Anti-proliferation activity of fucoidan on MKN45 gastric cancer cells and downregulation of phosphorylated ASK1, a cell-cycle-regulated cyclin-dependent kinase

Fucoidan is a high-molecular polysaccharide whose main constituent is sulfated fucose. Extensive studies have demonstrated numerous interesting biological activities for fucoidan. We specifically focused on the anti-proliferation activity of fucoidan and examined the underlying mechanism in MKN45 gastric cancer cells. BrdU assay revealed that fucoidan impeded the MKN45 cell cycle by approximately 50%, and clonogenic assay also showed that fucoidan inhibited cell proliferation. Preliminary examinations of fucoidan using LDH assay showed no immediate cytotoxic effects at 24-h exposure. However, longer time courses revealed inhibition of cell growth at 4 days in a dose-dependent manner. Microarray analysis in MKN45 cells treated with fucoidan identified genes that were upregulated and downregulated in response to fucoidan, including MAP3K5, or ASK1 (apoptosis signal-regulating kinase), which was upregulated by 1.38-fold. Western blot confirmed that fucoidan increased ASK1 protein levels, while reducing the levels of phosphorylated ASK1. Reduction of ASK1 by siRNA decreased proliferation of MKN45 cells. Our findings show that fucoidan may suppress cellular proliferation and DNA synthesis in MKN45 cells by suppressing the ASK1-p38 signaling pathway through reduction of phosphorylated ASK1 levels. To identify potential genes involved in mediating the cellular effects of fucoidan, which is a high-molecular polysaccharide whose main constituent is sulfated fucose, total RNA was collected from cultured MKN45 gastric cancer cells and microarray expression analyses were performed using the Agilent Human microarray kit (Palo Alto, CA, US). A total of 20278 sequences were examined at DNA Chip Research Inc. (Kanagawa, Japan), and 496 genes were found to be upregulated and 774 genes were downregulated by fucoidan.