Inhibition of epigenetic and cell cycle-related targets in glioblastoma cell lines: onametostat reduces proliferation and viability in both normoxic and hypoxic conditions
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ABSTRACT: The objective of the study was to compare the transcriptome of the glioblastoma cell line T98-G following the 48-h treatment with 1 microM onametostat or 0.1% DMSO (negative control) under normoxic or hypoxic conditions. It was found that treatment with onametostat lead to dramatic changes in the transcriptome profile by inducing the cell cycle arrest, suppressing RNA splicing, and down-regulating several major glioblastoma cell survival pathways. The effect of hypoxia was less pronounced.
Project description:Exosomes derived from NSCLC cells under normoxic or hypoxic conditions were compared to identify hypoxic associated enrichment of proteins in exosomes.
Project description:Inhibition of epigenetic and cell cycle-related targets in glioblastoma cell lines: onametostat reduces proliferation and viability in both normoxic and hypoxic conditions
Project description:The choice of targeted therapies for treatment of glioblastoma patients is currently limited, and most glioblastoma patients die from the disease recurrence. Thus, systematic studies in simplified model systems are required to pinpoint the choice of targets for further exploration in clinical settings. Here, we report screening of 5 compounds targeting epigenetic writers or erasers and 6 compounds targeting cell cycle-regulating protein kinases against 3 glioblastoma cell lines following incubation under normoxic or hypoxic conditions. The viability/proliferation assay indicated that PRMT5 inhibitor onametostat was endowed with high potency under both normoxic and hypoxic conditions in cell lines that are strongly MGMT-positive (T98-G), weakly MGMT-positive (U-251 MG), or MGMT-negative (U-87 MG). In U-251 MG and U-87 MG cells, onametostat also affected the spheroid formation at concentrations lower than the currently used chemotherapeutic drug lomustine. In T98-G cell line, treatment with onametostat led to dramatic changes in the transcriptome profile by inducing the cell cycle arrest, suppressing RNA splicing, and down-regulating several major glioblastoma cell survival pathways. Further validation by immunostaining in three cell lines confirmed that onametostat affects cell cycle and causes reduction in nucleolar protein levels. In this way, inhibition of epigenetic targets might represent a viable strategy for glioblastoma treatment even in the case of decreased chemo- and radiation sensitivity, although further studies in clinically more relevant models are required.
Project description:Outcome prediction classifiers were successfully constructed through expression profiling of a total of 1,329 miRNAs in MKN1, gastric cancer cell line under normoxic and hypoxic conditions.
Project description:MM1S cells have been cultured under normoxic and hypoxic conditions, and gene expression profiling has been performed using the Affymetrix Human Genome U133 Plus 2.0 array.
Project description:Outcome prediction classifiers were successfully constructed through expression profiling of a total of 1,329 miRNAs in MKN1, gastric cancer cell line under normoxic and hypoxic conditions. In the study presented here, MKN1 under normoxic and hypoxic conditions was used to acquire expression profiles of a total 1,329 unique miRNAs.
Project description:We used mass spectrometry-based proteomics to unravel anaplastic lymphoma kinase (ALK) signaling in the ALK and MYCN amplified neuroblastoma cell line, NB1. We specifically measured the ALK phosphoproteome upon siRNA depletion of ALK and upon ALK inhibition using the ALK-targeting small-molecule inhibitor lorlatinib. For quantitative phosphoproteomics we used a tandem mass tag (TMT)-based approach. Conditions for the TMT 11-plex setup is specified below. For each siRNA depletion experiment, NB1 cells were treated with siRNA (80 nM; as specified below) for 48 hours prior to stimulation with 0.1% DMSO for 30 minutes. For inhibitor treatment, NB1 cells were treated for 30 minutes with either 10 microM or 10 nM lorlatinib. The experimental treatment conditions and TMT11-plex labeling are specified below: 126: siControl replicate 1, 0.1% DMSO 127N: siControl replicate 2, 0.1% DMSO 127C: siControl replicate 3, 0.1% DMSO 128N: siALK sequence 1, 0.1% DMSO 128C: siALK sequence 2, 0.1% DMSO 129N: siALK mix of sequence 1 and 2, 0.1% DMSO 129C: 10 microM lorlatinib replicate 1 130N: 10 microM lorlatinib replicate 2 130C: 10 microM lorlatinib replicate 3 131N: 10 nM lorlatinib replicate 1 131C: 10 nM lorlatinib replicate 2
Project description:We hypothesize that changes in adrenal gene expression mediate the increased plasma corticosterone and steroidogenesis in rat pups exposed to hypoxia from birth. Experiment Overall Design: In the current study, rat pups (with their dams) were exposed to hypoxia from birth and compared to pups from normoxic dams fed ad libitum. Adrenal glands were collected from normoxic and hypoxic pups at PD7and pooled (N=24/sample; 4 samples per treatment group) for total RNA extraction. Microarray analysis was performed, followed by verification with real-time PCR. Furthermore, the expression of selected genes involved in adrenal function was analyzed by real-time PCR, regardless of microarray results.
Project description:MM1S cells have been cultured under normoxic and hypoxic conditions, and gene expression profiling has been performed using the Affymetrix Human Genome U133 Plus 2.0 array. We used microarrays to dissect the possible changes occurring at the mRNA level in normoxic cells versus hypoxic cells.
Project description:Hypoxia is a low oxygen condition that occurs in the developing tumor mass and that is associated with poor prognosis and resistance to chemo- and radio-therapy. The definition of the hypoxia gene signature is fundamental for the understanding of tumor biology, as in the case of neuroblastoma, the most common pediatric solid tumor. The issue of identifying a significant group of variables in microarray gene expression experiments is particularly difficult due to the typical high dimensional nature of the data and great effort has been spent in the development of feature selection techniques. Our main goal is to define a robust hypoxia gene signature in neuroblastoma cell lines. A set of 11 neuroblastoma cell lines were cultured under normoxic and hypoxic conditions for 18 hours, and their gene expression profiles were measured with Affymetrix GeneChip HG-U133 Plus 2.0. We used the l1-l2 regularization framework in order to select the significant probesets defining hypoxic versus normoxic cell lines.