ABSTRACT: Transcription profiling of human A549 lung cancer cells treated with actinomycin D and sapphyrin PCI-2050 a new class of tumor-selective inhibitors of gene expression.
Project description:Non-small cell lung cancer (NSCLC) is the most lethal and prevalent type of lung cancer. In almost all types of cancer, the levels of polyamines (putrescine, spermidine, and spermine) are increased, playing a pivotal role in tumor proliferation. Indomethacin, a non-steroidal anti-inflammatory drug, increases the abundance of an enzyme termed spermidine/spermine-N1-acetyltransferase (SSAT) encoded by the SAT1 gene. This enzyme is a key player in the export of polyamines from the cell. The aim of this study was to compare the effect of indomethacin on two NSCLC cell lines, and their combinatory potential with polyamine-inhibitor drugs in NSCLC cell lines. A549 and H1299 NSCLC cells were exposed to indomethacin and evaluations included SAT1 expression, SSAT levels, and the metabolic status of cells. Moreover, the difference in polyamine synthesis enzymes among these cell lines as well as the synergistic effect of indomethacin and chemical inhibitors of the polyamine pathway enzymes on cell viability were investigated. Indomethacin increased the expression of SAT1 and levels of SSAT in both cell lines. In A549 cells, it significantly reduced the levels of putrescine and spermidine. However, in H1299 cells, the impact of treatment on the polyamine pathway was insignificant. Also, the metabolic features upstream of the polyamine pathway (i.e., ornithine and methionine) were increased. In A549 cells, the increase of ornithine correlated with the increase of several metabolites involved in the urea cycle. Evaluation of the levels of the polyamine synthesis enzymes showed that ornithine decarboxylase is increased in A549 cells, whereas S-adenosylmethionine-decarboxylase and polyamine oxidase are increased in H1299 cells. This observation correlated with relative resistance to polyamine synthesis inhibitors eflornithine and SAM486 (inhibitors of ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase, respectively), and MDL72527 (inhibitor of polyamine oxidase and spermine oxidase). Finally, indomethacin demonstrated a synergistic effect with MDL72527 in A549 cells and SAM486 in H1299 cells. Collectively, these results indicate that indomethacin alters polyamine metabolism in NSCLC cells and enhances the effect of polyamine synthesis inhibitors, such as MDL72527 or SAM486. However, this effect varies depending on the basal metabolic fingerprint of each type of cancer cell.
Project description:Background: Lung cancer is the leading cause of cancer related death worldwide. Over the past 15 years no major improvement of survival rates could be accomplished. The recently discovered histone methyltransferase KMT9 as epigenetic regulator of prostate tumor growth has now raised hopes of enabling new cancer therapies. In this study we aimed to identify the function of KMT9 in lung cancer which has remained elusive so far. Methods: We linked full transcriptome and proteome analyses of A549 lung adenocarcinoma cells using RNA-Seq and mass spectrometry with functional cell culture, real-time proliferation and flow cytometry assays. Results: KMT9 is expressed in lung cancer tissue and cell lineswith high levels of KMT9 correlating with poor patient survival. We identified 460 overlapping genes and proteins that are deregulated upon knock-down of KMT9alpha in A549 cells. These genes cluster with proliferation, cell cycle and cell death gene sets as well as with subcellular organelles in gene ontology analysis. Knock-down of KMT9alpha inhibits lung cancer cell proliferation and induces non-apoptotic cell death in A549 cells. Conclusions: The novel histone methyltransferase KMT9 is crucial for proliferation and survival of lung cancer cells harboring various mutations. Small molecule inhibitors targeting KMT9 therefore should be further examined as potential milestones in modern epigenetic lung cancer therapy.
Project description:An orthotopic model of lung cancer was established in Rag-2 null mice by injecting 1 x 106 GFP-A549 cells into the mouse lung via the trachea. After 6 weeks a primary tumor develops in the right lung. RNA was collected from tumors established in the orthotopic model after 6 weeks in three separate mice and from the right lung of three sham control mice that were injected with PBS by the same method. RNA was also collected from the A549 cells in culture. RNA was applied to an Affymetrix Customized Protease Microarray (HuMu ProtIn chip). This custom chip is able to distinguish human and mouse genes by using oligonucleotides specific for each species. The chip contains all known human and mouse proteases and protease inhibitors. A distribution of gene expression from a comparison of all human and mouse genes present in the tumor vs. the control lung was indentified as well as a distribution of gene expression from a comparison of all human and mouse genes present in the tumor vs. the cells in culture. Human and mouse genes differentially expressed in each group were identifed.
Project description:Strongly induction of FOSB was observed in a cationic anticancer peptide TP4 treated human A549 lung cancer cell-line, causing obvious cell death. This work aims to address the gene expression profiling upon FOSB overexpression in A549 cell.
Project description:Tumor microenvironment plays an important role in regulating cell growth and metastasis. Recently we developed an ex vivo lung cancer model (4D) that forms perfusable tumor nodules on a lung matrix that mimics human lung cancer histopathology and protease secretion pattern. We compared the gene expression profile (Human OneArray v5 chip) of A549 cells, a human lung cancer cell line, grown on petri dish (2D), and of the same cells grown in the matrix of our ex vivo model (4D). Furthermore, we obtained gene expression data of A549 cells grown on petri dish (2D) and matrigel (3D) from a previous study and compared the 3D expression profile with that of 4D. Expression array analysis showed 2954 genes differentially expressed between 2D and 4D. Gene Ontology (GO) analysis showed up-regulation of several genes associated with extracellular matrix, polarity, and cell fate and development. Moreover, expression array analysis of 2D versus 3D showed 269 genes that were most differentially expressed, with only 35 genes (13%) having similar expression patterns as observed between 2D and 4D. Finally, the differential gene expression signature of 4D cells (versus 2D) correlated significantly with poor survival in patients with lung cancer (n=1492), while the expression signature of 3D versus 2D correlated with better survival in lung cancer patients. Since patients with larger tumors tend to have worse survival, the ex vivo 4D model may offer additional features over the 3D model, to better mimic of natural progression of tumor growth in lung cancer patients. We compared the gene expression profile (Human OneArray v5 chip) of A549 cells, human lung cancer cell line, grown on petri dish (2D) and same cells grown in the matrix of our ex vivo model (4D).
Project description:CTCs in cancer patients are thought to be responsible for metastasis. Currently, there is no ex vivo model that can isolate this group of cells. We have developed an ex vivo 4D lung cancer model that forms perfusable tumor nodules and form CTCs. Gene array analyses show 2504 differentially expressed genes when comparing CTCs from the 4D model seeded with A549 cells to the same cells grown on a petri dish (2D). We compared the gene expression profile (Human OneArray v5 chip) of A549 cells, human lung cancer cell line, grown on petri dish (2D) and same cells circulating as tumor cells in our ex vivo model (4D/CTC).
Project description:Ferroptosis is a new type of programmed cell death induced by iron-dependent lipid peroxidation that has been linked to several malignancies, including non-small cell lung cancer (NSCLC). Finding ferroptosis-associated genes is extremely helpful for guiding and improving ferroptosis-based anti-tumor therapy. To explore ferroptosis-associated genes, we treated A549 cells with DMSO, RSL3 alone, or co-treated with Fer-1 for 24h. Lung cancer-associated transcript 1 (LUCAT1) was identified as a novel ferroptosis suppressor via RNA-seq analysis.
Project description:The goal of this project was to characterize changes in gene expression in response to the anti-cancer agent sapphyrin PCI-2050. Cultured A549 human lung cancer cells were treated with sapphyrin PCI-2050 or actinomycin D, a known transcripitonal inhibitor. The gene expression profiles of drug-treated and control A549 cultures were determined using Human Genome U133 Plus 2.0 Arrays (Affymetrix, Santa Clara, CA). Further details are provided in our published manuscript: <http://www.molecular-cancer.com/content/6/1/9>. Keywords: responses to treatments with anti-cancer agents
Project description:Immune checkpoint inhibitors are increasingly used in combination with chemotherapy for treatment of non-small cell lung cancer, yet the success of combination therapies is relatively limited. Thus, more detailed insight regarding the tumour molecular markers that may affect the responsiveness of patients to therapy is required. Here, we set out to explore the proteome of two lung adenocarcinoma cell lines (HCC-44 and A549) treated with cisplatin, pemetrexed, durvalumab, and the corresponding mixtures to establish the differences in post-treatment protein expression that can serve as markers of chemosensitivity or resistance.
Project description:To assist in the identification of proteases, their endogenous inhibitors, and proteins that interact with proteases or proteolytic pathways in biological tissues, a dual species oligonucleotide microarray has been developed in conjunction with Affymetrix, Inc. To investigate the performance of the array, gene expression profiles were analyzed in pure mouse and human samples and orthotopically implanted xenografts of human A549 lung and MDA-MB-231 breast carcinomas. The Hu/Mu ProtIn array is a valuable discovery tool for the identification of components of human and murine proteolytic pathways in health and disease, and has particular utility in the determination of cellular origins of proteases and protease inhibitors in xenograft models of human cancer. Keywords: gene expression profiles were analyzed in pure mouse and human samples and orthotopically implanted xenografts of human A549 lung and MDA-MB-231 breast carcinomas Commercial pan human and mouse total RNA replicate samples (n=3) were profiled. In addition, human ductal stage II or III breast carcinoma biopsies (n=18) were compared to normal human breast biopsies (n=13). Spontaneous spontaneous murine mammary carcinomas from MMTV-PyMT+(FVB/n) (n=8) and MMTV-PyMT+/uPARAP-/-(FVB/n) (n=2) transgenic mice were compared to normal mouse mammary fat pads (n=4; female Rag-1 tm1Mom retired breeders at >6 weeks post lactation). In vitro cultures of MDA-MB-231 human breast carcinoma cells (n=3) and MDA-MB-231 orthotopic xenografts (n=3) were profiled. The xenografts were compared to normal mammary fat pads. A549 human lung adenocarcinoma cells (n=2), A549 orthotopic xenografts (n=3) and normal mouse lung tissue (n = 3) were profiled.