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Quantitative proteomic analysis of human lung tumor xenografts treated with the ectopic ATP synthase inhibitor citreoviridin.

ABSTRACT: ATP synthase is present on the plasma membrane of several types of cancer cells. Citreoviridin, an ATP synthase inhibitor, selectively suppresses the proliferation and growth of lung cancer without affecting normal cells. However, the global effects of targeting ectopic ATP synthase in vivo have not been well defined. In this study, we performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) and provided a comprehensive insight into the complicated regulation by citreoviridin in a lung cancer xenograft model. With high reproducibility of the quantitation, we obtained quantitative proteomic profiling with 2,659 proteins identified. Bioinformatics analysis of the 141 differentially expressed proteins selected by their relative abundance revealed that citreoviridin induces alterations in the expression of glucose metabolism-related enzymes in lung cancer. The up-regulation of enzymes involved in gluconeogenesis and storage of glucose indicated that citreoviridin may reduce the glycolytic intermediates for macromolecule synthesis and inhibit cell proliferation. Using comprehensive proteomics, the results identify metabolic aspects that help explain the antitumorigenic effect of citreoviridin in lung cancer, which may lead to a better understanding of the links between metabolism and tumorigenesis in cancer therapy.

SUBMITTER: Wu YH

PROVIDER: S-EPMC3749231 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Quantitative proteomic analysis of human lung tumor xenografts treated with the ectopic ATP synthase inhibitor citreoviridin.

Wu Yi-Hsuan YH Hu Chia-Wei CW Chien Chih-Wei CW Chen Yu-Ju YJ Huang Hsuan-Cheng HC Juan Hsueh-Fen HF

PloS one 20130821 8

ATP synthase is present on the plasma membrane of several types of cancer cells. Citreoviridin, an ATP synthase inhibitor, selectively suppresses the proliferation and growth of lung cancer without affecting normal cells. However, the global effects of targeting ectopic ATP synthase in vivo have not been well defined. In this study, we performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) and provided a comprehensive insight into the complic ...[more]

PMID: 23990911

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Project description:Non-small cell lung cancer is the leading cause of cancer death worldwide. Gefitinib, epidermal growth factor receptor tyrosine kinase inhibitor, is the first-line treatment of NSCLC, however, many patients eventually become resistant and experience progressive disease. Therefore, development of efficient therapeutic agents to overcome resistance is urgent. We previously found that citreoviridin, one of toxic mycotoxins derived from fungal species, can suppress lung cancer cell growth by inhibiting the activity of ectopic ATP synthase, but has limited effect on normal cells. Citreoviridin suppresses mitogen-activated protein kinase/extracellular signal-regulated kinase signaling by site-specific dephosphorylation of HSP90AB1 on Serine 255 in gefitinib non-resistant lung cancer CL1-0 cells and xenograft model. We are curious whether signaling pathways underlying citreoviridin-treated gefitinib-acquired resistant lung cancer cells are different. In this study, we showed that citreoviridin inhibited cell proliferation and anchorage-dependent growth of gefitinib-acquired resistance NCI-H1975 cells with EGFR T790M mutation. Furthermore, we explored the dynamic molecular response by temporal phosphoproteomic approach. We identified 1476 phosphopeptides corresponding to 738 phosphoproteins and quantified 1901 phosphorylation sites. There were 274 phosphosites corresponding to 174 phosphorylated proteins significantly differential expressed. Functional enrichment analysis demonstrated that citreoviridin treatment affected chromatin organization, cell cycle and apoptosis. Interestingly, we found that citreovirdin suppressed cell proliferation by site-specific phosphorylation of topoisomerase on serine 1106. Citreovirdin induced double strands breaks, and then leaded to DNA damage response. The DNA lesions triggered cells to cell cycle arrest at S phase for repairing or apoptosis for cell death. The results indicated that citreoviridin could potentially be a therapeutic agent against gefitinib-resistant NSCLC.

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.

Project description:Adenosine triphosphate (ATP) synthase is located on the inner membrane of mitochondria which generate ATP for various cellular processes. Our previous studies showed that ATP synthases existed on plasma membrane of several types of cancer cells, which was defined as ectopic ATP synthase. However, the trafficking pathway of ectopic ATP synthase is still unclear. To investigate how ATP synthase subunits are involved in the trafficking process, we performed shot-gun proteomic analysis to reveal the plasma membrane proteins of A549 lung cancer cells and identified four subunits of ATP synthases, ATP5A1, ATP5B, ATP5H, and MT-ATP6. With the results of gene set enrichment analysis, we inferred that ectopic ATP synthase subunits are assembled as complex consistently and then translocated to cell surface through the trafficking of mitochondria. On the other hand, microtubules are involved in many intracellular transport. During protein trafficking, mitochondria and vesicles are transported to their final destination along microtubules by motor proteins such as kinesins and dyneins. To explore the function of microtubules in the trafficking of ectopic ATP synthase, we treated MCF-7 breast and A549 lung cancer cells with nocodazole, a microtubule depolymerization agent, and found that the expression level of ectopic ATP synthase decreased using both immunocytochemistry and flow cytometry. Besides, since kinesin family member 5B (KIF5B) is an important motor proteins involved in the transport of mitochondria, we further performed the immunoprecipitation of KIF5B followed by liquid chromatography−tandem mass spectrometry (LC−MS/MS). The result revealed that one of the mitochondrial outer membrane proteins, dynamin-1 like proteins (Drp1), was able to associate to KIF5B. From our recent research results and other reports show that Drp1 is a mediator of mitochondrial dynamic through regulation of mitochondrial fission. Taken together, our findings suggested that Drp1-KIF5B complex-mediated mitochondrial trafficking along microtubules may play a critical role in the transport of ectopic ATP synthase

Dataset Information

Quantitative proteomic analysis of human lung tumor xenografts treated with the ectopic ATP synthase inhibitor citreoviridin.

Publications

Quantitative proteomic analysis of human lung tumor xenografts treated with the ectopic ATP synthase inhibitor citreoviridin.

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