Abstract 3282: Induction and characterization of cyclopamine resistance in SK-N-DZ neuroblastoma and DAOY medulloblastoma cell lines

Abstract

Abstract Neuroblastoma and medulloblastoma are malignant solid tumors of childhood and early adolescence, that often reappear after clinical treatment due of an acquired chemo-resistance. These tumors present an increased expression of the Sonic Hedgehog signaling pathway. In the last years, inhibitors of this pathway, such as cyclopamine are being studied as a possible alternative to current treatment. In this research, we aim at analyzing the appearance and characteristics of resistance derived from the treatment with mutation-selective concentrations of cyclopamine. The aim of this study is the design of different ways to bypass the resistance generated by the tumor once its biology is completely understood. Cells were exposed to several treatment cycles. A 24 h cyclopamine exposure was performed when cell culture reached 60-70% confluence. Basal half-maximal inhibitory concentration (IC50) was 10 μM. Hence, SK-N-DZ cells received 5 treatment cycles of cyclopamine at 12 μM dissolved in DMSO; whereas Daoy cells received 5 treatment cycles of cyclopamine at 10 μM. The control group only received the diluent. Anchorage-independent growth assays (in soft agar), clonogenicity assays, cell migration scratch (wound-healing) assays, and relative mRNA expression were studied during cycles 1-5. During the first 5 treatment cycles, mRNA expression of SMO remained constant. CD15 expression decreased. MYCN, Gli and expression showed an increasing trend. Decrease in the pro-apoptotic status was also described. Soft agar showed an increase tendency in relative colony formation capacity. No statistical differences were found in the clonogenicity assays for the SK-ND-Z cells. The results highlighted the evidence that cells became resistant because they were able to grow in soft agar without attachment, to migrate and to overexpress genes related to cancer stem cells. Results shown are congruent with a resistance generation possibly owing to multidrug efflux pumps. However, more clear results would be seen with 48 h treatment. Further analysis based on this work could include 48 h treatment, water-dissolvable cyclopamine performance study, a more complete gene analysis, or studies with other neurological tumors. Note: This abstract was not presented at the meeting. Citation Format: Javier de la Rosa, Amaia Paredes, Javier Asensio-Salazar, Xing Fan, Bárbara Meléndez, Juan A. Rey, Javier S. Castresana. Induction and characterization of cyclopamine resistance in SK-N-DZ neuroblastoma and DAOY medulloblastoma cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3282. doi:10.1158/1538-7445.AM2015-3282