Research Paper Volume 12, Issue 1 pp 611—627

Figure 5. Cotreatment with Aspirin and Cisplatin suppressed NF-κB/COX-2 signaling pathway in colon cancer cells. (A) The expression level of COX-2 in the whole cell lysate of human colon cancer cells RKO and LoVo treated with Cisplatin (15μM /5μM) and/or Aspirin(10mM /5mM) for 48 hours was analyzed by Western blot (n=3). (B) Human colon cancer cells RKO and LoVo were treated with Cisplatin (15μM /5μM) and/or Aspirin (10mM /5mM) for 48 h. The amount of PGE₂ in cell culture media was detected by Prostaglandin E₂ High Sensitivity in vitro competitive ELISA Kit (n=4). (C) Human colon cancer cells RKO and LoVo were incubated with combination of Aspirin and Cisplatin at indicated dose for 48 h after pretreatment with COX-2-selective inhibitor celecoxib (CB) at indicated dose for 8 h, and then cell viability was determined by MTT analysis (n=6). (D) Human colon cancer cells RKO and LoVo were treated with Cisplatin alone (15μM /5μM) or Aspirin alone (10mM /5mM) or their combination for 48 h. The expression levels of p65, p50, COX-2 and c-myc in nucleus, p65, p50, p-p65, IKKα, IKKβ, p-IKKα/β, p-IκBα and IκBα in cytoplasm were respectively detected by western blot assay (n=3). (E) Immunofluorescence assay of human colon cancer cell RKO treated with Cisplatin alone (15μM) or Aspirin alone (10mM) or their combination for 48 h was implemented to observe the subcellular localization of p65 and p50. The representative images were taken by laser scanning confocal microscope. Scale bars, 25 μm (n=3). (F) The streptavidin-biotin pulldown assay was performed to test the binding of p65 and p50 at COX-2 promoter region in human colon cancer cells RKO and LoVo treated with Cisplatin (15μM /5μM) and/or Aspirin (10mM /5mM) for 48 h (n=3). (G) Human colon cancer cells RKO and LoVo were transfected with COX-2 promoter (-892/+9 fragments) driven-luciferase plasmids and pRL-TK Renilla luciferase construct (Promega). After 24 hours, the cells were treated with Cisplatin (15μM /5μM) and/or Aspirin (10mM /5mM) for 48 hours. Then luciferase activities were measured according to the Dual-Luciferase Assay System protocol (Promega) (n=4). (H) Human colon cancer cells RKO and LoVo were pretreated with NF-κB activator LPS or NF-κB inhibitor QNZ at indicated dose for 8 h, and then incubated with combination of Aspirin and Cisplatin at indicated dose. After 48 h, cell viability was determined by MTT analysis (n=6). Data were presented as means ± SD, *P<0.05, **P<0.01, ***P<0.001.