Inhibition of cathepsin proteases attenuates migration and sensitizes aggressive N-Myc amplified human neuroblastoma cells to doxorubicin

Abstract

Neuroblastoma arises from the sympathetic nervous system and accounts for 15% of childhood cancer mortality. Amplification of the oncogene N-Myc is reported to occur in more than 20% of patients. While N-Myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of N-Myc in the aggressive progression of the disease is poorly understood. N-Myc being a transcription factor can modulate the secretion of key proteins that may play a pivotal role in tumorigenesis. Characterising the soluble secreted proteins or secretome will aid in understanding their role in the tumour microenvironment, such as promoting cancer cell invasion and resistance to treatment. The aim of this study is to characterise the secretome of human malignant neuroblastoma SK-N-BE2 (N-Myc amplified, more aggressive) and SH-SY5Y (N-Myc non-amplified, less aggressive) cells. Conditioned media from SK-N-BE2 and SH-SY5Y cell lines were subjected to proteomics analysis. We report a catalogue of 894 proteins identified in the secretome isolated from the two neuroblastoma cell lines, SK-N-BE2 and SH-SY5Y. Functional enrichment analysis using FunRich software identified enhanced secretion of proteins implicated in cysteine peptidase activity in the aggressive N-Myc amplified SK-N-BE2 secretome compared to the less tumorigenic SH-SY5Y cells. Protein-protein interaction-based network analysis highlighted the enrichment of cathepsin and epithelial-to-mesenchymal transition sub-networks. For the first time, inhibition of cathepsins by inhibitors sensitized the resistant SK-N-BE2 cells to doxorubicin as well as decreased its migratory potential. The dataset of secretome proteins of N-Myc amplified (more aggressive) and non-amplified (less aggressive) neuroblastoma cells represent the first inventory of neuroblastoma secretome. The study also highlights the prominent role of cathepsins in the N-Myc amplified neuroblastoma pathogenesis. As N-Myc amplification correlates with aggressive neuroblastoma and chemotherapy-based treatment failure, co-treatment with cathepsin inhibitors might be a better avenue for disease management.

Bovine milk-derived exosomes from colostrum are enriched with proteins implicated in immune response and growth

“…The relative protein abundance between the samples was obtained by estimating the ratio of normalized spectral counts (RSc) as previously described 29 .Where s is the significant MS/MS spectra for protein A, T is the total number of significant MS/MS spectra in the sample, c is the correction factor set to 1.25, and X or Y are the exosome samples at different time points (24 h, 48 h, 72 h and MM). When RSc is less than 1, the negative inverse RSc value was used.…”

Insulin Mediated Activation of PI3K/Akt Signalling Pathway Modifies the Proteomic Cargo of Extracellular Vesicles

“…EVs protein separation in SDS‐PAGE was carried out at constant voltage of 150 Volt for 1 h. The gel was stained with Coomassie for 1 h and destained (7.5% acetic acid, 20% ethanoic acid in milliQ) to obtain a clear background. Each protein lane was cut into 20 gel bands and subjected to a series of reduction, alkylation and trypsinization as previously described . Briefly, gel fragments were destained further using acetonitrile solution (50% (v/v) acetonitrile: 50 mM Amonium Bicarbonate (1:1)) overnight with constant shaking.…”

Cathepsin Inhibition Prevents Autophagic Protein Turnover and Downregulates Insulin Growth Factor-1 Receptor-Mediated Signaling in Neuroblastoma

“…A recent study indicates that cathepsin inhibition may sensitize neuroblastoma to commonly used therapeutic agents (Gangoda et al, 2015). Agents with good safety profiles that target multiple growth-related pathways simultaneously should improve cancer treatments by preventing tumor cells surviving by activating alternative signaling pathways.…”