Enhancement of radiosensitivity by proteasome inhibition: implications for a role of NF-kappaB.
OBJECTIVE
NF-kappaB is activated by tumor necrosis factor, certain chemotherapeutic agents, and ionizing radiation, leading to inhibition of apoptosis. NF-kappaB activation is regulated by phosphorylation of IkappaB inhibitor molecules that are subsequently targeted for degradation by the ubiquitin-proteasome pathway. PS-341 is a specific and selective inhibitor of the proteasome that inhibits NF-kappaB activation and enhances cytotoxic effects of chemotherapy in vitro and in vivo. The objective of this study was to determine if proteasome inhibition leads to enhanced radiation sensitivity.


METHODS
Inhibition of NF-kappaB activation in colorectal cancer cells was performed by treatment of LOVO cells with PS-341 or infection with an adenovirus encoding IkappaB super-repressor, a selective NF-kappaB inhibitor. Cells were irradiated at 0, 2, 4, 6, 8, and 10 Gy with or without inhibition of NF-kappaB. NF-kappaB activation was determined by electrophoretic mobility gel shift assay, and apoptosis was evaluated using the TUNEL assay. Growth and clonogenic survival data were obtained to assess effects of treatment on radiosensitization. In vitro results were tested in vivo using a LOVO xenograft model.


RESULTS
NF-kappaB activation was induced by radiation and inhibited by pretreatment with either PS-341 or IkappaBalpha super-repressor in all cell lines. Inhibition of radiation-induced NF-kappaB activation resulted in increased apoptosis and decreased cell growth and clonogenic survival. A 7-41% increase in radiosensitivity was observed for cells treated with PS-341 or IkappaBalpha. An 84% reduction in initial tumor volume was obtained in LOVO xenografts receiving radiation and PS-341.


CONCLUSIONS
Inhibition of NF-kappaB activation increases radiation-induced apoptosis and enhances radiosensitivity in colorectal cancer cells in vitro and in vivo. Results are encouraging for the use of PS-341 as a radiosensitizing agent in the treatment of colorectal cancer.
Russo
S M
SM
Department of Radiation Oncology, University of North Carolina School of Medicine, Chapel Hill, NC, USA.
Tepper
J E
JE
Baldwin
A S
AS
Liu
R
R
Adams
J
J
Elliott
P
P
Cusack
J C
JC
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D001897:Boronic Acids / Q000494:pharmacology*; D000069286:Bortezomib; D015179:Colorectal Neoplasms / Q000188:drug therapy / Q000473:pathology / Q000532:radiotherapy; D003546:Cysteine Endopeptidases; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology; D005260:Female; D006801:Humans; D020672:I-kappa B Proteins; D051379:Mice; D008819:Mice, Nude; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000502:physiology*; D011480:Protease Inhibitors / Q000494:pharmacology*; D046988:Proteasome Endopeptidase Complex; D011719:Pyrazines / Q000494:pharmacology*; D011836:Radiation Tolerance / Q000187:drug effects / Q000502:physiology*; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*; D012097:Repressor Proteins / Q000235:genetics / Q000502:physiology; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays
D001897:Boronic Acids; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D009097:Multienzyme Complexes; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D011480:Protease Inhibitors; D011719:Pyrazines; D011838:Radiation-Sensitizing Agents; D012097:Repressor Proteins; D000072000:NF-KappaB Inhibitor alpha; D000069286:Bortezomib; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex