蛇床子素抑制膀胱癌细胞生长和侵袭的机制

目的：探讨蛇床子素对表皮生长因子受体酪氨酸激酶(epidermal growth factor receptor tyrosine kinase，EGFR-TPK)、基质金属蛋白酶(matrix-metalloproteinase-2，MMP-2)和氨肽酶N(aminopeptidase N，APN)的抑制作用，并阐明蛇床子素抑制膀胱癌T24细胞生长和侵袭的机制。方法：利用分光光度法研究蛇床子素对T24细胞EGFR-TPK，APN，MMP-2和caspase-3活性的影响，利用MTT法测定蛇床子素对膀胱癌T24细胞和人正常膀胱上皮细胞SV-HUC-1生长的抑制作用，采用Transwell小室法分析蛇床子素对膀胱癌T24细胞转移能力；Western印迹检测培养液中加入蛇床子素后，人膀胱癌细胞株T24中COX-2，VEGF及β-actin的表达；采用非放射性NF-κB凝胶迁移或电泳迁移率实验(electrophoretic mobility shift assay，EMSA)试剂盒测定T24细胞中NF-κB活性。结果：蛇床子素对EGFR-TPK，APN和MMP-2的半数抑制浓度(IC50)分别为(45.33±3.98)，(28.21±3.23)和(8.11±0.54) µmol/L；随着蛇床子素浓度的增加，膀胱癌T24细胞生长抑制率不断增高，与空白对照组比较，差异均具有统计学意义(P<0.05)；但SV-HUC-1细胞的生长并未受到明显的抑制；与空白对照组比较，蛇床子素组穿膜细胞的数减少(P<0.05)；膀胱癌T24细胞中凋亡蛋白caspase-3活性显著增强(P<0.05)；蛇床子素能够下调NF-κB，COX-2及VEGF的表达。结论：低毒性的蛇床子素可能通过降低EGFR-TPK，APN和MMP-2活性和NF-κB，COX-2及VEGF的表达，激活caspase-3活性的方式，对膀胱肿瘤细胞的侵袭、增殖和血管生成产生抑制作用。

Mechanisms for effect of osthole on inhibiting the growth and invasion of bladder cancer cells

Objective: To investigate the effect of osthole on epidermal growth factor receptor tyrosine kinase (EGFR-TPK), matrix-metalloproteinase-2 (MMP-2), aminopeptidase N (APN) in bladder cancer cell and the underlying mechanism. Methods: The T24 cell lines were cultured. The inhibitory effects of osthole on EGFR-TPK, APN and MMP-2 were evaluated by spectrophotometric and MTT assay. The caspase-3 activity and the expression COX-2 and VEGF in T24 were examined. The activity of NF-κB was determined by electrophoretic mobility shift assay. Results: The half inhibition concentrations (IC50) of osthole on EGFR-TPK, APN and MMP-2 were (45.33±3.98), (28.21±3.23) and (8.11±0.54) µmol/L, respectively. The growth inhibitory rates for T24 cells were increased in a dose-dependent manner (P<0.05). The caspase-3 activities were significantly increased in T24 cells in the osthole group compared with control group, while the expression of angiogenesis related-protein COX-2, VEGF, and NF-κB in T24 cells were decreased. Conclusion: Through the inhibitory effect on EGFR-TPK, APN and MMP-2, osthole can decrease COX-2, VEGF and NF-κB expression while increase the activity of caspase-3, eventually blocking the growth and invasion of bladder cancer cell.