Nobiletin Induces Protective Autophagy Accompanied by ER-Stress Mediated Apoptosis in Human Gastric Cancer SNU-16 Cells
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
... 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. The endoplasmic reticulum (ER) performs several functions, including protein folding and transport, and regulation of the intracellular calcium concentration. Cells trigger the unfolded protein response (UPR) as a self-protective mechanism upon the disruption of ER functions via the accumulation of unfolded/misfolded proteins in the ER  . Under normal conditions, the ER stress sensors inositol-requiring-1α (IRE1-α), pancreatic ER kinase or PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6) bind to immunoglobulin heavy chain binding protein (BiP)/GRP78. These sensors are released from BiP/GRP78 under conditions of ER stress and transfer downstream signals to the cytoplasm. IRE1-α, a transmembrane protein in the endoplasmic reticulum (ER), which functions as a sensor and transducer of ER stress, activates X-box-binding protein-1 (XBP-1) and several UPR target genes. ER stress-mediated apoptosis is triggered by activation of the ER membrane resident caspase-12 (mice) and caspase-4 (humans) and the induction of C/EBP homology protein (CHOP)  . ER stress-induced apoptotic signaling has been studied to identify potential targets for therapeutic intervention in diseases associated with ER stress  . The CHOP-mediated downregulation of Bcl-2 can favorably influence pro-apoptotic Bcl-2 family proteins and cell death [8, 9] . Autophagy is a cellular defense mechanism involving the degradation and recycling of cytoplasmic constituents. Several cell signaling pathways regulate autophagy, including those involving phosphatidyl inositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR). Studies have shown that the inhibition of Akt and its downstream target mTOR contribute to autophagy initiation  . During autophagy, autophagosomes engulf cytoplasmic components, including cytosolic proteins and organelles. Concomitantly, a cytosolic form of LC3 (LC3-I) is conjugated to phosphatidylethanolamine to form LC3-phosphatidylethanolamine conjugate (LC3-II), which is recruited to autophagosomal membranes  . In the present study, we examined the changes in protein expression caused by nobiletin in human gastric cancer SNU-16 cells using 2-DGE and proteomic analysis to explore the mechanism of nobiletin-induced cell death. The ER-stress-related protein GRP78 was identified as a potential target for nobiletin, and our results demonstrate that nobiletin induces ER stress-mediated apoptosis in SNU-16 cells. Moreover, protective autophagy was followed by apoptosis. Thus, we propose that combined treatment with nobiletin and an autophagy inhibitor could be a promising treatment for gastric cancer patients.