Cancer Institute of Traditional Chinese Medicine (TCM), Longhua Hospital, School of Life Sciences, Shanghai University of TCM, Shanghai 200032, PR China.
Oncol Rep. 2012 May;27(5):1619-24. doi: 10.3892/or.2012.1647. Epub 2012 Jan 19.
The aim of the present study was to investigate the effects of cinobufocini on nuclear factor-κB (NF-κB), cyclooxygenase-2 (COX-2) and the production of cytokines induced by tumor necrosis factor-α (TNF-α) in the A549 cell line. A549 cells were incubated with cinobufocini at different concentrations for 24, 48 or 72 h. Cell proliferation was examined by the WST-8 assay. The expression of NF-κB, COX-2 and inhibitor κBα (IκBα) was studied by western blotting. The NF-κB-dependent luciferase rporter (3xκB-luc) was transfected for 24 h, the cells were treated with the reagents for 24 h, and the transcriptional activity of the NF-κB promoter was detected by a luciferase assay. The levels of IL-6 and IL-8 mRNA were detected by reverse transcription-polymerase chain reaction. We found that cinobufocini inhibited NF-κB p65 expression and the transcriptional activity of the NF-κB promoter induced by TNF-α compared with the control in the nuclei of A549 cells. Moreover, induced COX-2 expression was blocked by cinobufocini and was correlated with a reduction in the activated p65 subunit of NF-κB. Additionally, the levels of IL-6 and IL-8 mRNA induced by TNF-α were significantly suppressed by the addition of cinobufocini. In conclusion, these results suggest that the anti-inflammatory effects of cinobufocini are dependent on the NF-κB/COX-2 pathway in A549 cells, thereby providing a possible anticancer mechanism for the compound.
本研究旨在探讨华蟾毒精对 TNF-α 诱导的 A549 细胞中核因子-κB（NF-κB）、环氧化酶-2（COX-2）和细胞因子产生的影响。将 A549 细胞用不同浓度的华蟾毒精孵育 24、48 或 72 h。通过 WST-8 法检测细胞增殖。用 Western blot 法研究 NF-κB、COX-2 和抑制剂κBα（IκBα）的表达。用 NF-κB 依赖性荧光素酶报告基因（3xκB-luc）转染 24 h，用试剂处理细胞 24 h，通过荧光素酶检测 NF-κB 启动子的转录活性。用逆转录-聚合酶链反应检测 IL-6 和 IL-8 mRNA 的水平。我们发现，与对照组相比，华蟾毒精抑制 TNF-α诱导的 A549 细胞核中 NF-κB p65 表达和 NF-κB 启动子的转录活性。此外，华蟾毒精阻断了诱导型 COX-2 表达，并与 NF-κB 激活的 p65 亚基减少相关。此外，TNF-α 诱导的 IL-6 和 IL-8 mRNA 水平也显著受到华蟾毒精的抑制。总之，这些结果表明，华蟾毒精的抗炎作用依赖于 A549 细胞中的 NF-κB/COX-2 途径，从而为该化合物提供了一种潜在的抗癌机制。
