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Objective To investigate the effect of exosomes secreted from human lung adenocarcinoma A549 cells under hypoxic or normoxic conditions on the radiosensitivity and invasiveness of normoxia cells.Methods A549 cells were cultured in hypoxic (1％ O2) and normoxic (21％ O2) conditions,respectively.The exosomes (N-EXO and H-EXO) secreted from normoxic or hypoxic A549 cells were collected by ultracentrifugation and its number was measured using a NanoSight detector.The appearance and size distribution of exosomes were observed by a scanning electron microscopy.The exosomal marker protein CD63 was measured by Western blot.The proliferation of cells exposed to X-rays under hypoxic or normoxic conditions were detected by CCK8 assay.The cell uptake situation of exosomes labeled with PKH67 was observed by a fluorescence microscopy.Cell migration and invasiveness were detected by a cell scratch test and transwell assay.The expression of matrix metalloproteinase 2 (MMP2) and MMP9 was detected by ELISA.Cellular radioresistance effect of exosomes was evaluated by a colony formation assay.Results The NanoSight measurement showed the number of exosomes in cell culture medium was increased after hypoxia treatment.The H-EXO and N-EXO showed typical ring cake shape.The size distribution of H-EXO was mainly between 30 nm and 200 nm,smaller than that of N-EXO (50-220 nm).Western blot assay showed that CD63 was expressed in both H-EXO and N-EXO.At 4 and 6 days after 2 Gy X-rays irradiation,cell proliferation rate of hypoxia A549 cells was significantly higher than that of normoxia cells.The green fluorescent marker of exosomes,PKH67,was distributed inside of the cell.Cell scratch test showed that the width of H-EXO group was much smaller than that of N-EXO group at 12,24 and 48 hours after exosomes treatment (t =2.96,6.76,3.35,P ＜ 0.05).Transwell assay showed that the number of transmembrane cells in the H-EXO group was more than that in the N-EXO group and the control group (t =4.84,7.88,P ＜ 0.O1).The expression levels of MMP2 (t =4.70,3.21,P＜0.05) and MMP9 (t =5.61,3.76,P＜0.05) in the supernatant of H-EXO group were significantly higher than those in the control and N-EXO groups.Cell survival assay showed that the D0 values of control,N-EXO and H-EXO group were 2.614,2.552 and 4.50 respectively,indicating that H-EXO could enhance radioresistance of A549 cells significantly.Conclusions This study finds that the number of exosomes released from A549 cells was increased under hypoxic condition but its size becomes smaller than that under normoxia.Hypoxic exosomes can promote the migration of normoxia cells andenhance cell radioresistance as well.
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Objective To synthesize novel gold nanoparticles of GAL-PEG-GNPs,study its radiation effect on hepatocellular carcinoma cells HepG2 cells in vitro,and investigate the underlying mechanisms.Methods GAL-PEG-GNPs were synthesized and characterized successfully.HepG2 cells were divided into three groups of control,GNPs and GAL-PEG-GNPs.The cytotoxicities of these compounds were tested by the CCK-8 assay and their IC50 values of HepG2 cells were calculated.Cell uptake of nanoparticles was detected by TEM and ICP-MS.The radiosensitization effect of nanoparticles was tested by the colony formation assay.Cell cycle distribution was detected by FCM.The expressions of CAT,SOD,and total GSH were detected with a microplate reader,and the expressions of apoptosis-related proteins were tested by Western blot.Results The GNPs and GAL-PEG-GNPs had absorption peaks at 520 and 530 nm,respectively,and their diameters were (22.6-±2.12) and (32.0 ± 1.41) nm detected by ICP-MS.The GAL-PEG-GNPs and GNPs had similar cytotoxicity profiles (P ＞ 0.05),while GAL-PEG-GNPs could be more effectively uptaken by HepG2 cells than GNPs.The sensitive enhancement ratio (SER) of GNPs and GAL-PEG-GNPs to HepG2 cells were 1.46 和 1.95,respectively.The percentage of cells at phase of G2/M in HepG2 population treated with GNP was higher than that of untreated cells (t =14.20,P ＜0.05).The protein expressions of Cytochrome C,Bax,Caspase-3,and Caspase-9 were upregulated while Bcl-2 expression was down-regulated in the cells treated with GNPs/radiation or GAL-PEGGNPs/radiation.The expressions of CAT,SOD and total GSH in the GNP treated groups were significantly decreased compared with the control group(t =12.34,29.39,12.85,P ＜ 0.05).Conclusions GALPEG-GNPs has obvious radiosensitization effect on HepG2 cells,which is related to the induction of cell apoptosis and the generation of free radicals.
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Objective To study the radiosensitizing effect of nano-gold nano-silver particles in hepatocellular carcinoma cells (HepG2) in vitro and the possible mechanisms.Methods MTT assay and clonogenic assay were performed to determine the killing effect of nano-gold and nano-silver particles in HepG2 cells.Flow-cytometry was used to measure cell apoptosis and cell cycle distribution.Western blotting was used to measure the expression of Caspase-3,Bax and Bcl-2.ELIASA was used to determine the content of catalase (CAT),superoxide dismutase (SOD),and total glutathione (GSH).Results Nano-gold and nano-silver particles inhibited the proliferation of HepG2 cells with IC50 of 6.51 μg/ml and 2.47 μg/ml,respectively.Nano-gold and nano-silver particles significantly enhanced the radiosensitivity of HepG2 cells.Obtained by Dq,the SER of 1/5 IC50 nano-gold and nano-silver particles were 1.37 and 1.48,and 1/10IC50 with 1.11 and 1.09.Nano-gold and nano-silver particles increased the expression of Caspase-3 and Bax and reduced the exprcssion of Bcl-2.CAT,SOD and total GSH were significantly reduced.Conclusions Nano-gold and nano-silver particles can enhance the radiation sensitivity of HepG2 cells.Specific sensitizing mechanism may be the activation of the mitochondrial apoptosis pathway and the induction of reactive oxygen species in apoptotic pathways,which ultimately induces apoptosis.
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Objective:To investigate the inhibitory effect of inhibitor of differentiation 3(Id3)on growth of human lung adenocarcinoma cell line A549.Methods:Recombinant eukaryotic expression vector pEGFP/Id3 was constructed and transfected into A549 cells by liposome-mediated method.Expression of pEGFP/Id3 in A549 cells was analyzed by flow cytometry(FCM),fluorescence microscopy,semi-quantitative RT-PCR and immunocytochemistry.The growth inhibitory rate of A549 cells was examined by MTT assay;cell cycle change was evaluated by PI(propidium iodide)staining method.Cell apoptotic rate and nuclear morphology were detected by Annexin V/7-AAD and Hoechst33258 staining. Results:The recombinant eukaryotic expression vector pEGFP/Id3 was successfully constructed.The expression of EGFP reached the peak 48-72 h after transfection;the expresion of pEGFP-transfected group was higher than that of the pEGFP/ Id3 group.RT-PCR and immunocytochemistry staining showed that Id3 mRNA and protein were effectively expressed in pEGFP/Id3-transfected A549 cells.The growth of cells in pEGFP/Id3 transfeeted cells was significantly inhibited 48-72 h after transfection(P
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The ubiquitously expressed family of Id(inhibitor of differentiation) helix-loop-helix(HLH) proteins function as dominant negative regulators of basic HLH (bHLH) transcriptional regulators. In eukaryotic organisms, Id proteins have been implicated in development, cell differentiation, proliferation, angiogenesis, invasion and migration. Recent studies revealed that Id proteins are not only significantly correlated with cancer progression and prognosis, but also exploited as a tumor therapeutic target. The roles of Id proteins in tumorigenesis were reviewed and the opportunities of Id proteins in cancer targeted therapy were discussed.