Exosomes confer chemoresistance to pancreatic cancer cells by promoting ROS detoxification and miR-155-mediated suppression of key gemcitabine-metabolising enzyme, DCK

Abstract

Background:Chemoresistance is a significant clinical problem in pancreatic cancer (PC) and underlying molecular mechanisms still remain to be completely understood. Here we report a novel exosome-mediated mechanism of drug-induced acquired chemoresistance in PC cells.Methods:Differential ultracentrifugation was performed to isolate extracellular vesicles (EVs) based on their size from vehicle- or gemcitabine-treated PC cells. Extracellular vesicles size and subtypes were determined by dynamic light scattering and marker profiling, respectively. Gene expression was examined by qRT-PCR and/or immunoblot analyses, and direct targeting of DCK by miR-155 was confirmed by dual-luciferase 3′-UTR reporter assay. Flow cytometry was performed to examine the apoptosis indices and reactive oxygen species (ROS) levels in PC cells using specific dyes. Cell viability was determined using the WST-1 assay.Results:Conditioned media (CM) from gemcitabine-treated PC cells (Gem-CM) provided significant chemoprotection to subsequent gemcitabine toxicity and most of the chemoresistance conferred by Gem-CM resulted from its EVs fraction. Sub-fractionation grouped EVs into distinct subtypes based on size distribution and marker profiles, and exosome (Gem-Exo) was the only sub-fraction that imparted chemoresistance. Gene expression analyses demonstrated upregulation of SOD2 and CAT (ROS-detoxifying genes), and downregulation of DCK (gemcitabine-metabolising gene) in Gem-Exo-treated cells. SOD/CAT upregulation resulted, at least in part, from exosome-mediated transfer of their transcripts and they suppressed basal and gemcitabine-induced ROS production, and partly promoted chemoresistance. DCK downregulation occurred through exosome-delivered miR-155 and either the functional suppression of miR-155 or restoration of DCK led to marked abrogation of Gem-Exo-mediated chemoresistance.Conclusions:Together, these findings establish a novel role of exosomes in mediating the acquired chemoresistance of PC.

Cisplatin induces the release of extracellular vesicles from ovarian cancer cells that can induce invasiveness and drug resistance in bystander cells

“…The observation that heparin does not completely block this adaptive response could be because either additional non-vesicular factors are required for the response, or because some of the effects could be mediated by interaction of the EV with receptors at the cell surface without the need for EV uptake and cargo delivery. A recent study has also demonstrated that EVs released following treatment of pancreatic cancer cells with gemcitabine can induce an adaptive response in recipient cells, which may be mediated by enhanced reactive oxygen species detoxification and miR-155-induced suppression of a gemcitabine metabolising enzyme (75). Taken together, our results are consistent with the hypothesis that treatment of cells with cisplatin-EVs induces a survival mechanism that allows them to adapt to resist the effects of cisplatin.…”

Hypoxia alters the release and size distribution of extracellular vesicles in pancreatic cancer cells to support their adaptive survival

“…18,33 Furthermore, it has also been demonstrated that their biogenesis and shedding can be influenced by extracellular stimuli, which holds broader functional significance. Although initially believed to serve as trash bags to get rid of unwanted cellular material, EVs are now established as functional entities that are capable of altering the properties of the recipient cells through transfer of bioactive cargo.…”

“…17,18,24 Since we observed an increased release of EVs under hypoxia, we evaluated if they have a role in acute survival response of pancreatic cancer cells. 17,18,24 Since we observed an increased release of EVs under hypoxia, we evaluated if they have a role in acute survival response of pancreatic cancer cells.…”

“…12,14,15 It has been shown that EVs are not only capable of altering the properties of those cells that are in close proximity but also travel to distant sites to have functional consequences by transferring their bioactive cargo. [18][19][20] In the present study, we examined the effect of hypoxia on the release of EVs by the pancreatic tumor cells as an adaptive response mechanism. Emerging data from our lab and others have shown that EVs serve as sensors of the cellular microenvironment changes potentially helping the cells to adjust and respond to the environmental cues.…”

Resistin potentiates chemoresistance and stemness of breast cancer cells: Implications for racially disparate therapeutic outcomes

“…Cells (1×10 4 ) were grown in 96-well plates and treated with PBS or resistin (20 ng/ml) for 12 h. After treatment, cells were further treated with doxorubicin (0–2.0 μM) in presence or absence of resistin for 72 h, and cell viability was calculated using WST-1 assay kit (Roche, Indianapolis, IN), as described earlier [29, 30]. …”