SILAC Strategy for Analysis of Proteins Released To the Extracellular Medium and Penetrated

ABSTRACT: Exogenous signals from drug-stressed cancer cells accelerate the proliferation of neighboring tumor cells and promote them to acquire a more aggressive phenotype. We have recently found an exciting phenomenon: drug-stressed cancer cells secrete various spliceosomal proteins. We have observed this phenomenon both in vitro (culture media from cancer cell lines [Pavluykov M. et al.//Cancer Cell, 2018]) and in vivo (ovarian cancer ascites after chemotherapy [Shender V. et al//Mol. Сell. Proteomics, 2014]). The aim of this study was to elucidate which proteins from the extracellular vesicles can penetrate into recipient cells. SKOV3 donor cells were cultured on special medium containing isotope labeled lysine and arginine. When label inclusion was more than 98%, the cells were treated or untreated (control) with cisplatin. After 48 h, extracellular vesicles containing labeled proteins were isolated. These vesicles were added to unlabeled SKOV3 recipient cells and, after 10 or 48 h, the cells were lysed and subjected to LC-MS/MS to identify labeled proteins. We found that at least 189 heavy-labeled proteins entered recipient cells from the vesicles at 10-hour of incubation. Among them, 101 proteins were present at a higher level in cells incubated with apoptotic vesicles, and 29 proteins were present at a higher level in cells incubated with vesicles from non-apoptotic cells. Functional annotation of heavy-labeled proteins elevated in samples incubated with apoptotic vesicles revealed the highest enrichment in a cluster of spliceosome-related proteins. We suggest that the acquisition of a more aggressive phenotype of recipient cancer cells might be partially mediated by extracellular spliceosomal components.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Malignant Neoplasm Of Ovary

SUBMITTER: Georgij Arapidi  

LAB HEAD: Georgij Pavlovich Arapidi

PROVIDER: PXD027948 | Pride | 2024-05-29

REPOSITORIES: Pride