Nuclear factor-kappaB (NF-kappaB), a survival signal induced by tumor necrosis factor (TNF), contributes substantially to the resistance to TNF-induced cell death. Previous studies suggest that heat shock protein 90 (Hsp90) regulates the stability and function of receptor-interaction proteins (RIP) and IkappaB kinase beta (IKKbeta), the key components of the TNF-induced NF-kappaB activation pathway. In this study, we showed that the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17AAG) was synergistic with TNF to induce apoptotic cell death in a panel of lung tumor-derived cell lines. Treatment with 17AAG caused degradation of RIP and IKKbeta that, in turn, blocked TNF-induced NF-kappaB activation and antiapoptotic gene expression. The synergistic cytotoxicity was detected only when TNF treatment followed 17AAG preexposure. Importantly, the potentiation of cell death was abolished in NF-kappaB-disabled cells that express a nondegradable IkappaBalpha mutant (IkappaBalphaAA). These results suggest that the cytotoxicity seen with 17AAG and TNF treatment results from blocking TNF-induced NF-kappaB activation. The other components of the TNF receptor I signaling cascade were not altered, whereas TNF-induced c-Jun NH(2)-terminal kinase activation and apoptosis were potentiated. A similar synergism for inducing apoptosis was also observed in 17AAG-treated and TNF-related apoptosis-inducing ligand (TRAIL)-treated cancer cells. Our results suggest that NF-kappaB plays a key role in the resistance of lung cancer cells to TNF and TRAIL and that disabling this survival signal with 17AAG followed by TNF or TRAIL treatment could be an effective new therapeutic strategy for lung cancer.