Combined treatment wth HSP90 inhibitor and TRAIL down-regulated NFκB activity through their effects on the death receptor complex.

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HSP90 promote cell survival by stabilizing pro-survival, anti-apoptotic proteins indicating that its continued functional integrity favors cell proliferation. The expression of HSP90 is increased in cancer cells and has been correlated with poor prognosis and increased resistance to radiation and chemotherapy. Thus, the inhibition of HSP90 would down regulate the function of antiapoptotic proteins and promote apoptosis. We have reported that low, sublethal dose of Geldanamycin (Ld-GA), a HSP90 inhibitor, with TRAIL enhanced apoptosis. The induction of apoptosis followed degradation of IKK proteins and down-regulation of NFκB activity (Ma et al., 2005). In present study, we hypothesized that the effect on IKK-NFκB pathway is mediated through the death inducing signaling complex (DISC), particularly, the intracellular death domain kinases of death receptors. Prostate cancer cells (LNCaP and DU-145) were treated with Ld-GA and/or TRAIL and the proteins of the death receptor complex I and II were analyzed. Death receptor complex I is composed of TNF-R1, TRADD, RIP, TRAF2 and c-IAP1. RIP requires continued interaction with HSP90 for its activity and for recruiting IKK proteins leading to NFκB activity. Death receptor complex II includes FADD and pro-caspase 8, which are responsible for receptor-mediated apoptosis. Our results showed that GA down-regulated RIP and TRAF2 in a dose dependent manner, suggesting that RIP-HSP90 is necessary for its function. Immunoprecipitation data showed decreased binding of HSP90-RIP that supported the above results. Loss of RIP activity resulted in the degradation of IKK and decreased NFκB activity. Contrary to the loss of function of death receptor complex I, the pro-apoptotic death receptor complex II was activated as confirmed by the increased activation of caspase-8 in cells treated with both Ld-GA and TRAIL. In conclusion, our results demonstrate that in addition to IKK (Ma et al., 2005), HSP90 binds to RIP, and continued interaction with HSP90 is required for the recruitment of IKK by RIP. Combination of Ld-GA and TRAIL induced synergistic apoptosis by activating the pro-apoptotic death receptor complex II and inhibiting the anti-apoptotic death receptor complex I. Thus, the DISC plays a critical role in the apoptotic response of the cell and affects the activity of the RIP-IKK-NFκB pathway. The clinical implications of these observations are that the use of low dose GA would reduce its known adverse effects. Ma Y, Lakshmikanthan V, Lewis RW and Kumar MV. 2005. Sensitization of TRAIL-Resistant Cells by Inhibition Of Heat Shock Protein 90 with Low Dose Geldanamycin, Mol. Cancer Therap. In Press. This research was funded by VA Merit Review and the Medical College of Georgia.