Figure 3

MA inhibits TNFα-induced NF-κB activity in pancreatic cancer cells. (A) MA suppressed endogenous NF-κB DNA binding activity (left) in a time-dependent manner and TNFα-induced NF-κB activity (right) in a concentration-dependent manner. Panc-28 cells were pretreated with indicated concentrations of MA for 6 h, incubated with or without 0.1 nM TNFα for 15 minutes, and then subjected to EMSA to examine NF-κB activation. Arrowhead indicates the DNA-binding activity of NF-κB. (B)MA suppressed TNFα-induced NF-κB activity in different pancreatic cancer cell lines. Pancreatic cancer cells were pretreated with or without 25 microM MA for 6 h, and then incubated with 0.1 nM TNFα for 15 minutes. The DNA binding activity of NF-κB was evaluated by EMSA. (C) MA inhibited the binding of NF-κB to the COX-2 promoter in vivo. Panc-28 cells were pretreated with 25 microM MA for 12 h and treated with 0.1 nM TNFα for the indicated time. The proteins were cross-linked with DNA by formaldehyde and then subjected to ChIP assay using an anti-p65 antibody with the COX-2 primer. (D) MA inhibited TNFα-induced NF-κB dependant reporter gene expression. 293 cells were transiently transfected with a NF-κB luciferase reporter gene. After transfection, cells were pretreated with the indicated concentrations of MA for 24 h, and then incubated with 0.1 nM TNFα for another 24 h. Cell supernatants were collected and assayed for luciferase activity as described in Materials and Methods. Results are expressed as fold activity over the activity of the vector control. Column, means of three experiments carried out with triplicate; bar, SD. **, P < 0.01, ***, P < 0.001 versus TNFα alone treated group.