Global profiling of platinum drugs resistant genes in A549 cell lines
ABSTRACT: Non Small Cell Lung Cancer (NSCLC) causes the premature death of over 1 million people worldwide each year, but remains inadequately understood at the molecular level. To provide new insights for NSCLC treatment we performed a molecular characterisation of wild type and platinum drugs resistance in A549 cells. Transcriptome profiling revealed contrasting patterns of gene expression in sensitive and resistant cells and identified genes whose expression was highly correlated with the platinum drugs. Our results revealed a gene set of 15 transcripts whose expression was highly correlated with platinum-resistance in NSCLC A549 cell lines.
Project description:The transcribed ultraconserved regions (T-UCRs) are a family of long non-coding RNAs implicated in human carcinogenesis. The mechanism of action of T-UCRs and the factors regulating their expression in human cancers are poorly understood. In this study we show that high expression of uc.339 correlates with lower survival in 204 non-small cell lung cancer (NSCLC) patients. We also show that uc.339 found up-regulated in archival NSCLC samples, functions as a decoy for miR-339-3p, -663-3p and -95-5p. As a result, Cyclin E2, a direct target of these microRNAs is up-regulated, promoting cancer growth and migration. We provide evidence from cell lines and primary samples suggesting that TP53 directly regulates uc.339. Our results support a key role for uc.339 in lung cancer. 4 experimental groups in triplicate (total: 12 samples). A339= A549 infected with a lentivirus over-expressing uc.339; AE= A549 infected with an empty lentivirus (control of A339); L339= LoVo infected with a lentivirus over-expressing uc.330; LE= LoVo infected with an empty lentivirus (control of L339). 1-2-3 refer to each experiment.
Project description:We performed a time-course microarray experiment to define the transcriptional response to carboplatin in vitro, and to correlate this with clinical outcome in epithelial ovarian cancer (EOC). RNA was isolated from carboplatin and control-treated 36M2 ovarian cancer cells at several time points, followed by oligonucleotide microarray hybridization. Carboplatin induced changes in gene expression were assessed at the single gene as well as at the pathway level. Clinical validation was performed in publicly available microarray datasets using disease free and overall survival endpoints. Time-course and pathway analyses identified 317 genes and 40 pathways (designated time-course and pathway signatures) deregulated following carboplatin exposure. Both types of signatures were validated in two separate platinum-treated ovarian and NSCLC cell lines using published microarray data. Expression of time-course and pathway signature genes distinguished between patients with unfavorable and favorable survival in two independent ovarian cancer datasets. Among the pathways most highly induced by carboplatin in vitro, the NRF2, NF-kB, and cytokine and inflammatory response pathways were also found to be upregulated prior to chemotherapy exposure in poor prognosis tumors. Experiment Overall Design: we treated the 36M2 cell line with carboplatin 100μM or vehicle-control for 24hrs and cells were harvested and processed for RNA isolation at 24, 30 and 36hrs after treatment.
Project description:PPARγ is a member of the nuclear receptor family for which agonist ligands have anti-growth effects. However, clinical studies using PPARγ ligands as a monotherapy failed to show a beneficial effect. Here we have studied the effects of PPARγ activation with chemotherapeutic agents in current use for specific cancers. We observed a striking synergy between rosiglitazone and platinum-based drugs in several different cancers both in vitro and using transplantable and chemically induced “spontaneous” tumor models. The effect appears to be due in part to PPARγ-mediated downregulation of metallothioneins, proteins that have been shown to be involved in resistance to platinum-based therapy. These data strongly suggest combining PPARγ agonists and platinum-based drugs for the treatment of certain human cancers Experiment Overall Design: Cells were treated with either DMSO/control, rosiglitazone, carboplatin or combination or rosiglitazone and carboplatin in duplicate for 24 hr. RNA was isolated and microarray analysis carried out by the Dana-Farber Cancer Institute Microarray Core.
Project description:Azacitidine (AZA) and decitabine (DAC) are cytidine azanucleoside analogs with clinical activity in myelodysplastic syndromes (MDS) and potential activity in solid tumors. To better understand the mechanism of action of these drugs, we examined the effects of AZA and DAC in a panel of non-small cell lung cancer (NSCLC) cell lines. Of 5 NSCLC lines tested in a cell viability assay, all were sensitive to AZA (EC50 of 1.8–10.5 µM), while only H1299 cells were equally sensitive to DAC (EC50 of 5.1 µM). In the relatively DAC-insensitive cell line A549, both AZA and DAC caused DNA methyltransferase I depletion and DNA hypomethylation; however, only AZA significantly induced markers of DNA damage and apoptosis, suggesting that mechanisms in addition to, or other than, DNA hypomethylation are important for AZA-induced cell death. Cell cycle analysis indicated that AZA induced an accumulation of cells in sub-G1 phase, whereas DAC mainly caused an increase of cells in G2/M. Gene expression analysis of AZA- and DAC-treated cells revealed strikingly different profiles, with many genes distinctly regulated by each drug. In summary, while both AZA and DAC caused DNA hypomethylation, distinct effects were demonstrated on regulation of gene expression, cell cycle, DNA damage, and apoptosis. A549 and H1299 cells were treated with a dose range (0.3–3.0 μM) of AZA or DAC for 48 hours, and effects on gene expression were assessed by microarray analysis.
Project description: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.
Project description:Analysis of dexamethasone-stimulated A549 lung adenocarcinoma epithelial cells treated with a glucocorticoid response (GR) element (GRE) specific DNA binding polyamide. Polyamide designed to target the sequence 5'-WGWWCW-3' and disrupt GR-mediated gene expression. Effects of the GR antagonist mifepristone also examined. Experiment Overall Design: A549 cells were treated with compounds for 48 hours before RNA extraction and hybridization on Affymetrix microarrays.
Project description:Small RNAs were profiled during Sendai virus infection of human A549 cells to identify changes in microRNA abundance during the cellular antiviral response. Examination of microRNA abundance during Sendai virus infection.
Project description:This experiment is designed to study the effects to HMPV on A549 over time Experiment Overall Design: Confluent monolayers of A549 cells were infected with hMPV at MOI of 1 in serum-free media and harvested at 6, 12, 24, 48, or 72 hours post-infection to extract total RNA using RNAqueous®-Midi Kit (Ambion, Austin, TX), according to manufacturer’s instruction;3 Affymetrix HG-U133 plus 2.0 were used for hybridization.
Project description:Small RNAs were profiled during influenza A virus infection of human A549 cells to identify changes in microRNA abundance during the cellular antiviral response. Examination of microRNA abundance during influenza A virus infection.
Project description:Background: Current protocols for the treatment of ovarian cancer include combination chemotherapy with a platinating agent and a taxane. However, many patients experience relapse of their cancer and the development of drug resistance is not uncommon, making successful second line therapy difficult to achieve. The objective of this study was to develop a cell line resistant to both carboplatin and docetaxel (dual drug resistant ovarian cell line A2780CBNDXL), along with single agent resistant lines (docetaxel resistant A2780DXL and carboplatin resistant A2780CBN), to investigate the mechanisms which underlie the development of dual drug resistance. Methods: The A2780 epithelial ovarian cancer cell line was used to select for isogenic carboplatin, docetaxel and dual drug resistant cell lines. A selection method of gradually increasing drug doses was implemented to avoid clonal selection. Resistance was confirmed using a clonogenic assay. Changes in gene expression associated with the development of drug resistance were determined by microarray analysis compared to parental co-culture control A2780CC. Changes in selected genes were validated by QPCR and immunoblotting. Results: Three isogenic cell lines were developed and resistance to each drug or the combination of drugs was confirmed. Development of resistance was accompanied by a reduced growth rate. The microarray and QPCR analyses showed that unique changes in gene expression occurred in the dual drug resistant cell line and that genes known to be involved in resistance could be identified in all cell lines. Conclusions: Novel changes in gene expression can occur in the development of dual drug resistance, indicating that dual drug resistance is not a simple combination of the changes occurring in single agent resistance Carboplatin, docetaxel (GSE26129) and carboplatin/docetaxel dual resistant cell lines of ovarian A2780 were generated for gene expression profilling. Two colour microarray of Agilent whole human genome nucleotide arrays was conducted with two replicate of both forward and reverse labellings for each cell line. Four arrays were used for this experiments. And it was four replicate of both forward and reverse labellings for A2780 cells. Eight arrays were used for this experiments