Tumor Necrosis factor-related apoptosis-inducing ligand (TRAIL) is regarded as a potential anti-cancer agent, however, considerable numbers of cancer cells, especially glioblastoma multiforme (GBM), are resistant to apoptosis induction by TRAIL.
The aim of this proposal is to evaluate the naturally secreted Gaussia Luciferase (Gluc) as a reporter for high throughput drug screening in order to identify small molecules that sensitize glioma cells to TRAIL. Different glioma cells will be engineered by gene transfer to express Gluc which expression, and therefore cell viability, can be monitored over time by subjecting an aliquot of the conditioned medium to bioluminescence measurements using a 96-wells format plate luminometer. Initially, different components of the Gluc assay including well-to-well variation, as well as substrate stability over time and optimal substrate dose will be optimized for a 96-well plate format. Next, different glioma cell lines, including primary GBM cells dissociated from patient tumor sections, will be tested for their sensitivity or resistance to TRAIL. Finally, primary GBM cells will be screened for drugs that will sensitize them to TRAIL using small molecule libraries some of which are FDA-approved drugs known to cross the blood-brain barrier. The drug candidates that sensitize primary GBM cells to TRAIL will be further characterized in dose- and time-dependent experiments in vitro and potentially in our experimental glioma-Gluc models, in vivo. The Gluc assay time of 4 seconds makes it a valuable tool for HTS and can be easily applied into 384-well plate format to screen thousands of compounds over a relatively short period of time.

Public Health Relevance

The purpose of the work outlined in this proposal is to evaluate the naturally secreted Gaussia luciferase as a reporter for high throughput screening and to identify drugs that sensitize primary glioma cells to the tumor necrosis factor-related apoptosis-inducing ligand, a known tumor therapeutic. This work will provide an easy mean to monitor cell viability over time based on bioluminescence in high throughput manner using a 96-well plate format luminometer. The Gaussia luciferase assay time of few seconds makes it a valuable tool for HTS and can be easily translated into a 384-well plate format.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21NS061051-02
Application #
7762836
Study Section
National Institute of Neurological Disorders and Stroke Initial Review Group (NSD)
Program Officer
Fountain, Jane W
Project Start
2009-02-01
Project End
2011-01-31
Budget Start
2010-02-01
Budget End
2011-01-31
Support Year
2
Fiscal Year
2010
Total Cost
$191,657
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
073130411
City
Boston
State
MA
Country
United States
Zip Code
02199
Badr, Christian E; Van Hoppe, Stephanie; Dumbuya, Hawasatu et al. (2013) Targeting cancer cells with the natural compound obtusaquinone. J Natl Cancer Inst 105:643-53
Niers, Johanna M; Kerami, Mariam; Pike, Lisa et al. (2011) Multimodal in vivo imaging and blood monitoring of intrinsic and extrinsic apoptosis. Mol Ther 19:1090-6
Badr, Christian E; Wurdinger, Thomas; Tannous, Bakhos A (2011) Functional drug screening assay reveals potential glioma therapeutics. Assay Drug Dev Technol 9:281-9
Badr, Christian E; Wurdinger, Thomas; Nilsson, Jonas et al. (2011) Lanatoside C sensitizes glioblastoma cells to tumor necrosis factor-related apoptosis-inducing ligand and induces an alternative cell death pathway. Neuro Oncol 13:1213-24
Maguire, Casey A; Deliolanis, Nikolaos C; Pike, Lisa et al. (2009) Gaussia luciferase variant for high-throughput functional screening applications. Anal Chem 81:7102-6