TLR7 that are essential for GS-9620 <i>in vitro</i> activity.
<p>(a) Three dimensional molecular model of TLR7 endo-lysosomal domain (left) and magnified view of GS-9620 docked in TLR7 (right). (b,c) Fold increase in NF-κB-luciferase reporter activity upon GS-9620, resiquimod or DMSO control stimulation in Huh7 cells that were transfected with control vector, wild-type TLR7 or point mutants of TLR7. Four 2-fold dose titrations (left to right) were performed starting at 5μM for GS-9620, or 10 μM of resiquimod. Bar graphs show fold change of reporter activity relative to DMSO control, and error bars shown represent the mean of triplicate assay conditions and SEM. Representative data shown from three independent experiments with similar results. Area under the curve (AUC) calculations were performed to quantify reporter activity observed with titrated compound concentrations for each of the TLR7 variants. With GS-9620 stimulation the variants Y356S, F408A, D555A, L557D, and L557Y/T586G, and with R848 stimulation the variants Y356S, F408A, and D555A show a 4-fold or greater reduction in reporter activity compared to TLR7 WT, as assessed by AUC calculation. Therefore, these variants are viewed as significantly compromised in response to the respective compound.</p
Amino acid changes due to described single nucleotide polymorphisms (SNPs) do not impact GS-9620-dependent TLR7 activation.
<p>Fold increase in NF-kB-luciferase reporter activity upon GS-9620 stimulation in Huh7 cells that were transfected with TLR7 or SNPs rs179008 (Q11L), rs55907843 (V222D), or rs5743781 (A448V). Four 2-fold dose titration curves were performed starting at 5µM for GS-9620 (left to right). Bar graphs show fold change in reporter activity relative to DMSO, and error bars represent the mean of triplicate assay conditions and SEM. AUC calculations were performed as discussed for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146835#pone.0146835.g002" target="_blank">Fig 2</a>, above. By the same AUC criteria none of the three assessed TLR7 SNP variants elicited a significantly altered response, relative to TLR7 WT, after GS-9620 stimulation. Representative data are shown from three independent experiments with similar results.</p
Direct binding of ledipasvir to HCV NS5A: mechanism of resistance to an HCV antiviral agent.
Ledipasvir, a direct acting antiviral agent (DAA) targeting the Hepatitis C Virus NS5A protein, exhibits picomolar activity in replicon cells. While its mechanism of action is unclear, mutations that confer resistance to ledipasvir in HCV replicon cells are located in NS5A, suggesting that NS5A is the direct target of ledipasvir. To date co-precipitation and cross-linking experiments in replicon or NS5A transfected cells have not conclusively shown a direct, specific interaction between NS5A and ledipasvir. Using recombinant, full length NS5A, we show that ledipasvir binds directly, with high affinity and specificity, to NS5A. Ledipasvir binding to recombinant NS5A is saturable with a dissociation constant in the low nanomolar range. A mutant form of NS5A (Y93H) that confers resistance to ledipasvir shows diminished binding to ledipasvir. The current study shows that ledipasvir inhibits NS5A through direct binding and that resistance to ledipasvir is the result of a reduction in binding affinity to NS5A mutants