Identification of genes involved in enhancement of hyperthermia sensitivity by knockdown of BAG3 in human oral squamous cell carcinoma cells
ABSTRACT: Hyperthermia (HT) treatments in combination with either chemotherapy, radiotherapy or both are used for patients with cancer in various organs. However, the acquisition of thermotolerance in cancer cells due to the increase in cytoprotective proteins attenuates the therapeutic effects of HT. BAG3 (BCL2-associated athanogene 3) is a cytoprotective protein that acts against various stresses including heat stress. Recently, we demonstrated that the inhibition of BAG3 improves cell death sensitivity to HT in cancer cells. However, a detailed molecular mechanism involved in the enhancement of HT sensitivity by BAG3-knockdown (KD) in cancer cells is unclear. In this study, to identify genes involved in the enhancement of HT sensitivity by BAG3-knockdown (KD) in cancer cells, global-scale gene expression analysis was carried out using a GeneChip® system. Human oral squamous carcinoma cell HSC-3 cells were treated with a combination of HT (at 44°C for 90 min) and siRNA for BAG3 or luciferase. Total RNA samples were prepared from the cells, and quality of the RNA was analyzed using a Bioanalyzer 2100. Gene expression was analyzed by an Affymetrix GeneChip® system with a Human Genome U133-plus 2.0 array. Sample preparation for array hybridization was carried out as described in the manufacturer’s instructions.
Project description:Hyperthermia (HT) is widely used to treat patients with various cancers. In general, HT elicits a wide spectrum of stress responses, such as induction of heat shock proteins, protein aggregation and cell death in mammalian cells. Although many biological processes are affected by HT, the overall responses to HT in mammalian cells remain unknown. The effects of heat stress at 41°C for 30 min (mild hyperthermia) on the gene expression in human oral squamous cell carcinoma HSC-3 cells were investigated using an Affymetrix GeneChip system. Human oral squamous cell carcinoma HSC-3 cells were treated with heat stress (41°C for 30 min), followed by incubation for 0, 1, or 3 h at 37°C. Non-treated cells served as control. Total RNA samples were prepared from the cells, and quality of the RNA was analyzed using a Bioanalyzer 2100. Gene expression was monitored by an Affymetrix GeneChip® system with a Human Genome U133-Plus 2.0 array. Sample preparation for array hybridization was carried out as described in the manufacturer's instructions.
Project description:Analysis of BRMS1 KD-induced EMT in non-samll cell lung cancer at gene expression level. The hypothesis tested in the present study was that BRMS1 KD induces EMT through differential regulation of EMT genes and Twist1 KD restores the epithelial phenotype in cells with BRMS1 KD. Results provide important information of biological functions in lung cancer which BRMS1 KD involves in, such as EMT, signaling, biological adhesion, immune system process, response to stimulus, and so on. Overall design: Total RNA obtained from NSCLC H1993 cells infected with lentivirus encoding shRNA BRMS1 or shRNA BRMS1/shRNA Twist1, compared to shRNA control sequence. Illumina microarry were performed using Human HT-12 in duplicates.
Project description:HSC-3-5 cells, which drived from human HSC-3 cells via transwell invasion assay was high invasive.Tumor cells were injected into the tongue of SCID mice. After 1 week, transcriptional profiling of human HSC-3 tumor comparing with high invasive HSC-3-5 tumor. Goal was to determine the effects of invasion on global HSC-3-5 gene expression during tumor metastasis. HSC-3 and HSC-3-5 oral cancer cells were respectively injected into the tongue of SCID mice. After 1 weeks, total RNA was respectively extracted from the tumor tissue on tongue of SCID mice for gene expression profiling during metastasis. Briefly, two-condition experiment,HSC-3 vs. HSC-3-5 cells.
Project description:HSC-3-5 cells, which drived from human HSC-3 cells via transwell invasion assay was high invasive.Tumor cells were injected into the flank of SCID mice. After 6 weeks, transcriptional profiling of human HSC-3 tumor comparing with high invasive HSC-3-5 tumor. Goal was to determine the effects of invasion on global HSC-3-5 gene expression during tumor metastasis. HSC-3 and HSC-3-5 oral cancer cells were respectively injected into the flank and tongue of SCID mice. After 1 and 6 weeks, total RNA was respectively extracted from the tumor tissue on tongue and flank of SCID mice for gene expression profiling during metastasis. Briefly, two-condition experiment,HSC-3 vs. HSC-3-5 cells.
Project description:Achaete scute-like 2 (Ascl2), a basic helix-loop-helix (bHLH) transcription factor, controls the fate of intestinal stem cells. However, the role of Ascl2 in colon cancer progenitor cells remains unknown. The cell lines HT-29 (47.5-95% of CD133+ population) and LS174T (0.45% of CD133+ population) were chosen for functional evaluation of Ascl2 in colon cancer progenitor cells after gene knockdown by RNA interference. The microRNA (miRNA) microarrays identified 26 two-fold up-regulated miRNAs and 58 two-fold down-regulated miRNAs in shRNA-Ascl2/HT-29 cells and 178 two-fold up-regulated miRNAs and 172 two-fold down-regulated miRNAs in shRNA-Ascl2/LS174T cells. Two-condition experiment: shRNA-Ascl2/HT-29 cells vs. shRNA-Ctr/HT-29 cells, and shRNA-Ascl2/LS174T cells vs. shRNA-Ctr/LS174T cells. Biological replicates: 1 HT-29 cells stably transfected with shRNA-Ascl2/EGFP, 1 LS174T cells stably transfected with shRNA-Ascl2/EGFP, 1 HT-29 cells stably transfected with shRNA-Control/EGFP, and 1 LS174T cells stably transfected with shRNA-Control/EGFP, independently grown and harvested. One replicate per array.
Project description:The application of ketogenic diet (KD) (high fat/low carbohydrate/adequate protein) as an auxiliary cancer therapy is a field of growing attention. KD provides sufficient energy supply for healthy cells, while possibly impairing energy production in highly glycolytic tumor cells. Moreover, KD regulates insulin and tumor related growth factors (like insulin growth factor-1, IGF-1). In order to provide molecular evidence for the proposed additional inhibition of tumor growth when combining chemotherapy with KD, we applied untargeted quantitative metabolome analysis on a spontaneous breast cancer xenograft mouse model, using MDA-MB-468 cells. Healthy mice and mice bearing breast cancer xenografts and receiving cyclophosphamide chemotherapy were compared after treatment with control diet and KD. Metabolomic profiling was performed on plasma samples, applying high-performance liquid chromatography coupled to tandem mass spectrometry. Statistical analysis revealed metabolic fingerprints comprising numerous significantly regulated features in the group of mice bearing breast cancer. This fingerprint disappeared after treatment with KD, resulting in recovery to the metabolic status observed in healthy mice receiving control diet. Moreover, amino acid metabolism as well as fatty acid transport were found to be affected by both the tumor and the applied KD. Our results provide clear evidence of a significant molecular effect of adjuvant KD in the context of tumor growth inhibition and suggest additional mechanisms of tumor suppression beyond the proposed constrain in energy supply of tumor cells.
Project description:CD24 is the one of cell surface protein that anchored via glycosyl phosphatidylinositol that links to cell surface and modulates growth and differentiation signal many of cell types. A small population of cells, namely ‘Cancer Stem Cells (CSCs)’, charges highly aggressive and metastatic character of cancer cells and shows conformational change of ‘epithelial to mesenchymal transition (EMT). To understand the role of CD24 in CSC and EMT, CD24 was knocked down using siRNA in the MCF7 cell line (MCF-7 hCD24 KD) and analyzed transcriptome profiles by mRNA-sequencing. Corresponding author: Chul Geun Kim, Department of Life Science, Hanyang University, Seoul 133-791, Korea (e-mail, email@example.com). mRNA profiles of MCF-7 hCD24 KD cells [‘monolayer (m) cultured MCF7 wild type (WT) and mMCF7 CD24KD’ and ‘sphere-forming (s) MCF7 WT and sMCF7 CD24KD’] were generated by deep sequencing using Illumina GAIIx.
Project description:The cancer stem cell model maintains that tumors are organized in a hierarchy driven by tumor initiating cells (TICs), and that patient survival inversely correlates with TIC gene expression. Here we generated a prognostic signature for HER2+ breast cancer from TICs purified from MMTV-Her2/Neu mammary tumors. TICs from this model, identified as Lin-:CD24+:JAG1- at a frequency of 2-5% by serial and single cell transplantation assays, showed elevated expression of proliferation genes and low expression of differentiation genes (compared to non-TIC fraction CD24- of the same tumor). We used microarrays to detect differentially expressed genes in the TIC fraction compared to the non-TIC fraction of the same tumor Cells from each primary tumor were separated by FACS into TIC and non-TIC fractions and total RNA was extracted and hybridized on Affymetrix microarrays.
Project description:A mutant simian immunodeficiency (SIVmac239) virus, found to be selected within chronically SIV-infected Burmese rhesus monkeys with relatively enhanced SIV-specific antibody responses, was reconstituted as a molecular clone. The virus (SIV Nef G63E) was then subjected to a preliminary analysis for their intracellular signal transduction and gene expression modulation patterns (as compared with wild type SIVmac239) within infected CD4+ T cells. Analysis implicated that the mutant virus had a moderately enhanced cytopathic phenotype. A SIV mutation (SIVmac239 Nef G63E) found to be enriched in rhesus monkeys with enhanced SIV-specific antibody responses was reconstituted on a pBR-based SIV molecular clone pBRmac239. HSC-F cynomolgus macaque central memory Th2-like T cells were infected with mutant and wild type SIVmac239 at MOI 5 in triplicate along with uninfected controls (i.e. a total of 9 samples) for 24 hours and subjected to analysis of their gene expression patterns.
Project description:The level of trypsin-2 has been shown to correlate with the malignancy and metastatic potential of many cancer. We used microarrays to detail how tryspin-2 overexpression changes the gene profile of human tongue squamous cell carcinoma cell line (HSC-3). We compared trypsin-2 overexpressing HSC-3 line to HSC-3 which was transfected with control vector and because of that did not expressed trypsin-2 at all.