TRAIL POTENTIATES PANOBINOSTAT CYTOTOXICITY IN GLIOBLASTOMA MULTIFORME (GBM) IN VITRO

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Abstract

BACKGROUND: (blind field). METHODS: Low passage short-term cultures derived from GBM previously extensively characterised at a molecular level were used in this study. RNA was extracted using RNeasy Mini Kit. Quantitative RT-PCR (Q-PCR) was carried out using Solaris qPCR gene expression assay with FAM reporter on an ABI 7500 Sequence Detection System and protein expression confirmed used Western blotting. Cytotoxicity was determined in vitro using a sulforhodamine B assay, drug interaction was measured using the median-effect principle proposed by Chou-Talalay and a Cignal™ 45-pathway reporter assay was used to screen pathway activity following drug treatment. RESULTS: Q-PCR showed that TRAIL ligand was preferentially expressed in cultures that contained a p53 mutation or deletion whilst expression was low or absent in cultures with wild-type p53. TRAIL death receptors 1 (DR4) and 2 (DR5) were expressed in cultures irrespective of p53 status. TRAIL decoy receptor 3, 1 and 2 (DcR2) were highly expressed in cultures with wild-type p53 and either absent or at low levels in p53 mutant cultures. The ID50 of cultures to TRAIL ranged from 0.18–15.1 ng/ml. Most cultures with mutant p53 were resistant to TRAIL although one wild-type culture that expressed high levels of DcR2 was very resistant to TRAIL. Knocking down DR5 receptor produced resistance to TRAIL and knocking down DcR2 decoy receptor augmented response to TRAIL. Treatment of cultures with a non-toxic dose of TRAIL enhanced toxicity to panobinostat between 2 and 40-fold and that this was associated with increased reporter activity of the nanog, retinoic acid, wnt and xenobiotic pathways. CONCLUSIONS: A combination of low dose TRAIL and panobinostat are significantly more toxic to GBM cells in vitro than the single agents. Combinations of TRAIL and HDAC inhibitors that affect both cell death pathways should be evaluated in vivo with a view to clinical application in GBM. SECONDARY CATEGORY: n/a.

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