Proteomic signature of neuroblastoma cells UKF-NB-4 reveals key role of lysosomal sequestration and the proteasome complex in acquiring chemoresistance to cisplatin

Abstract

Cisplatin (CDDP) is a widely used agent in the treatment of neuroblastoma. Unfortunately, the development of acquired chemoresistance limits its clinical use. To gain a detailed understanding of the mechanisms underlying the development of such chemoresistance, we comparatively analyzed established cisplatin-resistant neuroblastoma cell line (UKF-NB-4(CDDP)) and its sensitive counterpart (UKF-NB-4). First, using viability screenings, we confirmed the decreased sensitivity of tested cells to cisplatin and identified a cross-resistance to carboplatin and oxaliplatin. Then, the proteomic signatures were analyzed using nano liquid chromatography with tandem mass spectrometry. Among the proteins responsible for UKF-NB-4(CDDP) chemoresistance, ion channels transport family proteins, ATP-binding cassette superfamily proteins (ATP = adenosine triphosphate), solute carrier-mediated trans-membrane transporters, proteasome complex subunits, and V-ATPases were identified. Moreover, we detected markedly higher proteasome activity in UKF-NB-4(CDDP) cells and a remarkable lysosomal enrichment that can be inhibited by bafilomycin A to sensitize UKF-NB-4(CDDP) to CDDP. Our results indicate that lysosomal sequestration and proteasome activity may be one of the key mechanisms responsible for intrinsic chemoresistance of neuroblastoma to CDDP.Cisplatin (CDDP) is a widely used agent in the treatment of neuroblastoma. Unfortunately, the development of acquired chemoresistance limits its clinical use. To gain a detailed understanding of the mechanisms underlying the development of such chemoresistance, we comparatively analyzed established cisplatin-resistant neuroblastoma cell line (UKF-NB-4(CDDP)) and its sensitive counterpart (UKF-NB-4). First, using viability screenings, we confirmed the decreased sensitivity of tested cells to cisplatin and identified a cross-resistance to carboplatin and oxaliplatin. Then, the proteomic signatures were analyzed using nano liquid chromatography with tandem mass spectrometry. Among the proteins responsible for UKF-NB-4(CDDP) chemoresistance, ion channels transport family proteins, ATP-binding cassette superfamily proteins (ATP = adenosine triphosphate), solute carrier-mediated trans-membrane transporters, proteasome complex subunits, and V-ATPases were identified. Moreover, we detected markedly higher proteasome activity in UKF-NB-4(CDDP) cells and a remarkable lysosomal enrichment that can be inhibited by bafilomycin A to sensitize UKF-NB-4(CDDP) to CDDP. Our results indicate that lysosomal sequestration and proteasome activity may be one of the key mechanisms responsible for intrinsic chemoresistance of neuroblastoma to CDDP.