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Transcription profiling of human A549 NSCLC cells cisplatin induced changes

ABSTRACT: Affymetrix high-density oligonucleotide microarray analysis was performed to analyse cisplatin-induced gene expression changes in A549 NSCLC cells. Cells were treated with 50 µM of cisplatin for 1 hour and incubated for a further 10 hours in drug-free media before the gene expression changes were investigated. Results show that cisplatin induced changes in the expression of genes involved in apoptosis, cell cycle control, DNA repair and transcription. Experiment Overall Design: Gene expression changes in response to cisplatin were analysed by Microarray technology in A549 NSCLC cells. Cells were treated with 50 µM of cisplatin (or drug-free media) for 1 hour and incubated for a further 10 hours in drug-free media before the cisplatin-induced gene expression changes were investigated. Control and cisplatin-treated samples were collected from three independent experiments.

ORGANISM(S): Homo sapiens

SUBMITTER: Almeida GM

PROVIDER: S-ECPF-GEOD-6410 | biostudies-other | 2008 Apr

REPOSITORIES: biostudies-other

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Project description:To identify genes associated with lung cancer progression, we examined gene expression profiles of tumor cells from 20 patients with primary, untreated non-small cell lung cancer (10 adenocarcinomas (AC) and 10 squamous cell carcinomas (SCC)) in comparison to lung tissue of 23 patients with stage IIIB or stage IV non-small cell lung cancer (15 AC and 8 SCC). Bronchoscopical biopsies from patient with recurrent lung tumor were taken after initial treatment. Cancer cells were isolated using laser capture microdissection in order to obtain pure samples of tumor cells. For expression analysis, microarrays covering 8793 defined genes (Human HG Focus Array, Affymetrix) were used. Array data were normalized and analysed for significant differences using variance stabilizing transformation (VSN) and significance analysis of microarrays (SAM), respectively. Genes were considered to be up- or down-regulated when the ratio between primary and recurrent tumor samples were at least 1.5-fold differentially expressed with an estimated false discovery rate: < 5%. Based on differentially expressed genes, primary cancer samples could be separated from recurrent tumor samples. We identified 115 and 124 significantly regulates genes in AC and SCC, respectively. For example, in recurrent AC we found increased expression of genes related to the wingless (FZD6, RYK, MYC) and calcium (CALM1, ATB2B1, S100A2) signalling pathways which might play a role in metastasis of tumor cells. Other differentially expressed genes were related to cell cycle (CCND1, CDK2), transcription factors (TTF1, TAF2, YY1), nuclear mRNA splicing and mRNA processing (SFRS1, HNRPL), protein-nucleus import (NUTF2, KPNB1, NUP50) and chromatin modification (HIST1H4C, SMARCC1). In SCC, we found an increased expression of CTNNB1, an important mediator in wingless signalling pathway. Among the down-regulated genes in SCC, the utmost fraction belonged to genes coding for ubiquitin mediated proteolysis (UCHL1, PSMA3, COPS6) and ribosomal proteins (RPS26, RPL7A, RPS15). Other down regulated genes were related to transcription factors (TCEA2, TAF10), nuclear mRNA splicing and mRNA processing (SNRPD2, HNRPM). In conclusion, a distinct pattern of gene expression is found during the progression from primary carcinoma to recurrent NSCLC. Our microarray-based expression profiling revealed interesting novel candidate genes and pathways that may contribute to lung cancer progression. Experiment Overall Design: - 20 patients with primary, untreated non-small cell lung cancer (10adenocarcinomas (AC) and 10 squamous cell carcinomas (SCC)) in comparison to lung tissue of 23 patients with stage IIIB or stage IV non-small cell lung cancer (15 AC and 8 SCC) Experiment Overall Design: - Human HG Focus Array, Affymetrix) were used Experiment Overall Design: - Array data were normalized and analysed for significant differences using variance stabilizing transformation (VSN) and significance analysis of microarrays (SAM) Experiment Overall Design: - Genes were considered to be up- or down-regulated when the ratio between primary and recurrent tumor samples were at least 1.5-fold differentially expressed with an estimated false discovery rate: < 5%

Project description:Hypoxia is commonly observed in solid tumors and contributes to the resistance of DNA damage drugs. However, the mechanisms behind this resistance are still unclear. In this study, we aimed to explore the effects of hypoxia-induced exosomes on non-small cell lung cancer (NSCLC). Methods: NSCLC cells were subjected to either normoxic or hypoxic conditions to assess cell survival and changes in the expression levels of key proteins. Comparative proteomics were performed to identify exosomal PKM2 in normoxic or hypoxic cisplatin-resistant NSCLC cells-derived exosomes. Functions of hypoxia induced-exosomal PKM2 in promoting cisplatin resistance to NSCLC cells were evaluated both in vitro and in vivo experiments and the molecular mechanisms of hypoxia induced-exosomal PKM2 were demonstrated using flow cytometry, immunoblotting, oxidative stress detection and histological examination. A series of in vitro experiments were performed to evaluate the function of hypoxia-induced exosomes on cancer-associated fibroblasts (CAFs). Results: Hypoxia exacerbated the cisplatin resistance in lung cancer cells due to the increased expression of PKM2 that was observed in the exosomes secreted by hypoxic cisplatin-resistance cells. We identified that hypoxia-induced exosomal PKM2 transmitted cisplatin-resistance to sensitive NSCLC cells in vitro and in vivo. Mechanistically, hypoxia-induced exosomal PKM2 promoted glycolysis in NSCLC cells to produce reductive metabolites, which may neutralize reactive oxygen species (ROS) induced by cisplatin. Additionally, hypoxia-induced exosomal PKM2 inhibited apoptosis in a PKM2-BCL2-dependent manner. Moreover, hypoxia-induced exosomal PKM2 reprogrammed CAFs to create an acidic microenvironment promoting NSCLC cells proliferation and cisplatin resistance. Conclusions: Our findings revealed that hypoxia-induced exosomes transmit cisplatin resistance to sensitive NSCLC cells by delivering PKM2. Exosomal PKM2 may serve as a promising biomarker and therapeutic target for cisplatin resistance in NSCLC.

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Transcription profiling of human A549 NSCLC cells cisplatin induced changes

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