Poster Presentation Advances in Neuroblastoma Research Congress 2016

Dextran-Catechin conjugate targets copper metabolism in neuroblastoma (#229)

Orazio Vittorio 1 2 , Miriam Brandl 1 , Giuseppe Cirillo 3 , Kathleen Kimpton 1 , Elizabeth Hinde 4 , Claudia Flemming 1 , Michelle Haber 1 , Murray D Norris 1 , Arvind Parmar 5 , Giancarlo Pascali 5 , Arnaud Charil 5 , Maria Kavallaris 1 2
  1. Children's Cancer Institute, Randwick, NSW, Australia
  2. ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Australian Centre for NanoMedicine, UNSW, Sydney, NSW, Australia
  3. University of Calabria, Catanzaro, Italy
  4. ARC Centre of Excellence in Advanced Molecular Imaging University of New South Wales, Sydney, Australia
  5. Australian Nuclear Science and Technology Organisation, Lucas Heights, NSW, Australia

Background: Despite the use of aggressive therapy survival rates for neuroblastoma patients are poor, and survivors often experience long-term side effects from the treatment. Catechin is an natural antioxidant under evaluation in multiple clinical trials, however, poor serum stability of catechin has limited its clinical application. We overcame this issue by conjugating catechin with dextran (Dextran-Catechin) and evaluated its potential as a treatment for neuroblastoma.

Methods: Viability assays were performed using Alamar-Blue. Apoptosis using PARP-cleavage and western-blotting. CTR1 gene and protein levels using qPCR and western-blotting, respectively. Copper levels measured using spectrophotometric analysis. Induction of oxidative stress using ROS-fluorescent probes and NADH/NAD+ ratio measured using FLIM. Levels of glutathione examined by colorimetric assay. Copper uptake in tumours quantitated using Cu64-PET-imaging. Dextran-Catechin anti-cancer activity assessed in xenograft and syngeneic models of neuroblastoma.

Results: Dextran-Catechin significantly decreased neuroblastoma cell viability in SH-SY5Y, IMR-32, BE(2)C and doxorubicin-resistant BE(2)C-ADR cell lines (IC50 9.7-18.2 µg/ml). Importantly, in non-malignant MRC-5 cells, no IC50 was reached at doses as high as 60μg/ml. In IMR-32-Cisplatin-resistant cells, Dextran-Catechin resistance was observed compared to parental cells prompting us to investigate copper-transporter-1 (Ctr1), the major cisplatin transporter. Mechanistically, Dextran-Catechin generated oxidative stress in neuroblastoma cells containing high intracellular copper, but not in MRC-5 with low copper levels. Dextran-Catechin was found to react with copper, generating ROS and inducing cell death. Decreased NADH/NAD+ ratio and GSH levels further confirmed induction of oxidative stress. To investigate copper metabolism in vivo, Cu64-PET-imaging of bio-distribution in a xenograft neuroblastoma model was performed and confirmed high accumulation of copper in the tumour mass. Finally, we showed that Dextran-Catechin significantly reduced tumour growth in human xenograft and syngeneic models of neuroblastoma.

Conclusion: Dextran-Catechin mediates its effects via copper metabolism and has the potential to be used as a treatment for neuroblastoma and other cancers.