Nobiletin, a major component of citrus fruits, is a polymethoxyflavone derivative that exhibits anticancer activity against several forms of cancer, including SNU-16 human gastric cancer cells. To explore the nobiletin-induced cell death mechanism, we examined the changes in protein expression caused by nobiletin in human gastric cancer SNU-16 cells by means of two-dimensional gel electrophoresis (2-DGE), followed by peptide mass fingerprinting (PMF) analysis. Seventeen of 20 selected protein spots were successfully identified, including nine upregulated and eight downregulated proteins. In nobiletin-treated SNU-16 cells the glucose-regulated protein 78 kDa (GRP78) mRNA level was induced most significantly among six proteins related to cell survival and death. Western blot analysis was used to confirm the expression of GRP78 protein. We detected increases in the levels of the ER-stress related proteins inositol requiring enzyme 1 alpha (IRE1-α), activating transcription factor 4 (ATF-4), and C/EBP homology protein (CHOP), as well as GRP78, in response to nobiletin in SNU-16 cells. Furthermore, the ER stress-mediated apoptotic protein caspase-4 was proteolytically activated by nobiletin. Pretreatment with chloroquine, an autophagy inhibitor, strongly augmented apoptosis in SNU-16 cells, as evidenced by decreased cell viability, an increased number of sub-G1 phase cells and increased levels of cleaved PARP. Our results suggest that nobiletin-induced apoptosis in SNU-16 cells is mediated by pathways involving intracellular ER stress-mediated protective autophagy. Thus, the combination of nobiletin and an autophagy inhibitor could be a promising treatment for gastric cancer patients. Full article