title: Resistance to TRAIL is mediated by DARPP-32 in gastric cancer.
abstract: OBJECTIVE
  Dopamine and cAMP-regulated phosphoprotein, Mr 32,000 (DARPP-32), is overexpressed during the gastric carcinogenesis cascade. Here, we investigated the role of DARPP-32 in promoting resistance to treatment with TRAIL.
  
  
  METHODS
  In vitro cell models including stable expression and knockdown of DARPP-32 were used. The role of DARPP-32 in regulating TRAIL-dependent apoptosis was evaluated by clonogenic survival assay, Annexin V staining, immunofluorescence, quantitative reverse transcriptase PCR, Western blot, and luciferase reporter assays.
  
  
  RESULTS
  Stable expression of DARPP-32 in MKN-28 cells enhanced cell survival and suppressed TRAIL-induced cytochrome c release and activation of caspase-8, -9, and -3. Conversely, short hairpin RNA-mediated knockdown of endogenous DARPP-32 sensitized the resistant MKN-45 cells to TRAIL-induced apoptosis and enhanced TRAIL-mediated activation of caspase-8, -9, and -3. DARPP-32 induced BCL-xL expression through activation of Src/STAT3 signaling, and treatment with the Src-specific inhibitor PP1 abrogated DARPP-32-dependent BCL-xL upregulation and cell survival in MKN-28 cells. The TRAIL treatment induced caspase-dependent cleavage of NF-κBp65 protein; this cleavage was prevented by DARPP-32, thus maintaining NF-κB activity and the expression of its target, FLIP(S) protein. This suggests that upregulation of BCL-xL could play a possible role in blocking the mitochondria intrinsic apoptosis pathway, whereas the DARPP-32 effect on the NF-κB/FLIP(S) axis could serve as an additional negative feedback loop that blocks TRAIL-induced activation of caspase-8.
  
  
  CONCLUSIONS
  Our findings uncover a novel mechanism of TRAIL resistance mediated by DARPP-32, whereby it inhibits the intrinsic apoptosis pathway through upregulation of BCL-xL, and the extrinsic apoptosis pathway through the NF-κB/FLIP(S) axis.
authors.affiliation: Department of Surgery and Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA. abbes.belkhiri@vanderbilt.edu
authors.forename: Abbes
authors.initials: A
authors.lastname: Belkhiri
authors.forename: Shoumin
authors.initials: S
authors.lastname: Zhu
authors.forename: Zheng
authors.initials: Z
authors.lastname: Chen
authors.forename: Mohammed
authors.initials: M
authors.lastname: Soutto
authors.forename: Wael
authors.initials: W
authors.lastname: El-Rifai
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic / Q000187:drug effects / Q000235:genetics; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32 / Q000235:genetics / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000235:genetics; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism; D015398:Signal Transduction; D013274:Stomach Neoplasms / Q000235:genetics / Q000378:metabolism; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000008:administration & dosage / Q000235:genetics; D051020:bcl-X Protein / Q000378:metabolism
chemical_list: C494811:BCL2L1 protein, human; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; D016328:NF-kappa B; C497928:PPP1R1B protein, human; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D051020:bcl-X Protein; D020169:Caspases