Characterization of hypoxia-induced proteome changes & exosomes bystander effect in H69 small-cell lung cancer cells
Author
Medina Fernandez, Jorge
Term
4. term
Publication year
2018
Submitted on
2018-05-31
Pages
60
Abstract
Småcellet lungecancer (SCLC) har dårlig prognose, og lavt iltindhold (hypoksi) og cellekommunikation via små vesikler (exosomer og andre ekstracellulære vesikler) kan bidrage til dens aggressivitet. Dette projekt undersøgte, hvordan hypoksi ændrer proteomet og vesikeludskillelsen i H69 SCLC-celler, og om exosomer dannet under hypoksi kan omprogrammere andre SCLC-celler (bystandereffekt). To in vitro-forsøg blev udført: (1) et hypoksiforsøg, hvor celler blev udsat for forskellige iltniveauer i 24 timer, efterfulgt af isolering af ekstracellulære vesikler og cellelysater; og (2) et co-kulturforsøg, hvor exosomer fra hypoksiske versus normoksiske forhold blev tilsat normoksiske celler i 48 timer. Vesikler blev karakteriseret med nanopartiklesporing (NTA), og proteomprofiler blev analyseret med tandem massespektrometri (MS/MS). Hypoksi øgede cellernes metaboliske profil og exosomeproduktion samt opregulerede GPI og P4HA1. Hypoksiske vesikler var beriget for pro-karcinogene proteiner, der understøtter angiogenese, metastase, proliferation og motilitet, herunder øget forekomst af ephrinreceptor (EPH) og HMGA1 sammenlignet med normoksiske vesikler. Hypoksisk afledte exosomer udløste en bystandereffekt med omprogrammering mod et mere aggressivt cellefænomen: nedsat apoptose og programmeret celledød, øget vækst, proliferation og overlevelse, ledsaget af hypermethylering af CpG-øer og overekspression af DDX6. Samlet peger resultaterne på, at hypoksi både direkte fremmer carcinogenese og tumorprogression i SCLC og indirekte forstærker sygdomsadfærd ved at ændre vesikelindholdet og dermed celle-til-celle kommunikation.
Small-cell lung cancer (SCLC) carries a poor prognosis, and both low oxygen (hypoxia) and cell-to-cell communication via small vesicles (exosomes and other extracellular vesicles) may contribute to its aggressiveness. This project examined how hypoxia alters the proteome and vesicle release in H69 SCLC cells, and whether exosomes produced under hypoxia can reprogram other SCLC cells (a bystander effect). Two in vitro experiments were conducted: (1) a hypoxia experiment in which cells were exposed to different oxygen conditions for 24 hours, followed by isolation of extracellular vesicles and cell lysates; and (2) a co-culture experiment in which exosomes from hypoxic versus normoxic conditions were added to normoxic cells for 48 hours. Vesicles were characterized by nanoparticle tracking analysis (NTA), and protein profiles were measured by tandem mass spectrometry (MS/MS). Hypoxia increased the cells’ metabolic profile and exosome production and upregulated GPI and P4HA1. Hypoxic vesicles were enriched in pro-carcinogenic proteins that promote angiogenesis, metastasis, proliferation, and motility, including higher levels of ephrin receptor (EPH) and HMGA1 compared with normoxic vesicles. Hypoxia-derived exosomes triggered a bystander effect that reprogrammed cells toward a more aggressive phenotype: decreased apoptosis and programmed cell death, increased growth, proliferation, and survival, along with CpG island hypermethylation and DDX6 overexpression. Overall, the findings indicate that hypoxia directly promotes carcinogenesis and tumor progression in SCLC and indirectly amplifies disease behavior by reshaping vesicle cargo and thereby cell-to-cell communication.
[This summary has been generated with the help of AI directly from the project (PDF)]
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