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Cytotoxicity Profiling of The Anticancer Agent Thiostrepton

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March 09, 2010

 

 

Authors: Paul Held, BioTek Instruments, Inc. Winooski, Vermont, USA; Kheng Newick, University of Vermont, Burlington, Vermont, USA; Wayne Patton and Dee Shen, Enzo Life Sciences, Farmingdale, New York, USA

 

Enzo

 

Abstract

 

Malignant mesothelioma (MM) is a form of cancer originating in the mesothelium that is almost always caused by the exposure to asbestos. Microarrays have previously demonstrated that MM cells had changes in gene expression indicative of enhanced production of reactive oxygen species, such as NADPH Oxidase 4 (Nox 4). The continual presence of ROS results in the increase and duration of pro-proliferative pathways, which lead to the up regulation of cell cycle genes. Different MM cell lines expressed isoforms of FoxM1 necessary for cell cycle transit in the absence of growth factors, yet were responsive to inhibitors of Nox4. Thiostrepton (TS) is a natural antibiotic, which has been shown to inhibit the oncogenic transcription factor FoxM1 and is cytotoxic to MM cells in a dose dependant fashion. Drugs elicit cytotoxic effects through several mechanisms. Drug accumulation in the mitochondria can result in a compromised membrane potential with a resultant disruption in electron transport. Lysosome perturbation has been shown to cause the formation of autophagosomes and autophagic cytopathology. Numerous compounds have been shown to initiate the apoptotic process in cells. Using CELLestial™ assay kits we have profiled the response of cells to thiostrepton. MM cells lines were exposed to TS along with agents known to perturb lysosomes, mitochondria, or induce apoptosis and their responses compared to normal LP9 cells. TS does not appear to induce apoptosis as measured by nuclear condensation in either normal or malignant cells. Interestingly marked differences between the normal LP9 cells and MM cells in the lysosomal response to thiostrepton are observed. To facilitate the process of cytotoxicity profiling of TS on mesothelioma cells, we have automated all of the fluid handling steps of these assays. These data and process steps serve as an example of how the cytotoxicity profile of other anticancer agents or potential drug compounds can be streamlined. Assay work flow along with performance results will be provided.

 

 

 
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