Nicotine increases oxidative stress, activates NF-kappaB and GRP78, induces apoptosis and sensitizes cells to genotoxic/xenobiotic stresses by a multiple stress inducer, deoxycholate: relevance to colon carcinogenesis.

Epidemiologic studies indicate that environmental (smoking) and dietary factors (high fat) contribute to carcinogenesis in many organ systems. The aim of our study was to test the hypothesis that nicotine, a component of cigarette smoke, and sodium deoxycholate (NaDOC), a cytotoxic bile salt that increases in concentration in the gastrointestinal tract after a high fat meal, induce similar cellular stresses and that nicotine may enhance some of the NaDOC-induced stresses. We found that nicotine, at 0.8 microM, the very low sub-micromolar level occurring in the tissues of smokers: (1). increases oxidative stress; (2). activates NF-kappaB, a redox-sensitive transcription factor; (3). activates the 78 kD glucose regulated protein promoter, an indication of endoplasmic reticulum stress; (4). induces apoptosis; (5). enhances the ability of NaDOC to activate the 153 kD growth arrest and DNA damage promoter, an indication of increased genotoxic stress; and (6). enhances the ability of NaDOC to activate the xenobiotic response element. Our findings have applicability to G.I. cancer, in general, since smoking is a risk factor in the development of esophageal, pancreatic, gastric and colon cancer, and these cancers are also promoted by bile acids.

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

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.