Untargeted Metabolomics analysis of A549 treated with 0.5 mM extracellular ATP and 10 ng/ml TGF-beta
Ontology highlight
ABSTRACT:
ORGANISM(S): Human Homo Sapiens
TISSUE(S): Lung
DISEASE(S): Cancer
SUBMITTER: 
Pratik Shriwas
PROVIDER: ST001779 | MetabolomicsWorkbench | Sun Apr 04 00:00:00 BST 2021
REPOSITORIES: MetabolomicsWorkbench
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Project description:The aim of this experiment was analyze the genes regulated by self-released extracellular ATP in gastric cancer-derived cell line AGS, for this we incubate the cells by 48h with apyrase, a potent ectonucleotidase thta hydrolyze the extracellular ATP to form ADP and AMP. Then cDNA microarray were performed using a human cancer library.
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Project description:Ectopic adenosine triphosphate synthase (eATP synthase) was found on the cell surface to generate extracellular ATP in various cancer cells. Recently using proteomics approach, several reports identified ATP synthase in extracellular vesicles (EVs) which are important in cell-cell communications. However, the functions of ATP synthase in EVs are still unclear. Here we increased the expression of eATP synthase on the plasma membrane of lung cancer A549 cells via 0.1% FBS starvation and collected EVs including microvesicles and exosomes from the culture medium using serial centrifugation. Both of them were assuredly isolated and confirmed by transmission electron microscopy, nano tracking analysis, and specific EV markers. We also used western blot to demonstrate the presence of ATP synthase in EVs. Besides, dot blot experiment revealed that the F1 portion faced towards extracellular space using anti-ATP beta subunit antibody. Under starvation, the expression of eATP synthase was enhanced on cell surface and exosomes as well as increased EV secretion. To further investigate the functions, we first elucidated whether eATP synthase on EV membrane produced ATP by ATP luciferase analysis. Our data showed that there was no significant ATP production. Moreover, we performed proteomic analysis using dimethyl labeling and LC-MS/MS to compare the difference between EVs released by A549 control and starvation. We identified a total of 1504 and 869 proteins in microvesicles and exosomes, respectively. Using 1.5- and 0.67-fold change as the thresholds, there were 171 and 46 differential expressed proteins in microvesicles and exosomes, respectively. Seven ATP synthase subunits in microvesicles and five in exosomes were identified. The proteomic data were further analyzed using gene set enrichment analysis (GSEA) within the C2 (curated gene sets) subclasses and setting 0.25 as FDR cutoff to find out biological functions related to eATP synthase. Among these up-regulated terms, metabolism, cell cycle and apoptosis were enriched, which indicating that starvation influences cell behavior. Additionally, the immune response is upregulated strikingly in exosomes within C2 gene set. One of these immune response pathways, MHC-1 antigen presentation, was previously proved to interact with eATP synthase which could be recognized by several immune cells.
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Project description:EMT is an early process in metastasis that is associated with up to 90% of cancer-related death. ATP has been known to play some roles in EMT. We previously described that internalization of extracellular ATP (eATP) by cancer cells in vitro and in vivo via macropinocytosis increases intracellular ATP levels, cell proliferation and resistance to anticancer drugs. Recently, we reported eATP also induces EMT and other early metastatic activities independent of TGF-b, a well-known inducer of EMT, in human lung cancer cells. However, exactly how eATP induces EMT at gene expression level is far from understood. In this study, we hypothesize that eATP acts as an inducer and regulator of EMT alternative to TGF-b. Transwell assays revealed that eATP treatment, alone, induced human lung cancer A549 and H1299 cells to invade at rates faster than TGF-b. eATP also induced formation of filopodia-like protrusions faster than TGF-b, and it restored viability of cancer cells treated with TGF-b-neutralizing antibodies. eATP significantly elevated intracellular ATP levels while TGF-b did not. RNA sequencing / western blots revealed that eATP induced changes in expression of genes/proteins involved in EMT similar to but with differences in number and induction time compared with those induced by TGF-b. Metabolomics analysis shows that eATP-induced major metabolic changes are consistent/correlated with those identified at the gene expression level. These differences could be accounted for by the multi-functional and multi-localization properties of eATP and macropinocytosis-internalized eATP, strongly suggesting that eATP is a previously unrecognized master inducer and regulator of EMT.
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Project description:Extracellular adenosine 5’-triphosphate (ATP) is a signaling molecule. To define the global transcriptional response to ATP, we performed DNA microarray analysis using RNA derived from wild-type (Col-0) Arabidopsis and the dorn1-1 mutant, Lectin receptor kinase I.9 (At5g60300) carrying a EMS point mutation, seedlings after 100 μM ATP. 12 samples ( 2 genotypes, 2 treatments, 3 biological replicates)
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Project description:Extracellular adenosine 5’-triphosphate (ATP) is a signaling molecule. To define the global transcriptional response to ATP, we performed DNA microarray analysis using RNA derived from wild-type (Col-0) Arabidopsis and the dorn1-1 mutant, Lectin receptor kinase I.9 (At5g60300) carrying a EMS point mutation, seedlings after 100 μM ATP.
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Project description:Nucleotides triphosphates are extracellular messengers binding to specific plasma membrane receptors (P2Rs) that modulate responses as different as proliferation, differentiation, migration or cell death on several cell types including hematopoietic stem cells. Little and controversial information is available on the role of extracellular nucleotides in human mesenchimal stem cells (hMSCs). In this study, we assessed whether P2Rs are expressed and functional in bone marrow-derived hMSCs. Our results demonstrated, at the mRNA and protein level, the expression of all P2X and P2Y receptor subtypes identified so far. P2R activation by their natural ligands adenosine triphosphate (ATP) and uridine triphosphate (UTP) induced in hMSCs, intracellular Ca2+ concentration changes, plasma membrane depolarization and permeabilization. hMSCs were resistant to the cytotoxic effects of high dose ATP despite the expression of permeabilizing P2Rs as demonstrated by the lack of morphological changes, significant release of intracellular markers of cell death or modification of the mitochondrial network. Gene expression profiling revealed the down-regulation of cell proliferation genes whereas genes involved in cell migration and cytokine production were strongly up-regulated by ATP. Functional studies confirmed the inhibitory activity of ATP on proliferation of hMSCs and clonogenic progenitors. Moreover, ATP exerted a chemotactic effect on hMSCs and increased their migration in response to the chemokine CXCL12. Finally, whereas ATP did not affect T-cell inhibitory activity of hMSCs, the nucleotide increased the production of pro-inflammatory cytokines by hMSCs. Thus, our data show that purinergic signaling modulates hMSC functions and point to a role for extracellular nucleotides on hMSCs biology.
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