Glycosaminoglycan Combined With Cisplatin Inhibits The Proliferation On Human Lung Adenocarcinoma Cell Line A549

Objective: Our objective was to investigate the effects of proliferation and apoptosis on human lung adenocarcinoma cell line A549 by Holothurian glycosaminoglycan(hGAG)and cisplatin(DDP).We also want to investigate the effects and mechanism of hGAG Promotes Sensitivity of of DDP on A549 cells.Methods: We cultured A549 cells in vitro.After cell cultures were 70% confluent,they were randomly divided into control group,hGAG group,DDP group,and combined treatment group(hGAG + DDP).The growth status and changes in morphogenesis of the cells was observed using an inverted microscope.CCK8 assay was used to determine the cell proliferation and inhibition.Morphological changes of apoptosis in A549 cell were observed by Hoechst33258 staining.Annexin V-FITC and propodium iodide staining flow cytometry was applied to detect apoptosis of cells.Propodium iodide staining flow cytometry was used to detect cell cycle.RT-PCR was used to examine the m RNA expression of Bax,Bcl-2,survivin and caspase-3.The protein expression of Bax,Bcl-2,survivin and caspase-3 were detected by Western blot.Results: Inverted microscope observed that the numbers were significantly lower,the density was decreased,the intercellular space was increased and some cells were maintained in resuspension of A549 cells after cultured in hGAG and DDP.The result of CCK8 showed that hGAG could promote the inhibition of A549 cells in DDP.The growth inhibition was with apparent differences between groups(P<0.05).Significant differences could be identified between the groups at 24 h,48 h and 72 h(P<0.05).The DDP +hGAG group was statistically significant compared to DDP group at 24(t=3.92,P<0.05),48(t=4.67,P<0.05)and 72 hours(t=4.14,P<0.05)respectively.We found no obviously apoptotic cells in control group.A549 cells in hGAG,DDP and hGAG+DDP groups were observed shrunken,hyperchromatic,and pyknotic.The cell density was the lowest and the apoptotic cells were the most common in hGAG+DDP group.Annexin V-FITC / PI double staining results indicated that the early and middle stage apoptosis rate between the groups was statistically significant(P<0.05 for all comparisons).A clear difference was observable between DDP and hGAG+DDP group(P<0.05).The result of RT-PCR showed that the expression of Bax and caspase-3 m RNA was increased,Bcl-2and Survivin m RNA were low expression,there was significant differences between the groups(all P<0.05).The result of western blot showed that the expression of Bcl-2 and Survivin protein was decreased,Bax and caspase-3 protein were overexpression(P<0.05 for all comparisons).The DDP +hGAG group was statistically significant compared to DDP group in the expression of four protein and m RNA(all P<0.05).Conclusion: HGAG can inhibit the human lung adenocarcinoma A549 cells.HGAG also can enhance the sensitivity of DDP in A549 cells.The mechanism of hGAG-sensitizing may be associated with the low expression of bcl-2 and survivin m RNA and protein,the overexpression of Bax and caspase-3 m RNA and protein.