Dataset Information

Induced-Decay of Glycine Decarboxylase Transcripts as an Anticancer Therapeutic Strategy for Non-Small-Cell Lung Carcinoma.

ABSTRACT: Self-renewing tumor-initiating cells (TICs) are thought to be responsible for tumor recurrence and chemo-resistance. Glycine decarboxylase, encoded by the GLDC gene, is reported to be overexpressed in TIC-enriched primary non-small-cell lung carcinoma (NSCLC). GLDC is a component of the mitochondrial glycine cleavage system, and its high expression is required for growth and tumorigenic capacity. Currently, there are no therapeutic agents against GLDC. As a therapeutic strategy, we have designed and tested splicing-modulating steric hindrance antisense oligonucleotides (shAONs) that efficiently induce exon skipping (half maximal inhibitory concentration [IC₅₀] at 3.5-7 nM), disrupt the open reading frame (ORF) of GLDC transcript (predisposing it for nonsense-mediated decay), halt cell proliferation, and prevent colony formation in both A549 cells and TIC-enriched NSCLC tumor sphere cells (TS32). One candidate shAON causes 60% inhibition of tumor growth in mice transplanted with TS32. Thus, our shAONs candidates can effectively inhibit the expression of NSCLC-associated metabolic enzyme GLDC and may have promising therapeutic implications.

SUBMITTER: Lin J

PROVIDER: S-EPMC5675722 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Induced-Decay of Glycine Decarboxylase Transcripts as an Anticancer Therapeutic Strategy for Non-Small-Cell Lung Carcinoma.

Lin Jing J Lee Jia Hui Jane JHJ Paramasivam Kathirvel K Pathak Elina E Wang Zhenxun Z Pramono Zacharias Aloysius Dwi ZAD Lim Bing B Wee Keng Boon KB Surana Uttam U

Molecular therapy. Nucleic acids 20171007

Self-renewing tumor-initiating cells (TICs) are thought to be responsible for tumor recurrence and chemo-resistance. Glycine decarboxylase, encoded by the GLDC gene, is reported to be overexpressed in TIC-enriched primary non-small-cell lung carcinoma (NSCLC). GLDC is a component of the mitochondrial glycine cleavage system, and its high expression is required for growth and tumorigenic capacity. Currently, there are no therapeutic agents against GLDC. As a therapeutic strategy, we have designed ...[more]

PMID: 29246305

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Project description:BackgroundGlycine decarboxylase (GLDC), a key metabolic enzyme, participates in the regulation of the glycine metabolic pathway. Differential expression of GLDC is linked to the malignant growth of renal cell carcinoma (RCC) and may regulate tumor progression through other genes. However, the regulatory function of GLDC in RCC is currently unknown. The purpose of this work was to evaluate the roles of GLDC in the invasion, proliferation, and migration of RCC cells and elucidate the processes underlying RCC development.MethodsThe expression of GLDC in RCC cell lines and tissues was identified by quantitative reverse transcription polymerase chain reaction (PCR) and western blot. A stably transfected cell line overexpressing GLDC was constructed using a lentiviral vector. Cell proliferation was detected using Cell Counting Kit-8 (CCK8) and EdU experiments, and scratch and transwell assays were used to determine migration and invasion capabilities. Furthermore, differential proteins were identified and obtained using high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) analysis. Finally, these differential proteins were analyzed by bioinformatics, including cluster analysis, subcellular localization, domain annotation, annotation of the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG), enrichment analysis, and study of protein-protein interactions.ResultsGLDC expression was found to be lower in six RCC cell lines (786-O, A498, Caki-1, 769-P, OSRC-2, and ACHN) than in 293T cells and decreased in kidney cancer tissues compared to neighboring normal tissues. Overexpression of GLDC inhibited the proliferation of RCC cells as well as their migration and invasion abilities. Tandem mass tag analysis showed that 317 and 236 genes were downregulated and upregulated, respectively, when GLDC was overexpressed in A498 cells. Tandem mass tag analysis showed that 317 and 236 genes were downregulated and upregulated, respectively, when GLDC was overexpressed in A498 cells. Volcano plot showed these upregulated and downregulated proteins. Cluster analysis showed that differentially expressed protein screening can represent the effect of biological treatment on samples. Subcellular localization analysis showed differential proteins are mainly distributed in the nucleus, cytoplasm, mitochondria, plasma membrane, extracellular matrix, and lysosome. GO annotation showed many biological processes in the cells were changed, including "positive regulation of histone H3-K4 methylation", "cofactor binding", and "nuclear body". KEGG pathway analysis showed key pathways have all undergone considerable alterations, such as "cell cycle", "glyoxylate and dicarboxylate metabolism", and "threonine, glycine, and serine metabolism". Finally, highly aggregated proteins with the same or similar functions were acquired by analysis of the protein-protein interaction (PPI) network.ConclusionsThese studies indicate that GLDC overexpression suppresses the invasion, proliferation, and migration of RCC cells and leads to the upregulation and downregulation of 236 and 317 genes, respectively.

Project description:DNA binding anticancer agents cause alteration in chromatin structure and dynamics. Here, we report the dynamic interaction of the DNA intercalator and potential anticancer, plant alkaloid, Sanguinarine (SGR) with chromatin. Association of SGR with different levels of chromatin structure was found to be enthalpydriven. Apart from DNA it binds with comparable affinity to histones and induces chromatin aggregation. The dual binding property of SGR leads to inhibition of core histone modifications. Although, it potently inhibits H3K9 methylation by G9a in vitro, H3K4 and H3R17 methylation are more profoundly inhibited in cells. SGR inhibits histone acetylation both in vitro and in vivo. It does not affect the in vitro transcription from DNA template but significantly represses acetylation dependent chromatin transcription. SGR mediated repression of epigenetic marks and the alteration of chromatin geography, also result in modulation of global gene expression. These data conclusively, show the anticancer DNA binding intercalator as a modulator of chromatin modifications and transcription in the chromatin context. The total RNA was isolated from control and treated cells using TRIZOL (Invitrogen) method. The Micromax direct labeling kit, MPS502 (PerkinElmer) was used to synthesize the labeled cDNA from 70 ug of total RNA and further process the hybridized cDNA on the array. All the steps were carried out according to manufacturerâs instructions (www.perkinelmer.com/lifesciences). The array slides were scanned immediately by PerkinElmer Scan array Gx Microarray scanner. The Scan array software (PerkinElmer) was used for grid wise normalization of array images. Two arrays were used with at least one biological treatment of cells and dye swap experiment were included in the final analysis. . The data was analysed by GeneSpring GX and Biointerpreter software from Genotypic Technology, Bangalore. The differential expression was considered if the Log 2 mean of at least -1 for the down regulated genes and +1 for the upregulated genes. We considered only the genes that were reproducible from both replicates.

Project description:DNA-binding anticancer agents cause alteration in chromatin structure and dynamics. Here, we report the dynamic interaction of the DNA intercalator and potential anticancer, plant alkaloid, sanguinarine (SGR) with chromatin. Association of SGR with different levels of chromatin structure was found to be enthalpy driven. Apart from DNA, it binds with comparable affinity to histones and induces chromatin aggregation. The dual binding property of SGR leads to inhibition of core histone modifications. Although it potently inhibits H3K9 methylation by G9a in vitro, H3K4 and H3R17 methylation are more profoundly inhibited in cells. SGR inhibits histone acetylation both in vitro and in vivo. It does not affect the in vitro transcription from DNA template but significantly represses acetylation-dependent chromatin transcription. SGR mediated repression of epigenetic marks and the alteration of chromatin geography also result in modulation of global gene expression. These data conclusively show that the anticancer DNA-binding intercalator as a modulator of chromatin modifications and transcription in the chromatin context. The total RNA was isolated from control and treated cells using the TRIzol (Invitrogen) method. The Micromax direct labeling kit, MPS502 (PerkinElmer), was used to synthesize the labeled cDNA from 70 ug of total RNA and further process the hybridized cDNA on the array. All the steps were carried out according to manufacturer’s instructions (www.perkinelmer.com/lifesciences). The array slides were scanned immediately by the PerkinElmer Scan array Gx Microarray scanner. The Scan array software (PerkinElmer) was used for grid wise normalization of array images. Five arrays were used with at least two biological treatments of cells, and dye-swap experiments were included in the final analysis. The data was analysed by GeneSpring GX and Biointerpreter software from Genotypic Technology, Bangalore. The differential expression was considered if the Log 2 mean of at least -1 for the downregulated genes and +1 for the upregulated genes. We considered only the genes that were reproducible from all five replicates.

Dataset Information

Induced-Decay of Glycine Decarboxylase Transcripts as an Anticancer Therapeutic Strategy for Non-Small-Cell Lung Carcinoma.

Publications

Induced-Decay of Glycine Decarboxylase Transcripts as an Anticancer Therapeutic Strategy for Non-Small-Cell Lung Carcinoma.

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