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

Carcinogenesis vol.23 no.12 pp.2063–2080, 2002 Development and molecular characterization of HCT-116 cell lines resistant to the tumor promoter and multiple stress-inducer, deoxycholate Cara L.Crowley-Weber1, Claire M.Payne1,3, Mary Gleason-Guzman3, George S.Watts3, Bernard Futscher3, Caroline N.Waltmire1, Cheray Crowley1, Katerina Dvorakova1, Carol Bernstein1, Mary Craven1, Harinder Garewal2,3,4 and Harris Bernstein1,3,5 Departments of 1Microbiology and Immunology, 2Internal Medicine, College of Medicine, and 3Arizona Cancer Center, University of Arizona, 85724–5049 and 4Tucson Veterans Affairs Medical Center, Section of Hematology/Oncology, Tucson, AZ 85723, USA Email: 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-κB(p50), NF-κB(p65), thioredoxin peroxidase (peroxiredoxin) 2, peroxiredoxin 4, maspin, guanylate cyclase activating protein-1, PKCζ, EGFR, Ras family Abbreviations: BH4, tetrahydrobiopterin; BNIP3, Bcl2/adenovirus EIB 19 kD-interacting protein 3; CAMK2D, calcium/calmodulin-dependent protein kinase II delta; DHAP, dihydroxyacetone phosphate; DOC, deoxycholate; EGFR, epidermal growth factor receptor; ER, endoplasmic reticulum; GAP, glyceraldehyde 3-phosphate; GC, guanylate cyclase; Grp78, 78 kD-glucoseregulated protein; IAP, inhibitor of apoptosis protein; IEF, isoelectric focusing; IKK-β, IκB-kinase-β; IP3, inositol triphosphate; MALDI-MS, matrix assisted laser desorption ionization mass spectroscopy; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; MEKK, MEK kinase; NIK, NF-κBinducing kinase; NLS, nuclear localization signal; NaDOC, sodium deoxycholate; NO, nitric oxide; NOS2, inducible NO synthase; ONOO–, peroxynitrite; PDTC, pyrrolidine dithiocarbamate; PKCζ, protein kinase C-zeta; PKG, cGMP-activated protein kinase; PN-1, protease nexin-1; QDPR, quinoid dihydropteridine reductase; SERPIN, serine protease inhibitor; TEM, transmission electron microscopy; TPI, triose phosphate isomerase; Trx, thioredoxin; TPx, Trx peroxidase (peroxiredoxin); TR, Trx reductase; TRAF, tumor necrosis factor receptor-associated factor; TTFA, thenoyl trifluoroacetone. [NB. An Excel file of all the data for the three resistant HCT-116 cell lines compared with the sensitive HCT-116 cell line is available as an Email attachment, upon request.] © Oxford University Press Introduction Bile acids are natural detergents synthesized in the liver and stored in the gall bladder. High levels of certain bile acids, however, are known to promote G.I. cancer (1–4), including colon cancer (1,5,6). The bile acids that promote colon cancer are secondary bile acids that have been deconjugated and dehydroxylated, resulting in an increase in hydrophobicity. Conversion of primary bile salts to their secondary bile acid counterparts is catalyzed by the bacterial enzyme 7αdehydroxylase which removes a hydroxyl group from the 7α position of the steroid nucleus. Cholic acid and chenodeoxycholic acid, respectively, are converted to deoxycholic acid (DOC) and lithocholic acid, the secondary bile acids found in greatest concentration in human fecal water. These secondary bile acids have been implicated in animal models of colon carcinogenesis as promoters of colon cancer (6), although the exact mechanism of tumor promotion by bile acids is unclear. The bile acid present in the highest concentration in the human colon and feces is DOC, and it is found at concentrations up to 0.78 mM in individuals consuming a high fat diet (7). Our laboratory previously reported that the sodium salt of DOC (NaDOC), at high physiological concentrations, induces apoptosis in colonic epithelial cells of the flat mucosa from normal subjects (8–11). We also found that resistance to NaDOC-induced apoptosis occurs in the normal appearing flat mucosa of patients with colon cancer (9–11). Decreased ability to undergo apoptosis is a risk factor in colon carcinogenesis (12–17) since apoptosis resistance creates a permissive environment for increasing genomic instability (e.g. aneuploidy, point mutations, loss of heterozygosity)(18–20), which can result in cancer. It has been calculated that sporadically arising polyps in the colon survive with nearly 11 000 genomic alterations per cell (21). It is assumed that some of these genomic alterations affect the expression of apoptosis and/or survival genes which may explain the further increase in apoptosis resistance during polyp development (22–24). We have proposed that excessive, frequent exposure of an individual’s 2063 5To whom correspondence should be addressed 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-κB 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. C.L.Crowley-Weber et al. Materials and methods Cell lines, media and chemicals HCT-116, a colon adenocarcinoma cell line [American Type Culture Collection (ATCC), Bethesda, MD; ATCC # CCL 247] and apoptosis resistant HCT-116 cell lines were maintained in DMEM supplemented with 10% fetal calf serum (Omega Scientific, Tarzana, CA), 1% MEM non-essential amino acids, 100 µg/ ml streptomycin, 100 U/ml penicillin, and 3.44 mg/ml L-glutamine. Unless otherwise indicated, (...truncated)