Background: Neuroblastoma is an aggressive childhood cancer that is poorly responsive to therapy. Moreover, survivors experience long term side effects from their treatment highlighting the need for effective and less toxic therapies. Catechin is a natural polyphenol with anti-cancer properties and limited side effects. Low serum stability of Catechin limits its clinical use that we overcame by chemical functionalization with Dextran. However its mechanism of action is unknown. Here, we investigated the mechanism of action of Dextran-Catechin, and its efficacy against neuroblastoma in vitro and in vivo.
Methods: Anticancer activity was tested in 4 independent neuroblastoma cell lines using the cell viability assay Alamar Blue and apoptosis was assessed using PARP cleavage. Gene expression was determined using qRT-PCR and protein expression of CTR1 by western blotting. Intracellular copper was measured by spectrophotometric analysis. Fluorescence-lifetime imaging microscopy was used to study NADH/NAD+ ratio to determine the induction of oxidative stress. Cellular levels of the antioxidant GSH was examined using a colorimetric assay. PET imaging studies with Cu64 were performed in a xenograft neuroblastoma model to monitor copper uptake in tumors. In vivo anticancer activity of Dextran-Catechin was assessed in human xenograft and syngeneic models of neuroblastoma.
Results: The neuroblastoma cell lines SH-SY5Y, IMR-32, BE(2)C and doxorubicin-resistant BE(2)C-ADR were sensitive to Dextran-Catechin (IC50 9.7 μg/ml, 17.83 μg/ml, 16 μg/ml and 18.2 μg/ml, respectively) at concentrations that were not toxic to non-malignant MRC-5 cells. However, Cisplatin-resistant neuroblastoma cells, IMR-32-CisRes, were 2.5-fold resistant against Dextran-Catechin compared to the parental cells. Copper transporter 1 (CTR1) mediates cisplatin uptake and IMR-32-CisRes exhibited 50% lower expression of CTR1 and lower intracellular copper compared to the IMR-32 cells. In contrast, Dextran-Catechin sensitive neuroblastoma cells had elevated intracellular copper implicating copper in the activity of this conjugate. We demonstrated that Dextran-Catechin reacts with copper generating reactive oxygen species and inducing cancer cell death. Dextran-Catechin treatment caused a decrease of NADH/NAD+ ratio and GSH levels, confirming oxidative stress. PET imaging analysis of the IMR-32-neuroblastoma xenograft model revealed high accumulation of copper in the tumor mass. The high levels of copper were maintained in the tumor mass for up to 48h, while Cu64 was cleared by the other organs. Importantly, we showed that Dextran-Catechin significantly reduced tumor growth in human xenograft and syngeneic models of neuroblastoma with no evidence of side effects.
Conclusion: Dextran-Catechin targets copper, inhibits tumour growth, and has therapeutic potential for cancers dependent on copper for their growth.
Citation Format: Orazio Vittorio, Miriam Brandl, Giuseppe Cirillo, Kathleen Kimpton, Elizabeth Hinde, Hien T. T. Duong, Cyrille Boyer, Claudia Flemming, Eugene M.H. Yee, Naresh Kumar, Arvind Parmar, Giancarlo Pascali, Arnaud Charil, Michelle Haber, Murray Norris, Maria Kavallaris. Dextran-Catechin conjugate: An anticancer nano-modified natural compound targeting copper metabolism in neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1340.