Resveratrol induced ER expansion and ER caspase-mediated apoptosis in human nasopharyngeal carcinoma cells
Autophagy and endoplasmic reticulum (ER) stress response is important for cancer cells to maintain malignancy and resistance to therapy. trans-Resveratrol (RSV), a non-flavonoid agent, has been shown to induce apoptosis in human nasopharyngeal carcinoma (NPC) cells. In this study, the involvements of tumor-specific ER stress and autophagy in the RSV-mediated apoptosis were investigated. In addition to traditional autophagosomes, the images of transmission electron microscopy (TEM) indicated that RSV markedly induced larger, crescent-shaped vacuoles with single-layered membranes whose the expanded cisternae contains multi-lamellar membrane structures. Prolonged exposure to RSV induced a massive accumulation of ER expansion. Using an EGFP-LC3B transfection and confocal laser microscopy approach, we found RSV-induced EGFP-LC3 puncta co-localized with ER-tracker red dye, implicating the involvement of LC3II in ER expansion. The proapoptotic effect of RSV was enhanced after suppression of autophagy by ATG7 siRNA or blocking the autophagic flux by bafilomycin A1, but that was not changed after targeted silence of IRE1 or CHOP by siRNA. Using caspase inhibitors, we demonstrated the upregulation of caspase-12 (casp12) and the activation of casp4 were associated with the proapoptotic induction of RSV through the caspase-9/caspase-3 pathway. Intriguingly, siRNA knockdown of casp12, but not caspase-4, decreased the susceptibility of the NPC cells to RSV-mediated apoptosis. Further, we showed that RSV dose-dependently increased the ceramide accumulation as assessed by LC–MS/MS system. Using serine palmitoyltransferase (SPT, a key enzyme of de novo ceramide biosynthesis) inhibitors (l-cycloserine and myriocin), we found the increased ceramide accumulation was strongly correlated with the proapoptotic potential of RSV. This study revealed the ER expansion and upregulation of ER casp12 together may indicate profound biological effects of RSV and contributed to NPC cell death. Targeting the different status of ER stress may provide a possible strategy for cancer treatments.
KeywordsResveratrol ER expansion Caspase-12 Apoptosis NPC Autophagy
This work was supported by Chang Gung Memorial Hospital & Chang Gung University. Contract Grant Number: CMRPD 1B0231, CMRPD 5C0011 and EMRPD 1C0271.
- 9.Hitomi J, Katayama T, Eguch Y, Kudo T, Taniguchi M, Koyama Y, Manabe T, Yamagishi S, Bando Y, Imaizumi K, Tsujimoto Y, Tohyama M (2004) Involvement of caspase-4 in endoplasmic reticulum stress-induced apoptosis and Aβ-induced cell death. J Cell Biol 165:347–356. doi: 10.1083/jcb.200310015 PubMedCrossRefGoogle Scholar
- 11.Yoneda T, Imaizumi K, Oono K, Yui D, Gom F, Katayama T, Tohyama M (2001) Activation of caspase-12, an endoplastic reticulum (ER) resident caspase, through tumor necrosis factor rceptor-associated factor 2-dependent mechanism in response to the ER stress. J Biol Chem 276:13935–13940PubMedGoogle Scholar
- 13.Saleh M, Vaillancourt JP, Graham RK, Huyck M, Srinivasula SM, Alnemri ES, Steinberg MH, Nolan V, Baldwin CT, Hotchkiss RS, Buchman TG, Zehnbauer BA, Hayden MR, Farrer LA, Roy S, Nicholson DW (2004) Differential modulation of endotoxin responsiveness by human caspase-12 polymorphisms. Nature 429:75–79PubMedCrossRefGoogle Scholar
- 25.Zhang K, Haynes T-AS, Filippova M, Filippov V, Duerksen-Hughes PJ (2011) Quantification of ceramide levels in mammalian cells by high performance liquid chromatography coupled to tandem mass spectrometry with multiple-reaction-monitoring mode (HPLC–MS/MS-MRM). Anal Method 3:1193–1197CrossRefGoogle Scholar