Whole genome expression microarray of cancer stem cells isolated from HT29 and HCT116 Human colorectal cancer cell lines
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ABSTRACT: In the present study we have isolated and characterized cancer stem cells and non-cancer stem cells (bulk tumor cells) from high grade human colorectal cancer cell line HCT116 and low grade human colorectal cancer cell line HT29. For this study, cancer stem cells and non-cancer stem cells (bulk tumor cells) were isolated from HCT116 and HT29 human colorectal cancer cell line. For isolating cancer stem cells by FACS, CD44 and CD166 tagged with V450 and PE respectively were used. CD44+CD166+ was the cancer stem cell population and CD44-CD166- was designated as the non-cancer stem cell (bulk tumor cells) population for this study. RNA was isolated from the isolated cell populations and microarray was done to study the whole genome transcriptomic changes.
Project description:This study was designed to understand the transcriptomic composition and the biological functions of cancer stem cells isolated from non-small cell lung cancer line (NSCLC) Putative lung cancer stem cells were isolated from cancer cell lines based on expression of known stem cell surface markers: CD166, CD44 and EpCAM using the Fluorescence Activated Cell Sorter (FACS).
Project description:Metformin is a drug used in the treatment of type 2 diabetes mellitus. Various studies have elucidated its anticancer properties. In this study, the effect of metformin on the differentiation and tumor microenvironment of colorectal cancer cells (CRC) was evaluated. For our study, we have used HCT116 colorectal cancer cell line and treated the cells with Metformin. Maximum tolerable non-toxic dose of metformin on HCT116 cells was determined by MTT assay. Cells were treated with 2.5 mM Metformin for 2 weeks. Analysis of apoptosis was done by flow cytometry using Annexin V / PI. CSC population was determined by flow cytometry using CSC markers CD44 and CD166. Metformin's ability to induce differentiation in CSC was assessed by analyzing Cytokeratin 20 (CK20) by flow cytometry and CDX1 (transcription factor for CK20), by RT-QPCR. Expression of Ki67 (proliferation marker) was done by RT-QPCR. RNA was isolated from 2.5 mM Metformin-treated and untreated cells populations. Microarray of untreated and 2.5 mM Metformin-treated RNA was done to study the whole genome transcriptomic changes.
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:Cancer stem cells (CSCs) are pluripotent cells that have been implicated in tumor proliferation, metastatic dissemination, and recurrence of various cancers. The prognosis of colorectal cancer, which often presents late due to absence of early symptoms, depends on the stage at presentation. In the present study we have isolated and characterized cancer stem cells from colorectal cancer tissues and normal stem cells from normal colon tissues. Only sporadic colorectal cancer cases were considered where the patient had not received any neo-adjuvant therapy to avoid any bias introduced in the cellular transcriptome by external agents. Normal colon tissues were taken from adjacent region of resected specimen as well as non-cancerous operative colon cases. For our study, we have used cancer and normal stem cell markers defined in established literature. For isolating cancer stem cells by FACS, CD44 and CD166 tagged to phycoerythrin (PE) and Alexa Fluor 647 respectively, were used. For isolating normal colon stem cells, CD29, tagged to PE, and Lgr5, tagged to Alexa Fluor 647 were used. Only dual marker-positive cells were isolated to get an enriched population. Tumor sphere assay was done on representative samples to validate the stemness of isolated cells. RNA was isolated from the isolated cell populations and microarray was done to study the whole genome transcriptomic changes.
Project description:This study was designed to understand the transcriptomic composition and the biological functions of cancer stem cells isolated from non-small cell lung cancer line (NSCLC) Putative lung cancer stem cells were isolated from cancer cell lines based on expression of known stem cell surface markers: CD166, CD44 and EpCAM using the Fluorescence Activated Cell Sorter (FACS). Affymetrix microarray were performed on cancer stem cells isolated from normal lung epithelial cells and lung cancer cell lines (A549 and NCI-H2170) using GeneChip Human Gene 1.0 ST array. The normal putative stem cells isolated from normal primary human bronchial/trachial epithelial cell line (PHBEC) was serve as control. Putative cancer stem cells isolated from A549 and NCI-H2170 cell lines are the treatment group. Each sample was performed in triplicate and total number of samples are five (n=5)
Project description:We prospectively isolated human colorectal CD44+ cancer stem cells (CSCs) from organoids and primary colorectal cancer. The gene expression of CD44+ CSCs are relevant to each other between organoids and primary tissues. The common gene signature in two different modality identifies CSCs' properties with the biological significance shown in original article.
Project description:Human colon cancer cells HT29, HCT116, LoVo robustly expressed PD-1. PD-1 signaling significantly decreased proliferation and promoted apoptosis in PD1+human colon cancer cells. The human anti-PD-1, Nivolumab (NIVO) ptomoted proliferation through pERK/pAKT signaling, reduced apoptosis and protected PD-1+ cells from Chemo/Radiotherapy (CRT). In vivo, NIVO promoted HT29 tumor growth reducing Oxaliplatin (OX) efficacy. As opposite to colon cancer cells, PD-1 signaling protects melanoma cells, thus NIVO treated human colon versus melanoma cancer cells, PES43 were evaluated for RNAseq. Among the commonly affected genes, opposite regulation was revealed between HT29 and HCT116 colon versus PES43 melanoma cells. BATF2, DRAM1, FXYD3, IFIT3, MT-TN, TNFRSF11A were upregulated in PES43 and downregulated in HT29 and HCT116 while CLK1, DCAF13, DNAJC2, MTHFD1L, PRPF3, PSMD7, SCFD1 were downregulated in PES43 and upregulated in HT29 and HCT116. DEGs were significantly enriched in the functional categories of the interferon pathway, innate immune, cytokine-mediated signaling pathway, neutrophil activation, immune effector process, granulocyte activation and cellular nitrogen compound metabolic process. Moreover, intrinsic PD-1 was expressed in 11/48 (22.9%) primary colorectal cancer and associated with tumor dimension (pT). Thus, PD-1 inhibition through NIVO protects human colon cancer cells and activate tumor survival pathways
Project description:The oncometabolite, 2-hydroxyglutarate (2HG), has been directly implicated in carcinogenesis; however, the underlying molecular mechanisms remain poorly understood. To elucidate the molecular mechanisms through which 2HG contributes to cancer progression, we performed transcriptome analysis in 2HG-treated colorectal cancer HCT116 and HT29 cells.