Folate deprivation and folate antagonist methotrexate treatment promote in vitro metastatic potential of human colorectal carcinoma cells through NF-κB-mediated activation of Hh signaling pathway

• Main Authors: Tz-Ping Wang, 王姿萍
• Other Authors: Rwei-Fen S. Huang
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/26556089771894430415

碩士 === 輔仁大學 === 營養科學系 === 98 === Previous studies have suggested that activation of Hedgehog (Hh) signaling pathway critical for embryogenesis development may mediate metastasis of pancreatic and gastric cancers. Transcriptional nuclear factor-κB (NF-κB) may involve in the regulation oh Hh signaling, yet the mechanisms remain unclear. Epidemiologic evidence has demonstrated increased risks of colon cancer as well as increased risks of developing advanced tumours associated with folate malnutrition. The casual effects and molecular mechanisms of folate deprivation (FD) to promote colorectal cancer metastasis are currently not known. The aims of the study were to investigate whether FD may promote in vitro metastasis potential of colon cancer cells, mediating by NF-κB-mediated activation of Hh signaling pathway. Using human epithelial colorectal carcinoma HCT116 cells with low invasive capability as the experimental model, the data revealed that FD induced by FD medium or treatment of methotrexate (MTX) to disturb intracellular one-carbon metabolism significantly enhanced the in vitro metastasis potential of HCT116 cells. Compared with the control, FD HCT16 cells have increased capabilities of migration, invasion and adhesion by 2.6~7.6- (P<0.05) , 2.1~2.8- (P<0.05) and 3- fold (P<0.05) , respectively, when cultured in the collagen-coated matrix gel. Using the zymography and western blotting analysis, FD HCT116 cells displayed increased proteolysis activities of matrix metalloproteinases (MMPs) by 1.2~2.1- fold with significantly higher levels of MMP-2 activities expression (P<0.05) . Expression of β1 integrin in FD HCT116 cells increased by 1.9~1.7- fold as compared to the control (P<0.05) . FD at the early stage induced increased protein levels of NF-κB, following by 1.8-fold and 3.5-fold increased expressions of Hh signaling ligand molecule sonic hedgehog (Shh) as well as Hh signaling target transcription factor Gli1 protein, respectively (P<0.05) . Blockade Hh pathway by cyclopamine significantly diminished FD-enhanced expression of Shh and Gli1 signal molecules, reduced MMP-2 proteiolytic activity, and abrogated metastasis phenotype of FD HCT116 cells. Blockade NF-κB activity by BAY11-7082 abrogated metastasis phenotype of FD HCT116 cells, which coincided with suppressions of Shh and Gli1 signal molecules, reduction of MMP-2 proteiolytic activity and β1 integrin expression. Using guantitative real time PCR methods, the data revealed that folate deprivation at the early stage up-regulate mRNA expression of NF-κB, following by activation of genetic expressions of Hh signaling molecules including Hh ligand (Shh and Ihh) , target receptor (Ptch) , secondary messenger (Smo) and target transcription factor (Gli) , and increased mRNA expression of Hh signaling-target genes such as MMP-2 and Snail. Suppressor molecules of Hh signaling (Hip), invasion (E-cadherin) and β-catenin were down-regulated. Treatment of BAY11-7082 or cyclopamine reversed FD-induced transcription regulation on Hh signaling molecules as well as its target genes without affecting mRNA expression of NF-κB, suggesting an upstream regulation of NF-κB upon Hh signaling in FD colorectal carcinoma cells. By electrophoretic mobility mobility shift assays, FD activated NF-κB complexes to bind with NF-κB-DNA binding sites of Shh promoter region. Taken together, our data suggest that folate deficiency promotes in vitro metastatic potential of human epithelian CRC HCT116 through NF-κB-mediated activation of Hh signaling pathway; enhanced metastatic protein and gene expression.