双氢青蒿素和顺铂对人肺腺癌A549细胞及其耐顺铂细胞株A549/CDDP的体外作用

Inhibition on the human lung adenocarcinoma cell line A549 and A549/CDDP in vitro by dihydroartemisinin in combination with cisplatin

目的:观察双氢青蒿素和顺铂在体外对A549及A549/CDDP细胞的作用,分析双氢青蒿素抗肺癌和逆转耐药效果。方法:①实验于2003-01/2003-09在解放军第四军医大学口腔医院临床药理基地实验室完成。②人肺腺癌A549细胞系亲代及耐顺铂细胞系A549/CDDP在含100g/L小牛血清,含青链霉素各1×105U/L的RPMI-1640培养液中,37℃,体积分数0.05CO2饱和湿度条件下培养,2.5g/L胰蛋白酶消化传代。③细胞增殖及增殖抑制实验采用四甲基偶氮唑盐检测法:取对数生长期A549和A549/CDDP细胞接种于96孔培养板中,每孔细胞数2×103培养12h后加入适当浓度药物,顺铂浓度0.032～10.000mg/L,单药组双氢青蒿素浓度3.2～1000nmol/L,联用组双氢青蒿素取100,320nmol/L2个浓度与顺铂联用,序贯治疗组先用双氢青蒿素100nmol/L处理24h后换液加顺铂,每组样本5个复孔,对照组加入等体积磷酸盐缓冲溶液,每孔中加入四甲基偶氮唑盐溶液(5g/L)20μL,在酶联免疫检测仪上测定各孔吸光度(A值),采用直线回归法计算药物的半数抑制浓度。④计量资料差异性比较采用方差分析。结果:①双氢青蒿素对A549细胞和A549/CDDP细胞的药物半数抑制浓度分别为(0.230±0.011)和(0.321±0.018)μmol/L;顺铂对A549细胞和A549/CDDP细胞药物半数抑制浓度分别为(0.270±0.014),(5.703±0.026)mg/L。②双氢青蒿素100,300nmol/L联合用药组顺铂对A549/CDDP细胞的顺铂半数抑制浓度明显低于对照组犤(2.255±0.114),(5.703±0.126)mg/L,P<0.01犦。③序贯治疗组对A549/CDDP细胞的顺铂半数抑制浓度明显低于对照组犤(2.523±0.115),(5.703±0.126)mg/L,P<0.01犦。结论:①双氢青蒿素具有抗癌作用,并与顺铂有协同作用。②体外实验条件下,双氢青蒿素可抑制肺癌细胞生长,并且逆转A549/CDDP细胞对顺铂的耐药。

AIM： To observe the inhibitory effect of dihydroartemisinin and eisplatin on human lung adenocarcinoma cell line A549 and A549/CDDP, and determine the effect of resistance reversion of dihydroartemisinin in vitro. METHODS： （1） The study was finished in the clinical pathological base, Medical College of Stomatology, the Fourth Military Medical University of Chinese PLA from June to September 2003. （2） The human lung adenocarcinoma cell line A549 and A549/CDDP cells were grown in PRMI 1640 medium supplemented with 100 g/L heat-inactivated fetal bovine serum （FBS was from TBD）, 100 kU/L penicillin and 1×10^5 U/L streptomycin. The cells were incubated at 37 ℃, in a humidified atmosphere at 0.05 CO2 carbon dioxide. Inhibition of proliferation in vitro was measured by MTT assay. The human lung carcinoma cell line A549 and A549/CDDP were assigned to one of the five groups： dihydroartemisinin group, cisplatin group combined group 1 （100 nM dihydroartemisinin and cisplatin）, combined group 2 （320 nM dihydroartemisinin and cisplatin）, pre-treated with dihydroartemisinin group. The cytotoxicity assay was performed as follows： 2×10^3 cells per mL were incubated in the presence of various concentrations of dihydroartemisinin. Co-treatment using cisplatin were performed. Cells （2 ×10^3 per mL） were incubated in the presence of various cisplatin concentrations and a fixed concentration of dihydroartemisinin ranging from 3.2 to 1000 nM. The viability of cells was determined by 3-（4,5-dimethyl- 2-thiazolyl）-2,5-diphenyl-2H-tetrazolium bromide （MTT）-reduction assay. A resistance factor （RF） was defined as the IC50 of resistant cells divided by the IC50 of the sensitive cells. （4） The enumeration data were analyzed by analysis of variance. RESULTS： （1） After 96 hours of treatment, the IC50 for dihydroartemisinin inhibition of A549 and A549/CDDP cell number was （0.230±0.011） and （0.321 ±0.018） μmol/L. The IC50 for cisplatin inhibition of A549 and A549/CDDP cell number was （0.270±0.014） and （5.703±0.026） mg/L. （2） Compared with control group [（5.703 ±0.026） mg/L], the IC50（cisplatin） of combined group （100 nM, 320 nM dihydroartemisinin） on A549/CDDP cells were both significantly decreased to （2.255 ±0.114） and （0.464 ±0.051） mg/L respectively. （3） There was a significant decrease in the IC50 （cisplatin） of pre-treated by 100 nM dihydroartemisinin compared with control group on A549/CDDP cells [（2.523±0.115）, （5.703±0.126） mg/L, P 〈 0.01]. CONCLUSION： （1） Dihydroartemisinin suppresses the growth of human lung adenocarcinoma cell line A53,9 and A549/CDDP in vitro, and dihydroartemisinin combined with cisplatin have synergistic antitumor activity in vitro. （2）Dihydroartemisinin can reverse cisplatin resistance of A549/CDDP in vitro.