title: Targeted RNA interference of PI3K pathway components sensitizes colon cancer cells to TNF-related apoptosis-inducing ligand (TRAIL).
abstract: BACKGROUND
  The phosphoinositide 3-kinase (PI3K/Akt) pathway transduces signals initiated from growth factors. Previously, we identified an important role for PI3K/Akt in colon cancer progression. The purpose of this study was to determine (1) whether short interfering RNA (siRNA) directed to PI3K/Akt components can render colon cancer cells sensitive to treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and (2) the cellular mechanisms contributing to the enhanced sensitivity.
  
  
  METHODS
  Human colon cancer cells KM20 and KM12C (both TRAIL resistant) were transfected with siRNA directed against the PI3K p85alpha regulatory subunit Akt1 or nontargeting control sequence and then treated with TRAIL (100 ng/mL) or vehicle. A ribonuclease protection assay was performed to assess changes in TRAIL receptor expression. Protein was extracted and analyzed by Western blot for expression of cleavage of TRAIL receptors (death receptor (DR) 4 and 5), caspase-3, caspase-8, and BID. Apoptosis was measured by enzyme-linked immunosorbent assay of DNA fragmentation.
  
  
  RESULTS
  Combination treatment with p85alpha or Akt1 siRNA and TRAIL increased apoptosis in KM20 and KM12C cells, compared with TRAIL alone; these results were corroborated further by complete inhibition of apoptosis by Z-acetyl-Asp-Glu-Val-Asp-(DEVD)-fmk, a caspase-3 inhibitor. Furthermore, siRNA-mediated PI3K pathway inhibition resulted in increased expression of the TRAIL death receptors 4 and 5.
  
  
  CONCLUSIONS
  Inhibition of PI3K/Akt by RNA interference sensitizes resistant colon cancer cells to TRAIL-induced cell death through the induction of TRAIL receptors and activation of caspase-3 and caspase-8. Agents that selectively target the PI3K/Akt pathway may enhance the effects of chemotherapeutic agents and provide novel adjuvant treatment for selected colon cancers.
authors.affiliation: Department of Surgery, The University of Texas Medical Branch, Galveston 77555-0536, USA.
authors.forename: Piotr G
authors.initials: PG
authors.lastname: Rychahou
authors.forename: Carlos A
authors.initials: CA
authors.lastname: Murillo
authors.forename: B Mark
authors.initials: BM
authors.lastname: Evers
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D051017:Apoptosis Regulatory Proteins; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000378:metabolism / Q000628:therapy*; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D034741:RNA, Small Interfering; D053221:TNF-Related Apoptosis-Inducing Ligand; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000494:pharmacology; D015854:Up-Regulation
chemical_list: D051017:Apoptosis Regulatory Proteins; D008562:Membrane Glycoproteins; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D034741:RNA, Small Interfering; D053221:TNF-Related Apoptosis-Inducing Ligand; D014409:Tumor Necrosis Factor-alpha; C505653:TNFSF10 protein, human; C494918:AKT1 protein, human