Objective To investigate the effect of extracellular vesicles derived from mitotic cell death PC3 cells(MEV)on prostate cancer cells.Methods After the phenotype of mitotic death was continuously induced by 1 μmol/L cisplatin for 5 days,nuclear damage was detected by immunofluorescence,cell stiffness was measured by Atomic Force Microscope,cell senescence marker(SA-β-Gal),proliferation,cycling,mitochondrial membrane potential,mitochondrial number by flow cytometry,and expression of senescence-secreting phenotype-associated factors including CDKNIA,CDKN2A,IL-6,IL-8,TNF-α,IFN-α/β/γ by qRT-PCR.Mitotic-death PC3 cells derived extracellular vesicles(MEV)were extracted,and the morphology and size of MEV were detected by electron microscopy and nanoparticle tracking analysis,and their stiffness and substance(β-actin and mtDNA)were separately detected by atomic force microscope and qRT-PCR.The effects of MEV on PC3 cells were further detected by immunofluorescence to detect phagocytosis,flow cytometry to detect proliferation and apoptosis,and qRT-PCR to detect the level of IFNα/β/γ.Finally,50 μg MEV were treated to PC3 cells combined with 15μmol/L adriamycin(Dox),and apoptosis was detected by flow assay.Results Mitotic cell death PC3 cells and MEV were both obtained successfully.In comparison with normal PC3's,the number of MEV group was significantly increased[(4 530.9±353.6)×106(particle)/1×106 cell and(33.7±5.4)×106 particle/1×106 cell,P＜0.01].Additionally,the particle size of the extracellular vesicles were significantly smaller[(122.0±2.6)nm and(163.6±2.6)nm,P＜0.01],along with significantly increased content of nuclear DNA[(111.0±20.7)/1×106 cell,P＜0.01]and mtDNA[(26.2±3.8)/1×106 cell,P＜0.01].MEV were also easier to absorb by PC3 for their softness[(0.11±0.01)MPa and(0.18±0.01)MPa,P＜0.01].MEV could significantly induce PC3 cell apoptosis[(641.0±42.5)MFI and(351.7±37.0)MFI,P＜0.01]and inhibit their proliferation[(1 523.0±64.9)MFI and(1 336.3±94.1)MFI,P＜0.05].Besides,they did not affect the level of IFNβ but down-regulated IFNα/γmRNA level[(0.6±0.1)and(0.8±0.1),P＜0.01].The combination of MEV and 15 μmol/L Dox can significantly promote PC3 cell apoptosis[(14 290.3±1315.9)MFI and(2 669.3±241.5)MFI,P＜0.01].Conclusions Mitotic cell death PC3 cells can efficiently secrete DNA-rich flexible extracellular vesicles.The MEV were more easily taken up by PC3 and significantly inhibit its cellular activity and promote the anticancer effect of Dox.
PC3 cell lineMitotic cell deathExtracellular vesiclesTherapy combined with adriamycin
维普
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