单端孢菌素逆转肺癌耐药A549/DDP细胞耐药性及其机制研究

Effects and Mechanisms of Trichothecenes in Reversing the Drug Resistance of A549/DDP

关键词:  单端孢菌素  A549/DDP  多药耐药

Key words:  trichothecenes  A549/DDP  multidrug resistance

摘要:

目的 探讨单端孢菌素对肺癌耐顺铂细胞系A549/DDP顺铂耐药性的逆转作用及机制。方法 应用MTS法检测单端孢菌素对A549/DDP细胞顺铂敏感性的影响，流式细胞术检测肿瘤细胞表面P-糖蛋白(P-Glycoprotein，P-pg)表达以及胞内罗丹明-123(Rhodamine-123，Rh-123)含量的变化，生化法检测细胞内SOD和GSH的水平，Western blot法和Real time PCR检测肿瘤细胞多药耐药蛋白MDR1、MRP1和Survivin表达的变化，Western blot法检测Akt磷酸化的变化，液相芯片法检测TGF-β，IL-6和IL-8的变化，应用双萤光报告基因技术检测细胞NF-κB和AP-1转录活性。结果 单端孢菌素可增加细胞对顺铂的敏感性，经5 μg·mL^-1和10 μg·mL^-1单端孢菌素作用72 h后，耐药逆转倍数(RF)为1.84和3.95倍。经5 μg·mL^-1和10 μg·mL^-1单端孢菌素作用24 h后，肿瘤细胞P-gp表达分别下降了62.6%和19.8%，细胞中Rh-123含量分别提高了183.3%和308.3%，细胞内SOD水平下降65.2%和50.1%，GSH水平下降 71.6%和46.3%，MDR1的mRNA水平下降了72.7%和52.3%，MRP1的mRNA水平下降了64.0%和22.5%，Survivin的mRNA水平下降了45.8%和14.7%，Akt磷酸化水平下降，TGF-β分泌水平下降了80.2%和51.5%，IL-6分泌水平下降了73.4%和37.2%，IL-8分泌水平下降了71.2%和43.2%，NF-κB转录活性下降42.3%和22.7%，AP-1的转录活性下降了57.4%和32.5%。结论 单端孢菌素逆转A549/DDP对顺铂的耐药性，这一作用与抑制药物外排，负调控肿瘤耐药相关蛋白的表达有关。

Abstract:

OBJECTIVE To investigate the effects and mechanisms of trichothecenes on reversing the cisplatin resistance of lung cancer drug-resistant cell line A549/DDP. METHODS MTS assay was employed to determine the effect of trichothecenes on A549/DDP sensitivity. The distribution of P-Glycoprotein (P-pg) and intracellular concentration of rhodamine-123 (Rh-123) were determined by flow cytometry. Intracellular level of SOD and GSH was determined by biochemistry assay. The expression of multi-drugs resistant proteins MDR1, MRP1 and Survivin were determined by Western blot and Real time PCR. The phosphorylation of Akt was analyzed by Western blot. Secretion of TGF-b, IL-6 and IL-8 were assayed by liquidchip. The transcriptional activities of NF-κB and AP-1 were detected by dual-luciferase reporter gene systems. RESULTS Trichothecenes was able to increase the cisplatin sensitivity of A549/DDP. After treatment with 5 and 10 μg·mL^-1 trichothecenes for 72 h, the reverse folds (RF) to adriamycin were 1.84 and 3.95 respectively. After treatment with 5 and 10 μg·mL^-1 trichothecenes for 24 h, the expression of P-gp was decreased 62.6% and 19.8%, the intracellular level of Rh-123 was elevated 183.3% and 308.3%, the intracellular level of SOD was decreased 65.2% and 50.1%, GSH was decreased 71.6% and 46.3%. The protein expressions of MDR1, MRP1 and Survivin were downregulated significantly, the mRNA expressions of MDR1 was decreased 72.7% and 52.3%, MRP1 decreased 64.0% and 22.5%, Survivin was decreased 45.8% and 14.7%. In addition, the phosphorylation of Akt decreased. The secretion of TGF-β was decreased 80.2% and 51.5%, IL-6 was decreased 73.4% and 37.2%, IL-8 was decreased 71.2% and 43.2%. Finally, transcriptional activity of NF-kB was decreased 42.3% and 22.7% and AP-1 was decreased 57.4% and 32.5%, significantly. CONCLUSION Trichothecenes is able to reverse the cisplatin resistance of A549/DDP. Trichothecenes processes this kind of effect by inhibiting the drug efflux, downregulating the expression of drug-resistant related proteins.