THE ANATOMICAL RECORD 00:00–00 (2014)

Inhibition of JNK3 Promotes Apoptosis Induced by BH3 Mimetic S1 in Chemoresistant Human Ovarian Cancer Cells XIAOCHUN YANG,1 XIYAN XIANG,1 MEIHUI XIA,2 JING SU,1 YAO WU,1 LUYAN SHEN,1 YE XU,3,4* AND LIANKUN SUN1 1 Department of Pathophysiology, Basic College of Medicine, Jilin University, Changchun 130021, People’s Republic of China 2 Department of Obstetrics and Gynecology, First Hospital, Jilin University, Changchun 130021, People’s Republic of China 3 Medical Research Laboratory, Jilin Medical College, Jilin 132013, People’s Republic of China 4 Department of Histology and Embryology, Jilin Medical College, Jilin 132013, People’s Republic of China

ABSTRACT Previous studies have suggested that the novel BH3 mimetic S1 could induce apoptosis in diverse tumor cell lines through endoplasmic reticulum (ER) stress or mitochondrial cell death pathways. The activation of c-Jun N-terminal kinase (JNK) through inositol requiring enzyme-1 (IRE1) is closely connected to ER stress-induced apoptosis. However, the role of JNK is complex, as there are different JNK subtypes and the function of each subtype is still not entirely clear. Here we found that the mRNA expression of JNK3 was continuously high in S1-treated human ovarian cancer SKOV3/DDP cells using a human unfolded protein response (UPR) pathway PCR array. Pharmacological inhibition of JNK3 increased cell sensitivity to apoptosis induced by S1. Furthermore, inhibition of JNK3 induced accumulation of both acidic compartment and p62, and upregulated ROS production. Our results suggest that JNK3 plays a pro-survival role during ER stress through preventing the block of autophagic flux and reducing oxidative stress in SKOV3/DDP cells. Inhibition of JNK3 may be a potential method to enhance the killing effect of the Bcl-2 inhibitor S1. Anat Rec, 00:000–000, 2014. VC 2014 Wiley Periodicals, Inc.

Key words: BH3 mimetic; JNK3; endoplasmic reticulum stress; autophagy; apoptosis; oxidative stress

Additional Supporting Information may be found in the online version of this article. Grant sponsor: National Natural Science Foundation of China; Grant numbers: 81272876, 81372793, 81202552. *Correspondence to: Dr. Ye Xu, Medical Research Laboratory, Jilin Medical College, 5 Jilin Street, Jilin 132013, PR China. E-mail: [email protected] or Dr. Liankun Sun, Department C 2014 WILEY PERIODICALS, INC. V

of Pathophysiology, Basic College of Medicine, Jilin University, 126 Xinmin Street, Changchun 130021, People’s Republic of China. E-mail: [email protected] Received 28 March 2014; Accepted 29 May 2014. DOI 10.1002/ar.22991 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com).

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Epithelial ovarian carcinoma is considered the most lethal gynecologic malignancy, as 70% of patients eventually develop recurrent tumors that are resistant to common chemotherapy drugs that induce apoptosis, such as cisplatin (Siegel et al., 2012). Apoptosis is strictly regulated by the interaction between anti-apoptotic and proapoptotic Bcl-2 family members via their BH3 domain. Levels of the anti-apoptotic Bcl-2 family proteins are high in most tumors, including ovarian carcinoma, which may contribute to apoptosis resistance (Herod et al., 1996; Brotin et al., 2010). Recently, specific inhibitors targeting the anti-apoptotic Bcl-2 family members were applied in tumor therapy, and studies of these inhibitors have been increasing. A series of BH3 mimetic small chemicals have been developed, such as ABT-737/ABT-263 (Wong et al., 2012), Obatoclax (Cruickshanks et al., 2012), TW-37 (Verhaegen et al., 2006) and AT-101 (Karaca et al., 2013), which can bind the anti-apoptotic Bcl-2 family members, thereby dissociating Bax/Bak from anti-apoptotic proteins and ultimately triggering apoptosis. The novel BH3 mimetic S1 could bind both Bcl-2 and Mcl-1 with high affinity (Zhang et al., 2011). Furthermore, studies have shown that S1 may induce apoptosis in many cancer cell lines, including U251 (Zhong et al., 2012), K562 (Song et al., 2012a), SMMC-7721 (Zhang et al., 2011), SCLC (Liu et al., 2013b), SKOV3 and SKOV3/DDP (Liu et al., 2013a). However, clarification of the mechanisms involved in the killing effects of S1 is required for better understanding of its function and to improve its therapeutic effect. Many factors may contribute to the induction of endoplasmic reticulum (ER) stress in cancer cells, and the extent of ER stress may control cell fate. A previous study found that pro-survival signaling pathways were activated by the mild ER stress process in glioma and smooth muscle cells (Raciti et al., 2012; Yi et al., 2012). However, in breast, colon and lung cancer cells, severe ER stress may induce apoptosis (Sanchez-Lopez et al., 2013). Our previous studies indicated that S1 could induce apoptosis through the ER stress pathway in glioma and ovarian cancer cells (Zhong et al., 2012; Liu et al., 2013a). Shore et al. proposed that the role of ER stress in apoptosis induction may depend on its extent and subsequent activation of different signaling pathways (Xu et al., 2005; Shore et al., 2011; Xu et al., 2012). Studies have shown that the main ER stress signaling pathway involving inositol-requiring kinase 1 (IRE1) may induce apoptosis though activating c-Jun N-terminal kinase (JNK) (Dasmahapatra et al., 2009; Kato et al., 2012). Other reports demonstrated that ER stressinduced autophagy depended on the activation of the IRE1-JNK pathway (Ogata et al., 2006; Kato et al., 2012). Additionally, JNK signaling pathway was closely linked to reactive oxygen species (ROS) regulation (Han et al., 2009). These studies suggested that JNK pathway may affect cell fate by regulating autophagy and ROS. Our previous studies indicated that S1 may trigger apoptosis through the IRE1-JNK pathway and activate autophagy, which may oppose the damaging effect of apoptosis (Zhong et al., 2012; Liu et al., 2013a). However, the exact JNK subtype that is involved in the regulation of autophagy and oxidative stress remains unclear. In this study, we used a specific inhibitor of JNK3 (SR-3576, a pyrazolourea compound, or XII) to precisely clarify the role of JNK3 (Kamenecka et al., 2009; Goeroegh et al., 2013). We evaluated changes in expression of genes in the UPR signaling pathway in S1-treated

SKOV3/DDP cells using a high-throughput screening method, and found that the expression of JNK3 gene was continuously high. To clarify the role of JNK3 upregulation in drug resistance, the specific inhibitor XII was used in combination with S1, and the results indicated that inhibition of JNK3 aggravated oxidative stress and blocked the autophagic flux, thereby sensitizing SKOV3/DDP cells to the apoptosis induced by S1.

MATERIALS AND METHODS Reagents and Antibodies The BH3 mimetic S1 was supplied by Professor Zhichao Zhang (Dalian University of Technology, Dalian, China) and dissolved in dimethyl sulfoxide (DMSO). LysoTracker Red DND-99, fetal bovine serum (FBS) and Roswell Park Memorial Institute (RPMI)-1640 culture medium were purchased from Invitrogen (Carlsbad, CA). The in situ cell death detection kit was purchased from Roche (Indianapolis, IN). Acridine orange (AO), 3-(4,5dimetrylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), N-acetylcysteine (NAC) and the JNK3 inhibitor XII were purchased from Sigma (St. Louis, MO). JC-1 fluorescent dye was purchased from KeyGen Biotechnology (Nanjing, China). The ROS indicator 5-(and -6)diacetate chloromethyl-20 ,70 -dichlorodihydrofluorescein acetyl ester (CM-H2DCFDA) was purchased from Molecular Probes (Eugene, OR). Enhanced chemiluminescence (ECL) reagents were from Thermo Scientific (Rockford, IL). Anti-CHOP, anti-p62, anti-Grp78, anti-LC3, and anti-caspase-3 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-JNK3, anti-b-actin and horseradish peroxidase-conjugated antirabbit- and anti-mouse-immunoglobulin were purchased from Proteintech (Chicago, IL). All reagents and antibodies were purchased by Changchun Baoxin Biotechnology Company (Changchun, China).

Cell Culture Human ovarian cancer cisplatin-resistant SKOV3/ DDP cells were obtained from the Chinese Academy of Medical Sciences and Peking Union Medical College. Cell lines were cultured at 37 C under 5% CO2 in RPMI1640 culture medium supplemented with 10% FBS, with a change of medium after each 2-day interval and split twice a week by trypsinisation. Cells were maintained as resistant in media containing 1 mg mL21 cisplatin. All experiments were performed at 70% of confluence.

Cell Viability Assays Cells were plated in 96-well plates at a density of 1 3 104 cells/well in 200 lL of complete medium. Each treatment was repeated in six separate wells. Cells were treated with 10 mM S1 and/or 2 mM JNK3 inhibitor XII for 24 h. Then, 20 lL of 5 mg mL21 MTT reagent in phosphate buffered saline (PBS) was added to each well and incubated for 4 h. Formazan crystals were dissolved in 150 lL DMSO. Absorbance was recorded at a wavelength of 490 nm using a Microplate Reader (Bio-Rad Instruments, Hercules, CA). The cell viability was calculated as (%) 5 absorbance of experimental group/absorbance of control group 3 100%. In each experiment, we calculated the mean value of six wells per treatment group.

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Real Time qPCR

TUNEL Assays

SKOV3/DDP cells were treated with 10 lM S1 for 0, 3, 6, 12, and 24 h, and then total RNA was extracted from cultured cells using an RNeasyV Mini Kit (SABiosciences-Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Single-stranded cDNA was obtained by reverse transcription of 1 lg of total RNA using the RT2 First Strand Kit (SABiosciences-Qiagen). cDNA was amplified by Real Time qPCR using RT2 SYBR Green Mastermix (SABiosciences-Qiagen). The primers were designed with Primer 5 software and the corresponding sequences are as follows: JNK3 (NM_002753) forward 50 -ATCCTGGGGATG GGCTACA-30 and reverse 50 -GAAGAGTTTGGGGAAG GTGAGT-30 ; GAPDH (NM_002046) forward 50 -CATCAAG AAGGTGGTGAAGCAG-30 and reverse 50 -CGTCAAAGGT GGAGG AGTGG-30 .

Apoptosis analysis was performed using the in situ cell death detection kit according to the manufacturer’s instructions. SKOV3/DDP cells were cultured on cover slips, washed three times with cold PBS, fixed in 4% (w/ v) paraformaldehyde/PBS for 30 min at room temperature, and then washed again three times with cold PBS. Cells were incubated with the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) reaction mix containing 10 U of terminal deoxyribosyl transferase, 10 mM dUTP biotin, and 2.5 mM cobalt chloride in 13 terminal transferase reaction buffer for 1 h at 37 C in a humidified atmosphere. Apoptotic cells with characteristic nuclear fragmentation (staining green) were counted in six randomly chosen fields. The experiment was repeated three times.

RT2 Profiler PCR Array System

Acidic Compartment Evaluation by AO

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The Human Unfolded Protein Response RT ProfilerTM PCR Array (SABiosciences-Qiagen, Hilden, Germany) profiles the expression of 84 key genes involved in unfolded protein accumulation in the ER. Total cellular RNA was extracted from cultured cells according to the manufacturer’s instructions. Singlestranded cDNA was obtained by reverse transcription of 1 lg of total RNA using the SABiosciences RT2 First Strand Kit. Real Time qPCRs were performed using Applied Biosystems 7300 Fast with SYBR Green Fluorophore. The reactions were carried out using RT2 SYBR Green Mastermix. cDNA was used as template and cycling parameters were 95 C for 10 min, followed by 40 cycles of 95 C for 15 s and 60 C for 1 min. Fluorescence intensities were analyzed using the manufacturer’s software, and relative quantification was calculated using the 22DDCt method. Change of expression of the 84 genes was shown by heat imaging. GAPDH was used as a reference gene.

Western Blot Analysis Cells were washed with PBS twice and harvested by scraping into 300 lL of RIPA lysis buffer. Cell lysates were ultrasonicated for 15 s on ice and then lysed at 4 C for 45 min and centrifuged at 12,000g for 10 min. Protein concentrations in the supernatants were determined by the Bio-rad reagent (Hercules, CA). Equal amounts of protein samples (30 lg) were separated by SDS-PAGE in duplicate and blotted onto PVDF membranes (Millipore, Billerica, MA). Transfer efficiency was checked with Ponceau staining. The blots were blocked in Trisbuffered saline containing 5% (w/v) nonfat dry milk and probed with specific primary antibodies overnight at 4 C. The membranes were washed with PBS-Tween-20 and then incubated with a peroxidase-conjugated secondary antibody for 2 h at room temperature. Duplicated membranes were probed for b-actin expression to ensure equal input of cell lysate proteins. The final dilutions and incubation times suggested by the manufacturer were used for each antibody. Immunodetection was performed using the ECL reagents and images were captured by Syngene Bio Imaging (Synoptics, Cambridge, UK). Densitometry quantitation of the bands was also performed using Syngene Bio Imaging.

Autophagy is characterized by the formation of acidic vesicular organelles (autophagosomes and autolysosomes). AO, a fluorescent weak base, causes these acidic compartments to fluoresce bright red, and the cytoplasm and nucleolus to fluoresce bright green and dim green, respectively. Cells were plated on coverslips. AO staining (100 lg mL21) was performed in the dark for 15 min at room temperature after cells were washed by PBS supplemented with 5% FBS. Staining was performed in the presence of drugs or vehicle. Cells were then washed twice with PBS, and examined by laser-scanning confocal microscopy (FV 1000, Olympus, Tokyo, Japan).

Acidic Compartment Evaluation by LysoTracker Red Cells were incubated for 15 min in PBS containing LysoTracker Red DND-99 (100 nM), a fluorescent acidotropic probe with high selectivity for acidic organelles, showing good retention after aldehyde fixation. PBSwashed cells were fixed with paraformaldehyde (4%) in PBS for 30 min at room temperature, washed twice with PBS, and analyzed by laser-scanning confocal microscopy (FV 1000, Olympus).

Detection of ROS Production SKOV3/DDP cells were seeded onto glass culture slides (BD Biosciences, Bedford, MA) and treated with the indicated drugs. At various time points, cells were loaded with 1 lM CM-H2DCFDA in PBS for 10 min at 37 C in the dark followed by a PBS wash step. DCFdependent fluorescence was examined by laser-scanning confocal microscopy (FV 1000, Olympus).

Assessment of Mitochondrial Depolarization Pretreated SKOV3/DDP cells were collected and suspended in 1 mL of complete medium containing 10 lg JC-1 (5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethyl mL21 benzimidazolyl carbocyanine iodide) for 30 min at 37 C. To assess the mitochondrial membrane potential (MMP), 1 3 104 cells/sample stained by JC-1 were evaluated using flow cytometry (BD Biosystems, Franklin Lakes, NJ).

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Fig. 1. BH3 mimetic S1 upregulated the expression of JNK3. (A) SKOV3/DDP cells were incubated with 10 mM S1 for 3 and 24 h, and the expression of the UPR pathway genes (84 genes) were examined using a PCR array. For genes name detail see Supporting Information. Changes are presented as heat images. Green indicates downregulation, and red indicates upregulation. Data are derived from three experiments. (B) JNK3 mRNA changes determined by real-time PCR

at different time points after 10 mM S1 treatment. Values represent mean and SD (n 5 3). *P < 0.05 compared with control group. (C) Western blot assay of JNK3 protein expression at different time points after 10 mM S1 treatment. (D) Quantitative analysis of JNK3 protein level. Values represent mean and SD (n 5 3), *P < 0.05 compared with control group.

Statistical Analysis

Unfolded Protein Response RT2 ProfilerTM PCR Array. The array results showed that many ER stress chaperone mRNA levels were significantly upregulated more than twofold at 3 h of treatment with S1, including glucose-regulated protein-78 (Grp78; position D4, HSPA5), IRE1 (position C2, ERN1), ATF6 (position A3) and PERK (position C1, EIF2AK3) (Fig. 1A, left panel). At 24 h, most of the genes were downregulated and only a few including JNK3 (position D11, MAPK10) were upregulated (Fig. 1A, right panel). The expression of JNK3 remained constantly high with an increase of more than 2-fold at both 3 and 24 h. We next confirmed changes of JNK3 mRNA levels by real-time PCR analysis at various times after treatment with S1, and these results showed higher than twofold changes in JNK3 mRNA expression from 3 to 24 h (Fig. 1B). The expression of JNK3 protein was also continuously upregulated (Fig. 1C,D). Together these results indicate that the BH3

Results are expressed as the mean 6 standard deviation (SD) of repeated experiments, as indicated in the figure legends. Data are representative of three independent experiments performed in triplicate. Statistical analysis of the data was performed using one-way ANOVA. The Tukey post hoc test was used to determine the significance for all pairwise comparisons of interest. Differences were considered statistically significant for values of P < 0.05.

RESULTS BH3 Mimetic S1 Upregulates the Expression of JNK3 in SKOV3/DDP Cells SKOV3/DDP cells were treated with 10 mM S1 for 3 h and 24 h. The expression levels of genes in the UPR pathway (84 genes) were examined using the Human

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mimetic S1 may induce ER stress and upregulates JNK3 expression.

Inhibition of JNK3 Promotes Apoptosis Induced by S1 To define the impact of JNK3 on the cytotoxic effect produced by S1, we used the specific JNK3 inhibitor XII and examined cellular morphological changes under an optical microscope. Compared with the group treated with S1 alone, there was a significant decrease in cell number and an increase in cell shrinkage and detachment in the combined S1 and XII treatment group (Fig. 2A). Cell viability was measured by MTT assay, and the results demonstrated that XII increased the cell death induced by S1 (Fig. 2B). The TUNEL assay was used to detect DNA fragmentation, which represents the occurrence of apoptosis. The apoptosis rate of S1-treated SKOV3/DDP cells was higher than that of the control group, and JNK inhibition further increased the rate of apoptotic cells after S1 treatment (Fig. 2C,D). Disturbance of ER homeostasis may lead to activation of the UPR pathway. Cell fate during UPR is often affected by the ratio of pro-survival Grp78 and proapoptotic CCAAT/enhancer-binding protein homologous protein (CHOP). Interestingly, the expressions of both Grp78 and CHOP were increased in S1-treated cells after 12 h, while inhibition of JNK3 altered their expression patterns: the expression of CHOP increased significantly after 12 h, while Grp78 levels declined. This suggests that JNK3 may have a potential role in regulating the expression of the two proteins (Fig. 2E,F). Therefore, inhibition of JNK3 may promote SKOV3/DDP cell sensitivity to apoptosis induced by S1 and lead to ER stress-mediated apoptosis.

Inhibition of JNK3 Promotes Acidic/Lysosomal Compartment Accumulation and Blocks the AutophagicFlux Previous studies showed that autophagy during ER stress induced by S1 may reduce apoptosis (Zhong et al., 2012). To further confirm the role of JNK3 in S1-induced autophagy, we evaluated the change of acidic compartments and autophagy markers. The fluorescent dye AO was used to indicate the acidic compartments. After entering these structures, the AO dye emits bright red light. Compared with the control group, red fluorescence was increased in the S1-treated group (Fig. 3A), while S1 combined with XII further increased the fluorescence (Fig. 3A). LysoTracker Red DND-99, a selective probe, was also used to stain the acidic organelles. The changes of LysoTracker Red fluorescence were similar to the AO staining: the fluorescence was increased in the S1-treated group and a stronger fluorescence was observed in the S1 combined with XII group (Fig. 3B). These results suggest that an increase of the acidic compartment is induced by S1, which is further increased after JNK3 inhibition, indicating that JNK3 is a key regulator of S1-induced autophagy in SKOV3/DDP cells. However, the cause of the observed increase in the acidic compartment remained unclear, as upregulated formation of autophagosomes and impaired autophagic degradation pathway could both lead to increased acidic

compartments. To confirm the regulatory role of JNK3 in the autophagy process, the expression of the autophagy marker microtubule-associated protein light chain 3 (LC3) after S1 and/or XII treatments was determined by western blot. The results showed that expression of phosphatidylethanolamine-conjugated microtubule-associated protein 1 light chain 3 (LC3-II) was increased (Fig. 3C,D), indicating that both S1 alone and S1 combined with XII treatment may upregulate the formation of autophagosomes. Because the upregulation of LC3-II may be due to increased formation of autophagosomes or reduced degradation of autophagic substrates, we examined protein p62, which is an important indicator of autophagic flux. The results showed a significant increase of p62 after S1 combined with XII treatment (Fig. 3C,D). Together these results indicate that JNK3 functions as a regulator of the final steps of autophagy, therefore preventing autolysosome and lysosome accumulation.

Inhibition of JNK3 Aggravating Oxidative Stress Contributes to Cell Death Previous studies showed that BH3 mimetics disturbed cellular redox homeostasis, as they promoted ROS generation, in which the JNK pathway may be involved (Han et al., 2009). To confirm the role of JNK3 in S1-induced ROS generation, the CM-H2DCFDA probe was used to detect ROS levels in SKOV3/DDP cells. Compared with the control group, there was an increase of green fluorescence in the S1 group (Fig. 4A), and a further increase was observed in the S1 combined with XII group (Fig. 4A). Accumulation of ROS may cause mitochondrial damage and lead to loss of MMP. We evaluated MMP by flow cytometry with JC-1 staining. In comparison with untreated control cells, S1 induced a decline in the MMP (Fig. 4B,C) and the S1 combined with XII group showed a further decline. During apoptosis, the loss of MMP results in an increase in mitochondrial membrane permeability and the release of cytochrome C followed by activation of caspase cascades. To examine the effect of ROS on cell survival, we evaluated cell viability. The results showed that the decline of cell viability in the S1 combined with XII group was reversed by pretreatment with the antioxidant N-acetylcysteine (NAC) for 1 h (Fig. 4D). These data suggested that inhibition of JNK3 aggravating oxidative stress contributed to cell death.

DISCUSSION Numerous studies have explored the role of JNK activation during stress; however no consensus has been reached. Indeed, stress-induced JNK activation is involved in various cellular processes, such as proliferation, cell survival and apoptosis (Heasley and Han, 2006; Kaminska et al., 2009; Tang et al., 2013). Even though multiple studies have uncovered the factors that may affect the outcome of JNK activation, the contribution of different JNK subtypes during these processes is worth considering. Experimental data of JNK are mostly derived from the use of broad-spectrum inhibitors such as SP600125, which made it difficult to distinguish the

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Fig. 2. Inhibition of JNK3 promoted apoptosis induced by S1. (A) Cell morphology was observed using an optical microscope. Treatment groups: 10 mM S1, 2 mM XII, and 10 mM S1 plus 2 mM XII for 24 h. Bar: 50 lm. (B) Apoptosis evaluated by TUNEL assays in SKOV3/ DDP cells treated with 10 mM S1, 2 mM XII, and 10 mM S1 plus 2 mM XII for 24 h (bar: 50 lm). (C) MTT assay was used to measure cell viability. Treatment groups: 10 mM S1, 2 mM XII, and 10 mM S1 plus 2 mM XII for 24 h. Values represent mean and SD (n 5 6), *P < 0.05 compared with the control group, #P < 0.05 compared with the S1 group.

(D) Apoptosis rate of SKOV3/DDP cells represented by the percentage of TUNEL-positive cells. Values represent mean and SD (n 5 3). *P < 0.05 compared with the control group, #P < 0.05 compared with the S1 group. (E) Western blot assay of Grp78 and CHOP protein expression. Treatment groups: 10 mM S1, 2 mM XII, and 10 mM S1 plus 2 mM XII for 12 h. (F) Quantitative analysis of Grp78 and CHOP protein expression. Values represent mean and SD (n 5 3). *P < 0.05 compared with the control group, #P < 0.05 compared with the S1 group.

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Fig. 3. Inhibition of JNK3 promoting acidic/lysosomal compartment accumulation and blocking the autophagic flux. (A) SKOV3/DDP cells stained by AO after treatment with 10 mM S1, 2 mM XII, or 10 mM S1 plus 2 mM XII for 12 h. Acidic compartments were observed by laserscanning confocal microscopy. Bar: 10 lm. (B) SKOV3/DDP cells stained by LysoTracker Red after treatment with 10 mM S1, 2 mM XII, or 10 mM S1 plus 2 mM XII for 12 h. Acidic compartments were

observed by laser-scanning confocal microscopy. Bar: 20 lm. (C) Western blot assay of LC3 and p62 protein expression. Treatment groups: 10 mM S1, 2 mM XII, and 10 mM S1 plus 2 mM XII for 12 h. (D) Quantitative analysis of LC3-II and p62 protein expression. Values represent mean and SD (n 5 3). *P < 0.05 compared with the control group, #P < 0.05 compared with the S1 group.

roles of the JNK subtypes (Cui et al., 2009; Zhao et al., 2012). JNK is one of the three mitogen-activated protein kinase (MAPK) family members, and several JNK subtypes are expressed in mammals. Specific inhibition of JNK1 induced apoptosis in ovarian and liver cancer cells and restrained the growth of xenograft tumors (Hui et al., 2008; Vivas-Mejia et al., 2010). The ER stress inducers tunicamycin and thapsigargin upregulate the expression of JNK2, which promoted cell survival through regulation of ER stress and autophagy (Raciti et al., 2012). Moreover, JNK2 negatively regulated the inhibitory signaling pathways of cell cycle (Ke et al., 2010). Deletion of JNK3 could lead to the development of brain tumors, while specific inhibition of JNK3 may conversely improve chemotherapeutic effects in head

and neck squamous cell carcinoma (Goeroegh et al., 2013). Together these data indicate that different subtypes of JNK may exert diverse roles in controlling cell fate. Additionally, a previous study reported the tissuespecific distribution of JNK subtypes: different from the wide expression of JNK1 and JNK2, JNK3 is mainly expressed in the brain, heart and testis (Gupta et al., 1996). Compared with JNK1 and JNK2, the understanding of JNK3 is still scarce. In this study, we found that ER stress induced by the BH3 mimetic S1 in SKOV3/DDP cells led to sustained upregulation of JNK3. Although activation of JNK3 in ischemia brain tissue and head and neck squamous cell carcinoma induced apoptosis (Song et al., 2012b; Goeroegh et al., 2013), pharmacological inhibition of JNK3 by XII promoted increased sensitivity of SKOV3/DDP cells

Fig. 4. Inhibition of JNK3 aggravating oxidative stress contributed to cell death. (A) Accumulation of ROS was evaluated by CM-H2DCFDA probe staining and observed by immunofluorescence microscopy. Treatment groups: 10 mM S1, 2 mM XII, and 10 mM S1 plus 2 mM XII for 12 h. Bar: 20 lm. (B) MMP evaluated by JC-1 staining and flow cytometry. Treatment groups: 10 mM S1, 2 mM XII, and 10 mM S1 plus 2 mM XII for 12 h. FL1-H: JC-1 green. FL2-H: JC-1 red. (C) Statistics

of MMP loss. Values represent mean and SD (n 5 3). *P < 0.05 compared with the control group, #P < 0.05 compared with the S1 group. (D) After pretreatment with 20 mM NAC for 1 h, cells were treated with 10 mM S1, 2 mM XII, or 10 mM S1 plus 2 mM XII for 24 h. MTT assay was used to measure cell viability. Values represent mean and SD (n 5 6). *P < 0.05 compared with the S1 group, #P < 0.05 compared with the S11XII group.

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to S1, which revealed a pro-survival role of JNK3 in S1induced ER stress. There are three interrelated pathways that are involved in the regulation of gene transcription during ER stress, and are regulated by PERK, ATF6 and IRE1, respectively. The sensors of these pathways monitor the accumulation of unfolded or misfolded proteins in the lumen of the ER and transmit this information to the nucleus. The IRE1 pathway may lead to downstream events such as autophagy or apoptosis and JNK activation (Oh and Lim, 2009; Kim et al., 2013). Previous reports have explored the link between IRE1 and JNK (Zhong et al., 2012), and in the current study we pinpointed the regulatory role of JNK3 in S1-induced ER stress. We found that the expression of Grp78 is positively correlated with JNK3, while the expression of CHOP was negatively correlated with JNK3. In fact, inhibition of JNK3 resulted in changes in both Grp78 and CHOP expression, downregulating the expression of the former and upregulating the latter. This suggests that JNK3 may act as a switch of cell fate decision, as the anti-apoptotic protein Grp78 inhibits cell death induced by ER stress and the transcription factor CHOP is involved in induction of apoptosis. Previous studies proposed that the phosphorylation of Bcl-2 induced by JNK may disrupt the Bcl-2/Beclin-1 complex and promote the formation of autophagosomes, and the LC3 punctate aggregation would decrease once the JNK pathway was inhibited (Ogata et al., 2006; Wei et al., 2008). These data indicate that JNK has a role in enhancing autophagy. We presumed that JNK3 inhibition may impair autophagic flux. Indeed, inhibition of JNK3 promotes the accumulation of the cellular acidic compartment. In addition, we also observed that the autophagic flux indicator p62 significantly increased after JNK3 inhibition. Together this suggests that JNK3 act as a specific regulator of autophagic flux in S1induced ER stress. Many BH3 mimetics such as ABT-737, Gossypol and Obatoclax can stimulate generation of ROS (Howard et al., 2009; Sung et al., 2010; Cruickshanks et al., 2012). ROS may aggravate ER stress and lead to the dysfunction of mitochondria, leading to induction of apoptosis (Appierto et al., 2009; Choi et al., 2013; Yu et al., 2013). Studies demonstrated that inhibition of JNK may increase the production of ROS (Han et al., 2009). As the JNK pathway may regulate ROS, we presume that inhibition of JNK3 could affect the generation of ROS and follow-up oxidative damage. In fact, we observed increased ROS generation in response to S1, and it significantly increased after S1 combined with XII treatment. Increased ROS damages mitochondrial membrane structures and causes the loss of MMP (Wong et al., 2006). Compared with S1 treatment, we detected significantly reduced MMP after S1 combined with XII treatment. Accordingly, blocking the synergistic effect of S1 combined with XII by the antioxidant NAC significantly reduced SKOV3/DDP cell death. Therefore, JNK3 may exert pro-survival signals by reducing oxidative stress injury. Together this study suggests that activation of JNK3 induced by S1 may increase autophagic flux and decrease oxidative stress, thus promoting SKOV3/DDP cell survival. This indicates that JNK3 may be a potential target of tumor suppression networks.

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