Search in the Abstract Database

Search Abstracts 2015

* = Presenting author

P017 Long-term stimulation with cytokines acquires irreversible accumulation of NF-κB signaling in colonic epithelial cells

S. Hibiya*1, K. Tsuchiya2, K. Fukushima1, R. Hayashi3, N. Horita1, S. Oshima1, R. Okamoto2, T. Nakamura2, M. Watanabe1

1Tokyo Medical and Dental University, Gastroenterology and Hepatology, Tokyo, Japan, 2Tokyo Medical and Dental University, Advanced Therapeutics for Gastrointestinal Diseases, Tokyo, Japan, 3Hiroshima University Hospital, Gastroenterology and Metabolism, Hiroshima, Japan


The patients with ulcerative colitis are at increased risk of developing colitis-associated cancer, because long-term Inflammation in colon leads to the development of carcinogenesis. The precise mechanism of the carcinogenesis in colitis-associated cancer however remains unknown. Recently, 3D primary spheroid culture of colon epithelial cells has been established in our group (Nature Medicine 2012). We therefore aimed to assess the effect of long-term stimulation with cytokines on colon epithelial cells in vitro, using 3D primary spheroid culture of colon epithelial cells.


Colonic crypts were isolated from 8-week old female mouse and were cultured by TMDU method. To mimic chronic inflammation on colon epithelial cells, the mixture of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), interleukin-6(IL-6), lipopolysaccharide(LPS) and flagellin was treated into primary colonic spheroids were added into medium every other day. The expression of NF-κB target genes IL-8, DUOX2 and DUOXA2 was assessed by quantitative RT-PCR. For the analysis of accumulation of NF-κB p65 in nuclei, 3-dimentional immunohistochemistry of whole spheroid was performed by stain with anti-NF-κB p65 antibody. To evaluate reactive oxygen species(ROS) in live spheroids, CellROX Deep Red Stress Reagents (Life echnologies) was added to medium for 1 day. Spheroids were visualized by confocal laser fluorescent microscopy.


The treatment with TNF-α, IL-1β, IL-6, LPS and flagellin showed the significant induction of NF-κB target genes in primary colonic spheroids. Interestingly, continuous treatment with all factors resulted in the elevated expression of DUOXA2 gene. suggesting that NF-κB signaling might be accumulated by the stimulating time.

3-dimentional immunostaining analysis showed stronger accumulation of NF-κB p65 in nuclei by the longer time of the stimulation, indicating that long-term stimulation with cytokines might lead to a stronger activation of NF-κB signaling. Interestingly, amplified NF-κB signaling by long-term stimulation was still active after the removal of all cytokines, whereas NF-κB signaling induced by short-term stimulation was completely shut down by the removal of all cytokines. ROS were induced by the stimulation with all cytokines. Moreover, ROS in the spheroid with long-term stimulation was shown at 11 weeks after the removal of cytokines, suggesting that oxidative stress in the spheroid with long-term stimulation is also irreversible.


Long-term inflammation leads to irreversible NF-κB signaling activation in colonic epithelial cells, suggesting that "signal spiral" might be crucial for the carcinogenesis of colitis-associated cancer.