The Radio-sensitizing Effects Of 17AAG-cypate Polymeric Micelle On Non-small-cell Lung Cancer A549 Cells

PurposeThis study investigated the radio-sensitizing effects of 17-AAG and the thermal damage of cypate on human Non-small-cell carcinoma A549 cells.As a targeting drug nano-carrier system,whether the 17AAG-cypate polymeric micelles had the superior performance of better anticancer efficacy than 17-AAG micelles and then discussed the possible mechanisms of these effects.Methods and Materials17AAG-cypate polymer micelles were prepared with organic solvent evaporation and solvent dialysis method;The standard curve and the concentration of 17-AAG and cypate were detected with ultraviolet spectrophotometry;The morphology and particle size distribution of micelles were observed with TEM;Size and dispersion of micelles were determined by the nano particle and zeta potential analyzer;Cellular uptake assay was used to detect the effect of drug intake by nano polymer carrier;The MTT assay was used to quantify the different growth inhibition of A549 cells between17AAG-cypate polymeric micelles.Using Multitarget-single hitting model fitting equation to analyze the radio-sensitizing effects caused by the drugs.Experimental methods included:β-Galactosidase Staining,cell cycle,cell apoptosis,immunofluoresc-ence,western-blot and nude mice model experiments.17-AAG and cypate were loaded by PEG-PCL₆₀,and self-assembled into 17AAG-cypate polymer micelles,which solved the solubility and stability problem of 17-AAG and cypate,the micelles entered into A549 cells through endocytosis,in combination with X-ray and near-infrared light irradiations to investigate the inhibition effect of polymer micelles on A549 cells.Results1)17AAG-cypate micelles were prepared with with PEG-PCL₆₀ polymer material carrying 17-AAG and cypate,the micellar morphology is rounded surrounding and approximately spherical under the transmission electron microscopy（TEM）,the average particle size is 63.42 nm,poly dispersion index（PDI）is less than 0.25,particle size distribution is narrow and all micelle size is between 20-150 nm.2)Cellular uptake assay showed that 17AAG-M and cypate-M all increased 4-5times about drugs intake compared with free 17-AAG/cypate after 24h incubation.3)MTT assay demonstrated that the cytotoxic effect of 17AAG-cypate-M was much higher than 17AAG-M under the laser and X-ray irradiations.The colony forming assay showed that efficiency of 17AAG-cypate-M had much more significantly radio-sensitizing effect on A549 cells.4)β-Galactosidase Staining assay showed that 17AAG-cypate-M and 17AAG-M all can cause obvious aging phenomenon under the laser and X-ray irradiations.The proportion of cell aging of 17AAG-cypate-M were higher than 17AAG-M.5)17AAG-cypate-M combined with the laser and X-ray irradiations can cause varying degrees of G2/M phase arrest and cell apoptosis phenomenon.The degree of G2/M phase arrest and cell apoptosis rate of 17AAG-cypate-M were higher than17AAG-M.6)Immunofluorescence analysis observed that 17AAG-cypate-M combined with the laser and X-ray irradiations can obviously increase the DNA-damage sites and delay the DNA damage repair,which was notably different with 17AAG-M.7)Western blot results showed that 17AAG-cypate-M could make the expression level of p-Erk1/2 and p-Akt suppressed in combination with the laser and X-ray irradiations,MAPK and PI3K signaling pathway were blocked.The activation and expression of Some proteins related to cell apoptosis such as PARP,caspase3,caspase9and Bcl-2 were also significantly affected.8)In nude mice model experiment,17AAG-M and 17-AAG both significantly inhibited A549 tumor growth and metastasis,and the inhibitory effect of same concentration revealed significant difference.ConclusionsThe vitro experiences indicate that 17AAG-cypate-M had higher inhibition efficiency than 17AAG-M on the human non-small-cell carcinoma A549 cells.The mechanism might be inducing the cell aging,cell cycle arrest,apoptosis and delaying the DNA damage repair.Western blot indicated that this may be related to inhibition of MAPKand PI3K signaling pathways and affect the activation of the proteins related to cell apoptosis such as PARP,caspase9,caspase3 and the expression of Bcl-2;In nude mice tumor exnograft model experiment,17AAG-M and 17-AAG both played obvious inhibitory effects on A549cells.