Tangeretin sensitises human lung cancer cells to TRAILinduced apoptosis via ROS-JNK/ERK-CHOP pathwaymediated up-regulation of death receptor 5

Purpose: To investigate the efficacy of tangeretin, a bioactive flavonoid, as an enhancer of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human lung cancer cells (H1299 and H1975).Methods: H1299 and H1975 cells were exposed to tangeretin (25, 50, or 100 μM), and its effect on cell viability was assessed by MTT assay. The cytotoxic effects of tangeretin in combination with TRAIL (25 ng/mL) were determined by live/dead assay and annexin V staining. Intracellular levels of reactive oxygen species (ROS) were determined by DHFDA-flow cytometry analysis. Western blotting was performed to assess the expression of death receptors, apoptosis pathway proteins, JNK and ERK1/2. The cell surface expression of death receptors was determined. RT-PCR was performed to assess the expression of death receptor 5 and CEBP homologous protein (CHOP) mRNA. The cytotoxic effects of tangeretin in the presence of DR5, CHOP siRNA and TRAIL were also detected.Results: MTT assay revealed that tangeretin exerted potent cytotoxic effects and, in combination with TRAIL, exhibited striking apoptosis. Down-regulation of cell survival proteins (Bcl-2, Bcl-xL, survivin, xIAP, c-IAP-1, and c-IAP-2) and elevated expression of Bax and caspases 3, 8, and 9 were observed upon tangeretin exposure. The expression of death receptors DR4 and DR5 was highly up-regulated by tangeretin, while that of decoy receptors DcR1 and DcR2 was down-regulated. DR5 and CHOP siRNA transfection suggested the involvement of CHOP in tangeretin-mediated up-regulation of DR5, leading to more pronounced apoptotic effects. Tangeretin increased ROS levels and effectively caused activation of the mitogen-activated protein kinases JNK, ERK, and p38MAPKs. Expression analysis in the presence of specific MAPK inhibitors suggested the involvement of JNK and ERK in tangeretinmediated TRAIL sensitisation.Conclusions: Tangeretin induces death receptors and enhances TRAIL-induced apoptosis through upregulation of the ROS-JNK/ERK-CHOP pathways.Keywords: Apoptosis, Death receptors, Lung cancer, Tangeretin, Reactive oxygen species, Tumour necrosis factor-related apoptosis-inducing ligan