CD13抑制剂乌苯美司对A549细胞顺铂敏感性的影响及其机制

  目的   检测CD13抑制剂乌苯美司联合顺铂对A549细胞增殖、细胞凋亡的影响，并探讨其机制。   方法   应用噻唑蓝比色法检测细胞增殖；流式细胞Annexin V-FITC/PI双染色法检测细胞凋亡；Western blot方法检测p53、PARP-1、Bcl-2、Bcl2-xL和Caspase蛋白水平；酶活性底物法检测CD13活性；Hoechst33342染色联合流式细胞术检测侧群细胞。   结果   乌苯美司单药对A549细胞增殖无显著影响（P>0.05），乌苯美司（100 μg/mL）联合顺铂作用A549细胞24、48、72 h后，乌苯美司可显著增强8、16 μmol/L顺铂对A549细胞的增殖抑制（P < 0.05），并呈时间依赖性，72h时，8、16μmol/L顺铂组和顺铂联合组的细胞增殖率分别为（62.06± 7.60）% vs.（27.92±5.84）%和（19.22±1.57）% vs.（0.67±0.42）%（P < 0.05）；但对64 μmol/L顺铂的细胞增殖率无明显影响（P>0.05）。16 μmol/L顺铂联合组细胞凋亡的比例明显增加（P < 0.05）。乌苯美司对P53、PARP-1、Bcl-2、Bcl2-xL及Capsize蛋白水平无明显影响，但显著抑制A549细胞CD13活性和降低A549侧群细胞比例（P < 0.05）。   结论   CD13抑制剂乌苯美司体外条件下显著提高低剂量顺铂对A549细胞的抑制作用，可能成为CD13阳性非小细胞肺癌患者顺铂的增敏剂。 

Effects of Ubenimex, a CD13 Inhibitor, on the Chemosensitivity of A549 Cells to Cisplatin

  Objective   This study was designed to investigate the effects of ubenimex, a CD13 inhibitor, (ubenimex alone and ubenimex with cisplatin), on A549 cell proliferation and apoptosis. This study also aimed to determine the possible underlying mechanisms.   Methods   A549 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. The cultured cells were then treated with ubenimex at varying concentrations or a combination of ubenimex (100 μg·mL–1) and cisplatin at varying concentrations. Methyl thiazolyl tetrazolium assay was used to determine the cell proliferation ratio. Flow cytometer was then used to analyze the cell apoptotic rates (AnnexinV-FITC/PI double-staining). The protein expressions of p53, Bcl-2, and Bcl2-xL, as well as the cleavage of PARP-1 and caspase were determined through Western blot. Spectrophotometrical assay was performed using alanine-p-nitroanilido as a substrate to detect CD13 activity. Hochest 33342 staining combined with flow cytometry analysis was performed to detect the side population (SP) cells in A549 cells.   Results   Ubenimex alone did not significantly inhibit A549 cell proliferation (P>0.05). By contrast, ubenimex significantly increased the chemosensitivity of A549 cells to cisplatin at 8 μM or 16 μM in a time-dependent manner. After the A549 cells were treated with different concentrations of cisplatin (i.e., 8, 16, 32, and 64 μM) and ubenimex (100 μg·mL–1) for 24, 48, and 72 h, the cell proliferation ratios were obtained. In particular, at a cisplatin concentration of 8 μM, the cell proliferation ratios of the cisplatin group against the combined treatment of cisplatin with ubenimex (100 μg·mL–1) were 89.44±15.84% versus 62.18±5.35% (24 h), 64.81±5.86% versus 33.09±3.14% (48 h), and 62.06±7.6% versus 27.92±5.84% (72 h). At 16 μM of cisplatin, the cell proliferation ratios (cisplatin against the combined treatment) were 84.61±3.73% versus 54.50±4.22% (24 h), 22.09±5.74% versus 3.62±3.28% (48 h), and 19.22±1.57% versus 0.67±0.42% (72 h). Ubenimex did not significantly increase the chemosensitivity of A549 cells to cisplatin (64 μM). Cellular apoptotic rates were also significantly increased in the combined treatment group [cisplatin (16 μM) and ubenimex] compared with that in the cisplatin group alone. Ubenimex alone cannot change the protein expression of p53, Bcl-2, and Bcl2-xL, as well as the cleavage of PARP-1 and caspase. By contrast, ubenimex can significantly inhibit the CD13 activity of A549 cells and decrease the percentage of SP cells of A549 cells (P < 0.05).   Conclusion   Ubenimex can significantly enhance the chemosensitivity of low-dose cisplatin on A549 cells in vitro. Therefore, ubenimex may be used as a sensitizer of cisplatin for human non-small cell lung cancer with CD13 expression.