Nimbolide sensitizes human colon cancer cells to TRAIL through reactive oxygen species- and ERK-dependent up-regulation of death receptors, p53, and Bax

TNF-related apoptosis-inducing ligand (TRAIL) shows promise as a cancer treatment, but acquired tumor resistance to TRAIL is a roadblock. Here we investigated whether nimbolide, a limonoid, could sensitize human colon cancer cells to TRAIL. As indicated by assays that measure esterase activity, sub-G(1) fractions, mitochondrial activity, and activation of caspases, nimbolide potentiated the effect of TRAIL. This limonoid also enhanced expression of death receptors (DRs) DR5 and DR4 in cancer cells. Gene silencing of the receptors reduced the effect of limonoid on TRAIL-induced apoptosis. Using pharmacological inhibitors, we found that activation of ERK and p38 MAPK was required for DR up-regulation by nimbolide. Gene silencing of ERK abolished the enhancement of TRAIL-induced apoptosis. Moreover, our studies indicate that the limonoid induced reactive oxygen species production, which was required for ERK activation, up-regulation of DRs, and sensitization to TRAIL; these effects were mimicked by H(2)O(2). In addition, nimbolide down-regulated cell survival proteins, including I-FLICE, cIAP-1, cIAP-2, Bcl-2, Bcl-xL, survivin, and X-linked inhibitor of apoptosis protein, and up-regulated the pro-apoptotic proteins p53 and Bax. Interestingly, p53 and Bax up-regulation by nimbolide was required for sensitization to TRAIL but not for DR up-regulation. Overall, our results indicate that nimbolide can sensitize colon cancer cells to TRAIL-induced apoptosis through three distinct mechanisms: reactive oxygen species- and ERK-mediated up-regulation of DR5 and DR4, down-regulation of cell survival proteins, and up-regulation of p53 and Bax.