Induction of ER stress-mediated apoptosis by ceramide via disruption of ER Ca2+ homeostasis in human adenoid cystic carcinoma cells
- Zhe Liu†1, 2,
- Yichao Xia†1, 2,
- Bo Li1, 2,
- Hui Xu2,
- Chenxing Wang1, 2,
- Ying Liu1, 2,
- Yi Li1, 2,
- Chunjie Li1, 2,
- Ning Gao1, 2 and
- Longjiang Li1, 2Email author
© Liu et al.; licensee BioMed Central Ltd. 2014
Received: 9 September 2014
Accepted: 15 November 2014
Published: 25 November 2014
Ceramides are a class of sphingolipids that form the structural component of the cell membrane and also act as second messengers in cell signaling pathways. Emerging results suggest that ceramide induces growth arrest and apoptosis in various human cancer cells. However, the mechanisms underlying its antitumor activity are yet to be identified. Endoplasmic reticulum stress (ER stress), a cellular adaptive response, is believed to initially compensate for damage but can eventually trigger cell death if the stimulus is severe or prolonged. In this study, we investigated whether ceramide induces cell death in human salivary adenoid cystic carcinoma (ACCs) through activation of the apoptotic ER stress.
RT-PCR, real-time PCR and western blot demonstrated that exogenous ceramide treatment up-regulated GRP78 and p-eIF2α expression and XBP1 splicing. Moreover, the ceramide synthase inhibitor FB1 abolished ceramide-induced ER stress. Up-regulation of the ER stress-associated apoptosis promoting transcription factor CHOP and p-JNK suggested that the antitumor activity of ceramide is owing to activation of apoptotic ER stress. Mechanistically, [Ca2+]ER depletion and SERCA inhibition by ceramide treatment suggested that it induces ER stress by disrupting [Ca2+]ER homeostasis. The chemical chaperone TUDCA inhibited ceramide-induced ER stress and cell death. In addition, the downstream metabolite of ceramide, S1P, cannot activate ER stress.
These results demonstrated that exogenous ceramide induces cancer cell death through a mechanism involving severe ER stress triggered by the disruption of ER Ca2+ homeostasis.
KeywordsCeramide ER stress ER calcium Apoptosis Cancer Unfolded protein response
Rapid proliferation of cancerous cells during cancer progression places a high demand on protein synthesis. The endoplasmic reticulum (ER) is a critical organelle in the synthesis, proper folding and assembly of secretory and membrane proteins. It is also the site of lipid synthesis and a major intracellular Ca2+ reservoir. Cellular stimuli that perturb ER homeostasis, including hypoxia, failure of protein synthesis, folding, transport or degradation, ER Ca2+ depletion and oxidative stress, may lead to ER stress. ER stress triggers the surveillance mechanism known as the unfolded protein response (UPR). The UPR involves the activation of inositol-requiring protein 1α (IRE1α), PKR-like ER kinase (PERK) and activating transcription factor 6 (ATF6). Activation of the UPR minimizes ER stress by improving the protein folding capacity of the ER, halting the rate of secretory protein synthesis and increasing the chaperone capacity in cells.
However, persistent or severe ER stress activates a UPR that results in apoptosis. Activated PERK, IRE1α and ATF6 under chronic ER stress regulate downstream targets, mainly the CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) and JNK, which play important roles in the commitment phase of ER stress-mediated apoptosis. CHOP inhibits expression of the anti-apoptotic protein Bcl-2 and induces the expression of the pro-apoptotic Bcl-2 family member Bim[2–4]. Activation of either IRE1α-TRAF2-ASK1 or CHOP-CAMK II pathways under ER stress induces JNK phosphorylation, which activates ER stress-mediated apoptosis through at least two mechanisms: induction of Fas and induction of Nox2 and subsequent oxidative stress[5–7]. Overwhelming ER stress eventually activates apoptosis through cleavage of caspase-12 in murine cells or caspase-4 in human cells, which subsequently activates executioner caspases such as caspase-3[8–10].
The interconvertable sphingolipid metabolites, ceramide and sphingosine-1- phosphate (S1P), constitute the sphingolipid rheostat. The dynamic balance of these two constituents has long been proposed to control the fate of the cell; with S1P promoting cell growth and survival, whereas ceramide drives apoptosis, autophagic responses and cell cycle arrest[11, 12]. Ceramide is produced by ceramide synthase through de novo biosynthesis in the ER. Recent studies suggested that alteration of ceramide synthase 6 (CerS6) activates the ATF6-CHOP arm of the UPR pathway and induces apoptosis[13, 14]. It was also reported that the combined treatment of sorafenib and vorinostat induces ER stress and apoptosis through elevation of ceramide level and CD95 activation. However, the mechanisms by which exogenous ceramide regulates ER stress and subsequent apoptosis remain unknown.
In this study, we have identified that exogenous ceramide triggers an apoptotic ER stress response by treating salivary adenoid cystic carcinoma cells (ACCs) with cell-permeable short chain C2-ceramide. We defined a novel mechanism that activates ER stress via SERCA inhibition and [Ca2+]ER depletion in response to ceramide treatment. Furthermore, we observed that ceramide induces apoptosis via activation of pro-apoptotic factors downstream of ER stress in ACCs.
Ceramide induces sustained activation of XBP1 mRNA splicing in ACCs
Ceramide activates eIF2α phosphorylation and increases GRP78 expression
Inhibition of ceramide synthase by FB1 impairs ceramide-induced ER stress
Ceramide induces [Ca2+]ER depletion and SERCA inhibition leading to ER stress
To determine the mechanism of [Ca2+]ER depletion, we evaluated changes in SERCA, which pumps cytoplasmic Ca2+ to ER lumen. Real-time PCR showed treatment with 100 μM ceramide for 12 h significantly inhibited SERCA2α, SERCA2β and SERCA3 mRNA expression in ACC-2 and ACC-M cells (Figure 4B). These results indicate that ceramide induces [Ca2+]ER depletion and disrupts Ca2+ homeostasis by inhibiting SERCA expression, thus further increasing ER stress.
The chemical chaperone TUDCA alleviates ceramide-induced ER stress
Ceramide induces cell death through ER stress-mediated apoptosis pathway
ER stress is induced by ceramide independent of its downstream metabolite S1P
In this study, we have shown that ceramide induces apoptosis in ACC-M and ACC-2 cells through a novel mechanism involving [Ca2+]ER depletion and SERCA inhibition, leading to ER stress and expression of downstream pro-apoptotic factors CHOP and p-JNK. The ceramide synthase inhibitor FB1 and chemical chaperone TUDCA inhibit ceramide-induced ER stress and subsequent cell death. In contrast to ER stress mediated by S1P elevation after SPP1 depletion, ER stress induced by exogenous ceramide is independent of its downstream metabolite S1P. These findings are summarized in Figure 7B. Delineating the ceramide-induced pro-apoptotic signaling cascades will provide potential therapeutic targets for cancer therapy.
Multiple stimuli under physiological or pathological conditions induce the accumulation of unfolded protein in the ER, which activates an evolutionarily conserved adaptive response termed the UPR which leads to cell death if the stimulus is severe or prolonged. The ER chaperone GRP78 acts as a major regulator of the UPR through direct interaction with UPR sensors PERK, IRE1α and ATF6. GRP78 maintains the three sensors in inactive forms under homeostatic conditions, and releasing them for activation upon ER stress. Increased GRP78 expression was observed in ACC-M and ACC-2 cells upon exogenous C2-ceramide treatment, indicating initiation of the ER stress and activation of the UPR cascades. Moreover, ceramide induced the ER stress response in a time- and dose-dependent manner, in support of the statement that the ER stress is increased as the stimulus is intensified and prolonged[1, 15, 19, 21].
In contrast to the selective activation of the ATF6/CHOP pathway of ER stress in response to CerS6/C16-ceramide down-regulation[13, 14], we found in this study that exogenous C2-ceramide treatment induced phosphorylation of eIF2α, suggesting the activation of the PERK/eIF2α arm of the ER stress response. IRE1α is a transmembrane protein that has both a Ser/Thr kinase domain and an endoribonuclease domain. Activated IRE1α uses its endoribonuclease activity to cleave a 26 base intron from XBP1 mRNA, resulting in a translational frameshift and translation of a spliced form of XBP1 (XBP1S), which is a more stable and potent transcription factor for target genes involved in protein folding and ER-associated degradation[1, 16]. Increased XBP1S expression was observed in ACC-M and ACC-2 cells upon C2-ceramide treatment, suggesting that ceramide also activates the IRE1α/XBP1S arm. The ceramide synthase inhibitor FB1 is reported to inhibit de novo biosynthesis of ceramide. In this study, FB1 treatment abolished ceramide-induced ER stress in ACC-M and ACC-2 cells, whereas interestingly, FB1 alone had no inhibitory effect on the splicing of XBP1 or phosphorylation of eIF2α. Consistently, other researchers reported that FB1 treatment alone does not impair the splicing of XBP1 in LPS-treated B cells or XBP1-deficient B cells. It might be due to the relatively low level of endogenous ceramide expression in ACC-M and ACC-2 cells, mitigating the inhibitory effect of FB1 on ceramide-induced ER stress response.
The ER is the major intracellular Ca2+ store, and perturbation of [Ca2+]ER homeostasis has been reported to induce ER stress. Ca2+ is pumped from the cytosol to the ER by SERCA and released through either the inositol-1,4,5-trisphosphate receptor/Ca2+ channels or ryanodine receptor/Ca2+ channels[28–30]. Although alteration of endogenous C16-ceramide levels by CerS6 knockdown has been reported previously to trigger ER stress by modulating SERCA expression and subsequently changing the [Ca2+]ER/[Ca2+]IN ratio, data presented here are novel because the role of exogenous ceramide in the induction of [Ca2+]ER depletion by SERCA2/3 inhibition have not been described previously. Our data showed that exogenous ceramide treatment disrupts Ca2+ homeostasis by inducing [Ca2+]ER depletion, which is in agreement with previous reports that release of [Ca2+]ER and the subsequent increase of Ca2+ concentration in the cytosol and mitochondrial matrix play an important role in exogenous ceramide-induced apoptosis[31, 32].
Both 4-PBA and TUDCA have been reported to alleviate ER stress, but by distinct mechanisms. Recent studies suggest that 4-PBA represses ER stress by stabilizing protein conformation in the ER[33–37], while TUDCA reduces [Ca2+]IN concentration after Thapsigargin treatment, thus inhibiting ER stress and apoptosis. TUDCA was reported to be more effective in inhibiting ER stress and protecting ER stress-mediated apoptosis than 4-PBA in steatotic and non-steatotic livers during partial hepatectomy under ischemia-reperfusion. In the present study, we observed that 4-PBA or TUDCA treatment alone reduced XBP1S and p-eIF2α expression, whereas TUDCA had more profound effects on impairing ceramide-induced ER stress than did 4-PBA, and only TUDCA is effective in inhibiting ceramide-induced cell death. These results suggest that exogenous ceramide triggers ER stress and apoptosis through mechanisms that can be largely inhibited by TUDCA. Recent studies suggest that both Ca2+ overload and [Ca2+]ER depletion result in changes in protein folding and ER stress[22, 39, 40]. Based on our findings and the mechanism by which TUDCA alleviate the ER stress, we speculated that ceramide-induced [Ca2+]ER depletion might play a major role in pro-apoptotic mechanisms in ACC-M and ACC-2 cells.
Activation of CHOP is a common point of convergence for all three arms of the UPR. Up-regulated ATF6, ATF4 or XBP1S expression induces apoptosis by interacting with binding sites within the promoter of the CHOP gene. In addition to mediating the down-regulation of Bcl-2 and up-regulation of Bim, CHOP also induces expression of the pro-apoptotic proteins ERO1α and Puma[1–3, 19, 21]. Pro-apoptotic ER stress eventually leads to mitochondria dysfunction, cytochrome c release and caspase-3 cleavage[10, 41]. It has been demonstrated that reduction of C16-ceramide by CerS6 knockdown activates CHOP expression. Our study shows for the first time that exogenous short chain ceramide activated CHOP expression in a time- and dose-dependent manner via induction of ER stress. We also identified activation of JNK and elevated expression of cleaved caspase-3 in ceramide-treated ACCs. These findings suggest that exogenous ceramide definitely activates the ER stress-mediated pro-apoptotic signaling pathways, and promotes the commitment phase of apoptosis.
Ceramide and its downstream metabolite S1P have long been reported to play opposing roles in the regulation of autophagy, angiogenesis and senescence. A recent report demonstrates that elevated intracellular S1P owing to S1P phosphohydrolase 1 depletion significantly activates ER stress and survival signaling via the Akt pathway. Our data showed that exogenous S1P treatment had no significant effect on ER stress, which suggests ER stress triggered by ceramide is independent of S1P. It might be interesting to further determine how the metabolic interconversion of ceramide and S1P regulates ER stress.
Materials and methods
Human salivary adenoid cystic carcinoma (ACC-M and ACC-2) cell lines were purchased from China Center for Type Culture Collection (Wuhan, China). Cells were cultured in DMEM containing 10% fetal bovine serum and antibiotics and maintained in a humidified chamber (5% CO2/95% air) at 37°C.
Chemicals and reagents
C2-ceramide (Avanti Polar Lipid, Alabaster, AL, USA), TUDCA (Calbiochem, EMD-Millipore, Billerica, MA, USA), Tunicamycin, Thapsigargin, FB1, 4-PBA, S1P, Pluronic F-127 (Sigma-Aldrich, St Louis, MO, USA), and Fluo 4-AM (Dojindo Laboratories, Kumamoto, Japan) were reconstituted as recommended by their respective manufacturers. Antibodies against p-eIF2α (Ser51), eIF2α, JNK, cleaved caspase-3, p-ERK (Thr202/Tyr204), ERK (Cell Signaling Technology, Beverly, MA, USA), p-JNK (Thr183/Tyr185) (Abcam, Cambridge, MA, USA), caspase-3 (Abgent, San Diego, CA, USA), and actin (Santa Cruz, Dallas, TX, USA) were used in this study. All secondary antibodies were purchased from Abcam.
RT-PCR and Real-time PCR
Sequences of primers used in RT-PCR and Real-time PCR
Forward primer sequence (5′–3′)
Reverse primer sequence (5′–3′)
Cells were rinsed with PBS and harvested in lysis buffer containing 20 mM HEPES (pH 7.4), 1% Triton X-100, 10% glycerol, 2 mM EGTA, 1 mM sodium vanadate, 2.5 mM sodium pyrophosphate, 25 mM sodium glycerophosphate, 50 mM NaF, complete EDTA-free protease inhibitor cocktail and the phosphatase inhibitor cocktail PhosStop (Roche, Mannheim, Germany). Equivalent amounts of protein (40–100 μg) were subjected to SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA). After blocking with 10% non-fat dry milk or BSA for 1 h, membranes were incubated with specific antibodies overnight at 4°C, followed by incubation with secondary antibodies for 1 h at room temperature. Proteins were detected using horseradish peroxidase-conjugated secondary antibodies and an enhanced chemiluminescence reagent (Millipore). The intensity of each band was quantified using Quantity One software (BioRad, Hercules, CA, USA) after normalization to corresponding loading controls.
Intracellular Ca2+ measurement
Cells were seeded on a confocal dish with a glass bottom. The cells were loaded with dye by incubating with 5 μM Ca2+-sensitive probe Fluo 4-AM in the presence of 0.05% Pluronic F-127 in HBSS for 30 min at 37°C, and then washed three times with HBSS to remove the extracellular Fluo 4-AM and incubated in HBSS containing 1% FBS for 20 min at 37°C. Cells were treated initially with HEPES buffer and then with buffer containing 100 μM C2-ceramide. Changes in fluorescent intensity were monitored using an Olympus FluoView FV1000 confocal laser scanning microscope (Olympus, Tokyo, Japan). Image was analyzed by Olympus FV10-ASW 3.1 Viewer software, using Time-series mode.
Colony formation assay
Cells were seeded into 60 mm culture dishes at 200 cells per dish. After 24 h, cultures were replaced with fresh medium containing 10% FBS with or without 10–100 μM ceramide. For ER stress inhibition, cells were pretreated for 3 h with 5 mM 4-PBA or 1 mg/ml TUDCA prior to ceramide addition. After 24 h incubation, culture dishes were rinsed three times with PBS. Cells were further grown in fresh medium containing 10% FBS for 3 weeks. Colonies were stained for 15 min with a solution containing 0.5% crystal violet and 25% methanol, followed by three rinses with tap water to remove excess dye. Colonies were counted only if a single clone contained more than 50 cells.
Statistical analysis was performed using SPSS 13.0 (Chicago, IL, USA). Statistical analyses were performed using the Student’s t-test or ANOVA for two-way analysis of variance. P-values of P <0.05 were defined as statistically significant.
Unfolded protein response
Sarcoplasmic/endoplasmic reticulum Ca2+- ATPase
Endoplasmic reticulum calcium
Spliced X-box binding protein 1
Unspliced X-box binding protein 1
Total X-box binding protein 1
Eukaryotic initiation factor 2α
CCAAT/enhancer-binding protein (C/EBP) homologous protein
c-Jun N-terminal kinase
4-phenyl butyric acid
This work was supported by the National Natural Science Foundation of China (No. 81472532).
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