Haynes Alfred, Shaik Madhu Sudhan, Chatterjee Abhijit, Singh M
College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee, Florida 32307, USA.
Pharm Res. 2003 Sep;20(9):1485-95. doi: 10.1023/a:1025774630993.
To evaluate the in vitro effects of an aerosolized cyclooxygenase-2 (COX-2) inhibitor, nimesulide, on the cytotoxicity and apoptotic response of doxorubicin against the human lung adenocarcinoma cell line A549.
Nimesulide was formulated into a metered dose inhaler (MDI) formulation and characterized for aerodynamic particle size and medication delivery. The in vitro cytotoxicity of nimesulide-MDI in the presence or absence of doxorubicin was assessed by using the six-stage viable impactor by an already standardized method. Induction of apoptosis in A549 cells by nimesulide (nonaerosolized or aerosolized) in combination with doxorubicin was evaluated by established techniques such as caspase-3 estimation and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining. Finally, to understand the mechanism of action, the influence of different treatments on the expression of COX-2 and peroxisome proliferator-activated receptor-gamma (PPAR-gamma) in A549 cells was studied by immunoblotting.
The nimesulide-MDI formulation had a mass median aerodynamic diameter (MMAD) of 1.1 microm, (GSD = 2.8) and a medication delivery of 51 microg/shot. Nimesulide-MDI (40 shots) in combination with doxorubicin (0.01 microg/ml) had a cell kill of more than 60% as determined by in vitro cytotoxicity assay. The specific caspase-3 activity in A549 cells treated with nimesulide (40 microg/ml) and doxorubicin (0.25 microg/ml) in combination was 3 and 5 times higher than doxorubicin and nimesulide, respectively. Further, TUNEL staining showed apoptosis in over 30% of A549 cells treated with aerosolized nimesulide and doxorubicin combination vs. negligible as seen in cells treated individually. The expression of COX-2 was not altered in control or treatments, whereas PPAR-gamma was expressed only in the combination treatment.
Our results indicate that aerosolized nimesulide significantly enhances doxorubicin activity against A549 cells, and the enhanced cytotoxicity was probably mediated via a COX-2-independent mechanism.
评估雾化的环氧化酶-2（COX-2）抑制剂尼美舒利对阿霉素针对人肺腺癌细胞系A549的细胞毒性和凋亡反应的体外作用。
将尼美舒利制成定量吸入器（MDI）制剂，并对其空气动力学粒径和药物递送进行表征。采用已标准化的方法，通过六级活体检波器评估尼美舒利-MDI在有或无阿霉素存在时的体外细胞毒性。通过诸如半胱天冬酶-3测定和末端脱氧核苷酸转移酶介导的缺口末端标记（TUNEL）染色等既定技术，评估尼美舒利（非雾化或雾化）与阿霉素联合对A549细胞凋亡的诱导作用。最后，为了解作用机制，通过免疫印迹研究不同处理对A549细胞中COX-2和过氧化物酶体增殖物激活受体-γ（PPAR-γ）表达的影响。
尼美舒利-MDI制剂的质量中值空气动力学直径（MMAD）为1.1微米，（几何标准差GSD = 2.8），每次喷射的药物递送量为51微克。通过体外细胞毒性试验测定，尼美舒利-MDI（40次喷射）与阿霉素（0.01微克/毫升）联合使用时细胞杀伤率超过60%。尼美舒利（40微克/毫升）与阿霉素（0.25微克/毫升）联合处理的A549细胞中，特异性半胱天冬酶-3活性分别比阿霉素和尼美舒利高3倍和5倍。此外，TUNEL染色显示，雾化的尼美舒利与阿霉素联合处理的A549细胞中，超过30%出现凋亡，而单独处理的细胞中凋亡可忽略不计。对照组或各处理组中COX-2的表达未发生改变，而PPAR-γ仅在联合处理组中表达。
我们的结果表明，雾化的尼美舒利显著增强阿霉素对A549细胞的活性，增强的细胞毒性可能通过非COX-2依赖机制介导。
