Development and molecular characterization of HCT-116 cell lines resistant to the tumor promoter and multiple stress-inducer, deoxycholate

Abstract

Evidence from live cell bioassays shows that the flat mucosa from patients with colon cancer exhibits resistance to bile salt-induced apoptosis. Three independent cell lines derived from the colonic epithelial cell line HCT-116 were selected for resistance to bile salt-induced apoptosis. These cell lines were developed as tissue culture models of apoptosis resistance. Selection was carried out for resistance to apoptosis induced by sodium deoxycholate (NaDOC), the bile salt found in highest concentrations in human fecal water. Cultures of HCT-116 cells were serially passaged in the presence of increasing concentrations of NaDOC. The resulting apoptosis resistant cells were able to grow at concentrations of NaDOC (0.5 mM) that cause apoptosis in a few hours in unselected HCT-116 cells. These cells were then analyzed for changes in gene expression. Observations from cDNA microarray, 2-D gel electrophoresis/MALDI-mass spectroscopy, and confocal microscopy of immunofluorescently stained preparations indicated underexpression or overexpression of numerous genes at either the protein or mRNA level. Genes that may play a role in apoptosis and early stage carcinogenesis have been identified as upregulated in these cell lines, including Grp78, Bcl-2, NF-kappaB(p50), NF-kappaB(p65), thioredoxin peroxidase (peroxiredoxin) 2, peroxiredoxin 4, maspin, guanylate cyclase activating protein-1, PKCzeta, EGFR, Ras family members, PKA, PI(4,5)K, TRAF2 and BIRC1 (IAP protein). Under-expressed mRNAs included BNIP3, caspase-6, caspase-3 and serine protease 11. NF-kappaB was constitutively activated in all three resistant cell lines, and was responsible, in part, for the observed apoptosis resistance, determined using antisense oligonucleotide strategies. Molecular and cellular analyses of these resistant cell lines has suggested potential mechanisms by which apoptosis resistance may develop in the colonic epithelium in response to high concentrations of hydrophobic bile acids that are associated with a Western-style diet. These analyses provide the rationale for the development of hypothesis-driven intermediate biomarkers to assess colon cancer risk on an individual basis.

Letter to the Editor: The response to Dr. Yang

“…Hydrophobic bile acids, such as deoxycholic acid (DCA, model bile acid), induce apoptosis after acute exposure (3). In contrast, chronic exposure of epithelial cells to bile acids leads to the selection of clones with an apoptosis-resistant phenotype (1). Similarly, we reported in our recent paper, the acute exposure to relatively low concentrations of DCA leads to normal cellular response, the induction of autophagy, and increased Beclin-1 expression, but chronic exposure to DCA does not affect Beclin-1 expression and results in defective autophagy (5).…”

EphA2 Up-regulation induced by deoxycholic acid in human colon carcinoma cells, an involvement of extracellular signal-regulated kinase and p53-independence

“…An increasing amount of evidence suggest that bile acids may be a multiple stress-inducer (Crowley-Weber et al 2002), and one of the best-documented pathways is the one involving protein kinase C (PKC) (Debruyne et al 2001). Recently, many researchers reported that DCA might activate the Ras/Raf/ERK kinase pathway, then subsequently up-regulate some genes downstream (Yoon et al 2002).…”

Cyclooxygenase-independent Induction of Apoptosis by Sulindac Sulfone Is Mediated by Polyamines in Colon Cancer

“…Target Preparation-Microarray analysis was done as described before (41). In short, the RNeasy total RNA kit (Qiagen, Valencia, CA) and protocol was used to isolate total RNA from the Caco-2 cells treated with either vehicle or sulindac sulfone.…”