The Experimental Study Of Streptococcal Preparation Mannatide Combined With Radiotherapy For A549Lung Adenocarcinoma Cells

ObjectiveTo investigate cytotoxicity of streptococcal preparation mannatide, the combining effect and related mechanism of mannatide and radiation on the A549lung adenocarcinoma cell line,provides the theoretical basis for the union treatment of lung cancer.Methods1.A549lung adenocarcinoma cells were exposed to different concentrations of mannatide, MTT experiments on drug were done to obtain the cell proliferation inhibition and IC50; A549cells were exposed to different doses of X-xay, MTT experiments on radiation were done to select the appropriate radiation doses.2.Colony formation assay were done to observe the optimal radiation time and the radiosensitivity of mannatide on A549cells. Cell survival curves were drawn to calculate the sensitization enhancement ratio.3.Flow cytometry were done to analyze the effects on cell apoptosis and cycle of drug and radiation alone and combined group.4.Immunocytochemical staining were done to analyze the effects on p53(mutant type) expression of drug and radiation alone and combined group.Results1.MTT experiment results showed the cell inhibition rates were11.11%17.00%26.75%、42.67%、51.01%、54.94%、67.02%、75.61%,after the effects of different concentrations(100、200、300、400、500、600、700、800μg/ml)on A549cells for24hours. Inhibition rate of mannatide to A549cells was positively correlated with the concentration(r=0.994,P<0.001),and the IC50was475.91μg/ml. In a certain dose range, inhibition rate of radiation to A549cells was positively correlated with the dose(r=0.988,P<0.001)2.The results of colony forming assay showed that the optimal radiaion time was36hours. Do values of radiation alone group,50μg/ml concentration of drug combined with radiation group and100μg/ml concentration of drug combined with radiation group were2.58Gy、2.47Gy、2.35Gy, Dq values were3.14Gy、2.93Gy、2.60Gy in turn. The sensitization enhancement ratios(Do) of50μg/ml and100μug/ml concentration of mannatide were ultimately achieved for1.06、1.18,and the sensitization enhancement ratios(Dq) were1.07、1.20, respectively.3.The results of flow cytometry:The apoptosis rate increased along with the enhancement of radiation dose. Furthermore, the apoptosis rate of drug combined with radiation group was higher than that of radiation alone group at the same dose. When mannatide effected on the cells, the percentage of cells in G2/M phase increased, mannatide could cooperate with the blockage of phase G2/M caused by radiation, but it couidn’t influence the percentage of cells in S phase obviously.4. The results of immunocytochemical staining showed that both mannatide and radiation could down-regulated the expression of mutant type p53in A549cells. Compared with radiation alone group, p53expression was lower in drug combined with radiation group.ConclusionsLow-dose mannatide followed by radiation could down-regulated the expression of mutant type p53in A549cells,inhibit the proliferation of cells, cause the blockage of G2/M phase, and induce cell apoptosis.Mannatide played a role in radiosensitivity of A549lung adenocarcinoma cells in certain degree.