Adult brain tumors represent 1.3% of all the cancers and 2.2% of all the cancer related deaths. Adult brain cancers cause 22,000 deaths every year in the USA. These tumors derive from glial cells and seem to be multifocal. Surgery is unsuccessful and invariably the initial resection is accompanied by relapses at different anatomic locations in the brain. These tumors are resistant to radio- and chemo-therapy. Mortality rate are high and treatment option have not significantly improved over the course of the years. It is clear that more research is needed to increase treatment options in this type of cancer whose diagnosis, even today, represent the equivalent of a death sentence. Our laboratory has produced a large amount of preliminary data indicating that glioma cells are sensible to cell death induced by triggering endoplasmic reticulum stress response (ERSR). On the other hand, normal glial cells and neurons seem to be resistant to ERSR-mediated cell death. Human and rat glioma cells during ERSR produce higher levels of GRP78, the key architect of ERSR and a chaperone involved in protein folding in the ER than astrocytes. This latter finding indicates the failure to manage protein unfolding in glioma cells. Failure of ERSR repair mechanisms in turns is responsible for ER-associated caspase (4/12) activation, and consequent apoptosis. Based on these evidences, we engineered U87-MG to express luciferase under the control of the GRP78 promoter and generated a stable cell line so that we could identify novel ERSR inducers. We have validated the assay with two positive controls known activators of ERSR such as tunicamycin and thapsigargin. These two positive controls are effective and inexpensive. We have set all the parameters needed to successfully run the assay, such as growth medium, cell number, incubation time, drugging protocols. Z factor were assessed above 0.67 on average, CV were significantly below 10%, for both negative and positive controls exposed cells, S/B was in excess of 100. The dynamic range was wide and stimulation reached 8-10 folds with submaximal concentrations of the positive controls. We screened the assay using automated compounds plating, manual cell dispensing and automated Bright Glow addition and reading, against the Prestwick II library (1120 FDA approved compounds + some natural molecules) and identified 12 hits. Our follow up testing has shown that most of the compounds were able to upregulate GRP78-Luc. We focused post screening validation on 3 of the hits. Spiperone does indeed activate ERSR, upregulate native GRP78, activate ER-associated caspases and cause marked death of glioma cells. A second class of compounds has been developed from ibuprofen the third hit. Quinacrine modulates GRP78 resulting in glioma cell death. We have also tested quinacrine and a compound derived from initial SAR in a skin U87-MG xenograft in mice and found a significant reduction of tumor growth in a two week single agent administration. Also target modulation was observed in the animal tumor specimens. We propose to use this assay to perform automated high throughput screen of larger small molecules libraries such as the MLSPCN (200,000). We will screen these libraries to 1) identify small molecules able to induce ERSR;to support the center performing hit clustering analysis, commercial analoging and initial basic structure- activity relationship analysis, looking for interesting chemical scaffolds to refine our probes 2) to validate selected compounds using tertiary assay for efficacy and mechanism of action identification. By the end of this project we will have characterized the hit compounds, organized them for chemical classes of interest, tested the effect of prototypes scaffolds on gliotoxicity, and gathered insight on their mechanism of action. The added value of our work could be identified in the possibility that some of the hit compounds will have the potential to initiate a larger effort aiming to develop and test active and safe molecules as possible therapeutics to use in the battle against adult primary brain tumors of the glial lineage.
Brain tumors represent 1.3% of all the cancers and 2.2% of all the cancer related deaths. It is assessed that brain cancers will cause a total of 22,000 deaths yearly only in the USA (www.cancer.org). These neoplasms seem to be multifocal and even complete macroscopic initial resection is often accompanied by relapses at different anatomic locations in the brain. These tumors are quite resistant to radio- and chemo-therapy and the brain is not accessible by many drugs due to the blood brain barrier. Mortality rate are high and treatment option have not significantly improved. It is clear that more research is needed to achieve better results in this type of cancer. Our laboratory has produced a large amount of preliminary data indicating that glioma cells are sensible to cell death induced by triggering endoplasmic reticulum stress response (ERSR) Based on these evidences, we engineered U87-MG to express luciferase under the control of the GRP78 promoter and generated a stable cell line. We have validated the assay with two positive controls known activators of ERSR such as tunicamycin and thapsigargin. We propose to use this assay to perform automated high throughput screen of larger small molecules libraries such as the MLSPCN (200,000). We will screen these libraries to 1) identify small molecules able to induce ERSR;to support the center performing hit clustering analysis, commercial analoging and initial basic structure- activity relationship analysis, looking for interesting chemical scaffolds to refine our probes 2) to validate selected compounds using tertiary assay for efficacy and mechanism of action identification. By the end of this project we will have characterized the hit compounds, organized them for chemical classes of interest, tested the effect of prototypes scaffolds on gliotoxicity, and gathered insight on their mechanism of action. The added value of our work could be identified in the possibility that some of the hit compounds will have the potential to spin off a larger effort aiming to develop and test active and safe molecules as possible therapeutics to use in the battle against adult primary brain tumors of the glial lineage.
| Johnson, Guyla G; White, Misti C; Wu, Jian-He et al. (2014) The deadly connection between endoplasmic reticulum, Ca2+, protein synthesis, and the endoplasmic reticulum stress response in malignant glioma cells. Neuro Oncol 16:1086-99 |
| White, M C; Johnson, G G; Zhang, W et al. (2013) Sulindac sulfide inhibits sarcoendoplasmic reticulum Ca2+ ATPase, induces endoplasmic reticulum stress response, and exerts toxicity in glioma cells: relevant similarities to and important differences from celecoxib. J Neurosci Res 91:393-406 |
| Johnson, Guyla G; White, Misti C; Grimaldi, Maurizio (2011) Stressed to death: targeting endoplasmic reticulum stress response induced apoptosis in gliomas. Curr Pharm Des 17:284-92 |
