No abstract

Background: Neuroblastoma (NB) is one of the most common malignant solid tumors to occur in children, characterized by a wide range of genetic and epigenetic aberrations. We studied whether modifications of the latter with a 5-aza-2′-deoxycytidine (decitabine, Dac) DNA methyltransferase inhibitor can provide a therapeutic advantage in NB. Methods: NB cells with or without MYCN amplification were treated with Dac. We used flow cytometry to measure cell apoptosis and death and mitochondrial reactive oxygen species (mtROS), microarray to analyze gene expression profile and bisulfite pyrosequencing to determine the methylation level of the DDX58/RIG-I promoter. Western blot was used to detect markers related to innate immune response and apoptotic signaling, while immunofluorescent imaging was used to determine dsRNA. We generated mtDNA depleted ρ0 cells using long-term exposure to low-dose ethidium bromide. Results: Dac preferentially induced a RIG-I-predominant innate immune response and cell apoptosis in SK-N-AS NB cells, significantly reduced the methylation level of the DDX58/RIG-I promoter and increased dsRNA accumulation in the cytosol. Dac down regulated mitochondrial genes related to redox homeostasis, but augmented mtROS production. ρ0 cells demonstrated a blunted response in innate immune response and apoptotic cell death, as well as greatly diminished dsRNA. The response of NB cells to CDDP and poly(I:C) was potentiated by Dac in association with increased mtROS, which was blunted in ρ0 cells. Conclusions: This study indicates that Dac effectively induces a RIG-I-related innate immune response and apoptotic signaling primarily in SK-N-AS NB cells by hypomethylating DDX58/RIG-I promoter, elevated mtROS and increased dsRNA. Dac can potentiate the cytotoxic effects of CDDP and poly(I:C) in NB cells.

Ceramide is a sphingolipid which regulates a variety of signaling pathways in eukaryotic cells. Exogenous ceramide has been shown to induce cellular apoptosis. In this study, we observed that exogenous ceramide induced two distinct morphologies of cell fate following C2-ceramide treatment between the two breast cancer cell lines MCF-7 (wild type p53) and MDA-MB-231 (mutant p53) cells. The growth assessment showed that C2-ceramide caused significant growth inhibition and apoptosis in MDA-MB-231 cells through down-regulating the expression of mutant p53 whereas up-regulating the expression of pro-apoptotic Bad, and the proteolytic activation of caspase-3. However, senescence-associated (SA)-β-galactosidase (β-gal) was regulated in MCF-7 cells after C2-ceramide treatment. The results of proliferation and apoptosis assays showed that MCF-7 cells were more resistant to C2-ceramide treatment compared to MDA-MB-231 cells. Furthermore, C2-ceramide treatment induced a time-responsive increase in Rb protein, a key regulator of senescence accompanied with the upregulation of both mRNA level and protein level of SA-genes PAI-1 and TGaseII in MCF-7 but not in MDA-MB-231 cells, suggesting that some cancer cells escape apoptosis through modulating senescence-like phenotype. The results of our present study depicted the mechanism of C2-ceramide-resistant breast cancer cells, which might benefit the strategic development of ceramide-based chemotherapeutics against cancer in the future.