amanitins and Colonic-Neoplasms

Pretreatment of HT29-D4 epithelial adenocarcinoma colic cells with des-IGF-1 upregulated TNF alpha-mediated activation of IL-8 expression at different levels (protein, mRNA, and hnRNA). RNA transcription but not RNA stabilization was found to be involved. In this cell line, cooperation of NF-kappa B with other factors appeared essential for IL-8 expression. Indeed, TNF alpha-induced NF-kappa B translocation was not sufficient to support enhancement of the transcription and des-IGF-1 did not promote but partly inhibited both the TNF alpha-induced NF-kappa B activation and I kappa B alpha degradation through a PI-3K-dependent pathway. A CCAAT/enhancer binding protein (C/EBP) site located on the IL-8 gene enhancer cooperated with a NF-kappa B binding site and led to the upregulation of IL-8 expression. Binding of C/EBP alpha to this sequence disappeared in IGF-1 treated cells. This event may be important for the cross-talk between IGF-1- and TNF alpha-mediated pathways leading to the control of inflammatory processes and the decision concerning apoptosis or cell survival.

Topics: Adenocarcinoma; Amanitins; Binding Sites; CCAAT-Enhancer-Binding Protein-alpha; CCAAT-Enhancer-Binding Proteins; Colonic Neoplasms; Down-Regulation; Humans; I-kappa B Proteins; Insulin-Like Growth Factor I; Interleukin-8; Kinetics; NF-kappa B; NF-KappaB Inhibitor alpha; Peptide Fragments; Phosphatidylinositol 3-Kinases; RNA, Messenger; Transcription Factor AP-1; Transcriptional Activation; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Up-Regulation

The C-24 oxidation pathway plays a major role in the degradation of vitamin D metabolites in kidney and other target tissues. The aim of the present study was to establish an intestinal cell culture system to study the mechanisms regulating the vitamin D catabolic pathway. 25-Hydroxyvitamin D3-24-hydroxylase (24-hydroxylase), the first enzyme in the catabolic sequence, was examined in Caco-2 cells, a human colon adenocarcinoma cell line which exhibits differentiated functions of absorbing intestinal epithelial cells. While untreated Caco-2 cells did not exhibit 24-hydroxylase activity, significant catabolic activity was induced by prior treatment of cell monolayers with 1,25-dihydroxyvitamin D3(1,25-(OH)2D3). Induced 24-hydroxylase D3 (25OHD3) and 1,25-(OH)2D3 was detected 6 h after treatment of cells with 10(-8)M 1,25-(OH)2D3, peaked at 16 h, and decreased thereafter. Treatment of cells with 10(-7) M 1,25-(OH)2D3 elicited a maximal 24-hydroxylase response. Comparable time courses of induction by 1,25-(OH)2D3 and 1,25-(OH)2D3-dose response curves were observed in cultured human skin fibroblasts and Caco-2 cells. 25OHD3 was not as good an inducer of the vitamin D catabolic pathway in Caco-2 cells as 1,25-(OH)2D3. Induction of 24-hydroxylase activity by 1,25-(OH)2D3 was inhibited by pretreatment of Caco-2 cells with either actinomycin D, alpha-amanitin, or cycloheximide suggesting that mRNA and protein synthesis are required for induction. The present study demonstrates that 1,25-(OH)2D3-treated Caco-2 cells express the vitamin D catabolic pathway and, therefore, constitute a useful in vitro model to study the mechanism of induction by 1,25-(OH)2D3.

Topics: Adenocarcinoma; Amanitins; Calcifediol; Calcitriol; Colonic Neoplasms; Cycloheximide; Cytochrome P-450 Enzyme System; Dactinomycin; Enzyme Induction; Fibroblasts; Humans; Hydroxylation; Intestines; Kinetics; Steroid Hydroxylases; Tumor Cells, Cultured; Vitamin D; Vitamin D3 24-Hydroxylase