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 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 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.

Metallothionein-3 promotes cisplatin chemoresistance remodelling in neuroblastoma

“…In addition, the up-regulation of ATP-binding cassette transporters (ABC family members) provides a supporting mechanism for direct efflux of CDDP from the cell via energy-fuelled active transport. CDDP can further activate the proteasome complex pathway, which was also demonstrated by our previous results 24,46 . Interestingly, we also identified a pronounced up-regulation of numerous proteasome subunits in UKF-NB-4 cells overexpressing hMT3.…”

“…This phenomenon might be due to CDDP action on several signalling pathways, thereby triggering resistance mechanisms by establishing a complicated self-defence system to escape exogenous cytotoxic compounds of different origins 38 . By contrast, MTs have been implicated in the metastasis in several types of cancers 29,30 , with evidence that MT3 regulated the genes involved in metastasis 25,46 . In this study, we elucidated the potential mechanisms of hMT3-mediated induction of Nbl invasion and CDDP-chemoresistance in a CAM xenograft tumour model.…”

“…After harvesting the cells by trypsin, the suspension was centrifuged (10,000 rpm, 10 min). Subsequent lysis of the pellet, separation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis, electroblotting on poly(vinylidene fluoride) membrane and blocking of non-specific binding was performed according to our previously published study 46 . For experiments, primary anti-MT3 antibody (ab214314; dilution 1:750, Abcam, Cambridge, UK), anti-GFP antibody (ab183734, dilution 1:2,000, Abcam), anti-GAPDH (ab181602, dilution 1:5,000, Abcam) and anti-α-tubulin (ab7291, dilution 1:5,000, Abcam) were utilized.…”

“…Sample preparation and nLC-MS/MS analysis. Protein extraction and processing and proteomic profiling using nLC-MS/MS was carried out according to Merlos Rodrigo et al 46 . The MS analyses were carried out using Q-Exactive mass spectrometer (Thermo Fisher Scientific) utilizing 2000 V of liquid junction voltage for sample ionization and capillary temperature of 270 °C.…”

Getting Lost in the Cell–Lysosomal Entrapment of Chemotherapeutics

“…For example, in pancreatic ductal adenocarcinoma (PDA), constitutive nuclear import of MiT/TFE factors leads to upregulated lysosomal catabolism, generating autolysosome-derived pools of amino acids to sustain robust tumor growth [84]. Proteomic analysis of cisplatin-sensitive and resistant neuroblastoma cells also reveals remarkable lysosomal enrichment and proteasomal activity [85]. Autophagy inhibition, on the other hand, restores chemosensitivity to previously BRAF inhibitor-resistant brain tumor cells, highlighting the need for upregulated autophagic machineries in drug-resistant cancer cells [86].…”