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Therapeutic Modulation of Akt Activity
and Antitumor Efficacy of Interleukin-12 Against Orthotopic Murine
Neuroblastoma
Tahira Khan, Julie
A. Hixon, Jimmy K. Stauffer, Erin Lincoln,
Timothy C. Back, Jason Brenner, Stephen
Lockett, Kunio Nagashima, Douglas Powell,
Jon M. Wigginton
Affiliations of authors: Pediatric
Oncology Branch, Center for Cancer Research (TK, JAH, JKS, JMW), Data
Management Services (DP), National Cancer Institute at Frederick,
Frederick, MD; Intramural Research Support Program (EL, TCB), Image
Analysis Laboratory (JB, SL, KN), SAIC-Frederick, National Cancer
Institute at Frederick, Frederick, MD. Correspondence to: Jon M.
Wigginton, MD, Pediatric Oncology Branch, NCI-CCR, Bldg. 560, Rm.
31–93, Frederick, MD 21702–1201 (e-mail: jw121b@nih.gov
).
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-α and interferon-γ 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.
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