Project description:Preclinical cancer drug discovery efforts have employed two-dimensional (2D)-cell-based assay models, which fail to forecast in vivo efficacy and contribute to a lower success rates of clinical approval. Three-dimensional (3D) cell culture models are recently expected to bridge the gap between 2D and in vivo models. We have developed a novel 3D culture method that improves the growth of spheroid-forming cancer cells under anchorage-independent condition by leveraging a feature of FP001, a bacteria-derived polysaccharide. Gene microarrays were used to observe the global gene expression in SKOV3 cells cultured with adhesion condition (2D, control) or with low adhesion condition (FP001) and identified distinct classes of up or down-regulated genes. SKOV3 cells were cultured for 11 days in normal attachment plates with normal medium (as control) or low-attachment plates with FP001 containing medium. Each sample was collected three times.
Project description:Preclinical cancer drug discovery efforts have employed two-dimensional (2D)-cell-based assay models, which fail to forecast in vivo efficacy and contribute to a lower success rates of clinical approval. Three-dimensional (3D) cell culture models are recently expected to bridge the gap between 2D and in vivo models. We have developed a novel 3D culture method that improves the growth of spheroid-forming cancer cells under anchorage-independent condition by leveraging a feature of FP001, a bacteria-derived polysaccharide. Gene microarrays were used to observe the global gene expression in SKOV3 cells cultured with adhesion condition (2D, control) or with low adhesion condition (FP001) and identified distinct classes of up or down-regulated genes.
Project description:3D cell culture models are recognized for representing the physiological microenvironment and exhibiting higher concordance with in vivo conditions, when compared to a conventional 2D cell culture model. However, cells grown in 3D cultures are likely to exhibit slower growth than those in 2D cultures. We found that addition of a novel small molecule named GA-017 to culture media promotes the cell proliferation particularly under 3D conditions. Gene microarrays were used to observe the global gene expression in Skov3 cells cultured under 3D condition with DMSO or GA-017 and identified distinct classes of up or down-regulated genes.
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
Project description:This experiment investigates transcriptomic differences between two SKOV3 ovarian cancer cell strains, S1 (CW1) and S2 (SM), to assess how clonal evolution and culture conditions influence gene expression. Bulk RNA sequencing (RNA-seq) was performed on cells cultured in both 2D monolayers and 3D spheroids. The study aims to identify differentially expressed genes and pathways associated with epithelial-mesenchymal transition (EMT), proliferation, and metabolic regulation. The sequencing data were processed using standard bioinformatics pipelines, including quality control, alignment, normalization, and differential expression analysis.
Project description:HCV proliferation is closely related to three-dimentional cellular condition. In case of blood-borne (bb) HCV culture in HuS-E2 cells, bbHCV was reproduced only from 3D-cultured cells in hollow fibers. Thus, in order to identify novel factors which support HCV proliferation under three-dimentional condition, we compared gene expression profile between 2D- and 3D-cultured HuS-E/2 cells with 3D-gene Human oligo chip 25k (Toray, Tokyo, Japan).