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13. Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation

Author

Vinci Maria, Gowan Sharon, Boxall Frances, Patterson Lisa, Zimmermann Miriam, Court William, Lomas Cara, Mendiola Marta, Hardisson David, and Eccles Suzanne A

Subjects
3D, angiogenesis, drug response, high throughput, invasion, migration, tumor spheroids, Biology (General), QH301-705.5
Abstract

Abstract Background There is overwhelming evidence that in vitro three-dimensional tumor cell cultures more accurately reflect the complex in vivo microenvironment than simple two-dimensional cell monolayers, not least with respect to gene expression profiles, signaling pathway activity and drug sensitivity. However, most currently available three-dimensional techniques are time consuming and/or lack reproducibility; thus standardized and rapid protocols are urgently needed. Results To address this requirement, we have developed a versatile toolkit of reproducible three-dimensional tumor spheroid models for dynamic, automated, quantitative imaging and analysis that are compatible with routine high-throughput preclinical studies. Not only do these microplate methods measure three-dimensional tumor growth, but they have also been significantly enhanced to facilitate a range of functional assays exemplifying additional key hallmarks of cancer, namely cell motility and matrix invasion. Moreover, mutual tissue invasion and angiogenesis is accommodated by coculturing tumor spheroids with murine embryoid bodies within which angiogenic differentiation occurs. Highly malignant human tumor cells were selected to exemplify therapeutic effects of three specific molecularly-targeted agents: PI-103 (phosphatidylinositol-3-kinase (PI3K)-mammalian target of rapamycin (mTOR) inhibitor), 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) (heat shock protein 90 (HSP90) inhibitor) and CCT130234 (in-house phospholipase C (PLC)γ inhibitor). Fully automated analysis using a Celigo cytometer was validated for tumor spheroid growth and invasion against standard image analysis techniques, with excellent reproducibility and significantly increased throughput. In addition, we discovered key differential sensitivities to targeted agents between two-dimensional and three-dimensional cultures, and also demonstrated enhanced potency of some agents against cell migration/invasion compared with proliferation, suggesting their preferential utility in metastatic disease. Conclusions We have established and validated a suite of highly reproducible tumor microplate three-dimensional functional assays to enhance the biological relevance of early preclinical cancer studies. We believe these assays will increase the translational predictive value of in vitro drug evaluation studies and reduce the need for in vivo studies by more effective triaging of compounds.

Published
2012
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16. Discovery of Potent, Orally Bioavailable, Small-Molecule Inhibitors of WNT Signaling from a Cell-Based Pathway Screen

Author

Mallinger, Aurélie, Crumpler, Simon, Pichowicz, Mark, Waalboer, Dennis, Stubbs, Mark, Adeniji-Popoola, Olajumoke, Wood, Bozena, Smith, Elizabeth, Thai, Ching, Henley, Alan T., Georgi, Katrin, Court, William, Hobbs, Steve, Box, Gary, Ortiz-Ruiz, Maria-Jesus, Valenti, Melanie, De Haven Brandon, Alexis, TePoele, Robert, Leuthner, Birgitta, Workman, Paul, Aherne, Wynne, Poeschke, Oliver, Dale, Trevor, Wienke, Dirk, Esdar, Christina, Rohdich, Felix, Raynaud, Florence, Clarke, Paul A., Eccles, Suzanne A., Stieber, Frank, Schiemann, Kai, and Blagg, Julian

Subjects
Models, Molecular, Dose-Response Relationship, Drug, Molecular Structure, Pyridines, Drug Evaluation, Preclinical, Administration, Oral, Biological Availability, Antineoplastic Agents, Crystallography, X-Ray, Xenograft Model Antitumor Assays, Article, Small Molecule Libraries, Disease Models, Animal, Mice, Structure-Activity Relationship, Cell Line, Tumor, Animals, Humans, Biological Assay, Spiro Compounds, Colorectal Neoplasms, Luciferases, Wnt Signaling Pathway, Cell Proliferation
Abstract

WNT signaling is frequently deregulated in malignancy, particularly in colon cancer, and plays a key role in the generation and maintenance of cancer stem cells. We report the discovery and optimization of a 3,4,5-trisubstituted pyridine 9 using a high-throughput cell-based reporter assay of WNT pathway activity. We demonstrate a twisted conformation about the pyridine-piperidine bond of 9 by small-molecule X-ray crystallography. Medicinal chemistry optimization to maintain this twisted conformation, cognisant of physicochemical properties likely to maintain good cell permeability, led to 74 (CCT251545), a potent small-molecule inhibitor of WNT signaling with good oral pharmacokinetics. We demonstrate inhibition of WNT pathway activity in a solid human tumor xenograft model with evidence for tumor growth inhibition following oral dosing. This work provides a successful example of hypothesis-driven medicinal chemistry optimization from a singleton hit against a cell-based pathway assay without knowledge of the biochemical target.

Published
2015

18. PAK4 promotes kinase-independent stabilization of RhoU to modulate cell adhesion

Author

Dart, Anna E., Box, Gary M., Court, William, Gale, Madeline E., Brown, John P., Pinder, Sarah E., Eccles, Suzanne A., and Wells, Claire M.

Subjects
rho GTP-Binding Proteins, Ubiquitin-Protein Ligases, Ubiquitination, Article, p21-Activated Kinases, Cell Movement, Cell Line, Tumor, Enzyme Stability, Cell Adhesion, Humans, Protein Interaction Domains and Motifs, Paxillin, Phosphorylation, cdc42 GTP-Binding Protein, Research Articles
Abstract

PAK4, via a novel kinase-independent mechanism, protects RhoU from a Rab40A/Cullin 5 ubiquitin ligase complex–driven K48 ubiquitination to regulate breast cancer cell adhesion., P21-activated kinase 4 (PAK4) is a Cdc42 effector protein thought to regulate cell adhesion disassembly in a kinase-dependent manner. We found that PAK4 expression is significantly higher in high-grade human breast cancer patient samples, whereas depletion of PAK4 modifies cell adhesion dynamics of breast cancer cells. Surprisingly, systematic analysis of PAK4 functionality revealed that PAK4-driven adhesion turnover is neither dependent on Cdc42 binding nor kinase activity. Rather, reduced expression of PAK4 leads to a concomitant loss of RhoU expression. We report that RhoU is targeted for ubiquitination by the Rab40A–Cullin 5 complex and demonstrate that PAK4 protects RhoU from ubiquitination in a kinase-independent manner. Overexpression of RhoU rescues the PAK4 depletion phenotype, whereas loss of RhoU expression reduces cell adhesion turnover and migration. These data support a new kinase-independent mechanism for PAK4 function, where an important role of PAK4 in cellular adhesions is to stabilize RhoU protein levels. Thus, PAK4 and RhoU cooperate to drive adhesion turnover and promote cell migration.

Published
2015
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20. A Cyclic Peptide Inhibitor of HIF-1 Heterodimerization That Inhibits Hypoxia Signaling in Cancer Cells

Author

Miranda, Elena, primary, Nordgren, Ida K., additional, Male, Abigail L., additional, Lawrence, Charlotte E., additional, Hoakwie, Franciane, additional, Cuda, Francesco, additional, Court, William, additional, Fox, Keith R., additional, Townsend, Paul A., additional, Packham, Graham K., additional, Eccles, Suzanne A., additional, and Tavassoli, Ali, additional

Published
2013
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25. Nonvolatile organic acids of flue-cured tobacco as affected by production practices

Author

Court, William A. and Hendel, John G.

Subjects
Agricultural research -- Analysis -- Research, Acidity function -- Research -- Analysis, Agricultural industry, Business
Abstract

NONVOLATILE ORGANIC ACIDS OF FLUE-CURED TOBACCO AS AFFECTED BY PRODUCTION PRACTICES INTRODUCTION Flue-cured tobacco (Nicotiana tabacum L.) contains significant amounts of nonvolatile organic acids. Malic, citric, and oxalic acids are [...]

Published
1989

37. Overexpression of epidermal growth factor receptor in human head and neck squamous carcinoma cell lines correlates with matrix metalloproteinase-9 expression and <TOGGLE>in vitro</TOGGLE> invasion

Author

O-Charoenrat, Pornchai, Rhys-Evans, Peter, Modjtahedi, Helmout, Court, William, Box, Gary, and Eccles, Suzanne

Abstract

Numerous reports have shown an association between overexpression of the epidermal growth factor receptor (EGFR), and poor prognosis in head and neck squamous cell carcinomas (HNSCC), however, the underlying mechanisms are still unclear. In the present study, we set out to determine whether EGFR expression was associated with in vitro invasive capacity in a panel of four established and ten newly derived HNSCC lines. Ten of the cell lines expressed high levels of EGFR as determined by a ligand-binding assay and dot blot analysis, whereas the remaining four showed weak overexpression or normal levels of EGFR. The ability of cells to invade through Matrigel was found to be higher in the EGFR overexpressing cell lines (p &lt; 0.0001). Expression levels of matrix metalloproteinases (MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, MMP-11, MMP-13, MT1-MMP) and tissue inhibitors of MMP (TIMP-1, TIMP-2) were evaluated by semiquantitative RT-PCR, substrate zymography and western blot. We found a strong positive correlation between EGFR levels and the expression of MMP-9 mRNA (r2 = 0.95; p &lt; 0.0001), MMP-9 enzyme activity (r2 = 0.8099; p &lt; 0.0001) and an inverse correlation with TIMP-1 (r2 = 0.48; p = 0.0059). In six selected HNSCC lines, in vitro invasion was assayed in the presence of an anti-EGFR monoclonal antibody, ICR62. A significant reduction of invasion in four selected EGFR-overexpressing cell lines was found with 30 nM ICR62 (from 50% to 70%; p &lt; 0.001) but there was no effect in two cell lines with normal EGFR levels. Our results show that the in vitro invasive phenotype of HNSCC lines correlates with high EGFR and MMP-9 expression, and it is therefore suggested that the EGFR signaling pathway might play an important role in the invasive behavior of HNSCC via specific upregulation of MMP-9 and downregulation of TIMP-1. Int. J. Cancer 86:307–317, 2000. © 2000 Wiley-Liss, Inc.

Published
2000
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38. Ginseng, the Genus Panax

Author

Court, William E. and Court, William E.

Subjects
Ginseng, Ginseng--Therapeutic use
Abstract

Panax Ginseng has been commonly used in indigenous medicine in the Far East for over 5,000 years. Its man-like appearance ensured that it was quickly accepted as a tonic maintaining the body in good health, inducing rejuvenation and retarding ageing. The vast output of modern research suggests uses for this ancient drug in today's medicine.This volume endeavours to present a well referenced review of many aspects of ginseng literature including history, botany, horticulture, tissue culture, chemistry, pharmacology, therapeutics, side effects, quality control, patents and related or substituted species.

Published
2000

47. African RauwolfiaSpecies

Author

Court, William E

Abstract

A substitute or adulterant for the roots of R. vomitoriaAfz. is R. mombasianaStapf, an East African shrub with a high reserpine yield. The anatomy of the root and stem is described and illustrated, and compared with published data about other African species.

Published
1962
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