Anti-Cancer Roles of Probiotic-Derived P8 Protein in Colorectal Cancer Cell Line DLD-1.

A novel probiotics-derived protein, P8, suppresses the growth of colorectal cancer (CRC). P8 can penetrate the cell membrane via endocytosis and cause cell cycle arrest in DLD-1 cells through down-regulation of CDK1/Cyclin B1. However, neither the protein involved in the endocytosis of P8 nor the cell cycle arrest targets of P8 are known. We identified two P8-interacting target proteins [importin subunit alpha-4 (KPNA3) and glycogen synthase kinase-3 beta (GSK3ß)] using P8 as a bait in pull-down assays of DLD-1 cell lysates. Endocytosed P8 in the cytosol was found to bind specifically to GSK3ß, preventing its inactivation by protein kinases AKT/CK1ε/PKA. The subsequent activation of GSK3ß led to strong phosphorylation (S33,37/T41) of ß-catenin, resulting in its subsequent degradation. P8 in the cytosol was also found to be translocated into the nucleus by KPNA3 and importin. In the nucleus, after its release, P8 binds directly to the intron regions of the GSK3ß gene, leading to dysregulation of GSK3ß transcription. GSK3ß is a key protein kinase in Wnt signaling, which controls cell proliferation during CRC development. P8 can result in a cell cycle arrest morphology in CRC cells, even when they are in the Wnt ON signaling state.

Zaluzanin C Inhibits Differentiation of 3T3-L1 Preadipocytes into Mature Adipocytes.

BACKGROUND: An excess storage of body fat causes obesity. Since obesity increases risk of chronic diseases, it is important to inhibit excessive storage of fat. Zaluzanin C is a sesquiterpene lactone isolated from Ainsliaea acerifolia. The aim of this study was to demonstrate the effect of zaluzanin C on differentiation of 3T3-L1 preadipocytes into mature adipocytes. METHODS: The cytotoxicity of zaluzanin C and its effect on cell proliferation was determined. For the induction of adipocyte differentiation, 3T3-L1 preadipocytes were treated with differentiating medium containing 10 µg/mL insulin, 115 µg/mL methylisobutylxanthine, and 1 µM dexamethasone. Differentiated 3T3-L1 cells were subjected to Oil red O solution or used for Western blot analysis. Zaluzanin C was added to the cell culture medium at concentrations of 0, 1, 2.5, 5, and 10 µM. RESULTS: Zaluzanin C did not inhibit cell proliferation and showed no cytotoxicity at 10 µM concentration in 3T3-L1 cells. Therefore, concentration range of 0-10 µM zaluzanin C was used for subsequent experiments. Zaluzanin C inhibited accumulation of lipid droplets in 3T3-L1 adipocytes. To understand the underlying mechanism of zaluzanin C, expression of adipogenesis regulators was determined by Western blot analysis. Zaluzanin C suppressed peroxisome proliferator-activated receptor gamma (PPARγ) expression, an adipogenesis related transcription factor, and inhibited aP2/fatty acid-binding protein-4 expression, a target gene of PPARγ. However, it did not affect expression of CCAAT/enhancer-binding protein alpha related with acquisition of insulin sensitivity. CONCLUSION: These data suggest that inhibitory effect of zaluzanin C on adipogenesis of 3T3-L1 adipocytes could be partially caused by suppressing PPARγ.