Increased radiation-induced apoptosis of Saos2 cells via inhibition of NFkappaB: a role for c-Jun N-terminal kinase.
To elucidate the possible effect of NFkappaB on radioresistance, we used the osteosarcoma cell line Saos2, stably expressing the NFkappaB constitutive inhibitor, mIkappaB (Saos2-mIkappaB) or stably transfected with the empty vector (Saos2-EV). Ionizing radiation induced "intrinsic" apoptosis in Saos2-mIkappaB cells but not in Saos2-EV control cells, with intact NFkappaB activity. We find as expected, that this NFkappaB activity was enhanced following irradiation in the Saos2-EV control cells. On the other hand, inhibition of NFkappaB signaling in Saos2-mIkappaB cells led to the upregulation of the pro-apoptotic systems, such as Bax protein and c-Jun N-terminal Kinase (JNK)/c-Jun/AP1 signaling. Inhibition of NFkappaB resulted in decreased expression of the DNA damage protein GADD45beta, a known inhibitor of JNK. Subsequently, JNK activation of c-Jun/AP-1 proteins increased radiation-induced apoptosis in these mutants. Radiation-induced apoptosis in Saos2-mIkappaB cells was inhibited by the JNK specific inhibitor SP600125 as well as by Bcl-2 over-expression. Furthermore, release of cytochrome-c from mitochondria was increased and caspase-9 and -3 were activated following irradiation in Saos2-mIkappaB cells. Antisense inhibition of GADD45beta in Saos2-EV cells significantly enhanced apoptosis following irradiation. Our results demonstrate that radioresistance of Saos2 osteosarcoma cells is due to NFkappaB-mediated inhibition of JNK. Our study brings new insight into the mechanisms underlying radiation-induced apoptosis of osteosarcoma, and may lead to development of new therapeutic strategies against osteosarcoma.
Eliseev
Roman A
RA
Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, New York 14642, USA.
Zuscik
Michael J
MJ
Schwarz
Edward M
EM
O'Keefe
Regis J
RJ
Drissi
Hicham
H
Rosier
Randy N
RN
D000943:Antigens, Differentiation / Q000378:metabolism / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology / Q000528:radiation effects*; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011836:Radiation Tolerance / Q000187:drug effects*; D015398:Signal Transduction; D013997:Time Factors; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein / Q000378:metabolism; D000097642:GADD45 Proteins
D000943:Antigens, Differentiation; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases; D045304:Cytochromes c; D020672:I-kappa B Proteins; D048031:JNK Mitogen-Activated Protein Kinases; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B; D051028:bcl-2-Associated X Protein; C086470:GADD45B protein, human; D000097642:GADD45 Proteins; C000606204:NFKBIA protein, human; C505407:CASP3 protein, human; C506433:CASP9 protein, human