Bakhos A Tannous
Molecular Neurogenetics Unit, Neuroscience Program, Harvard Medical School Center for Molecular Imaging Research, Massachusetts General Hospital Boston, MA 02115

Abstract:

We have developed a novel functional screening assay based on bioluminescence monitoring of the naturally secreted Gaussia luciferase (Gluc) in the conditioned medium of cultured cells in vitro and in blood of animals ex vivo. glioma cells were engineered by lentivirus vector to stably express Gluc, a marker for cell viability, which can be monitored over time by bioluminescence measurements using a plate luminometer. We have optimized the Gluc assay and showed it to be a robust tool for high throughput screening with a Z'' factor of 0.75 and a coefficient of variation (CV) <10%. Further, we showed that 5 µL blood assayed ex vivo can be used to monitor the effect of novel drugs against mice-bearing tumors in vivo. We have applied the Gluc assay to screen for drugs which act against glioma cells as well as primary glioblastoma stem-like cells cultured as neural spheres. We found the cardiac glycosides family to sensitize these cells to TRAIL-induced apoptosis in glioblastoma xenograft model in vivo. More importantly, we showed that lanatoside C on its own serves as a therapeutic agent against glioblastoma by activating a caspase-independent cell death pathway. Cells treated with lanatoside C showed necrotic cell morphology with absence of caspase activation, low mitochondrial membrane potential as well as early intracellular ATP depletion. Moreover, lanatoside C penetrated the mouse brain efficiently and inhibited glioblastoma stem-like cells tumor growth and invasion. A recent 28-year long study in human showed that drugs from the cardiac glycosides family, including lanatoside C, have a neuroprotection role against stroke (http://www.medicalnewstoday.com/articles/47200.php). These studies provide a further evidence of the use of cardiac glycosides in brain treatment and the lack of toxicity of these drugs on normal brain cells. In conclusion, by using the Gluc-based drug screen, we show that lanatoside C sensitizes glioblastoma cells to TRAIL-induced cell death and mitigates apoptosis resistance of glioblastoma cells by inducing an alternative necrosis-like cell death pathway. To our knowledge, this is one of the first examples of using caspase-independent cell death inducers to trigger tumor regression in vivo. Activation of such mechanism may be a useful strategy for counter resistance of cancer cells to apoptosis.

Keywords: drug screening, assay development, glioblastoma, glioblastoma stem cells