Suppression of IKK, but not activation of p53 is responsible for cell death mediated by naturally occurring oxidized tetranortriterpenoid.

Tetranortriterpenoids (limonoids) obtained from the neem tree (Azadirachta indica) have gained significant attention due to their anti-proliferative properties. Here we are investigating the role of a highly oxidized tetranortriterpenoid, azadirachtin on induction of the cell death. Using various apoptotic assays, we show that azadirachtin induces cell death independent of cell types. Although azadirachtin-treated cells show increased expression of p53, but no phosphorylation of p53 (at Ser15 and Ser46) is detected. In silico analysis reveals that azadirachtin interacts with Mdm2 in the p53 binding site, postulating the mutually exclusive interaction of p53 and azadirachtin with Mdm2. Surprisingly, azadirachtin potentiates cell death efficiently in both p53 wild-type and p53 negative cells. In addition, we find azadirachtin suppresses nuclear transcription factor kappaB (NF-κB) by inhibiting the phosphorylation of upstream inhibitory subunit of NF-κB (IκB) kinase (IKK). Further, azadirachtin is unable to potentiate apoptosis in NF-κB-downregulated (IκB-DN) cells, whereas ectopic expression of p65 rescues azadirachtin-mediated apoptosis, regardless of their p53 status. Hence, our data suggest that azadirachtin mediates cell death through inhibition of NF-κB, but not due to the activation of p53. In conclusion, this study proposes azadirachtin as a potential therapeutic agent where insensitivity toward chemotherapy occurs due to the inactivation or mutations in p53.