Modification of cysteine 179 of i kappa B alpha kinase by nimbolide leads to down-regulation of NF-kappa B-regulated cell survival and proliferative proteins and sensitization of tumor cells to chemotherapeutic agents

Abstract

Reverse pharmacology, also called the "bedside to bench" approach, that deals with new uses for a well known molecular entity has been used extensively in cancer drug development to identify novel compounds and delineate their mechanisms of action. Here, we show that nimbolide, a triterpenoid isolated from Azadirachta indica, enhanced the apoptosis induced by inflammatory cytokines and chemotherapeutic agents in tumor cells. This limonoid abrogated the expression of proteins associated with cell survival (Bcl-2, Bcl-xL, IAP-1, and IAP-2), proliferation (cyclin D1), invasion (MMP-9), and angiogenesis (VEGF), all regulated by nuclear factor (NF)-kappa B. Nimbolide inhibited the activation of NF-kappa B induced by carcinogens and inflammatory stimuli. Constitutively active NF-kappa B found in most tumor cells was also inhibited. We found that suppression of NF-kappa B activation by nimbolide was caused by inhibition of I kappa B kinase (IKK), which led to suppression of I kappa B alpha phosphorylation and degradation, nuclear translocation, DNA binding, and gene transcription. Reducing agent reversed the action of the limonoid, suggesting the involvement of a cysteine residue. Replacement of Cys(179) of IKK-beta with alanine abolished the effect of nimbolide, suggesting that Cys(179) plays a critical role in inhibiting the NF-kappa B activation. Overall, our results indicate that nimbolide can sensitize tumor cells to chemotherapeutic agents through interaction with IKK, leading to inhibition of NF-kappa B-regulated proteins.