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. 2016 Jul 14;21(7):914.
doi: 10.3390/molecules21070914.

Nobiletin Induces Protective Autophagy Accompanied by ER-Stress Mediated Apoptosis in Human Gastric Cancer SNU-16 Cells

Jeong Yong Moon  1 , Somi Kim Cho  2   3
Affiliations

Nobiletin Induces Protective Autophagy Accompanied by ER-Stress Mediated Apoptosis in Human Gastric Cancer SNU-16 Cells

Jeong Yong Moon et al. Molecules. .

Abstract

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.

Keywords: ER stress; GRP78; SNU-16; apoptosis; autophagy; nobiletin.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Representative protein maps from human gastric cancer SNU-16 cells that were treated with (A) DMSO or (B) 50 μM nobiletin for 24 h. Spots with arrows represent proteins that changed their expression after nobiletin treatment; the PMF-based identification of these spots is summarized in Table 1.
Figure 2
Figure 2
Effect of nobiletin on mRNA levels in SNU-16 cells. (A) Expression levels of six genes related to cell survival and death were determined by RT-PCR in SNU-16 cells treated with or without 50 μM nobiletin for 24 h; (B) The intensities of RT-PCR bands were quantified using ImageJ software. * p < 0.01.
Figure 3
Figure 3
Effect of nobiletin on the expression of ER stress-related genes and proteins. (A) GRP78 expression and the splicing of endogenous XBP1 (XBP1u, unspliced XBP1; XBP1s, spliced XBP1) was examined by RT-PCR in SNU-16 cells treated with various concentrations of nobiletin for 24 h; (B) The intensities of RT-PCR bands were quantified using the ImageJ software; (C) ER stress- related protein expression was analyzed by western blotting in SNU-16 cells treated with various concentrations of nobiletin for 24 h; (D) The intensities of western blot bands were quantified using the ImageJ software. Data represent the means ± SD of at least three independent experiments. * Compared with the vehicle group, p < 0.01.
Figure 4
Figure 4
Autophagy induction due to nobiletin and inhibition of autophagy enhance the anticancer activity of nobiletin. (A) Western blotting for Akt, p-Akt, mTOR, p-mTOR, LC3, p62, and β-actin after treatment of cells with the indicated concentrations of nobiletin for 24 h; (B) The intensities of western blot bands were quantified using ImageJ software. * p < 0.01; (C) Cell viability (MTT) assay and (D) western blotting were performed after pretreatment with (+) or without (−) 40 μM chloroquine (CQ) for 2 h followed by treatment with 25 μM nobiletin (NT) for 24 h; (E) The intensities of western blot bands were quantified using ImageJ software. * p < 0.01.
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