Microarray analysis of MCF-7 and MDA-MB-231 breast cancer cells treated or not with phytoestrogen Emodin
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ABSTRACT: Estrogen receptor positive MCF-7 and estrogen receptor negative MDA-MB-231 breast cancer cells treated with 10µg/ml phytoestrogen Emodin (1,3,8-trihydroxy-6-methylanthraquinone) and their control cells (untreated with Emodin) were incubated for 48 hours. All samples were prepared as duplicates to have biological replicates. Isolated RNA samples were used for microarray analysis. Through this experiment gene expression profiles of breast cancer cells having estrogen receptor and not were revealed upon phytoestrogen Emodin treatment.
Project description:Estrogen receptor positive MCF-7 and estrogen receptor negative MDA-MB-231 breast cancer cells treated with 10µg/ml phytoestrogen Emodin (1,3,8-trihydroxy-6-methylanthraquinone) and their control cells (untreated with Emodin) were incubated for 48 hours. All samples were prepared as duplicates to have biological replicates. Isolated RNA samples were used for miRNA microarray analysis. Through this experiment miRNA profiling of breast cancer cells having estrogen receptor and not were revealed upon phytoestrogen Emodin treatment.
Project description:We used gene expression profiling to investigate whether the molecular effects induced by estrogens of different provenance are intrinsically similar. In this article we show that the physiologic estrogen 17-beta-estradiol, the phytoestrogen genistein, and the synthetic estrogen diethylstilbestrol alter the expression of the same 179 genes in the intact immature mouse uterus under conditions where each chemical has produced an equivalent gravimetric and histologic uterotrophic effect, using the standard 3-day assay protocol. Data are also presented indicating the limitations associated with comparison of gene expression profiles for different chemicals at times before the uterotrophic effects are fully realized. We conclude that the case has yet to be made for regarding synthetic estrogens as presenting a unique human hazard compared with phytoestrogens and physiologic estrogens. Key words: diethylstilbestrol, estrogen, gene expression, genistein, microarray, phytoestrogen, toxicogenomics, uterus.
Project description:By silencing of RALA, a downstream member of the RAS signal transduction pathway, we aimed to determine whether genes downstream of a mutated KRAS (codon 12 or 13) or a mutated BRAF can have significant functions in colorectal cancer carcinogenesis. RALA was silenced in three colorectal cancer cell lines (SW480, HCT116 and HT29). Effects were normalized to mock-transfected cells and the effects of scramble siRNA were excluded. SW480, HCT116 and HT29 cell lines were treated with the PI3K inhibitor LY294002 or DMSO.
Project description:Inhibitor of apoptosis (IAP) proteins are expressed at high levels in CLL cells and may contribute to evasion of cell death leading to poor therapeutic outcome. Of note, prognostic unfavourable cases with e.g. non-mutated VH-status and TP53 mutation responded significantly better to BV6 than samples with unknown or favourable prognosis e.g. 13q deletion. The majority of cases with 17p deletion (10/12) and Fludarabine refractory cases were sensitive to BV6, indicating that BV6 acts independently of the p53 pathway. Importantly, BV6 dose-dependently induced cell death in 28 of 51 (54%) investigated patient samples while B cells from healthy donors were largely unaffected. BV6 also triggered cell death under survival conditions mimicking the microenvironment e.g. by adding CD40 ligand or in conditioned medium. Gene expression profiling identified cell death- and NF-kB-signaling among the top pathways regulated by BV6. This was confirmed by data showing that BV6 causes degradation of cIAP1 and cIAP2 and NF-kB pathway activation. BV6 induced cell death depended on production of reactive oxygen species, since addition of ROS scavengers significantly rescued BV6-triggerd cell death. In contrast, BV6 induced cell death independently of caspase activity, RIP1 activity or TNF-alpha, since zVAD.fmk, necrostatin-1 or TNF-alpha-blocking antibody Enbrel failed to protect against cell death. Of note, transcripts of ROS regulatory proteins were modulated by BV6. Thus, these data have important implications for developing new therapeutic strategies to overcome cell death resistance in CLL especially in poor prognostic subgroups. Gene expression was profiled in 12 CLL samples [n=3 CLL cases for 4 hours and 20 hours with either DMSO or BV6].
Project description:Bromodomain and extra terminal domain (BET) inhibition reduces occupancy of BET-family proteins at promoter and enhancer sites resulting in changes in the transcription of specific genes. We used microarray profiling to investigate the transcriptional changes induced by BET inhibitor JQ1 treatment in DV90 cells to identify the underlying changes of gene regulation that lead to JQ1 sensitivity. DV90 cells (JQ1 sensitive non-small cell lung cancer cell line) were treated with 135 nM (IC50) or 785 nM (IC90) of JQ1 for 4h and 24h. DMSO treated controls served as reference and at least four replicates per condition were collected. RNA was extracted and hybridized to Affymetrix HuGene-2.1ST microarrays to identify treatment induced transcriptional changes.
Project description:Cadmium sulfide quantum dots (CdS QDs) are widely used in novel equipment. The relevance of the research lies in the need to develop risk assessments for nanomaterials, using as basis a model plant species. Here a screen of Arabidopsis thaliana mutant lines was performed in an attempt to identify plants tolerant to CdS QDs. Two tolerant Ds insertion mutant lines (atnp01 and atnp02) were identified. A whole-genome microarray experiment showed how genes were regulated by CdS QDs. Most of the genes involved in the response to CdS QDs were related to detoxification and general metabolism. The two mutant lines treated with CdS QDs showed different patterns of gene expression. The genome-wide expression profile of seedlings of the two selected resistant mutants were acquired and compared to that of wild type seedlings using the Arabidopsis ATH1 Genome Array. The wild type seedlings were exposed to either 0 mg/L, 40 mg/L (1/2 MIC) or 80 mg/L (MIC wt) CdS QDs, and each of the resistant/tolerant mutant line plants to either 0 mg/L or 80 mg/L CdS QDs for 21 days.
Project description:We used microarrays to detail the global transcriptional response mediated by ERalpha or ERbeta to the phytoestrogen genistein in the MCF-7 human breast cancer cell model. Experiment Overall Design: MCF-7 human breast cancer cells expressing endogenouse Estrogen Receptor Alpha (ERalpha) were infected with adenovirus carrying either estrogen receptor beta (AdERb) or no insert (Ad) at multiplicity of infection (moi) of 20. Cells were then treated with either vehicle control (veh), 6nM 17beta-estradiol (E2), 6nM genistein (LG), 300nM genistein (HG), 300nM S-Equol (EQ), HG+3uM ICI182,780 (IG), EQ+3uM ICI 182,780(IE) for a additional periods of 4h or 24hr before RNA extraction and hybridization on Affymetrix microarrays. We sought to determine if genistein and S-Equol, phytoestrogens selective for the ERbeta can elicit transcriptional response distinctive from those mediated by the ERalpha.
Project description:Background: Global gene expression profiling has been widely used in lung cancer research to identify clinically relevant molecular subtypes as well as to predict prognosis and therapy response. So far, the value of these multi-gene signatures in clinical practice is unclear and the biological importance of individual genes is difficult to assess as the published signatures virtually do not overlap. Methods: Here we describe a novel single institute cohort, including 196 non-small lung cancer (NSCLC) cases with clinical information and long-term follow-up, which was used as a training set to screen for single genes with prognostic impact. The top 450 gene probe sets identified using a univariate Cox regression model (significance level p<0.01) were tested in a meta-analysis including five publicly available independent lung cancer cohorts (n=860). Results: The meta-analysis revealed that 17 probe sets were significantly associated with survival (p<0.0005) with a false discovery rate of 1%. The prognostic impact of one of these genes, the cell adhesion molecule 1 (CADM1), was confirmed by use of immunohistochemistry on a tissue microarray including 355 NSCLC samples. Low CADM1 protein expression was associated with shorter survival (p=0.028), with particular influence in the adenocarcinoma patient subgroup (p=0.002). Conclusions: We were able to validate single genes with independent prognostic impact using a novel NSCLC cohort together with a meta-analysis approach. CADM1 was identified as an immunohistochemical marker with a potential application in clinical diagnostics. Fresh frozen tissue of 196 consecutive NSCLC patients, operated between 1995 and 2005 were analyzed using Affymetrix microarrays HG-U133-Plus2. Clinical data were retrieved from the regional lung cancer registry.
Project description:To comparatively analyze the transcriptional responses of yeast cells to the presence of rapamycin or caffeine the homozygous hoΔ/hoΔ strain of S. cerevisiae BY4743 were grown in fully controlled fermenters in the presence of 200 nM rapamycin or 5 mM caffeine. Cells were cultured overnight in YPD medium (2% [w/v] D-glucose, 2% [w/v] peptone, 1% [w/v] yeast extract) at 30°C in an orbital shaker at 180 rpm prior to the fermentations. For the cultivations in the fermenters defined synthetic medium was used. The batch cultivations were carried out in duplicates in 2L B-Braun Biostat B Plus fermenters with 1.5L working volume kept at 30°C with the rate of agitation at 800rpm. pH was controlled at 5.5 with 0.5M NaOH and HCl. Sampling was carried out at the mid-exponential phase of growth at an OD range of 0.6-0.8. Samples harvested for the transcriptome analysis were immediately frozen in liquid nitrogen and were stored at -80oC until RNA isolation.
Project description:Hypothesis: Non-small cell lung cancer (NSCLC) is characterized by a multitude of genetic aberrations with unknown clinical impact. In this study, we aimed to identify gene copy number changes that correlate with clinical outcome in NSCLC. To maximize the chance to identify clinically relevant events, we applied a strategy involving two prognostically extreme patient groups. Results: Genetic aberrations were strongly associated with tumor histology. In adenocarcinoma (n=50), gene copy number gains on chromosome 8q21-q24.3 (177 genes) were more frequent in long-term survivors. In squamous cell carcinoma (n=28), gains on chromosome 14q23.1-24.3 (133 genes) were associated with shorter survival, whereas losses in a neighboring region, 14q31.1-32.33 (110 genes), correlated with favorable outcome. In accordance with copy number gains and losses, mRNA expression levels of corresponding genes were increased or decreased, respectively. Conclusion: Comprehensive tumor profiling permits the integration of genomic, histologic and clinical data. We identified gene copy number gains and losses, with corresponding changes in mRNA levels, that were associated with prognosis in adenocarcinoma and squamous cell carcinoma of the lung. Short-term (<20 months; n=53) and long-term survivors (>58 months;n=47) were selected from a clinically well-characterized NSCLC patient cohort with available fresh-frozen tumor specimens. The samples were analyzed using high-resolution SNP-array technology. The molecular data was combined with information on clinical parameters.