In vitro evaluation of P-glycoprotein functions in human neuroblastoma cell lines

Abstract

Background: Neuroblastoma (NB) remains one of the most puzzling of paediatric cancers in which most patients develop progressive disease that is refractory to chemotherapy. Effective treatment is hampered by drug resistance related to the expression of multidrug-resistant proteins belonging to the ATP-binding cassette transporters family, especially P-glycoprotein (P-gp). Most previous studies focused on molecular evidence of P-gp expression, however, functional studies of P-gp efflux have not clearly demonstrated. Objectives: The aim of this study was to determine whether human neuroblastoma cell lines (SH-SY5Y and SK-N-SH) express the functionally active P-gp efflux pump. Materials and methods: Functional studies on P-gp–mediated pumping were performed using pirarubicin, a P-gp substrate, with verapamil, a multidrug resistance inhibitor, and analyzed by a spectrofluorometer. To confirm the gene expression, reverse transcription polymerase chain reaction (RT-PCR) was performed with specific primers for human multidrug resistance 1 (MDR1). Results: MDR1 expression was observed in neuroblastoma cell line (SH-SY5Y) in the same degree of expression as in the sensitive K562 cell line, a negative P-gp model. Kinetic analysis showed that there was no difference in drug accumulation in the presence or absence of verapamil, indicating that no function of P-gp influenced the accumulation of pirarubicin (PIRA) in both human neuroblastoma cell lines. Combination treatment of verapamil and PIRA was also not found to increase the sensitivity of PIRA. Conclusion: This study suggests that the existence of P-gp in neuroblastoma cell lines is not significant function.

Keywords

• Chemotherapy
• drug resistance
• neuroblastoma
• P-glycoprotein