Studies On The Mechanisms Of The Lobaplatin-induced Apoptosis In Nsclc By P53/ROS/p38MAPK Pathway

OBJECTIVELung cancer is the leading cause of cancer-related death worldwide.Platinum-based chemotherapy is recommended as the first-line treatment of patients with advanced NSCLC.However their clinical applications are limited by the side effects and intrinsic or acquired of DDP and CBP.Lobaplatin(LBP),as the third generation of platinum anti-neoplastic agent,has shown a better efficacy against lung cancer.The aim of this study was to investigate the mechanisms of the LBP-induced apoptosis in A549 with wild-type p53 on NSCLC.METHODSThe CCK-8 assay was performed to evaluate the effects of LBP on the proliferation of SK-MES-1(mutant p53),NCI-H1299(deficient p53)and A549(wild-type)cells.P53 gene was knocked out by performing RNAi technology.The production of intracellular Reactive oxygen species(ROS)was measured using the oxidation-sensitive fluorescent dye carboxy-DCFDA with fluorescent and flow cytometry(FCM).Cell apoptosis was detected by FCM after staining with Annexin-V/PI.FCM analysis after PI staining was used to detect the cell cycle arrest induced by LBP.Cell lysates were subjected to western-blot analysis for the detection of p53,Bax,Bcl-2,PARP,Caspase3,Caspase8,Caspase9 and p-p38MAPK protein level.The thiol antioxidant N-acetylcysteine(NAC)and SB203580(a specific inhibitor of MAPK/p38)were used to detect the role of LBP in inducing apoptosis and its effect on ROS,p53 and p-p38MAPK.Human NSCLC xenograft of A549 cells was established by inoculating viable A549 cells into the right flank of nude mice and measured the anti-tumor effect of LBP.And cell apoptosis in the excised xenografts was measured by TUNEL assay.RESULTS1.The IC50 of SK-MES-1(mutant p53)was 63.47�1.03?M,15.77�1.09?M and 12.0�1.08?M for 24h,48h and 72h,respectively.The IC50 of H1299(deficient p53)was67.26�1.06?M,17.98�1.05?M ? 7.85�1.01?M respectively.The IC50 of A549 was 11.69�1.05?M,5.02�1.11?M and 2.43�1.01?M.LBP triggered G1 cell cycle arrest on the concentration of 6?M and 12Mm in A549.The percentages of A549 in G1 phase was 72.03%�1.53%,and 73.78%�1.26%after treatment of 6?M,12?M lobaplatin for 24h,and that was 47.47%�1.59%in control group,respectively.The apoptosis rates were 20.2%�1.3%and 44.5%�1.6%when the A549 cells were treated with 12 ?M and 24 ?M of LBP for 24h,whereas they increased up to 55.3%�1.2%and 70.0%�0.9%when treated for 48h,respectively.Despite that there were less cells to be stained due to higher LBP concentration,chromatin condensation was more evident on higher lobaplatin concentration.Apoptosis bodies were seen when A549 cells were treated with 24?M LBP for 24h.A significant generation of ROS was observed after exposure of LBP for 4h.LBP increased the level of PARP,Bax,Clv-caspase3,8,9 level,whereas that of Bcl-2 significantly decreased in a dose-dependent manner.LBP inhibited tumor growth of A549 xenograft models and induced apoptosis in vivo.2.The IC50 of A549/p53-shRNA was 39.54�1.02?M,27.89�1.13?M and 10.93�1.08?M and those of A549/NS-shRNA was 11.48�1.12?M,5.14�0.94?M ?2.49�10.90pM,respectively.The proliferation of A549/p53-shRNA and A549/NS-shRNA cells were inhibited in a time-and concentration-dependent manners.The apoptosis rates were 45.0%�1.1%when A549/NS-shRNA cells were treated with 24?M of LBP for 24h,whereas there was no obvious apoptosis of A549/p53-shRNA cells at the same condition,respectively.Thus A549/NS-shRNA cells were more sensitive than A549/p53-shRNA cells.A significant generation of ROS was observed after exposure of LBP in A549/NS-shRNA whereas there is a slight increase in A549/p53-shRNA.The expression level of p53 and p-p38MAPK upregulate by LBP in A549/NS-shRNA cells.The p-p38MAPK protein level in control was elevated and its level was no change treated with LBP.The lobaplatin-induced apoptosis can be inhibited by NAC and SB203580 in A549 cells.The generation of ROS was inhibited by NAC but not by SB203580 in A549 cells.The elevated expression of p53 can not be inhibited by NAC and SB203580 in A549 cells.CONCLUSIONS1.A549 cells with wildtype p53 are more sensitive to LBP.LBP trigger A549 cells arrest at G1 phase at lower concentration of LBP.LBP can induce apoptosis in A549.LBP increases the levels of PARP,Bax and Clv-caspase3,8,9.The expression of Bcl-2 significantly decreased.The intrinsic and extrinsic apoptosis pathways are involved in LBP-induced apoptosis.2.LBP inhibits tumor growth of A549 xenograft models and induce apoptosis in vivo.3.We for the first time demonstrate p53/ROS/p38MAPK pathway appears to mediate the LBP-induced apoptosis in A549 cells with wild type-p53.4.LBP is a promising candidate for the treatment of NSCLC with wild type-p53.