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1.
SLAS Discov ; 22(5): 473-483, 2017 06.
Article in English | MEDLINE | ID: mdl-28288283

ABSTRACT

The NCI60 cell line panel screen includes 60 human tumor cell lines derived from nine tumor types that has been used over the past 20+ years to screen small molecules, biologics, and natural products for activity. Cells in monolayer culture in 96-well plates are exposed to compounds for 48 h, and Sulforhodamine B is used to determine cell viability. Data analysis tools such as COMPARE allow classification of compounds based on the pattern of cell line response. However, many compounds highly active in monolayer cell culture fail to show efficacy in vivo. Therefore, we explored 3D culture of the NCI60 panel as a strategy to improve the predictive accuracy of the screen. 3D cultures more closely resemble tumors than monolayer cultures with tighter cell-cell contact and nutrient and oxygen gradients between the periphery and the center. We optimized the NCI60 cell line panel for generating 3D spheroids of a prespecified diameter (300-500 µm) in ultra-low attachment (ULA) plates. Spheroids were classified into four categories based on imaging, and concentration response of select agents in 2D and 3D models is presented.


Subject(s)
Antineoplastic Agents/pharmacology , Cell Line, Tumor/cytology , Cell Line, Tumor/drug effects , Drug Screening Assays, Antitumor/methods , Cell Culture Techniques/methods , Cell Survival/drug effects , Humans , Spheroids, Cellular/cytology , Spheroids, Cellular/drug effects
2.
Mol Biol Cell ; 22(18): 3344-54, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21795396

ABSTRACT

The calcium-binding protein S100A4 is a central mediator of metastasis formation in colon cancer. S100A4 is a target gene of the Wnt/ß-catenin pathway, which is constitutively active in the majority of colon cancers. In this study a high-throughput screen was performed to identify small-molecule compounds targeting the S100A4-promoter activity. In this screen calcimycin was identified as a transcriptional inhibitor of S100A4. In colon cancer cells calcimycin treatment reduced S100A4 mRNA and protein expression in a dose- and time-dependent manner. S100A4-induced cellular processes associated with metastasis formation, such as cell migration and invasion, were inhibited by calcimycin in an S100A4-specific manner. Calcimycin reduced ß-catenin mRNA and protein levels despite the expression of Δ45-mutated ß-catenin. Consequently, calcimycin inhibited Wnt/ß-catenin pathway activity and the expression of prominent ß-catenin target genes such as S100A4, cyclin D1, c-myc, and dickkopf-1. Finally, calcimycin treatment of human colon cancer cells inhibited metastasis formation in xenografted immunodeficient mice. Our results demonstrate that targeting the expression of S100A4 with calcimycin provides a functional strategy to restrict cell motility in colon cancer cells. Therefore calcimycin may be useful for studying S100A4 biology, and these studies may serve as a lead for the development of treatments for colon cancer metastasis.


Subject(s)
Antineoplastic Agents/pharmacology , Calcimycin/pharmacology , Cell Movement/drug effects , S100 Proteins/metabolism , Wnt Signaling Pathway/drug effects , Animals , Antineoplastic Agents/therapeutic use , Calcimycin/therapeutic use , Cell Proliferation , Cell Survival/drug effects , Colonic Neoplasms/drug therapy , Colonic Neoplasms/pathology , Gene Expression/drug effects , Genes, Reporter , HCT116 Cells , Humans , Liver Neoplasms/prevention & control , Liver Neoplasms/secondary , Luciferases/biosynthesis , Luciferases/genetics , Mice , Mice, Inbred NOD , Mice, SCID , Neoplasm Invasiveness , Promoter Regions, Genetic , S100 Calcium-Binding Protein A4 , S100 Proteins/antagonists & inhibitors , S100 Proteins/genetics , Xenograft Model Antitumor Assays
3.
J Natl Cancer Inst ; 103(13): 1018-36, 2011 Jul 06.
Article in English | MEDLINE | ID: mdl-21685359

ABSTRACT

BACKGROUND: Metastasis formation in colon cancer severely reduces the survival rate in patients. S100A4, a calcium-binding protein, is implicated in promoting metastasis formation in colon cancer. METHODS: To identify a transcription inhibitor of S100A4, high-throughput screening of 1280 pharmacologically active compounds was performed using a human colon cancer cell line expressing a S100A4 promoter-driven luciferase (LUC) reporter gene construct (HCT116-S1004p-LUC). Niclosamide, an antihelminthic agent, was identified as a potential candidate. Colon cancer cell lines (HCT116, SW620, LS174T, SW480, and DLD-1) were treated with 1 µM niclosamide to analyze the effect on S100A4 mRNA and protein expression by quantitative reverse transcription-polymerase chain reaction and immunoblot assays, and effects on cell migration, invasion, proliferation, and colony formation were also assessed in vitro. The effect of niclosamide on liver metastasis was assessed in a xenograft mouse model of human colon cancer (n = 8 mice) by in vivo imaging. The long-term effect of niclosamide on metastasis formation after discontinued treatment was quantified by scoring, and overall survival (n = 12 mice) was analyzed by Kaplan-Meier method after discontinuation of treatment. All statistical tests were two-sided. RESULTS: Reduced S100A4 mRNA and protein expression, and inhibited cell migration, invasion, proliferation, and colony formation were observed in niclosamide-treated colon cancer cells in vitro. In vivo imaging of niclosamide-treated mice showed reduced liver metastasis compared with solvent-treated control mice (n = 4 mice per group). Compared with the control group, discontinuation of treatment for 26 days showed reduced liver metastasis formation in mice (n = 6 mice per group) (control vs discontinuous treatment, mean metastasis score = 100% vs 34.9%, mean difference = 65.1%; 95% confidence interval [CI] = 18.4% to 111.9%, P < .01) and increased overall survival (n = 6 mice per group; control vs discontinuous treatment, median survival = 24 vs 46.5 days, ratio = 0.52, 95% CI = 0.19 to 0.84, P = .001). CONCLUSION: Niclosamide inhibits S100A4-induced metastasis formation in a mouse model of colon cancer and has therapeutic potential.


Subject(s)
Antineoplastic Agents/pharmacology , Biomarkers, Tumor/metabolism , Colonic Neoplasms/drug therapy , Colonic Neoplasms/metabolism , Liver Neoplasms/prevention & control , Niclosamide/pharmacology , S100 Proteins/metabolism , Animals , Anthelmintics/pharmacology , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/chemistry , Biomarkers, Tumor/genetics , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Chromatin Immunoprecipitation , Colonic Neoplasms/pathology , Disease Progression , Electrophoretic Mobility Shift Assay , Gene Expression Regulation, Neoplastic/drug effects , Humans , Immunoblotting , Infusions, Parenteral , Kaplan-Meier Estimate , Liver Neoplasms/secondary , Mice , Mice, Inbred NOD , Mice, SCID , Neoplasm Invasiveness , Niclosamide/administration & dosage , Niclosamide/chemistry , Prognosis , RNA, Messenger/metabolism , RNA, Neoplasm/metabolism , Random Allocation , Reverse Transcriptase Polymerase Chain Reaction , S100 Calcium-Binding Protein A4 , S100 Proteins/genetics , Signal Transduction/drug effects , Time Factors , Transplantation, Heterologous , Tumor Stem Cell Assay , Wnt Proteins/drug effects , Wnt Proteins/metabolism , beta Catenin/drug effects , beta Catenin/metabolism
4.
Cancer Res ; 62(15): 4316-24, 2002 Aug 01.
Article in English | MEDLINE | ID: mdl-12154035

ABSTRACT

Hypoxia-inducible factor 1 (HIF-1) is a master regulator of the transcriptional response to oxygen deprivation. HIF-1 has been implicated in the regulation of genes involved in angiogenesis [e.g., vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase] and anaerobic metabolism (e.g., glycolytic enzymes). HIF-1 is essential for angiogenesis and is associated with tumor progression. In addition, overexpression of HIF-1 alpha has been demonstrated in many common human cancers. Therefore, HIF-1 is an attractive molecular target for development of novel cancer therapeutics. We have developed a cell-based high-throughput screen for the identification of small molecule inhibitors of the HIF-1 pathway. We have genetically engineered U251 human glioma cells to stably express a recombinant vector in which the luciferase reporter gene is under control of three copies of a canonical hypoxia-responsive element (U251-HRE). U251-HRE cells consistently expressed luciferase in a hypoxia- and HIF-1-dependent fashion. We now report the results of a pilot screen of the National Cancer Institute "Diversity Set," a collection of approximately 2000 compounds selected to represent the greater chemical diversity of the National Cancer Institute chemical repository. We found four compounds that specifically inhibited HIF-1-dependent induction of luciferase but not luciferase expression driven by a constitutive promoter. In addition, these compounds inhibited hypoxic induction of VEGF mRNA and protein expression in U251 cells. Interestingly, three compounds are closely related camptothecin analogues and topoisomerase (Topo)-I inhibitors. We show that concomitant with HIF-1 and VEGF inhibition, the activity of the Topo-I inhibitors tested is associated with induction of cyclooxygenase 2 mRNA expression. The luciferase-based high-throughput screen is a feasible tool for the identification of small molecule inhibitors of HIF-1 transcriptional activation. In addition, our results suggest that altered Topo-I function may be associated with repression of HIF-1-dependent induction of gene expression.


Subject(s)
Antineoplastic Agents/pharmacology , DNA-Binding Proteins/antagonists & inhibitors , Nuclear Proteins/antagonists & inhibitors , Transcription Factors , Transcriptional Activation/drug effects , Camptothecin/analogs & derivatives , DNA, Neoplasm/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/physiology , Drug Screening Assays, Antitumor , Endothelial Growth Factors/antagonists & inhibitors , Endothelial Growth Factors/biosynthesis , Endothelial Growth Factors/genetics , Enzyme Inhibitors/pharmacology , Gene Expression/drug effects , Glioma/drug therapy , Glioma/genetics , Glioma/metabolism , Humans , Hypoxia-Inducible Factor 1 , Hypoxia-Inducible Factor 1, alpha Subunit , Luciferases/antagonists & inhibitors , Luciferases/biosynthesis , Luciferases/genetics , Lymphokines/antagonists & inhibitors , Lymphokines/biosynthesis , Lymphokines/genetics , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nuclear Proteins/physiology , Promoter Regions, Genetic/drug effects , Promoter Regions, Genetic/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Response Elements/drug effects , Response Elements/genetics , Topotecan/pharmacology , Transcriptional Activation/physiology , Transfection , Tumor Cells, Cultured , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors
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