title: Specific down-regulation of XIAP with RNA interference enhances the sensitivity of canine tumor cell-lines to TRAIL and doxorubicin.
abstract: BACKGROUND
  Apoptosis resistance occurs in various tumors. The anti-apoptotic XIAP protein is responsible for inhibiting apoptosis by reducing caspase-3 activation. Our aim is to evaluate whether RNA inhibition against XIAP increases the sensitivity of canine cell-lines for chemotherapeutics such as TRAIL and doxorubicin. We used small interfering RNA's (siRNA) directed against XIAP in three cell-lines derived from bile-duct epithelia (BDE), mammary carcinoma (P114), and osteosarcoma (D17). These cell-lines represent frequently occurring canine cancers and are highly comparable to their human counterparts. XIAP down-regulation was measured by means of quantitative PCR (Q-PCR) and Western blotting. The XIAP depleted cells were treated with a serial dilution of TRAIL or doxorubicin and compared to mock- and nonsense-treated controls. Viability was measured with a MTT assay.
  
  
  RESULTS
  All XIAP siRNA treated cell-lines showed a mRNA down-regulation over 80 percent. Western blot analysis confirmed mRNA measurements. No compensatory effect of IAP family members was seen in XIAP depleted cells. The sensitivity of XIAP depleted cells for TRAIL was highest in BDE cells with an increase in the ED50 of 14-fold, compared to mock- and nonsense-treated controls. The sensitivity of P114 and D17 cell-lines increased six- and five-fold, respectively. Doxorubicin treatment in XIAP depleted cells increased sensitivity in BDE cells more than eight-fold, whereas P114 and D17 cell-lines showed an increase in sensitivity of three- and five-fold, respectively.
  
  
  CONCLUSIONS
  XIAP directed siRNA's have a strong sensitizing effect on TRAIL-reduced cell-viability and a smaller but significant effect with the DNA damaging drug doxorubicin. The increase in efficacy of chemotherapeutics with XIAP depletion provides the rationale for the use of XIAP siRNA's in insensitive canine tumors.
authors.affiliation: Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands. B.Spee@vet.uu.nl
authors.forename: Bart
authors.initials: B
authors.lastname: Spee
authors.forename: Martijn D B
authors.initials: MD
authors.lastname: Jonkers
authors.forename: Brigitte
authors.initials: B
authors.lastname: Arends
authors.forename: Gerard R
authors.initials: GR
authors.lastname: Rutteman
authors.forename: Jan
authors.initials: J
authors.lastname: Rothuizen
authors.forename: Louis C
authors.initials: LC
authors.lastname: Penning
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004285:Dogs; D015536:Down-Regulation; D004317:Doxorubicin / Q000494:pharmacology*; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006706:Homeostasis; D051034:Inhibitor of Apoptosis Proteins / Q000145:classification / Q000235:genetics; D008562:Membrane Glycoproteins / Q000494:pharmacology*; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D012680:Sensitivity and Specificity; D053221:TNF-Related Apoptosis-Inducing Ligand; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000235:genetics* / Q000378:metabolism*
chemical_list: D051017:Apoptosis Regulatory Proteins; D051034:Inhibitor of Apoptosis Proteins; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; D051636:X-Linked Inhibitor of Apoptosis Protein; D004317:Doxorubicin