Abstract 3553: Cepharanthine reverses multidrug resistance sensitizing neuroblastoma cell lines to vincristine-induced cell death
Authors: Graham Regina M. , Guest James D. , Thompson John W. , Webster Keith A. , Vanni Steven 
Published: February 2011

Abstract
Neuroblastoma (NB) is the most common extra cranial solid tumor of childhood and accounts for approximately 15% of all cancers in children less than 5 years of age. At time of diagnosis, most children already have metastatic disease. Despite multimodality treatment for advanced NB, the survival rate is only 30%; therefore there is a clinical need for more effective therapeutic strategies. Cepharanthine (CEP), a natural alkaloid extracted from Stephania Cepharantha Hayata, has been widely used in Japan for years to treat both acute and chronic diseases without serious side effects. We examined the potential of cepharanthine to induce cell death in several NB cell lines. We exposed SMSKCNR (SMSR), SK-N-BE2c (BE2c), and SH-SY5Y (SY5Y) NB cells to increasing concentrations of CEP and examined cell viability with MTS assay. While 1μM treatment had no significant effect on cell viability at 72 hours, 10μM CEP significantly reduced cell viability compared to vehicle-treated controls (SMSR 11±2.2%; BE2c 8.2±1.1%; SY5Y 1.2±4.1%, p&lt;0.01). In comparison 10μM CEP did not affect PC3 prostate cancer cell viability (98±2.9% of non-treated controls). Next we examined the effect of CEP in combination with vincristine (Vin), a vinca alkaloid commonly used for NB treatment. NB cells were treated with 3.0μM CEP, 50nM Vin, or both. At 72 hours, 3.0μM CEP alone reduced viability to 72±3.4% (SMSR), 83±3.7% (BE2c), and 82±7.5% (SY5Y) of vehicle-treated controls. While Vin treatment alone had no affect on viability, 92±4.3% (BE2c), 104±4.7% (SMSR), and 94±7.5% (SY5Y), the combination of Vin and CEP significantly reduced viability to less than 10% of non-treated controls (SMSR 7.2±3.8%, BE2c 8.8±1.0%, and SY5Y 2.8±7.0%, p&lt;0.01). To determine if CEP treatment increased Vin-induced cell death by reversing multidrug resistance, we examined the effect of CEP on doxorubicin (DOX) cellular accumulation. DOX is an anthracycline commonly used in chemotherapy and possesses fluorescent characteristics. In addition, both DOX and Vin are known substrates for multidrug drug resistance proteins. Be2c NB cells were treated with 3.0μM DOX with and without 10μM CEP for one hour and the level of DOX (n = 100 nuclei) evaluated with fluorescent microscopy. Nuclear retention of DOX was evaluated 18 hours after removing DOX and CEP from the culture media. Using fluorescent microscopy, the intensity of DOX signal was approximately 3-fold higher in cells treated with CEP (CEP+DOX: 59±19.2 vs. DOX: 20±19.1 arbitrary units; p&lt;0.01) suggesting an inhibition of multidrug resistance proteins by CEP. Our data indicates that CEP can directly induce NB cell death as well as sensitize cells to Vin-induced cell death at concentrations that are clinically achievable. CEP represents a novel addition to NB treatment regimens and may potentially improve clinical outcome.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3553.