title: Therapeutic modulation of Akt activity and antitumor efficacy of interleukin-12 against orthotopic murine neuroblastoma.
abstract: BACKGROUND
  Patients with advanced neuroblastoma have a poor prognosis. The antiapoptotic protein Akt has been implicated as a possible mediator of the resistance of human neuroblastoma cells to apoptosis; the proapoptotic protein Bid, is inhibited by activated Akt. Neuroblastoma has demonstrated responsiveness to immunotherapeutic approaches in preclinical studies, prompting investigation of new therapeutic strategies based on potentiation of the host immune response, including the use of systemic cytokines.
  
  
  METHODS
  We examined the antitumor efficacy and mechanisms of action of the central immunoregulatory cytokine interleukin-12 (IL-12) in mice bearing established orthotopic neuroblastoma tumors derived from murine TBJ and Neuro-2a cells. Cohorts of mice (10 mice/group) bearing established orthotopic neuroblastoma tumors were injected intraperitoneally with IL-12 or vehicle and monitored for survival. IL-12-induced apoptosis within the tumor microenvironment was investigated using ribonuclease protection assays, nuclear staining, and electron microscopy. Protein expression was determined via Western blot analysis and enzyme-linked immunosorbent assays. Confocal microscopy was used to examine the distribution of overexpressed Bid-enhanced green fluorescent protein fusion protein (Bid-EGFP) in TBJ cells. All statistical tests were two-sided.
  
  
  RESULTS
  IL-12 induced complete tumor regression and long-term survival of 8 (80%) of 10 mice bearing established neuroblastoma tumors compared with 1 (10%) of 10 control mice (P = .0055) and profound tumor cell apoptosis in vivo despite the fact that TBJ and Neuro-2a cells were resistant to receptor-mediated apoptosis in vitro. These cells expressed high levels of phosphorylated Akt, a key prosurvival molecule, and Akt inhibitors sensitized neuroblastoma cells to apoptosis mediated by IL-12-inducible cytokines including tumor necrosis factor-alpha and interferon-gamma in vitro. IL-12 increased the expression of proapoptotic genes and decreased Akt phosphorylation within established TBJ tumors in conjunction with activation and subcellular translocation of Bid.
  
  
  CONCLUSIONS
  Our results suggest that IL-12 overcomes a potentially critical mechanism of tumor self-defense in vivo by inhibiting Akt activity and imply that IL-12 may possess unique therapeutic activity against tumors that express high levels of activated Akt.
authors.affiliation: Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702-1201, USA.
authors.forename: Tahira
authors.initials: T
authors.lastname: Khan
authors.forename: Julie A
authors.initials: JA
authors.lastname: Hixon
authors.forename: Jimmy K
authors.initials: JK
authors.lastname: Stauffer
authors.forename: Erin
authors.initials: E
authors.lastname: Lincoln
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Back
authors.forename: Jason
authors.initials: J
authors.lastname: Brenner
authors.forename: Stephen
authors.initials: S
authors.lastname: Lockett
authors.forename: Kunio
authors.initials: K
authors.lastname: Nagashima
authors.forename: Douglas
authors.initials: D
authors.lastname: Powell
authors.forename: Jon M
authors.initials: JM
authors.lastname: Wigginton
mesh_terms: D000310:Adrenal Gland Neoplasms / Q000188:drug therapy / Q000201:enzymology; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D051023:BH3 Interacting Domain Death Agonist Protein / Q000187:drug effects / Q000378:metabolism; D015153:Blotting, Western; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D005456:Fluorescent Dyes; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000187:drug effects / Q000378:metabolism; D007150:Immunohistochemistry; D007211:Indoles; D007371:Interferon-gamma / Q000187:drug effects / Q000378:metabolism; D018664:Interleukin-12 / Q000494:pharmacology*; D051379:Mice; D018613:Microscopy, Confocal; D009368:Neoplasm Transplantation; D009447:Neuroblastoma / Q000188:drug therapy* / Q000201:enzymology*; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000187:drug effects* / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000187:drug effects / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D051023:BH3 Interacting Domain Death Agonist Protein; C494815:Bid protein, mouse; D005456:Fluorescent Dyes; D007211:Indoles; D014409:Tumor Necrosis Factor-alpha; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D018664:Interleukin-12; C007293:DAPI; D007371:Interferon-gamma; D051057:Proto-Oncogene Proteins c-akt