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.
关键词
PC3细胞/细胞裂亡/细胞外囊泡/联合阿霉素治疗
Key words
PC3 cell line/Mitotic cell death/Extracellular vesicles/Therapy combined with adriamycin
维普
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