Ursolic acid induces autophagy in U87MG cells via ROS-dependent endoplasmic reticulum stress.

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Ursolic acid induces autophagy in U87MG cells via ROS-dependent endoplasmic reticulum stress

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Shuying Shen a, Yi Zhang a, Rui Zhang b, Xintao Tu a, Xingguo Gong a,⇑ a b

The Institute of Biochemistry, Zhejiang University, Hangzhou 310058, China Department of Neurosurgery, The First Affiliated Hospital of Nanjing Medical University, No. 300 Guangzhou Road, Nanjing 210029, China

a r t i c l e

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Article history: Received 7 January 2014 Received in revised form 20 March 2014 Accepted 23 April 2014 Available online xxxx Keywords: ER stress Autophagy Ursolic acid Calcium

a b s t r a c t Malignant gliomas are the most common primary brain tumors, and novel ways of treating gliomas are urgently needed. Ursolic acid (UA), a pentacyclic triterpenoid, has been reported to exhibit promising antitumor activity. Here, we evaluated the effects of UA on U87MG cells and explored the underlying molecular mechanisms. The results demonstrated that both G1-phase arrest and autophagy were induced by UA in U87MG cells. Evidence of UA-induced autophagy included the formation of acidic vesicular organelles, increase of autophagolysosomes and LC3-II accumulation. UA was also found to induce ER stress and an increase in intracellular calcium accompanied by ROS production. The increase in free cytosolic calcium induced by UA activated the CaMKK-AMPK-mTOR kinase signaling cascade, which ultimately triggered autophagy. Western blot analysis showed that UA promoted the phosphorylation of PERK and eIF2a; this was followed by the upregulation of the downstream protein CHOP, implying the involvement of the ER stress-mediated PERK/eIF2a/CHOP pathway in glioma cells. Meanwhile, UA activated IRE1a and subsequently increased the levels of phosphorylated JNK and Bcl-2, resulting in the dissociation of Beclin1 from Bcl-2. Furthermore, TUDCA and the silencing of either PERK or IRE1a partially blocked the UA-induced accumulation of LC3-II, suggesting that ER stress precedes the process of autophagy. Additionally, NAC attenuated the UA-induced elevation in cytosolic calcium, ER stress markers and autophagy-related proteins, indicating that UA triggered ER stress and autophagy via a ROS-dependent pathway. Collectively, our findings revealed a novel cellular mechanism triggered by UA and provide a molecular basis for developing UA into a drug candidate. Ó 2014 Published by Elsevier Ireland Ltd.

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1. Introduction

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The endoplasmic reticulum (ER) is a highly dynamic organelle in eukaryotic cells, responsible for Ca2+ storage, protein folding, and lipid synthesis. During tumorigenesis, ER stress develops in response to harsh environmental cues including nutrient starvation, oxidative stress and other metabolic dysregulations of cells [1]. A group of ER stress-induced signal transduction pathways, collectively known as the unfolded protein response (UPR), has evolved to facilitate adaptation to the changing environment and re-establish ER function and homeostasis [2]. The UPR is primarily regulated by three ER-located sensors: activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1), and PKR-like ER kinase (PERK). In response to ER stress, ATF6 is cleaved by site 1

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⇑ Corresponding author. Tel.: +86 571 88206475; fax: +86 571 88206549. E-mail addresses: [email protected] (S. Shen), zhangyiqiaoqiao @126.com (Y. Zhang), [email protected] (R. Zhang), [email protected] (X. Gong).

and site 2 proteases (S1P and S2P) and then migrates to the nucleus to activate the transcription of GRP78. IRE1 initiates gene expression by processing the mRNA of the transcription factor X boxbinding protein 1 (XBP1) via its endonuclease activity. PERK phosphorylates a subunit of the eukaryotic translation initiation factor 2a (eIF2a), leading to a general reduction in protein synthesis as a means to counteract ER stress [3]. PERK-eIF2a pathway also mediates the transcriptional activation of the Atg5 and LC3 proteins via the C/EBP homologous protein (CHOP) and activating transcription factor 4 (ATF4), respectively [4]. Recent studies have shown that intense or persistent ER stress may induce cell death through an autophagic mechanism [4]. Autophagy is the cellular process of ‘‘self-digestion’’ and is mediated by the lysosomal degradation pathway [5]. Early studies identified autophagy as an adaptive response of cells to nutrient deprivation to ensure minimal housekeeping functions. Over the last decade, a tremendous amount of information has been gained about autophagy, proving that cellular injury, the accumulation of damaged organelles or membranes, and intracellular inclusions

http://dx.doi.org/10.1016/j.cbi.2014.04.017 0009-2797/Ó 2014 Published by Elsevier Ireland Ltd.

Please cite this article in press as: S. Shen et al., Ursolic acid induces autophagy in U87MG cells via ROS-dependent endoplasmic reticulum stress, ChemicoBiological Interactions (2014), http://dx.doi.org/10.1016/j.cbi.2014.04.017

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can activate the autophagic pathway [6]. The intricate molecular mechanism of autophagy is associated with multiple, complex pathways under various conditions. Notably, these signaling pathways may undergo crosstalk and regulation at different levels in the autophagic cascade [7]. Recent work by different laboratories has revealed that several signaling pathways regulate autophagy, such as the mammalian target of rapamycin (mTOR) pathway [8,9] and the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway [10]. The upstream regulators of mTOR, such as AMP-activated protein kinase (AMPK) and tuberous sclerosis protein 2 (TSC2), have been shown to inhibit the mTOR complex 1 (mTORC1), resulting in the activation of autophagy, while class I phosphoinositide 3-kinase (PI3K) and Akt activate mTORC1 to initiate tumor promoter signaling pathways and therefore inhibit autophagy [11]. In mammalian cells, AMPK can be activated by upstream factors, including LKB1, TAK1 and Ca2+/calmodulindependent kinase kinase (CaMKK). A recent report demonstrated that ROS activates AMPK by activating ataxia-telangiectasia mutated (ATM), which is an upstream regulator of AMPK [12]. Malignant gliomas are the most common primary brain tumors and usually infiltrate deep into the normal tissue. The complete surgical removal of the glioma is almost impossible, resulting in a high incidence of tumor recurrence [13]. Therefore, there is an urgent need to develop novel therapeutic approaches. Ursolic acid (UA), a pentacyclic triterpenoid derived from the roots of Catharanthus trichophyllus and Chamobates pusillus, has shown anti-inflammatory, hepatoprotective and antitumor properties [14]. The potential mechanisms of its anticancer action include cell cycle arrest and apoptosis induction through the inhibition of STAT3 [15] and NFjB51[16] and the activation of the MAPK8/9/ 10 [17] or TP53 [18] signaling pathways. However, no detailed studies have yet been reported on the effects of UA and the underlying mechanisms in human glioma cells. In this study, we present the first report that UA initiates the autophagic pathway through a novel mechanism; UA increased Ca2+ release from the ER lumen and then activated the CaMKKAMPK-mTOR kinase signaling cascade. Meanwhile, UA-triggered ER stress is also responsible for the autophagic response, especially PERK/eIF2a/CHOP and IRE1a/JNK pathways. Furthermore, the increased Ca2+ release by UA in turn enhanced ER stress and UPR activation. We provide evidence that the generation of reactive oxygen species (ROS) was necessary for the activation of these pathways as well.

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2. Materials and methods

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2.1. Materials and reagents

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Ursolic acid (UA), acridine orange, monodansylcadaverine (MDC), N-acetyl-cysteine (NAC) were purchased from Sigma– Aldrich (St. Louis, MO). The fluorescent probes dihydrorhodamine 123 (DHR123) and dihydroethidium (DHE) were from Molecular Probes (Eugene, OR, USA). 2-aminoethoxydiphenyl borate (2-APB) was from Calbiochem (San Diego, CA). 1,2-bis(2-amino-phenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA-AM), Fluo-3 AM and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were from Sigma Chemical (St. Louis, MO). Tauroursodeoxycholic acid (TUDCA) was obtained from Shanghai Jing-Chun Industrial (Shanghai, China). Primary antibodies were purchased from these companies: Santa Cruz (CHOP, GRP78, PERK, phosphoPERK(Thr980), IRE1a, eIF2a, CaMKK, p27 and p21), Cell Signaling (phospho-eIF2a(Ser51), Cyclin D1, Cyclin D3, Cdk4, CyclinE, LC3B, AMPK, phospho-AMPKa(Thr172), Akt, phospho-Akt (Thr308), mTOR, p-mTOR, p-4EBP1 (Ser65), JNK, phospho-JNK (Thr183/ Tyr185), phospho-p70S6K (Thr389), Atg5, Beclin1, ACC,

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phospho-ACC (Ser79), Bcl-2, phospho-Bcl-2 (Ser70) and GAPDH). Horseradish peroxidase-conjugated goat anti-rabbit secondary antibody was obtained from Santa Cruz Biotechnology.

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2.2. Cell culture

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Human glioma cell line U87MG, obtained from American Type Culture Collection (Manassas, VA, USA), were cultured in monolayers at 37 °C in a humidified atmosphere of 95% air and 5% CO2 using Dulbecco’s Modified Eagle’s Medium supplemented with 10% FBS, 50 U/ml penicillin and 50 lg/ml streptomycin.

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2.3. Cytotoxicity

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U87MG cells (2 104) were grown to 80% confluence in 96-well plates, and following treatment with various concentrations of UA for 24–72 h. MTT was then added to each well and the cells were incubated for 4 h at 37 °C. The formazan precipitate was dissolved in 150 ll DMSO and absorbance at 490 nm was measured using an ELISA plate reader. Each test was repeated at least three times.

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2.4. Clonogenic survival assay

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U87MG cells were cultured overnight in a 6-well plate (1500 cells per well) and treated with UA (0–40 lM) for 24 h. Then the medium was replaced with drug-free medium, and cells were incubated for 10 days to form colonies. After 10 days, cells were fixed and stained with crystal violet (0.2%) to visualize cell colonies. Colonies were counted, and results were normalized to the colonies of the control cells. Each sample was repeated in 3 wells.

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2.5. Cell cycle analysis

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U87MG cells were treated with different concentrations of UA for 24 h. The cells were harvested and washed twice with PBS, then fixed in ice-cold 70% (v/v) ethanol for 16 h at 4 °C. Before analysis, cells were washed with PBS, suspended in 1 ml of cold PI solution (50 lg/ml PI, 100 lg/ml RNase A) and incubated for 30 min in darkness at 37 °C. The samples were analyzed by flow cytometry (Becton–Dickinson LSR II, San Jose, CA, USA) using Cell Quest software.

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2.6. Measurement of intracellular calcium

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After treatment with or without UA, U87MG cells were harvested and incubated with 500 nM Fluo-3 AM dye for 30 min at 37 °C and then immediately analyzed on a flow cytometer using FL-1 as a detector. The relative intracellular calcium concentrations were calculated from the ratio of the geographic mean values of the FL-1 peak generated from UA-treated cells over each respective control as indicated in the figure legend.

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2.7. Determination of the cellular redox state by ROS analyses

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The oxidative stress of cells was determined by detection of cellular ROS. After incubation with 5–40 lM UA for 24 h, the cells were washed and incubated with 20 lM DHR123 (for H2O2) or 20 lM DHE (for O2 ) at 37 °C for 30 min. The cells were then washed three times with PBS. The fluorescence intensity was measured by flow cytometry.

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2.8. Electron microscopy

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To morphologically observe the induction of autophagy in UAtreated U87MG cells, we performed an ultrastructural analysis according to published procedures [10]. After being treated with

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40 lM UA for 24 h, cells were washed twice with PBS and fixed with ice-cold glutaraldehyde (3% in 0.1 M cacodylate buffer, pH 7.4) for 30 min. Cells were postfixed in osmium tetroxide and embedded in Epon, before being cut and stained with uranyl acetate/lead citrate (Fluka, Chemie AG, Buchs, Switzerland) and viewed with a Hitachi H600 electron microscope (Hitachi Instrument, Tokyo, Japan).

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2.9. Detection of acidic vesicular organelles

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The formation of acidic vesicular organelles (AVOs), a morphological characteristic of autophagy, was detected by acridine orange staining. Cells were stained with 1 lg/ml acridine orange for 15 min, and the samples were observed under a Zeiss Axiovert 200 inverted microscope (excitation, 546 nm; emission, 575/640 nm).

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2.10. Visualization of autophagic vacuoles

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The autofluorescent agent MDC was recently introduced as a specific autophagolysosomal marker. U87MG cells were treated with temozolomide (TM) or UA for 24 h. The autophagic vacuoles were labeled with MDC by incubating the cells with 50 lM MDC in PBS at 37 °C for 15 min. Following incubation, the cells were washed three times with PBS and immediately analyzed using a Zeiss Axiovert 200 inverted microscope (excitation, 390 nm; emission, 460 nm).

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2.11. RNA interference

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For the knockdown of gene expression, siRNAs targeting Beclin1, CaMKK, AMPK, Atg5, eIF2a, CHOP, JNK, PERK or IRE1a were used. Their respective scrambled siRNAs were used as controls. Cells were transfected with each siRNA at final concentration of 100 pM using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) for 48 h according to the manufacturer’s instructions. The efficacy of RNA interference was determined by western blotting. All above siRNAs were purchased from Dharmacon (Lafayette, CO).

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2.12. Western blot analysis

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The treated cells were collected, washed in PBS and then lysed with lysis buffer on ice. The protein concentration in the cell lysate was measured by the Lowry method. Approximately 30 lg of the lysed protein was separated by SDS–PAGE and transferred to nitrocellulose membranes. The membranes were then blocked in blocking buffer (5% bovine serum albumin and 0.1% Tween 20 in Tris-buffered saline) and probed with the indicated antibodies at 1/500–1/1000 dilution overnight at 4 °C. After incubation with an appropriate second antibody, membranes were visualized using an enhanced chemiluminescent detection kit (Pierce, Rockford, IL, USA).

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2.13. Immunoprecipitation

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U87MG cells (1 106/dish) were seeded in 100-mm culture dishes and cultured for 24 h. After treatment with UA (5 mM) for 6 h, the cells were harvested and then resuspended in lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 50 mM NaF, 2 mM EGTA, 10% Glycerol, and 0.25% NP-40). The protein extracts (1 mg) were incubated with antibodies against Bcl-2 (2 mg) or GRP78 (2 mg) at 4 °C overnight. After this incubation, Protein G beads (BD Biosciences, Franklin Lakes, NJ, USA) were added and incubated at 4 °C for 2 h. The immunoprecipitated complexes were collected and washed three times with ice-cold cell lysis buffer. The beads were resuspended in 50 ml of 2 SDS sample buffer and then boiled for

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5 min. After gentle centrifugation, the supernatant was subjected to SDS–PAGE and subsequent immunoblotting analysis with antiBeclin1 or anti-PERK antibody.

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2.14. Statistical analyses

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All experiments were performed in triplicate with means ± S.D. subjected to Student’s t test for pairwise comparison or one-way ANOVA followed by Tukey post hoc test for multivariate analysis. Differences with p < 0.05 were considered significant. The figures in this article are representative of at least three independent experiments with a similar pattern.

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3. Results

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3.1. UA inhibits the proliferation and induces the cell cycle arrest of U87MG cells

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To investigate the growth inhibitory properties of UA (Fig. 1A), we performed a MTT assay based on the ability of viable cells to reduce MTT to formazan crystals. As shown in Fig. 1B, cell growth was inhibited by UA in a dose-and time-dependent manner. The IC50 was calculated as 71.02, 51.20, and 45.66 lM for cells treated at 24, 48 and 72 h, respectively. In addition, a colony-formation assay further confirmed that UA inhibited the proliferation of U87MG cells in a dose-dependent manner (Fig. 1C). To determine whether UA induced a cell cycle arrest, we analyzed the effect of UA on the cell cycle distribution using PI staining. Exponentially growing U87MG cells were treated with UA for 24 h and then subjected to cell cycle analysis. As shown in Fig. 1D, the cells were arrested in the G1 phase. Specifically, 57.60% of the untreated U87MG cells were in the G1 phase, while the cells treated with 20 or 40 lM UA showed a significantly greater proportion of cells in the G1 phase (65.89% and 75.94%, respectively) (Fig. 1D and E). The increased number of cells in the G1 phase following UA exposure was related to a decrease in the number of cells in the S and G2/M phases relative to the control. To elucidate the molecular mechanisms responsible for UAinduced G1-phase arrest, we examined the expression of cell cycle-related proteins, including cyclin E, cyclin D1, cyclin D3 and Cdk4 in U87MG cells. Compared to the control, the UA-treated cells exhibited concentration-dependent decreases in the levels of cyclin E, cyclin D1, cyclin D3 and Cdk4, which is consistent with the role of these proteins in the regulation of the G1 phase transition. Then, we assessed the effect of UA on the expression of key regulators of G1 phase progression, including the Cdk inhibitors p21Waf1/Cip1 and p27Kip1. P21 is known to inhibit Cyclin E/ Cdk2 and Cyclin D1/Cdk4 complexes that lead to G1 arrest, and both CyclinE and Cdk4 appear to be down-regulated at the protein level. As shown in Fig. 1F, western blot analysis revealed that UA treatment resulted in a dose-dependent upregulation of the p21Waf1/Cip1 and p27Kip1 proteins, indicating that UA increases the levels of Cdk inhibitors, which in turn induce a G1 phase arrest.

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3.2. UA activates the process of autophagy in U87MG cells

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It is reported that the cytotoxicity of some chemo-therapeutic drugs in cancer cells is mediated by the activation of autophagy rather than the induction of apoptosis. To examine whether UA induced the autophagic process, we performed a series of experiments to detect autophagy in UA-treated U87MG cells. The occurrence of autophagy was supported by the direct observation of autophagosomes using electron microscopy. As shown in Fig. 2, the control U87MG cells exhibited normal nuclei with uniform and finely dispersed chromatin, whereas the cytoplasm of

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Fig. 1. UA inhibits the proliferation and induces the cell cycle arrest of U87MG cells. (A) The chemical structure of UA. (B) U87MG cells were treated with various concentrations of UA for 24–72 h. Cell viability was determined using MTT assay. (C) The clonogenicity of U87MG cells after UA treatment was measured using a colony formation assay. (D) DNA content analysis of U87MG cells treated with 20 or 40 lM UA for 24 h. (E) Histogram of the cell cycle phase distribution of U87MG cells treated with UA for 24 h. (F) U87MG cells were treated with the indicated concentrations of UA for 24 h. The total cell extracts (containing 30 lg protein) were subjected to 12% SDS–PAGE and immunoblotted with antibodies against cyclin D1, cyclin D3, Cdk4, p21, p27, cyclin E and GAPDH (loading control) as indicated. The data are presented as the means ± S.D. of three separate experiments. ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001 vs. control cells; #p < 0.05, ##p < 0.01 vs. the preceding group.

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UA-exposed U87MG cells had abundant autophagosomes. The UAinduced autophagy in U87MG cells was further examined by staining with acridine orange and MDC. The number of acidic vesicular organelles stained by acridine orange and MDC-labeled mature autophagosomes was increased in a dose-dependent manner in

UA-treated U87MG cells (Fig. 3A and B). LC3-II, a hallmark of autophagy, was upregulated in U87MG cells after UA treatment; this accumulation was both dose- and time-dependent (Fig. 3C and D). In conclusion, our findings reveal that UA activates the process of autophagy in U87MG cells.


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Fig. 2. Morphological observation of autophagy in U87MG cells. U87MG cells treated with or without 40 lM UA for 24 h were visualized by transmission electron microscopy. White arrows indicate autophagosomes.

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3.3. UA induces autophagy through the activation of the CaMKKAMPK-mTOR kinase signaling cascade Having clearly established that UA can induce autophagy in U87MG cells, we set out to investigate the molecular mechanism underlying this biological effect. Nutrient deprivation can activate autophagy through the AMP-activated protein kinase (AMPK) dependent pathway [19]. Fig. 4A showed that AMPK, which regulates metabolism and cell proliferation through the phosphorylation of acetyl CoA carboxylase (ACC), was involved in UA-induced autophagy. The levels of phosphorylated AMPK and ACC were dramatically increased in a dose-dependent manner (Fig. 4A). Subsequently, U87MG cells were transfected with siRNA specific to AMPK. As a result, the UA-induced LC3-II formation was attenuated by si-AMPK transfection (Fig. 4B), confirming that an AMPK signal was involved in the UA-induced autophagy pathway. Several upstream kinases including LKB1, CaMKK, and TAK1 can activate AMPK by the phosphorylation of Thr172 in the kinase domain of the a-subunit [20]. Our results revealed that the knockdown of CaMKK markedly reduced the accumulation of LC3-II and p-AMPK (Fig. 4C), suggesting that the CaMKK-AMPK pathway participates in the autophagic process induced by UA. mTOR is one of the major downstream targets regulated by AMPK [21]. Western blot analysis showed that p-mTOR was downregulated after UA exposure in U87MG cells (Fig. 4D). The downstream targets of mTOR, including p70 ribosomal S6 kinase (p70S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), also decreased following UA treatment (Fig. 4D). Furthermore, the blockage of either CaMKK or AMPK by siRNA transfection prevented the UA-induced decrease in p-mTOR level (Fig. 4E and F). Taken together, these data support the conclusion that the activation of the CaMKK-AMPK-mTOR

pathway plays an important role in UA-induced autophagy. In addition to AMPK, Akt also functions as an upstream regulator of mTOR. We therefore evaluated the level of Akt activation in U87MG cells and found that the phosphorylation of Akt decreased following UA treatment (Fig. 4D). To investigate the role of the key autophagy proteins Beclin-1 and Atg5 in UA-induced autophagy, we used an RNA interference approach to achieve specific knockdown of these proteins. As shown in Fig. 4G and H, the knockdown of either Beclin-1 or Atg5 expression led to a reduction in LC3-II accumulation in UA-treated cells, indicating that both Beclin-1 and Atg5 are required for UA-induced autophagy.

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3.4. UA raises the cytosolic Ca2+ level and induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR)

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Ca2+-mobilizing agents have been shown to induce autophagy by activating the CaMKK-AMPK-mTOR signaling cascade [22]. Therefore, we hypothesized that the concentration of Ca2+ may be elevated in the autophagic process. A calcium indicator dye, Fluo-3 AM was used to detect variations in calcium after UA treatment. As shown in Fig. 5A and B, there was a marked increase in cytosolic calcium levels in U87MG cells, climbing 3.4-fold compared to the control after treatment with 60 lM UA for 24 h. A rise in cytosolic calcium levels is reported to be an important marker of ER stress [23]. As shown in Fig. 5C, the expression of IRE1a, CHOP, GRP78 and p-PERK was increased in a dose-dependent manner. Fig. 5C shows a transient increase in eIF2a phosphorylation by UA. Western blotting revealed that the increase was significant at 10 lM, peaked at 20 lM, and returned to baseline at 40 lM after treatment with UA. The induction of representative UPR markers GRP78 and CHOP indicates that UA is an inducer of

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Fig. 3. UA induces autophagy in U87MG cells. (A) Acridine orange staining was used to detect autophagic vacuoles in U87MG cells treated in the nutrient-free (NF) condition or with UA for 24 h. Following acridine orange staining, the cytoplasm and the nucleoli fluoresce green, whereas the acidic compartments fluoresce bright red or orange–red. (B) U87MG cells were treated with UA or temozolomide for 24 h, and the autophagolysosomes were observed using MDC staining. Temozolomide (TM) treatment was presented as a positive control. (C) U87MG cells were exposed to the indicated concentrations of UA for 24 h and then analyzed by western blotting for the expression of LC3-I and LC3-II. (D) U87MG cells were treated with 40 lM UA for 12–36 h, and the total cell extracts were then analyzed by western blotting for the expression of LC3-I and LC3-II. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 378 379 380 381 382 383 384 385 386 387

the ER stress response. GRP78 normally binds to PERK and inhibits its activation. When unfolded proteins accumulate in the ER lumen, GRP78 dissociates from PERK to bind the unfolded proteins, leading to PERK activation and the phosphorylation of eIF2a [24]. Therefore, we performed immunoprecipitation with an antibody that targets PERK. As shown in Fig. 5D, a substantial amount of GRP78 was bound to PERK, and this binding became quite weak after treatment with UA. Furthermore, a specific siRNA directed against PERK was utilized in the U87 glioma cells. The knockdown of PERK attenuated the UA-induced increase of p-PERK and p-eIF2a

(Fig. 5E). Similarly, transfecting the cells with si-eIF2a lowered the UA-induced expression of CHOP (Fig. 5F). In conclusion, all of these results indicate that UA raises the cytosolic Ca2+ level and induces ER stress involving the PERK-eIF2a-CHOP pathway.

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3.5. ER stress mediates UA-induced autophagy in U87MG cells

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Emerging evidence has indicated that ER stress is a potent inducer of autophagy [25–28]. We then tested whether alleviating ER stress affected UA-induced autophagy. U87MG cells were

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Fig. 4. The molecular mechanism of autophagy following UA treatment. (A) U87MG cells exposed to the indicated concentrations of UA for 24 h were lysed to determinate the levels of phosphorylated AMPK, total AMPK, phosphorylated ACC and total ACC. (B) U87MG cells were transfected with siRNA targeting AMPK and then exposed to 40 lM UA. After 24 h of incubation, the expression levels of AMPK and LC3-II were analyzed by western blotting. (C) U87MG cells were transfected with siRNA targeting CaMKK and then exposed to 40 lM UA. After 24 h of incubation, the expression levels of p-AMPK, CaMKK and LC3-II were analyzed by western blotting. (D) Following UA exposure for 24 h, the cell lysates were probed for p-Akt, Akt, p-mTOR, mTOR, p-p70S6K and p-4EBP1. The phosphorylated mTOR and total mTOR levels were also analyzed after transfecting the cells with siRNA targeting CaMKK (E) and AMPK (F). GAPDH was used as a loading control. (G and H) U87MG cells were transfected with Beclin-1 siRNA (G) or Atg5 siRNA (H) as indicated. The cells were exposed to 40 lM UA for 24 h and then lysed for immunoblotting with antibodies against Beclin-1, Atg5, LC3 II or GAPDH.

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pretreated with TUDCA, an ER stress inhibitor, for 2 h. As expected, TUDCA inhibited eIF2a phosphorylation and GRP78 and CHOP expression in glioma cells (Fig. 6A). In addition, the UA-induced increase of p-AMPK and decrease of p-mTOR levels were both alleviated by TUDCA, and the accumulation of the LC3-II isoform was also partially inhibited (Fig. 6A). Accordingly, the MDC-positive vacuoles were relatively sparse and weak in fluorescence intensity in cell cultures co-treated with UA + TUDCA when compared to cultures treated with UA (Fig. 6B), indicating that the incorporation of MDC into vacuoles was inhibited by TUDCA. Similarly, the blockage of either PERK or IRE1a by siRNA transfection inhibited the UA-induced accumulation of LC3-II (Fig. 6D). Moreover, Fig. 6E illustrates that the knockdown of PERK or IRE1a effectively

decreases UA-induced cell death, which is in parallel with the results of cotreatment with TUDCA (Fig. 6C). To further investigate the relationship between autophagy and ER stress, an autophagy inhibitor, 3-MA was introduced. As shown in Fig. 6F, the addition of 3-MA did not alter the upregulation of GRP78 and CHOP by UA. We then examined whether PERK/eIF2a/CHOP pathway was responsible for UA-induced autophagy. Western blot analysis revealed that transfection with either si-eIF2a or si-CHOP significantly attenuated the UA-induced LC3-II conversion (Fig. 6G and H), indicating the participation of PERK/eIF2a/CHOP pathway in UA-induced autophagy. The kinase domain of IRE1 has been reported to activate JNK, and activated JNK can phosphorylate Bcl-2 to initiate autophagy


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Fig. 5. UA activates UPR signaling pathways in response to ER stress. (A and B) U87MG cells were treated with the indicated concentrations of UA for 24 h, incubated with 0.5 mM Fluo-3 AM dye for a total of 30 min, and then analyzed by flow cytometry. The cytosolic calcium was elevated significantly after treatment with UA. The data presented in panel A are representative of three independent experiments, and the statistical results from these experiments are presented in panel B (⁄⁄p < 0.01 vs. the control; #p < 0.05, ##p < 0.01 vs. the preceding group). (C) U87MG cells were treated with 10–40 lM UA for 24 h. Cell lysates were resolved by SDS–PAGE and probed with antibodies against p-PERK, PERK, IRE1a, GRP78, p-eIF2a, eIF2a, and CHOP. (D) U87MG cells were treated with UA for 6 h, and GRP78 was immunoprecipitated from the resulting lysates and then immunoblotted with PERK antibody. (E) U87MG cells were transfected with PERK siRNA or (F) eIF2a siRNA as indicated. The cells were then exposed to 40 lM UA for 24 h and lysed for immunoblotting with the indicated antibodies.


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Fig. 6. UA induces autophagy by the stimulation of ER stress in U87MG cells. (A) U87MG cells were treated with UA (40 lM) and/or TUDCA (500 lM) for 24 h and then subjected to western blotting. (B) The number of MDC-labeled vacuoles was observed in U87MG cells after UA treatment (40 lM) for 24 h with or without TUDCA using a laser scanning confocal microscope (63). (C) The viability of U87MG cells after UA treatment (40 lM) with or without TUDCA, was calculated by the MTT assay. The data are presented as the means ± S.D. of three separate experiments. ⁄p < 0.05 vs. the U87MG cells exposed to UA alone. (D) U87MG cells were transfected with PERK siRNA or IRE1a siRNA as indicated. The cells were exposed to 40 lM UA for 24 h and then lysed for immunoblotting with the indicated antibodies. (E) U87MG cells were transfected with siRNA specifically targeting PERK or IRE1a as indicated. The cells were then treated with 40 lM UA for 24 h, and cell viability was measured by the MTT assay. The data are presented as the means ± S.D. of three separate experiments. ⁄p < 0.05 vs. U87MG cells transfected with scrambled siRNA and exposed to UA. (F) Western blot analysis of the expression of LC3, GRP78 and CHOP in U87MG cells treated with 40 lM UA and/or 3 mM 3-MA for 24 h. (G and H) U87MG cells transfected with sieIF2a/siCHOP were treated with 40 lM UA for 24 h and then harvested for protein analysis. (L) Representative blots illustrating the expression levels of JNK, p-JNK and p-Bcl-2 are shown. (M) Co-immunoprecipitation of Beclin1 with Bcl-2 in U87MG cells exposed to UA (40 lM) for 6 h. Representative figures illustrating the expression of Beclin1-Bcl-2 complex are shown. (N) JNK, Beclin1 and LC3 expression in U87MG cells treated with UA (40 lM) in the presence or absence of siRNA against JNK were detected by western blotting.


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[29,30]. Therefore, we examined the expression of typical proteins in the IRE1a-JNK pathway. Consistent with the increase of IRE1a (Fig. 5C), western blot analysis showed that there was a significant upregulation in the levels of phosphorylated JNK and Bcl-2 (Fig. 6L). Generally speaking, Bcl-2 exerts its anti-autophagy effect via an inhibitory interaction with Beclin1, and phosphorylated Bcl2 dissociates from Beclin1 to trigger autophagy [31]. As expected, the expression level of the Beclin1-Bcl-2 complex was decreased in U87MG cells exposed to UA for 6 h, suggesting the dissociation of Beclin1 from Bcl-2 (Fig. 6M). Moreover, si-JNK transfected cells exhibited attenuated LC3-II expression, confirming the significant role of JNK in UA-induced autophagy (Fig. 6N). Taken together, all these results suggest that UA-induced ER stress activates autophagy via the PERK-eIF2a-CHOP and IRE1a-JNK pathways in U87MG cells.

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3.6. UA-induced ER stress and autophagy is mediated by calcium

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Variations in cytosolic calcium concentration have been shown to participate in apoptosis in several cell lines [32,33], but the role of calcium in UA-induced autophagy is still unclear. To evaluate the effects of variations in cytoplasmic calcium on UA induced cell death, we measured the viability of cells exposed to UA with or without BAPTA-AM, a chelator of cytosolic calcium. As shown in Fig. 7A and C, pretreatment with 10 mM BAPTA-AM before exposure to 40 lM UA resulted in markedly reduced cytosolic calcium and decreased the percentage of cell death. Moreover, the addition of BAPTA-AM to the cells abolished the formation of LC3-II (Fig. 7E). The ER is one of the major calcium storage units in cells, and reports have demonstrated that blockers of the ER calcium channel effectively inhibit autophagy induced by various stimuli, suggesting that the release of calcium from the ER may lead to cell death. Therefore, we explored the effects of 2-APB, a blocker of the ER calcium channel (IP3R), on UA-induced cell death. As shown in Fig. 7, we observed that 2-APB was sufficient to suppress the UAinduced upregulation of LC-3 II (Fig. 7F) and cell death (Fig. 7D); additionally, the increase in [Ca2+]i induced by UA was suppressed by 2-APB from 2.6- to 1.6-fold after treatment with UA for 24 h (Fig. 7B), indicating that 2-APB partially blocked Ca2+ leakage from the ER. Importantly, the levels of p-AMPK, CHOP and GRP78 were all decreased by both BAPTA-AM (Fig. 7E and G) and 2-APB (Fig. 7F and H) treatment, indicating that the increased intracellular calcium levels caused by UA affect both ER stress and autophagy. Taken together, these results demonstrate that the autophagy induced by UA in U87MG cells is linked to the release of calcium from the ER.

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3.7. UA-induced ER stress and autophagy are mediated by ROS production ER stress, autophagy and oxidative stress are closely linked events [34]. To gain further insight into the mechanism of UAinduced ER stress and autophagy, we assessed the involvement of two types of ROS, superoxide and hydrogen peroxide. The exposure of cells to UA resulted in a significant increase in intracellular ROS production, as measured by the O2 -sensitive probe DHE (Fig. 8A). However, there was no obvious changes in the levels of hydrogen peroxide in UA-exposed U87MG cells (Fig. 8A). The UAinduced increase in ROS was inhibited by pretreatment with the ROS scavenger NAC (5 mM) (data not shown). We also provided evidence for the robust downregulation of the cell death rate after preincubation with NAC compared to UA exposure alone (Fig. 8B). We then focused our attention on the effect of ROS on UA-induced ER stress. NAC attenuated the UA-induced upregulation of the intracellular calcium level (Fig. 8F) and the ER stress markers GRP78 and CHOP (Fig. 8D and E), indicating that NAC inhibits

UA-induced ER stress. We further analyzed the expression of autophagy-related proteins to address the role of ROS in the activation of autophagy. When NAC was added to cells, the level of pAMPK was reduced, and this correlated with reduced LC-3 II expression (Fig. 8D and E). These results indicate that ROS mediate UA-induced autophagy via AMPK signaling.

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UA has been shown to have anticarcinogenic potential by inducing apoptosis of tumor cells, such as hepatocellular cancer cell HepG2 [35], cervical carcinoma cell Hela [36] and gastric cancer cell line BGC-803 [37]. It also has been reported to promote autophagy through PI3-K signaling in TC-1 cervical cancer cells [38]. In the present study, we demonstrate that the activation of ER stress triggers autophagy in malignant glioma cells exposed to UA. This is supported by several lines of evidence. First, UA-induced ER stress resulted in increased Ca2+ release from the ER lumen and subsequently activated the CaMKK-AMPK-mTOR pathway, which ultimately led to autophagy. In addition, increased Ca2+ in turn enhanced UA-induced ER stress. Second, UA treatment led to UPR activation involving an IRE1a signal, which initiated autophagy by promoting the phosphorylation of JNK and Bcl-2. Third, UA increased the phosphorylation of PERK and eIF2a and subsequently upregulated the expression of CHOP. In conclusion, our results demonstrated that three pathways, the CaMKK/ AMPK/mTOR, PERK/eIF2a/CHOP and IRE1a/JNK pathways, are involved in UA induced autophagy in U87MG cells. ER stress activation is frequently accompanied by the elevation of cytoplasmic calcium which leads to the activation of several regulated signaling pathways. Different agents (including ER stress inducers) have been shown to generate an increase in cytosolic calcium concentration and activate autophagy. Our results also demonstrated that the release of calcium played a crucial role in the autophagy induced by UA. Consistent with our results, vitamin D, Saikosaponin-d, ionomycin, and photodynamic therapy are known to induce calcium-mediated autophagy [39,40], suggesting that the disturbance of calcium homeostasis is associated with the induction of autophagy. More importantly, this study reports for the first time that UA exhibited its cytotoxicity via autophagy through ERassociated Ca2+ signaling. Two types of ER-resident Ca2+ release channels are reported to exist in the ER, namely the ryanodine receptors and the IP3 receptors (IP3R) [41]. In this study, we noticed that the inhibition of IP3R by 2-APB significantly reduced UA-induced autophagy and cell death. Although our data showed that UA-induced autophagy required IP3R in U87MG cells, Criollo et al. demonstrated that the knockdown of IP3R isoforms using RNA interference is capable of inducing autophagy in mammalian cells [42]. Additionally, it has been reported that a rise in the free cytosolic calcium through ATP stimulation that causes IP3 production is a potent inducer of macroautophagy [43]. These conflicting results may due to the fact that IP3R activation and inhibition are able to induce autophagy through different signaling pathways [41]. Therefore, the role of calcium release by IP3R in regulating cell death is still controversial and requires further elucidation. In addition to IP3R, another mechanism connecting Ca2+ release from the ER and autophagy is the stimulation of AMPK, which inhibits mTORC1 by activating TSC2 [44]. It is well established that AMPK senses energetic stress (a high AMP/ATP ratio) and is activated to modulate metabolism for cellular adaptation [45]. Indeed, in addition to its role in sensing energy deprivation, AMPK has evolved to sense stress signals from the ER relayed by one of its upstream kinases, known as calmodulin-dependent kinase protein kinase (CaMKK) [46]. Jäättelä [47] and Tong [48] showed that an increase in cytosolic Ca2+ concentration following treatment with

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Fig. 7. UA-induced ER stress and autophagy is mediated by calcium. (A and B) The UA-induced elevation of cytosolic calcium was diminished by 10 mM BAPTA-AM or 60 mM 2-APB. The cells were pretreated with BAPTA-AM (A) or 2-APB (B) for 1 h and then treated with 40 lM UA for another 24 h. The variations in cytosolic calcium were analyzed by Fluo-3 AM staining using flow cytometry. ⁄p < 0.05 vs. the respective control. (C and D) The viability of U87MG cells after UA treatment (40 lM) with or without BAPTA-AM (C) or 2-APB (D). The data are presented as the means ± S.D. of three separate experiments. ⁄p < 0.05 vs. the respective control. (E and F) U87MG cells were pretreated with BAPTA-AM (E) or 2-APB (F) for 1 h and then treated with UA (40 lM) for another 24 h. The expression of p-AMPK and LC3 was detected by western blot. (G and H) U87MG cells were pretreated with BAPTA-AM (G) or 2-APB (H) for 1 h and then treated with UA (40 lM) for another 24 h. CHOP and GRP78 expression were detected by western blot.

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ER stress inducers stimulates CaMKK, resulting in the phosphorylation of AMPK, inhibition of mTORC1, and activation of autophagy. Consistent with these observations, results presented here suggest that UA induced autophagy involving a mechanism of AMPK activation via CaMKK and the suppression of mTOR signaling. Recent studies also indicate that AMPK can directly induce autophagy by phosphorylating Ulk1, a key regulator in autophagy initiation [49]. Further studies are needed to explore whether Ulk1 is involved in UA-induced autophagy. Moreover, Grotemeier et al.

[50] demonstrated that Ca2+ mobilization permits LC3 lipidation in AMPK double-deficient cells, suggesting that Ca2+ signaling switch on autophagy by either AMPK-dependent or AMPK-independent pathway. Increasing evidence has indicated that autophagy can be activated by eIF2a phosphorylation during starvation and viral infection [51]. There are four eIF2a kinases (PKR, GCN2, HRI and PERK); these kinases are activated by viral infection, amino acid starvation, heme depletion and ER stress, respectively [52].


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Fig. 8. Reactive oxygen species are involved in UA-induced ER stress and autophagy. (A) Effects of UA on intracellular ROS. DHE and DHR123 (20 lM) were used to detect superoxide and hydrogen peroxide, respectively. Results are expressed as a ratio of relative fluorescent intensity. Each bar represents the means ± SD obtained from three experiments. ⁄p < 0.05, ⁄⁄p < 0.01 vs. control cells; #p < 0.05, ##p < 0.01 vs. the preceding group. (B) The viability of U87MG cells after UA treatment (5–60 lM) with or without the ROS scavenger NAC. The data are presented as the means ± S.D. of three separate experiments. ⁄p < 0.05, ⁄⁄p < 0.01 vs. the U87MG cells exposed to UA alone. (C and E) U87MG cells were treated with NAC (C), UA (D) or the combination of UA and NAC (E) for 24 h. Western blotting was performed to detect the expression of phosphorylated AMPK, total AMPK, GRP78, CHOP and LC3. (F) The UA-induced elevation of cytosolic calcium was attenuated by NAC. U87MG cells were pretreated with NAC for 1 h and then exposed to UA for another 24 h. The variations in cytosolic calcium were analyzed by Fluo-3 AM staining using flow cytometry. ⁄⁄p < 0.01 vs. the respective control.

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Recently, Kouroku et al. reported that expanded polyglutamine (polyQ)-induced ER stress activates autophagosome formation and LC3 conversion from LC3-I to LC3-II via the PERK-eIF2a pathway [53]. It is known that CHOP is the best characterized mediator in the transition of ER stress to apoptosis, which is a key proapoptotic transcription factor induced during ER stress. However, Rouschop et al. [54] and Ito et al. [55] independently revealed that the PERK-eIF2a pathway triggers the transcriptional activation of Atg5 in hypoxic responses through the action of CHOP,

suggesting that CHOP may play a role in autophagy as well. All these previous studies suggest that the PERK-eIF2a-CHOP pathway may be associated with the activation of autophagy, which is consistent with our results that the knockdown of PERK, eIF2a or CHOP attenuated LC3-II conversion. In addition, a recent report demonstrated that the activation of PERK causes a decrease in cyclin D1 and that this downregulation requires eIF2a phosphorylation at serine 51. Stockwell et al. identified a novel small molecule (CCT020312) that achieves the G1/S checkpoint through


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program triggered by UA. These findings and a safe track record make UA a promising agent for the treatment of malignant gliomas.

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Conflict of Interest

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The authors declare that there are no conflicts of interest.

Fig. 9. A proposed model of molecular interaction to delineate the mechanisms of UA action in glioma cells.

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PERK/eIF2a signaling, which is also linked to the control of CDK4 activation [56,57]. Consistent with these studies, we demonstrated that both cell cycle arrest and PERK/eIF2a activation were induced by UA treatment, suggesting that the G1-phase arrest in U87MG cells may be related to a PERK/eIF2a signal. Nevertheless, more evidence is required to support this hypothesis. Upon ER stress, IRE1ais activated after GRP78 dissociates from its luminal domains. Activated IRE1a upregulates the XBP-1s protein, which initiates the expression of a broad spectrum of UPRassociated genes to restore ER homeostasis. Alternatively, activated IRE1a can also recruit TNFR-associated factor 2 (TRAF2) to phosphorylate JNK. The activation of JNK initiates the process of autophagy in part by phosphorylating Bcl-2, which is widely distributed in the ER [58]. In the present study, UA treatment led to the activation of the IRE1a-JNK pathway, with Beclin1 dissociating from Bcl-2. However, it remains unclear whether the activation of the JNK pathway affects the function of mTOR, which occupies a central position in the signaling cascade of autophagy [59] or constitutes a novel signaling pathway in the activation of autophagy. In fact, in the presence of TUDCA, LC3II formation was still sustaining in UA-treated U87MG cells. This may be explained by the fact that some of the key regulatory steps in the activation of autophagy upon stimulation of ER stress can also receive signals derived from different inputs including those not directly related to ER stress [60]. Since autophagy and ER stress have been implicated in various human cancers, an exploration of the novel signaling pathways relevant to ER stress and autophagy would shed light on the development of new therapeutic strategies for diseases. In summary, we demonstrate that UA induces autophagy primarily through the induction of ROS-mediated ER stress in glioma cells (Fig. 9). The close relationship between autophagy and ER stress is also revealed in the execution of cell death

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The Transparency document associated with this article can be found in the online version.

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Acknowledgments

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This work was supported by Key Laboratory of Microbial Biochemistry and Metabolism Engineering of Zhejiang Province and the State Agricultural S&T Result Transforming Fund, the Ministry of Science and Technology of China (No. 2012C2202001).

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References

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[1] A. Gorostizaga, M.M. Mori Sequeiros García, A. Acquier, N.V. Gomez, P.M. Malobert, C.F. Mendez, C. Paz, Modulation of albumin-induced endoplasmic reticulum stress in renal proximal tubule cells by upregulation of mapk phosphatase-1, Chem. Biol. Interact. 206 (1) (2013) 47–54. [2] X. Li, K. Zhang, Z. Li, Unfolded protein response in cancer: the physician’s perspective, J. Hematol. Oncol. 4 (2011) 8. [3] S. Srinivasan, M. Ohsugi, Z. Liu, S. Fatrai, E. Bernal-Mizrachi, M.A. Permutt, Endoplasmic reticulum stress-induced apoptosis is partly mediated by reduced insulin signaling through phosphatidylinositol 3-kinase/Akt and increased glycogen synthase kinase-3beta in mouse insulinoma cells, Diabetes 54 (4) (2005) 968–975. [4] A. Lugea, D. Tischler, J. Nguyen, J. Gong, I. Gukovsky, S.W. French, F.S. Gorelick, S.J. Pandol, Adaptive unfolded protein response attenuates alcohol-induced pancreatic damage, Gastroenterology 140 (3) (2011) 987–997. [5] C. Bincoletto, A. Bechara, G.J. Pereira, C.P. Santos, F. Antunes, J. Peixoto da-Silva, M. Muler, R.D. Gigli, P.T. Monteforte, H. Hirata, A. Jurkiewicz, S.S. Smaili, Interplay between apoptosis and autophagy, a challenging puzzle: new perspectives on antitumor chemotherapies, Chem. Biol. Interact. 206 (2) (2013) 279–288. [6] J. Li, L. Zhang, Z. Jiang, B. Shu, F. Li, Q. Bao, L. Zhang, Toxicities of aristolochic acid I and aristololactam I in cultured renal epithelial cells, Toxicol. In Vitro 24 (4) (2010) 1092–1097. [7] S. Periyasamy-Thandavan, M. Jiang, P. Schoenlein, Z. Dong, Autophagy: molecular machinery, regulation, and implications for renal pathophysiology, Am. J. Physiol. Renal Physiol. 297 (2) (2009) F244–F256. [8] M. Djavaheri-Mergny, M. Amelotti, J. Mathieu, F. Besancon, C. Bauvy, P. Codogno, Regulation of autophagy by NFkappaB transcription factor and reactive oxygen species, Autophagy 3 (4) (2007) 390–392. [9] I. Tanida, Autophagosome formation and molecular mechanism of autophagy, Antioxid. Redox Signal. 14 (11) (2011) 2201–2214. [10] P. Codogno, S. Pattingre, C. Bauvy, Amino acids interfere with the ERK1/2dependent control of macroautophagy by control-ling the activation of Raf-1 in human colon cancer HT-29 cells, J. Biol. Chem. 278 (19) (2003) 16667– 16674. [11] T. Opriessnig, M.J. Meng, P.G. Halbur, Porcine circovirus type 2 associated disease: update on current terminology, clinical manifestations, pathogenesis, diagnosis, and intervention strategies, J. Vet. Diagn. Invest. 19 (6) (2007) 591– 615. [12] A. Alexander, S.L. Cai, J. Kim, A. Nanez, M. Sahin, K.H. MacLean, K. Inoki, K.L. Guan, J. Shen, M.D. Person, D. Kusewitt, G.B. Mils, M.B. Kastan, C.L. Walker, ATM signals to TSC2 in the cytoplasm to regulate mTORC1 in response to ROS, Proc. Natl. Acad. Sci. U.S.A. 107 (9) (2010) 4153–4158. [13] T. Okazaki, T. Kageji, K. Kuwayama, K.T. Kitazato, H. Mure, K. Hara, R. Morigaki, Y. Mizobuchi, K. Matsuzaki, S. Nagahiro, Upregulation of endogenous PML induced by a combination of interferon-beta and temozolomide enhances p73/ YAP-mediated apoptosis in glioblastoma, Cancer Lett. 323 (2) (2012) 199–207. [14] M. Niikawa, H. Hayashi, T. Sato, H. Nagase, H. Kito, Isolation of substances from glossy privet (Ligustrum lucidum Ait) inhibiting the mutagenicity of benzo[a] pyrene in bacteria, Mutat. Res. 319 (1) (1993) 1–9. [15] A.K. Pathak, M. Bhutani, A.S. Nair, K.S. Ahn, A. Chakraborty, H. Kadara, S. Guha, G. Sethi, B.B. Aggarwal, Ursolic acid inhibits STAT3 activation pathway leading to suppression of proliferation and chemosensitization of human multiple myeloma cells, Mol. Cancer Res. 5 (9) (2007) 943–955. [16] S. Shishodia, S. Majumdar, S. Banerjee, B.B. Aggarwal, Ursolic acid inhibits nuclear factor-kappaB activation induced by carcinogenic agents through suppression of IkappaBalpha kinase and p65 phosphorylation: correlation

631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686


624 623

627 628 629

CBI 7038


8 May 2014 14 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24] [25]

[26]

[27]

[28]

[29]

[30]

[31] [32]

[33]

[34]

[35]

[36]

[37]

[38]

Q3 [39]

S. Shen et al. / Chemico-Biological Interactions xxx (2014) xxx–xxx with down-regulation of cyclooxygenase 2, matrix metalloproteinase 9, and cyclin D1, Cancer Res. 63 (15) (2003) 4375–4383. Y. Zhang, C. Kong, Y. Zeng, L. Wang, Z. Li, H. Wang, C. Xu, Y. Sun, Ursolic acid induces PC-3 cell apoptosis via activation of JNK and inhibition of Akt pathways in vitro, Mol. Carcinog. 49 (4) (2010) 374–385. Y.L. Hsu, P.L. Kuo, C.C. Lin, Proliferative inhibition, cell-cycle dysregulation, and induction of apoptosis by ursolic acid in human non-small cell lung cancer A549 cells, Life Sci. 75 (19) (2004) 2303–2316. K. Sato, K. Tsuchihara, S. Fujii, M. Sugiyama, T. Goya, Y. Atomi, T. Ueno, A. Ochiai, H. Esumi, Autophagy is activated in colorectal cancer cells and contributes to the tolerance to nutrient deprivation, Cancer Res. 67 (20) (2007) 9677–9684. A. Gruzman, G. Babai, S. Ssasson, Adenosine monophosphate-activated protein kinase (AMPK) as a new target for antidiabetic drugs: a review on metabolic, pharmacological and chemical considerations, Rev. Diabetic Stud. 6 (1) (2009) 13–36. H.J. Chung, H.K. Rhee, S.K. Lee, H.Y. Park Choo, Antitumor effects of a novel benzonaphthofurandione derivative (8e) on the human colon cancer cells in vitro and in vivo through cell cycle arrest accompanied with the modulation of EGFR and mTOR signaling, Chem. Biol. Interact. 193 (1) (2011) 43–49. V.K. Wong, T. Li, B.Y. Law, E.D. Ma, C.N. Yip, F. Michelangeli, C.K. Law, M.M. Zhang, K.Y. Lam, P.L. Chan, L. Liu, Saikosaponin-d, a novel SERCA inhibitor, induces autophagic cell death in apoptosis-defective cells, Cell Death Dis. 4 (2013) e720. Y.L. Hsu, L.Y. Wu, P.L. Kuo, Dehydrocostuslactone a medicinal plant-derived sesquiterpene lactone, induces apoptosis coupled to endoplasmic reticulum stress in liver cancer cells, J. Pharmacol. Exp. Ther. 329 (2) (2009) 808–819. J. Li, A.S. Lee, Stress induction of GRP78/BiP and its role in cancer, Curr. Mol. Med. 6 (1) (2006) 45–54. H.P. Harding, Y. Zhang, D. Ron, Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase, Nature 397 (6716) (1999) 271– 274. S. Lépine, J.C. Allegood, M. Park, P. Dent, S. Milstien, S. Spiegel, Sphingosine-1phosphate phosphohydrolase-1 regulates ER stress-induced autophagy, Cell Death Differ. 18 (2) (2011) 350–361. C.W. Younce, P.E. Kolattukudy, MCP-1 causes cardiomyoblast death via autophagy resulting from ER stress caused by oxidative stress generated by inducing a novel zinc-finger protein, MCPIP, Biochemistry 426 (1) (2010) 43–53. T. Rzymski, M. Milani, L. Pike, F. Buffa, H.R. Mellor, L. Winchester, I. Pires, E. Hammond, I. Ragoussis, A.L. Harris, Regulation of autophagy by ATF4 in response to severe hypoxia, Oncogene 29 (31) (2010) 4424–4435. M. Ogata, S. Hino, A. Saito, K. Morikawa, S. Kondo, S. Kanemoto, T. Murakami, M. Taniguchi, I. Tanii, K. Yoshinaga, S. Shiosaka, J.A. Hammarback, F. Urano, K. Imaizumi, Autophagy is activated for cell survival after endoplasmic reticulum stress, Mol. Cell. Biol. 26 (24) (2006) 9220–9231. D. Rodriguez, D. Rojas-Rivera, C. Hetz, Integrating stress signals at the endoplasmic reticulum: the BCL-2 protein family rheostat, Biochim. Biophys. Acta 1813 (4) (2011) 564–574. R.T. Marquez, L. Xu, Bcl-2:Beclin 1 complex: multiple, mechanisms regulating autophagy/apoptosis toggle switch, Am. J. Cancer Res. 2 (2) (2012) 214–221. D. Lu, J. Liu, J. Jiao, B. Long, Q. Li, W. Tan, P. Li, Transcription factor Foxo3a prevents apoptosis by regulating calcium through the apoptosis repressor with caspase recruitment domain, J. Biol. Chem. 288 (12) (2013) 8491–8504. R.M. Kluck, C.A. McDougall, B.V. Harmon, J.W. Halliday, Calcium chelators induce apoptosis–evidence that raised intracellular ionised calcium is not essential for apoptosis, Biochim. Biophys. Acta 1223 (2) (1994) 247–254. J.D. Malhotra, R.J. Kaufman, Endoplasmic reticulum stress and oxidative stress: a vicious cycle or a double-edged sword?, Antioxid Redox Signal. 9 (12) (2007) 2277–2293. L. Yang, X. Liu, Z. Lu, J. Yuet-Wa Chan, L. Zhou, KP. Fung, P. Wu, S. Wu, Ursolic acid induces doxorubicin-resistant HepG2 cell death via the release of apoptosis-inducing factor, Cancer Lett. 298 (1) (2010) 128–138. E.K. Yim, K.H. Lee, S.E. Namkoong, S.J. Um, J.S. Park, Proteomic analysis of ursolic acid-induced apoptosis in cervical carcinoma cells, Cancer Lett. 235 (2) (2006) 209–220. S. Prasad, V.R. Yadav, R. Kannappan, B.B. Aggarwal, Ursolic acid, a pentacyclin triterpene, potentiates TRAIL induced apoptosis through p53-independent upregulation of death receptors: evidence for the role of reactive oxygen species and JNK, J. Biol. Chem. 286 (7) (2011) 5546–5557. S. Leng, Y. Hao, D. Du, S. Xie, L. Hong, H. Gu, X. Zhu, J. Zhang, D. Fan, H. Kung, Ursolic acid promotes cancer cell death by inducing Atg5-dependent autophagy, Int. J. Cancer 133 (12) (2013) 2781–2790. E. Buytaert, G. Callewaert, N. Hendrickx, L. Scorrano, D. Hartmann, L. Missiaen, J.R. Vandenheede, I. Heirman, J. Grooten, P. Agostinis, Role of endoplasmic

[40]

[41]

[42]

[43] [44] [45]

[46]

[47] [48]

[49]

[50]

[51]

[52] [53]

[54]

[55]

[56]

[57]

[58] [59] [60]

reticulum depletion and multidomain proapoptotic Bax and Bak proteins in shaping cell death after hypericin-mediated photodynamic therapy, FASEB J. 20 (6) (2006) 756–758. M. Hoyer-Hansen, L. Bastholm, P. Szyniarowski, M. Campanella, G. Szabadkai, T. Farkas, K. Bianchi, N. Fehrenbacher, F. Elling, R. Rizzuto, I.S. Mathiasen, M. Jäättelä, Control of macroautophagy by calcium, calmodulindependent kinase kinase-b, and Bcl-2, Mol. Cell 25 (2) (2007) 193–205. D.V. Gordienko, T.B. Bolton, Crosstalk between ryanodine receptors and IP(3) receptors as a factor shaping spontaneous Ca(2+)-release events in rabbit portal vein myocytes, J. Physiol. 542 (Pt 3) (2002) 743–762. A. Criollo, M.C. Maiuri, E. Tasdemir, I. Vitale, A.A. Fiebig, D. Andrews, J. Molgó, J. Díaz, S. Lavandero, F. Harper, G. Pierron, D. di Stefano, R. Rizzuto, G. Szabadkai, G. Kroemer, Regulation of autophagy by the inositol trisphosphate receptor, Cell Death Differ. 14 (5) (2007) 1029–1039. T. Verfaillie, M. Salazar, G. Velasco, P. Agostinis, Linking ER stress to autophagy: potential implications for cancer therapy, Int. J. Cell Biol. 2010 (2010) 930509. K. Inoki, T. Zhu, K.L. Guan, TSC2 mediates cellular energy response to control cell growth and survival, Cell 115 (5) (2003) 577–590. L. Ma, N. Niknejad, I. Gorn-Hondermann, K. Dayekh, J. Dimitroulakos, Lovastatin induces multiple stress pathways including LKB1/AMPK activation that regulate its cytotoxic effects in squamous cell carcinoma cells, PLoS One 7 (9) (2012) e46055. J. Merlin, B.A. Evans, R.I. Csikasz, T. Bengtsson, R.J. Summers, D.S. Hutchinson, The M3-muscarinic acetylcholine receptor stimulates glucose uptake in L6 skeletal muscle cells by a CaMKK-AMPK-dependent mechanism, Cell Signal. 22 (7) (2010) 1104–1113. M. Høyer-Hansen, M. Jäättelä, AMP-activated protein kinase: a universal regulator of autophagy?, Autophagy 3 (4) (2007) 381–383 X. Tong, K.A. Smith, J.C. Pelling, Apigenin, a chemopreventive bioflavonoid, induces AMP-activated protein kinase activation in human keratinocytes, Mol. Carcinog. 51 (3) (2012) 268–279. C.W. Park, S.M. Hong, E.S. Kim, J.H. Kwon, K.T. Kim, H.G. Nam, K.Y. Choi, 25BNIP3 is degraded by ULK1-dependent autophagy via MTORC1 and AMPK, Autophagy 9 (3) (2013) 345–360. A. Grotemeier, S. Alers, S.G. Pfisterer, F. Paasch, M. Daubrawa, A. Dieterle, B. Viollet, S. Wesselborg, T. Proikas-Cezanne, B. Stork, AMPK-independent induction of autophagy by cytosolic Ca2+ increase, Cell Signal. 22 (6) (2010) 914–925. Z. Tallóczy, W.X. Jiang, H.W. Virgin, D.A. Leib, D. Scheuner, R.J. Kaufman, E.L. Eskelinen, B. Levine, Regulation of starvation- and virus-induced autophagy by the eIF2a kinase signaling pathway, Proc. Natl. Acad. Sci. U.S.A. 99 (1) (2002) 190–195. M.A. Garcia, E.F. Meurs, M. Esteban, The dsRNA protein kinase PKR: virus and cell control, Biochimie 89 (6–7) (2007) 799–811. Y. Kouroku, E. Fujita, I. Tanida, T. Ueno, A. Isoai, H. Kumagai, S. Ogawa, R.J. Kaufman, E. Kominami, T. Momoi, ER stress (PERK/eIF2a phosphorylation) mediates the polyglutamine-induced LC3 conversion, an essential step for autophagy formation, Cell Death Diff. 14 (2) (2007) 230–239. K.M. Rouschop, T. van den Beucken, L. Dubois, H. Niessen, J. Bussink, K. Savelkouls, T. Keulers, H. Mujcic, W. Landuyt, J.W. Voncken, P. Lambin, A.J. van der Kogel, M. Koritzinsky, B.G. Wouters, The unfolded protein response protects human tumor cells during hypoxia through regulation of the autophagy genes MAP1LC3B and ATG5, J. Clin. Invest. 120 (1) (2010) 127–141. M. Ito, H. Nakagawa, T. Okada, S. Miyazaki, S. Matsuo, ER-stress caused by accumulated intracistanal granules activates autophagy through a different signal pathway from unfolded protein response in exocrine pancreas cells of rats exposed to fluoride, Arch. Toxicol. 83 (2) (2009) 151–159. J.F. Raven, D. Baltzis, S. Wang, Z. Mounir, A.I. Papadakis, H.Q. Gao, A.E. Koromilas, PKR and PKR-like endoplasmic reticulum kinase induce the proteasome-dependent degradation of cyclin D1 via a mechanism requiring eukaryotic initiation factor 2alpha phosphorylation, J. Biol. Chem. 283 (6) (2008) 3097–3108. S.R. Stockwell, G. Platt, S.E. Barrie, G. Zoumpoulidou, R.H. Te Poele, G.W. Aherne, S.C. Wilson, P. Sheldrake, E. McDonald, M. Venet, C. Soudy, F. Elustondo, L. Rigoreau, J. Blagg, P. Workman, M.D. Garrett, S. Mittnacht, Mechanism-based screen for G1/S checkpoint activators identifies a selective activator of EIF2AK3/PERK signalling, PLoS One 7 (1) (2012) e28568. C. Hetz, L.H. Glimcher, Fine-tuning of the unfolded protein response: assembling the IRE1alpha interactome, Mol. Cell 35 (5) (2009) 551–561. T. Shintani, D.J. Klionsky, Autophagy in health and disease: a double-edged sword, Science 306 (5698) (2004) 990–995. J.H. Lee, E.J. Kwon, H. Kim do, Calumenin has a role in the alleviation of ER stress in neonatal rat cardiomyocytes, Biochem. Biophys. Res. Commun. 439 (3) (2013) 327–332.

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Baicalein induces apoptosis and autophagy via endoplasmic reticulum stress in hepatocellular carcinoma cells.

Palmitate induces autophagy in pancreatic β-cells via endoplasmic reticulum stress and its downstream JNK pathway.

Endoplasmic reticulum stress induces epithelial-mesenchymal transition through autophagy via activation of c-Src kinase.

Ursolic acid prevents endoplasmic reticulum stress-mediated apoptosis induced by heat stress in mouse cardiac myocytes.

Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis.

Bufalin induces the interplay between apoptosis and autophagy in glioma cells through endoplasmic reticulum stress.

Trinitrotoluene Induces Endoplasmic Reticulum Stress and Apoptosis in HePG2 Cells.

CHOP favors endoplasmic reticulum stress-induced apoptosis in hepatocellular carcinoma cells via inhibition of autophagy.

Sertraline induces endoplasmic reticulum stress in hepatic cells.

Naloxone induces endoplasmic reticulum stress in PC12 cells.

Glycolaldehyde induces endoplasmic reticulum stress and apoptosis in Schwann cells.

Choline kinase inhibition induces exacerbated endoplasmic reticulum stress and triggers apoptosis via CHOP in cancer cells.

Dictyopteris undulata Extract Induces Apoptosis via Induction of Endoplasmic Reticulum Stress in Human Colon Cancer Cells.

Helicobacter pylori vacuolating cytotoxin induces apoptosis via activation of endoplasmic reticulum stress in dendritic cells.

Ursolic acid inhibits the development of nonalcoholic fatty liver disease by attenuating endoplasmic reticulum stress.

Emodin induces apoptosis of human osteosarcoma cells via mitochondria- and endoplasmic reticulum stress-related pathways.

Homocysteine Induces Apoptosis of Human Umbilical Vein Endothelial Cells via Mitochondrial Dysfunction and Endoplasmic Reticulum Stress.

T. gondii rhoptry protein ROP18 induces apoptosis of neural cells via endoplasmic reticulum stress pathway.

Fluoxetine induces cytotoxic endoplasmic reticulum stress and autophagy in triple negative breast cancer.

Palmitate induces endoplasmic reticulum stress and autophagy in mature adipocytes: implications for apoptosis and inflammation.

Efavirenz Causes Oxidative Stress, Endoplasmic Reticulum Stress, and Autophagy in Endothelial Cells.

Interplay of endoplasmic reticulum stress and autophagy in neurodegenerative disorders.

Antitumor agent 25-epi Ritterostatin GN1N induces endoplasmic reticulum stress and autophagy mediated cell death in melanoma cells.

H+ exchanger-1 reduces podocyte injury caused by endoplasmic reticulum stress via autophagy activation.