Combination Of Holothurian Glycosaminoglycan And Radiation Has Synergistically Inhibitory Effects On Human Lung Adenocarcinoma A549

Objective: To investigate whether there are synergistically inhibitory effects of Holothurian Glycosaminoglycan(HGAG)with cisplatin(DDP)on pulmonary carcinoma cell line A549 and to study the mechanism of these effects.Methods: The human lung adenocarcinoma A549 cells were cultured in appropriate culture medium.Pre-experiment was carried out by CCK-8 method.A series of treatment doses were set up and detected,then,the most suitable experimental treatment dose was selected.The experimental cells were grouped according to the results: control group,HGAG group,radiation group,combined group(HGAG combined radiation).For each group,the following observations or experiments were done.Inverted microscope was used to observe the growth and morphology of cells in each group during each time period.CCK-8 method was used to detect the inhibition rate of cell proliferation in each group of each time period.Hochest 33258 staining was used to stain the experimental cells and observe the morphological changes of cell apoptosis.AnnexinV-FITC/PI double staining combined with flow cytometry was used to detect cell apoptosis.Western blot was used to detect the expression of four apoptotic proteins Bcl-2,Bax,Survivin and caspase-3.Results: Under inverted microscope,it was found that after HGAG and radiation treatment,the number of cells in the culture medium decreased,the cells were sparse,the density was low,the suspended cells were visible,and the combined group was the most obvious.CCK-8 experimental results showed that HGAG could promote the growth inhibition of A549 cells by radiation and had radiosensitization effect on radiation.The proliferation inhibition rates were significantly different between groups at 24,48 and 72hours(all P < 0.05).The proliferation inhibition rates of the combined group at each time period were significantly different from those of the radiation group(all P < 0.05).Hochest 33258 staining experiment showed that dense apoptotic cells,nuclear fragmentation and nuclear pyknosis could be observed in HGAG group,radiation group and combination group.The combination group was the most obviousone.Annexin V-FITC/PI double-staining flow pattern experiment showed that the total apoptotic rate of the three experimental groups was significantly higher than that of the control group and the total apoptotic rate of the combination group was significantly higher thanthat of the radiation group(all P < 0.05).Western blot results showed that the expression of protein Bax and caspase-3 increased and the expression of protein Bcl-2 and Survivin decreased in the three experimental groups compared with the control group and the combined groupcompared with the radiotherapy group(all P < 0.05).Conclusion: HGAG can inhibit the growth and proliferation of A549 cells and enhance the radiosensitivity of A549 cells.Its mechanism may be related to up-regulation of Bax and caspase-3 protein expression and down-regulation of Bcl-2 and Survivin protein expression.