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.
Crowley-Weber
Cara L
CL
Department of Microbiology and Immunology, College of Medicine, University of Arizona, Tucson 85724-5049, USA.
Payne
Claire M
CM
Gleason-Guzman
Mary
M
Watts
George S
GS
Futscher
Bernard
B
Waltmire
Caroline N
CN
Crowley
Cheray
C
Dvorakova
Katerina
K
Bernstein
Carol
C
Craven
Mary
M
Garewal
Harinder
H
Bernstein
Harris
H
D017209:Apoptosis; D002352:Carrier Proteins / Q000378:metabolism; D003106:Colon / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D003840:Deoxycholic Acid / Q000494:pharmacology*; D003902:Detergents / Q000494:pharmacology*; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm; D015180:Electrophoresis, Gel, Two-Dimensional; D000091342:Endoplasmic Reticulum Chaperone BiP; D004847:Epithelial Cells; D006360:Heat-Shock Proteins; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D008954:Models, Biological; D018832:Molecular Chaperones / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D052002:NF-kappa B p50 Subunit; D020411:Oligonucleotide Array Sequence Analysis; D009841:Oligonucleotides / Q000494:pharmacology; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D018384:Oxidative Stress; D011401:Promoter Regions, Genetic; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D051996:Transcription Factor RelA; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D002352:Carrier Proteins; D018076:DNA, Complementary; D003902:Detergents; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D051996:Transcription Factor RelA; D003840:Deoxycholic Acid