Transcription profiling by array of the effect of hypoxia in tumour-initiating cells derived from a colorectal cancer patient
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ABSTRACT: Transcriptome profiling of tumour-initiating cells was performed under normoxia and hypoxia conditions for two time points: 3 and 7 days. The cell culture was established from fresh patient material (colorectal tumour) and maintained in spheroid culture condition.
Project description:The aim of this study is to characterize generated stable spheroid cultures (SC) directly derived from fresh surgical specimens of three patients and compare them to their differentiated counterpart as well as to SC derived from established colorectal cancer cell lines HT-29 and HCT-116.
Project description:Drug target identification is a critical step towards the understanding of the mechanism of action of a drug, which will help to improve the current therapeutic regime and to expand the drug’s therapeutic potential. However, current in vitro affinity chromatography-based and in vivo activity- based protein profiling (ABPP) approaches generally face difficulties discriminating specific drug targets from non-specific ones. Here we describe a novel approach combining isobaric tag for relative and absolute quantitation (iTRAQ) with Clickable ABPP, named ICABPP, to specifically and comprehensively identify the protein targets of andrographolide (Andro), a natural product with known anti-inflammation and anti-cancer effects, in live cancer cells. We identified a spectrum of specific targets of Andro, which furthered our understanding of the mechanism of action of the drug. We found that Andro has a potential novel application as the tumor metastasis inhibitor, which was validated through cell migration and invasion assays. Moreover, we have unveiled the target binding mechanism of Andro with a combination of drug analogue synthesis, protein engineering and mass spectrometry-based approaches and determined the drug-binding sites of two protein targets, NF-kappaB and actin.
Project description:Introduction: Breast radiotherapy is currently â??one size fits allâ?? regardless of breast cancer subtype (eg. luminal, basal). However, recent clinical data suggests that radiation response may vary significantly among subtypes. Therefore, current practice leads to over- or under-treatment of women whose tumors are more or less radiation responsive. We hypothesized that this clinical variability may be due, in part, to differences in cellular radiation response. Methods: We exposed 16 biologically-diverse breast tumor cell lines to 0 or 5GY radiation. Microarray analysis was performed on RNA harvested from those cell lines. Samples were run in triplicate. Following quality assessment, differential gene expression analysis was performed using a two-way multiplicative linear mixed-effects model. A candidate radiation response biomarkers with biologically plausible role in radiation response, were identified and confirmed at the RNA and protein level with qPCR and Western blotting assays. Induction in human breast tumors was confirmed in 32 patients with paired pre- and post-radiation tumor samples using IHC and microarray analysis. Quantification of protein was performed in a blinded manner and included positive and negative controls. The objective of our study was to identify genomic determinants of radiation sensitivity using clinical samples as well as breast tumor cell lines. In order to identify differences in the radiation response gene expression profiles of specific breast cancer subtypes, we exposed 16 biologically-diverse breast tumor cell lines to 0 or 5GY radiation. Microarray analysis was performed on RNA harvested from those cell lines. Samples were run in triplicate. Following quality assessment, differential gene expression analysis was performed using a two-way multiplicative linear mixed-effects model. Candidate radiation response biomarker with a biologically plausible role in radiation response, were identified and confirmed at the RNA and protein level with qPCR and Western blotting assays. Induction of the genes of interest were further evaluated and confirmed in human breast tumors in 32 breast cancer patients with paired pre- and post-radiation tumor samples using IHC and microarray analysis assays.
Project description:Increasing evidence supports the existence of a subpopulation of cancer cells capable of self-renewal and differentiation into diverse cell lineages. These cancer stem-like or cancer initiating cells (CICs) also demonstrate resistance to chemo- and radiotherapy and may function as a primary source of cancer recurrence. We report here on the isolation and in vitro propagation of multicellular ovarian cancer spheroids from a well-established ovarian cancer cell line (OVCAR-3). Cells forming these spheroids display self-renewal potential, the ability to produce differentiated progeny and increased expression of genes previously associated with CICs. The spheroid-derived cells (SDCs) also demonstrate higher invasiveness, migration potential and enhanced resistance to standard anticancer agents relative to progenitor OVCAR-3 cells. SDCs display up-regulation of genes associated with epithelial-to-mesenchymal transitions (EMT), anticancer drug resistance and/or decreased susceptibility to apoptosis, as well as, down-regulation of genes typically associated with the epithelial cell phenotype and pro-apoptotic genes. Dataset includes 3 replicate cultures of parental OVCAR-3 cells and 3 replicate cultures of stem cell-like spheroid-derived cells
Project description:Although fibrosis depicts a reparative mechanism, maladaptation of the heart due to excessive production of extracellular matrix accelerates cardiac dysfunction. The anthraquinone Rhein was examined for its anti-fibrotic potency to mitigate cardiac fibroblast-to-myofibroblast transition (FMT). Primary human ventricular cardiac fibroblasts were subjected to hypoxia and characterized with proteomics, transcriptomics and cell functional techniques. Knowledge based analyses of the omics data revealed a modulation of fibrosis-associated pathways and cell cycle due to Rhein administration during hypoxia, whereas p53 and p21 were identified as upstream regulators involved in the manifestation of cardiac fibroblast phenotypes. Mechanistically, Rhein-mediated cellular effects were linked to the histone deacetylase (HDAC)-dependent acetylation status of p53 a posttranslational modification that acts protein stabilizing. Direct enzymatic testing revealed an inhibitory potency of Rhein for HDAC classes I/II. Functionally, Rhein inhibited collagen contraction in response to protein abundance of SMAD7, thus demonstrating its anti-fibrotic property in cardiac remodeling. In conclusion, this study identifies Rhein as a novel potent HDAC inhibitor and provides evidence that Rhein may contribute to the treatment of cardiac fibrosis as anti-fibrotic agent. As readily available drug with approved safety, repurposing of Rhein constitutes a promising potential therapeutic approach in the supplemental and protective intervention of cardiac fibrosis.
Project description:Senescence can be transmitted in a paracrine way from cells undergoing Oncogene Induced Senescence (OIS) to naM-CM-/ve normal cells. We define this phenomenon as M-bM-^@M-^\paracrine senescenceM-bM-^@M-^] We used microarrays to compare the trancriptome of cells undergoing paracrine senescence to the transcriptome of cells suffering OIS to unveil the common signatures defining both events and the similarities between them IMR90 cells were co-cultured with IMR90 ER:RAS undergoing OIS or IMR90-Vector control cells using 0.2 M-NM-<m pore transwell (anopore) to allow communication of soluble factors but physical separation of the two cell populations. The total mRNA of IMR90, IMR90 ER:RAS or IMR90 cells cultured in Transwells together with IMR90 vector or IMR90 ER:RAS cells during 7 days in the presence of 200 nM 4OHT and 0.5 % FBS was extracted and hybridized on Affymetrix microarrays to compare paracrine senescence to OIS.
Project description:OVDM1, a new ovarian cell line derived from a serous ovarian metastatic adenocarcinoma sample, was established by immortalization with SV40 large-T antigen and hTERT and then characterized Expression microarrays were used for molecular profiling of early and late passages of OVDM1 cell line, as well as the original metastatic tumor. Total RNA was extracted from early and late passages of OVDM1 and from the metastatic tumor for hybridization on Affymetrix microarrays
Project description:Tendon is a hypocellular tissue that contains functional cable-like units of type I collagen responsible for the transmission of force from muscle to bone. In the setting of injury or disease, patients can develop chronic tendinopathies that are characterized by pain, loss of function and persistent inflammatory changes that are often difficult to treat. Platelet-rich plasma (PRP) has shown promise in the treatment of chronic tendinopathy, but little is known about the mechanisms by which PRP can improve tendon healing. PRP contains many different growth factors and cytokines, and since these proteins can both activate and inhibit various signaling pathways it has been challenging to determine precisely which signaling pathways and cellular responses are most important. Using state-of-the-art bioinformatics tools and genome wide-expression profiling, the purpose of this study was to determine the signaling pathways activated within cultured tendon fibroblasts in response to PRP treatment. Tendon fibroblasts were isolated from rat tail tendons and embedded in 3D type I collagen gels. Cells were treated with PRP or PPP for 24 hours, and total RNA was extracted for hybridization on Affymetrix arrays.