Proteomic Signature of Neuroblastoma Cells UKF-NB4 Reveals Key Role of Lysosomal Sequestration and the Proteasome Complex in Acquiring Chemoresistance to Cisplatin
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 analysed 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
... tested cells to cisplatin and identified a cross-resistance to carboplatin and oxaliplatin. Then, the proteomic signatures were analysed using nanoLC MS/MS. Among the proteins responsible for UKF-NB-4 CDDP chemoresistance, ion channels transport family proteins, ABC superfamily proteins, SLC-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 key mechanisms responsible for intrinsic chemoresistance of neuroblastoma to CDDP. identified that the regulation of sensitivity to CDDP in SH-SY5Y cells may be driven by Nrf2 pathway 5 . Similarly, Tabata et al. identified that acute application of CDDP can alter expression of enzymes involved in DNA methylation status 18 . However, due to the limitations of 2D-electrophoresis, it can be expected that the low abundance, very large or small proteins cannot be properly identified. Therefore, to unravel the putative mechanisms responsible for acquired chemoresistance to CDDP in UKF-NB-4 cells, we carried out comparative deep proteome survey using nanoLC MS/MS. The obtained results indicate that due to the induced chemoresistance, UKF-NB-4 CDDP cells acquire a highly specific proteomic signature that promotes a remarkable lysosomal enrichment together with the increased activity of proteasome complex. Materials and Methods Chemicals All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) in ACS purity, unless noted otherwise. Cell lines and culture conditions The parental UKF-NB-4 cell line, established from recurrent bone marrow metastases of H-R Nbl and the cisplatin-resistant line UKF-NB-4 CDDP were a kind gift from Prof. J. Cinatl (Goethe University in Frankfurt am Main, Germany). The cells were cultured in IMDM supplemented with 10% foetal calf serum (Thermo Fisher Scientific, Waltham, MA, USA). The cell cultures were incubated at 37°C in a humidified 5% CO 2 atmosphere. The cell lines were passaged at regular intervals twice a week. The morphology of cells was observed under phase contrast using EVOS FL Auto 2 (Thermo Fisher Scientific).