Ultraviolet Induced Tumor Cell Microparticles Enhance The Sensitivity Of Non-small Cell Lung Cancer Cells To Cisplatin By Inhibiting Autophagy

Objective:The purposes of this study were to investigate whether Ultraviolet induced tumor cell microparticles?UV-TMPs?could enhance the sensitivity of non-small cell lung cancer cells to cisplatin;whether cisplatin could induce autophagy in tumor cells;whether UV-TMPs enhanced the sensitivity of non-small cell lung cancer cells to cisplatin by inhibiting autophagy.Methods:After extensively culturing A549 cells,they were irradiated with ultraviolet(UVB,300 J m^-2),and then the supernatant was collected to extract UV-TMPs.The particle size distribution,size,morphology and source of UV-TMPs were identified by NTA technique?Nanoparticle Tracking Analysis?,electron microscopy and WB?Western Blot?.Different staining of A549 cells and UV-TMPs were performed,and then the uptake of UV-TMPs by A549 cells was observed under confocal microscopy.A549 cells were infected with StubRFP-sensGFP-LC3 lentivirus,and then A549 cells stably expressing StubRFP-sensGFP-LC3 protein were selected with puromycin.The effects of UV-TMPs alone on A549 cells and the function of UV-TMPs to enhance the toxicity of cisplatin were detected by flow cytometry kit,CCK8 kit,and WB.Using WB and A549 cells stably expressing StubRFP-sensGFP-LC3 protein to detect whether UV-TMPs affect the expression of LC3 protein in A549 cells,and whether UV-TMPs affect the autophagy process of A549 cells.Results:1.NTA detection and electron microscopy observations proved that the UV-TMPs prepared by our method had diameters between 100-1000 nm,and its shapes were irregular spherical and cup-shaped.The WB results showed that the UV-TMPs were indeed microparticles derived from A549 cell membrane.2.The A549 cells infected with the StubRFP-sensGFP-LC3 lentivirus screened and purified with puromycin could stably express GFP and RFP fluorescence.3.Under the confocal microscope,the green UV-TMPs and the A549 cells?whose membrane was stained red,and the nuclei were stained blue?were co-localized,indicating that UV-TMPs can be taken up by the A549 cells.4.Flow cytometry experiments,CCK8,WB,migration experiments showed that UV-TMPs alone did not affect the apoptosis and survival of A549 cells,but decreased the migration ability of A549 cells.5.Flow cytometry demonstrated that UV-TMPs could increase the apoptosis rate of A549 cells induced by cisplatin.6.The results of the CCK8 test demonstrated that UV-TMPs reduced the survival rate of A549 cells after cisplatin treatment.7.The results of WB demonstrated that UV-TMPs could increase the expression of cleaved caspase-3 induced by cisplatin in A549 cells.8.Fluorescence experiments confirmed that UV-TMPs accumulated the autophagosome or autolysosome in A549 cells.9.WB demonstrated that the UV-TMPs and cisplatin could increase the quantity of LC3-?in A549 cells.Conclusions:UV-TMPs alone could not affect the apoptosis or survival of tumor cells,but could affect the migration ability of tumor cells.UV-TMPs could enhance the sensitivity of non-small cell lung cancer cells to cisplatin.Cisplatin could induce autophagy in tumor cells.UV-TMPs enhanced the sensitivity of non-small cell lung cancer cells to cisplatin by inhibiting autophagy.