The role of extracellular vesicles in the transfer of multidrug resistance in human ovarian cancer cells
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Ovarian cancer is the fifth most common cancer in Canadian women and has the highest mortality rate of all gynecologic malignancies. First-line treatment is typically cytoreductive surgery followed by a combination of paclitaxel and carboplatin. Unfortunately, patients frequently relapse with drug resistant disease, and only 45% of patients survive beyond 5 years. Drug resistance results from multiple mechanisms, one of which is mediated by one or more of the ATP-binding cassette (ABC) drug efflux transporters. The two ABC transporters considered clinically relevant in ovarian cancer are P-glycoprotein (P-gp) and multidrug resistance protein 1 (MRP1). These plasma membrane transporters can efflux an array of solutes from the cell, including paclitaxel. Extracellular vesicles (EVs) are a collective term for nano-sized membrane vesicles released from all mammalian cells that can serve as cell-free vehicles to deliver a variety of biomolecules to recipient cells. The human ovarian cancer cell lines A2780 and 2008, and their drug resistant variants, AD645 and 2008/MRP1, which overexpress P-gp and MRP1, respectively, were used to investigate whether transporter-containing EVs can transfer drug resistance to sensitive cells. All four cell lines were shown to release EVs isolated by differential ultracentrifugation (DUC) and size-exclusion chromatography (SEC), as indicated by the presence of EV markers CD63, CD81, and syntenin-1 in immunoblots of EV extracts. P-gp and MRP1 were also detected in EV extracts from AD645 and 2008/MRP1 cells, respectively. DUC- isolated AD645 EVs appeared toxic to A2780 recipient cells and there was no detectable transfer of paclitaxel resistance after co-culture. SEC-isolated AD645 EVs were more enriched than DUC-isolated EVs for CD63, CD81, and syntenin-1, and appeared less toxic to A2780 cells. However, P-gp was not enriched in SEC-isolated AD645 EVs and there was no detectable ii transfer of paclitaxel resistance or P-gp to A2780 cells. These observations suggest that the amount of functional P-gp transferred to recipient cells was insufficient to confer detectable resistance. These studies suggest that although P-gp and MRP1 can be detected in cellular material consistent with the presence of EVs, additional experiments are needed to optimize isolation of transporter-enriched EVs and co-culture conditions to detect the transfer of drug resistance.