Synergistic potentiation of tumor necrosis factor-induced lung cancer cell death by 17-Allylamino-17-Demethoxygeldanamycin through blocking the nuclear factor-κB pathway

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Tumor necrosis factor (TNF) is a potential cancer therapeutic agent because of its ability to induce cancer cell death. However, most lung cancer cells are resistant to TNF-induced death, the mechanism of which has not been well elucidated. Nuclear factor κB (NF-κB), a survival signal induced by TNF, has been implicated in resistance to TNF-induced apoptosis in cancer cells. Heat shock protein 90 (Hsp90) regulates the stability and function of receptor-interaction proteins (RIP) and IκB kinase β (IKKβ), the key components of the TNF-induced NF-κB activation pathway. Therefore, suppression of HSP90 function may sensitize cancer cells to TNF-induced death. In this study, we examined the effect of the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17AAG) on TNF-induced cell death in lung cancer cells. Exposure to 17AAG caused a significant reduction of RIP and IKKβ levels in H460 cells. A synergistic cytotoxicity was detected when TNF treatment followed 17AAG pre-exposure that caused RIP and IKKβ degradation, suggesting that the 17AAG sensitizes cells to TNF-induced cell death by blocking the TNF-induced NF-κΒ pathway. This was supported by further results, including suppressed IκBα phosphorylation and degradation, and decreased NF-κΒ activity detected by Western blot and reporter assay, respectively. The induction of NF-κB targeted anti-apoptosis genes was also suppressed in 17AAG-treated cells. Importantly, the potentiation of cell death by 17AAG was abolished in NF-κB-disabled cells expressing a nondegradable IκBα mutant (IκBαAA). These results suggest that the synergistic cytotoxicity seen with 17AAG and TNF treatment is resulted from blockage of TNF-induced NF-κB activation. The other components of the TNF receptor-I signaling cascade such as TRADD, TRAF2 and FADD were not altered in 17 AAG-treated cells. When treated with 17AAG, the TNF-induced c-Jun N-terminal protein kinase (JNK) activation was enhanced and sustained. 17AAG and TNF co-treatment-induced cytotoxicity is mainly apoptotic, which was demonstrated both morphologically and biochemically, suggesting that the TNF-induced apoptosis pathway was triggered by 17AAG. A similar synergism for inducing apoptosis was also observed in 17AAG and TNF-related apoptosis-inducing ligand (TRAIL) treated lung cancer cells. Our results suggest that NF-κB 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 this deadly malignancy.