title: Downregulation of active caspase 8 as a mechanism of acquired TRAIL resistance in mismatch repair-proficient colon carcinoma cell lines.
abstract: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers the apoptotic cascade in various colon cancer cell lines after binding to the membrane receptors DR4 and DR5. However, not all cancer cell lines are sensitive to the therapeutic recombinant human TRAIL (rhTRAIL). To investigate the causes of TRAIL resistance in colon cancer cell lines, models have been developed, mostly in mismatch repair-deficient cells. These cells are prone to mutations in genes containing tandem repeat, including pro-apoptotic protein Bax. We therefore investigated the mechanism underlying TRAIL resistance acquisition in a mismatch repair-proficient colon carcinoma cell line. The TRAIL-resistant cell line SW948-TR was established from the TRAIL-sensitive cell line SW948 by continuous exposure to rhTRAIL, and exhibited 140-fold less sensitivity to rhTRAIL in a cell viability assay. Resistance was stable for over a year in the absence of rhTRAIL. Both cell lines had similar TRAIL receptor cell membrane expression levels. Treatment with the protein synthesis inhibitor cycloheximide sensitized SW948-TR to rhTRAIL-induced apoptosis, indicating that the functionality of the TRAIL receptors was maintained. In SW948-TR, procaspase 8 protein levels but not mRNA levels were notably lower than in SW948. Downregulation of c-FLIP with short interfering RNA (siRNA) sensitized SW948-TR cells to rhTRAIL while caspase 8 siRNA decreased rhTRAIL sensitivity in SW948, indicating the importance of the caspase 8/c-FLIP ratio. Proteasome inhibition with MG132 did not restore basic procaspase 8 levels but stabilized cleaved caspase 8 in rhTRAIL-treated SW948-TR cells. Altogether, our results suggest that colon cancer cells can acquire rhTRAIL resistance by primarily reducing the basal procaspase 8/c-FLIP ratio and by increasing active caspase 8 degradation after rhTRAIL treatment. Proteasome inhibitors can effectively overcome acquired rhTRAIL resistance in mismatch repair-proficient colon cancer cells.
authors.affiliation: Department of Medical Oncology, University Medical Center Groningen, 9713 GZ, Groningen, The Netherlands.
authors.forename: Caroline M M
authors.initials: CM
authors.lastname: Van Geelen
authors.forename: Bodvael
authors.initials: B
authors.lastname: Pennarun
authors.forename: Wytske Boerma-Van
authors.initials: WB
authors.lastname: Ek
authors.forename: Phuong T K
authors.initials: PT
authors.lastname: Le
authors.forename: Diana C J
authors.initials: DC
authors.lastname: Spierings
authors.forename: Elisabeth G E
authors.initials: EG
authors.lastname: De Vries
authors.forename: Steven
authors.initials: S
authors.lastname: De Jong
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000378:metabolism; D002277:Carcinoma / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D053181:Caspase 8 / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003110:Colonic Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D003513:Cycloheximide / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D053843:DNA Mismatch Repair / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D004789:Enzyme Activation; D006801:Humans; D020128:Inhibitory Concentration 50; D007976:Leupeptins / Q000494:pharmacology; D010641:Phenotype; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D011994:Recombinant Proteins / Q000494:pharmacology; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000494:pharmacology*; D013997:Time Factors
chemical_list: D000970:Antineoplastic Agents; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D061988:Proteasome Inhibitors; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D003513:Cycloheximide; C505530:CASP8 protein, human; D053181:Caspase 8; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde