Figure 5. NFκB activation and TNFα secretion during SM-164 radiosensitization.

(A) NFκB activity: Cells were transfected with a NF-κB luciferase reporter (pNifty plasmid), along with Renilla, in 6-well plates, and split into a 96-well plate 24 hr later. Cells were then left untreated (control), or treated with TNFα (10 ng/ml) for 3 hrs as a positive control, or irradiated with 4 Gy. SM-164 or SM-173 (100 nM) were added immediately after radiation. Cells were lysed 24 hrs later for luciferase activity assay (Promega). Results (n=3) are presented as the fold activation after normalization with Renilla (*, p<0.05). (B) Increased TNFα secretion: TNFα levels in conditioned media were measured by ELISA. The values were normalized with protein concentrations of the cell lysates. Mean ± SEM (n=3). (C) Induction of TNFα mRNA: UMSCC-1 cells were treated with SM-164, or radiation (6 Gy) or the combination. Cells were harvested 24 hrs later for RT-PCR analysis to determine mRNA expressions of TNFα with TRAIL as a control. (D). Partial blockage of SM-164 radiosensitization by TNFα Ab: UMSCC-1 cells were left untreated or treated with SM-164 (200 nM) for 2 hr with or without TNFα antibody (50 ng/ml) before irradiation. Cells were grown in media containing SM-164±TNFα Ab for 9 days before the colonies were counted (mean ± SEM (n=2).