PP54. NANO-ENCAPSULATED DISULFIRAM TARGETS GLIOBLASTOMA STEM CELLS IN VITRO AND IN VIVO BY MODULATION OF HYPOXIA-NF-KB PATHWAY
Authors: Dr Vinodh Kannappan, Dr Zhipeng Wang, Dr Peng Liu, Dr Sarah Brown, Ms Kate Butcher, Mr Karim Azar, Prof Xiuwu Bian, Dr Angel Armesilla, Prof John Darling, Prof Weiguang Wang
Journal: Neuro-Oncology (2017)

BACKGROUND: Despite decades of research the therapeutic outcome of Glioblastoma Multiforme (GBM) remains dismal as no contemporary chemotherapeutic regimen is effective. Recent understanding of the molecular mechanisms behind chemoresistance has focussed on a small population (~1%) of GBM cancer stem cells (CSCs) which promotes therapeutic resistance and local invasion of GBM cells. Evidence indicates that intra-tumoral hypoxia drives CSC phenotypes in solid tumours via epithelial-to-mesenchymal transition (EMT). Hypoxia inducible factors (HIFs), NF-κB and aldehyde dehydrogenase (ALDH) activity are highly up-regulated in hypoxia-induced CSCs, but the insights on how HIFs, NF-κB and ALDH together coordinate the stemness and chemoresistance still remain obscure. Development of drugs that can cross blood brain barrier (BBB) and effectively target the CSCs is of significant importance for GBM chemotherapy. Disulfiram (DS), a clinically used anti-alcoholism drug, is a strong inhibitor of NF-κB and ALDH. Our previous studies show that DS in combination with copper (Cu) can effectively reverse chemoresistance and block metastasis in multiple cancers. In this study we used an in vitro GBM CSC model of NF-κB-p65 and HIFs transfected GBM cell lines to investigate the relationship between hypoxia induced HIFs, NF-κB activation and ALDH activity and their role in chemoresistance. We also examined the in vitro cytotoxicity and the molecular mechanism of action of DS/Cu on GBM CSCs. Although DS is very effective in suppressing GBM in vitro, its clinical application is severely limited by its short half-life in the bloodstream. We recently developed and characterized a poly lactic-co-glycolic acid (PLGA)-encapsulated DS nanoparticles, protecting DS from the degradation in the bloodstream. We evaluated the in vivo efficacy of the PLGA-DS nanoparticles in subcutaneous and orthotopic xenograft glioma mouse models. RESULTS: GBM cells grown as spheres showed vast proportion of hypoxic cells with elevated CSC and EMT markers suggesting hypoxia induced EMT. GBM-CSCs are chemoresistant and displayed increased levels of HIFs, NF-κB and ALDH activity. GBM cells transfected with NFκB-p65 and HIFs exhibited CSC markers and chemoresistance indicating their pivotal role in maintaining CSC phenotypes. DS/Cu is cytotoxic to GBM cell lines and completely eradicated CSCs at low nanomolar levels. DS/Cu inhibits NF-κB and ALDH activity and triggers intrinsic apoptotic pathway. PLGA encapsulation extended the half-life of DS from shorter than 2 minutes to 7 hours in serum. In combination with copper, DS-PLGA significantly inhibited GBM tumour in both subcutaneous and orthotopic xenograft mouse models at a very low dose (<1/10 of antialcoholism dose). CONCLUSIONS: DS/Cu can effectively suppress GBM tumour both in vitro and in vivo with low/no toxicity to normal tissues and can freely pass through the BBB. Both DS and PLGA are FDA approved products for clinical application. Our study may repurpose DS into cancer indication and lead to a breakthrough in GBM treatment.