Functional Drug Screening Assay Reveals Potential Glioma Therapeutics

Abstract

Here we describe a novel functional screening assay based on bioluminescence monitoring of the naturally secreted Gaussia luciferase (Gluc) in the conditioned medium of cultured cells. Using this assay, we identified small-molecule drugs that sensitized brain tumor cells to the tumor necrosis factor-related apoptosis-inducing ligand-induced cell death. Human glioblastoma multiforme cells were engineered by gene transfer to express Gluc as a reporter for cell viability, which can be monitored over time by bioluminescence measurements using a plate luminometer. We have optimized the Gluc assay for screening and validated it using the National Institute of Neurological Disorders and Stroke (NINDS) custom collection II library consisting of 1,040 drugs and bioactive compounds, most of which are Food and Drug Administration-approved and are able to cross the blood-brain barrier. We found that the cardiac glycosides family sensitized glioblastoma multiforme cells to the tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. In conclusion, the Gluc secretion assay is a robust tool for functional drug screening and can be applied to many different fields including cancer.

Identification of Mitoxantrone as a TRAIL-sensitizing agent for Glioblastoma Multiforme

“…In addition, it has been shown by different groups that the combination of low dose cardiac glycosides with TRAIL can trigger cell death in various cancers, including GBM. 37,42,48 Our studies are in line with these previous findings, validating the utility and consistency of our screen. However, when we applied the low dosages of the cardiac glycoside Digitoxigenin, Proscillaridin A and Digoxin in combination with TRAIL on non-malignant cells such as human fibroblasts and normal human astrocytes, we observed equal amount of cell death in these cells, highlighting a possible toxicity issue.…”

“…Nine of these hit drugs (Doxorubicin, Daunorubicin, Camptothecine (S,C), Azacytidine-5, Vorinostat, Topotecan, Mitoxantrone, Cycloheximide and Quinacrine dihydrochloride) were previously indicated as TRAIL-sensitizing agents in several cancers; 17,27-36 and 6 of them (Cycloheximide, Monensin sodium salt, Doxorubicin, Topotecan, Digoxin and Lanatoside C) were also studied as TRAIL-sensitizers in GBM. [37][38][39][40][41][42] The novel TRAIL-sensitizers for GBM were Alexidine dihydrochloride, Daunorubicin, Methyl benzethonium chloride, Benzethonium chloride, Amphotericin B, Camptothecine (S,C), Azacytidine-5, Vorinostat, Mitoxantrone, Digitoxigenin, Proscillaridin A, Digoxigenin, Cyclosporin A, Pyrivinium pamoate, Niclosamide, Quinacrine dihydrochloride, Terfenadine, Astemizole, Thonzonium bromide, and Pinaverium bromide in our screen.…”

Bioluminescence imaging: progress and applications

“…Luciferase expression correlates with the number of tumor cells both in vitro and in vivo , hence its use as a reporter for cell proliferation and cell death, compatible with high-throughput screening [54, 55]. Typically, tumor cells are engineered ex vivo to stably express luciferase under the control of a constitutively active, tissue-specific or gene/process-specific promoter (e.g.…”

A chemical screen for medulloblastoma identifies quercetin as a putative radiosensitizer

“…This resulted in a four-day assay, using 750 DAOY cells per well with 4 Gy irradiation. In addition, a drug concentration of 1 μM was chosen, since this showed good results in a pilot experiment using eight different, randomly chosen small molecules (Figure 1D), and yielded positive hits in a drug screen performed previously by our group [18]. To identify putative radiosensitizers, cells were treated with compounds from the ActiTarg-K960 drug library consisting of 960 putative kinase inhibitors, or with 0.1% DMSO as an internal control, either as monotherapy, or in combination with irradiation.…”

“…Treatment was performed with drugs in paired 96-well plates, where one plate was exposed to 4 Gy in a Gammacell ® 220 Research Irradiator (MDS Nordion, Canada) 30 minutes after addition of the compounds, and the other plate was a non-irradiated control. Four days later, cell survival was evaluated by measuring Gaussia luciferase (Gluc) activity [18], or by means of the Acumen e X3 laser scanning cytometer (TTP LabTech, UK). Results were analyzed using Acumen Explorer software, calculating the percentage survival for each compound tested with the assay.…”