LY303511 Enhances TRAIL Sensitivity of SHEP-1 Neuroblastoma Cells via Hydrogen Peroxide-Mediated Mitogen-Activated Protein Kinase Activation and Up-regulation of Death Receptors

We recently reported that LY294002 (LY29) and LY303511 (LY30) sensitized tumor cells to drug-induced apoptosis independent of the phosphoinositide 3-kinase/Akt pathway. Here, we investigated the mechanism of LY304-induced sensitization of human neuroblastoma cells to TRAIL-mediated apoptosis. We provide evidence that LY30-induced increase in intracellular H2O2 up-regulates the expression of TRAIL receptors (DR4 and DR5) in SHEP-1 cells by activating mitogen-activated protein kinases, resulting in a significant amplification of TRAM-mediated caspase-8 processing and activity, cytosolic translocation of cytochrome c, and cell death. Involvement of the death receptors was further confirmed by the ability of blocking antibodies against DR4 and/or DR5 to inhibit LY30-induced TRAIL sensitization. Pharmacologic inhibition of c-Jun NH2 terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) activation by SP600125 and PD98059, respectively, blocked LY30-induced increase in sensitization to TRAIL-mediated death. Finally, small interfering RNA-mediated gene silencing of JNK and ERK inhibited LY30-induced increase in surface expression of DR4 and DR5, respectively. These data show that JNK and ERK are two crucial players involved in H2O2-mediated increase in TRAIL sensitization of tumor cells upon exposure to LY30 and underscore a novel mode of action of this inactive analogue of LY29. Our findings could have implications for the use of LY30 and similar compounds for enhancing the apoptotic sensitivity of neuroblastoma cells that often become refractory to chemotherapy. [Cancer Res 2009;69(5):1941-50]