Abstract 2479: Curcumin mediated apoptosis in human neuroblastoma cells via ROS and sphingolipid generation

Background: Neuroblastoma is the most common solid tumor of infancy and third leading cause of cancer death in children. It has one of the lowest survival rates of all pediatric cancers with less than 50% survival in “high risk” disease. Curcumin is the active compound of the yellow spice turmeric. Curcumin induces apoptosis and inhibits proliferation, angiogenesis, invasion and metastasis in many human cancer cells. The mechanism of cytotoxicity in neuroblastoma is unclear. Objectives: In this study we investigated the effects of curcumin on SMS-KCNR and CHLA-20 human neuroblastoma cells. Methods: SMS-KCNR and CHLA-20 neuroblastoma, cells were treated with increasing concentrations of curcumin. Cell viability was determined by Alamar Blue assays. Real-time PCR analysis of ER stress markers and apoptotic pathway genes was performed. Western Blotting was also performed to examine the downstream signaling pathways. Measurement of endogenous sphingolipids was performed by LC/MS. Sphingolipid pathway enzyme activities were also determined. Results: Curcumin was cytotoxic to both cell lines at 10 and 20 uM concentrations. PARP cleavage was noted at 24 hours, but cleavage of caspases 3, 8 and 9 was not observed. Treatment with the pancaspase inhibitor z-VAD did not reverse the cytotoxicity in curcumin treated cells, confirming that curcumin induced cell death was caspase-independent. Since perturbation in complex sphingolipid levels is associated with apoptosis, LC/MS measurement of endogenous sphingolipids was performed and showed increases in both dihydroceramides and ceramides. The increases in endogenous dihydroceramides, indicated that the dihydroceramide desaturase (DEGS-1) enzyme was inhibited. DEGS-1 activity was inhibited in-situ in a dose dependent manner in SMS-KCNR cells. There was no change in the mRNA or protein levels of DEGS-1, supporting that curcumin inhibited this enzyme indirectly. Next, the mechanism of ceramide generation was investigated by measuring the activity of sphingomyelin synthase (SMS) glycosylceramide synthase (GCS), acid ceramidase, neutral ceramidase, acid sphingomyelinase and neutral sphingomyelinase (SMase). At 6hrs, curcumin downregulated SMS activity by 30% and 54% GCS activity by 40% and 42% at concentrations of 10 and 20uM respectively. Curcumin has been demonstrated to induce ROS generation. Pre-treatment with the antioxidants N-acetylcysteine or glutathione abrogated curcumin mediated apoptosis and sphingolipid generation in SMS-KNCR cells. Furthermore, curcumin mediated SMS and GCS inhibition was blocked by these antioxidants. Conclusions: ROS plays a key role in sphingolopid and curcumin induced-cytotoxicity in neuroblastoma cells. Modulation of sphingolipid signaling pathways may provide a more effective and novel approach for the treatment of pediatric solid tumors. Curcumin is a potential novel therapy for neuroblastoma. Citation Format: Mehrdad Rahmaniyan, Li Li, Julio Garcia, A A. Qudeimat, Jacqueline M. Kraveka. Curcumin mediated apoptosis in human neuroblastoma cells via ROS and sphingolipid generation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2479.