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p38 MAPK pathway
Journal of Pharmacological Sciences xxx (2015) 1e9
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Diarachidonoylphosphoethanolamine induces apoptosis of malignant pleural mesothelioma cells through a Trx/ASK1/p38 MAPK pathway Ayako Tsuchiya a, Yoshiko Kaku a, Takashi Nakano b, Tomoyuki Nishizaki a, * a b
Division of Bioinformation, Department of Physiology, Hyogo College of Medicine, Nishinomiya 663-8501, Japan Division of Respiratory Medicine, Department of Internal Medicine, Hyogo College of Medicine, Nishinomiya 663-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 July 2015 Received in revised form 20 August 2015 Accepted 6 October 2015 Available online xxx
1,2-Diarachidonoyl-sn-glycero-3-phosphoethanolamine (DAPE) induces both necrosis/necroptosis and apoptosis of NCI-H28 malignant pleural mesothelioma (MPM) cells. The present study was conducted to understand the mechanism for DAPE-induced apoptosis of NCI-H28 cells. DAPE induced caspaseindependent apoptosis of NCI-H28 malignant pleural mesothelioma (MPM) cells, and the effect of DAPE was prevented by antioxidants or an inhibitor of NADPH oxidase (NOX). DAPE generated reactive oxygen species (ROS) and inhibited activity of thioredoxin (Trx) reductase (TrxR). DAPE decreased an association of apoptosis signal-regulating kinase 1 (ASK1) with thioredoxin (Trx), thereby releasing ASK1. DAPE activated p38 mitogen-activated protein kinase (MAPK), which was inhibited by an antioxidant or knocking-down ASK1. In addition, DAPE-induced NCI-H28 cell death was also prevented by knockingdown ASK1. Taken together, the results of the present study indicate that DAPE stimulates NOXmediated ROS production and suppresses TrxR activity, resulting in the decrease of reduced Trx and the dissociation of ASK1 from a complex with Trx, allowing sequential activation of ASK1 and p38 MAPK, to induce apoptosis of NCI-H28 MPM cells. © 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: 1,2-Diarachidonoyl-sn-glycero-3phosphoethanolamine Reactive oxygen species Thioredoxin Apoptosis signal-regulating kinase 1 p38 Mitogen-activated protein kinase
1. Introduction MPM is an aggressive malignant tumor with very poor prognosis, and exposure to asbestos fibers is the major pathogenic factor for MPM as well as fibrotic lung disease and lung cancer (1). Asbestos causes DNA double strand breaks, chromosomal aberrations, and abnormal chromosome segregation (2,3). Asbestos induces genotoxicities directly or indirectly by producing ROS (4). Asbestos affects expression of genes involved in integrin-mediated signaling pathways, MAPK pathways, and IkB/NFkB pathways (5). Asbestos stimulates secretion of tumor necrosis factor a (TNFa) and activates NFkB cascades, to prevent MPM cells from apoptosis, i.e., to promote proliferation/differentiation of MPM cells (6). MPM cells are resistant to ROS-induced oxidative stress or radiation. This may be due to the potential antioxidant mechanisms in MPM cells. Expression of mitochondrial manganese superoxide
* Corresponding author. E-mail address: [email protected] (T. Nishizaki). Peer review under responsibility of Japanese Pharmacological Society.
dismutase and enzymes associated with glutathione metabolism such as glutathione-S-transferase and g-glutamylcysteinyl synthetase is upregulated in MPM cells (7e11). In addition, Trx may be implicated in the resistance to oxidants and cytotoxic drugs. In response to a variety of factors such as oxidative stress, tumorpromoting agents, and cytotoxic drugs expression of Trx is induced (12e14). Expression of Trx and TrxR is upregulated in MPM cells (15), and higher concentrations of serum Trx are found in MPM patients (16). The Trx system, which is composed of NADPH, cytosolic and mitochondrial Trx, and TrxR, plays a crucial role in the regulation of cancer cell proliferation and invasion (17e20). TrxR reduces and activates Trx, and thioredoxin-interacting protein (TXNIP) negatively regulates Trx. Trx is a 12 kD multifunctional protein and has an active site with two redox-active cysteine residues (-Trp-CysGly-Pro-Cys-) catalyzing protein disulfide reductions. Trx keeps the cellular redox state and regulates cell proliferation by acting as an electron donor of ribonucleotide reductase, essential for DNA synthesis (21,22). Trx, alternatively, regulates p53-mediated p21 activity and modulates gene expression through thiol redox control over transcription factors such as NFkB and AP-1 (23e26).
http://dx.doi.org/10.1016/j.jphs.2015.10.003 1347-8613/© 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Tsuchiya A, et al., Diarachidonoylphosphoethanolamine induces apoptosis of malignant pleural mesothelioma cells through a Trx/ASK1/p38 MAPK pathway, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.10.003
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A. Tsuchiya et al. / Journal of Pharmacological Sciences xxx (2015) 1e9
In our previous study, the phosphatidylethanolamine 1,2dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (DPPE) induced apoptosis of NCI-H28 MPM cells by enhancing activities of protein phosphatase 2A (PP2A) and protein tyrosine phosphatase 1B (PTP1B) (27). We have lately found that another phosphatidylethanolamine DAPE induced both necrosis/necroptosis and apoptosis of NCI-H28 MPM cells (28). DAPE-induced necrosis/necroptosis was mediated by RIP1 kinase and caused by cyclophilin D-dependent mitochondrial damage in association with ROS generation, to reduce intracellular ATP levels (28). The mechanism underlying DAPE-induced apoptosis of NCI-H28 cells, however, remains to be elucidated as yet. The present study was designed to answer this issue. We show here that DAPE induces apoptosis of NCI-H28 cells through a pathway along an ASK1/MKK(3/6)/p38 MAPK axis under the regulation of Trx. 2. Materials and methods 2.1. Cell culture NCI-H28 cell line was purchased from American Type Culture Collection (Manassas, VA, USA). Cells were grown in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated FBS, 0.003% (w/v) L-glutamine, penicillin (final concentration, 100 U/ml), and streptomycin (final concentration, 0.1 mg/ml), in a humidified atmosphere of 5% CO2 and 95% air at 37 C. 2.2. Assay of cell viability 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was performed by the method as previously described (27).
before and after treatment with DAPE for periods of time, and then centrifuged at 800 g for 5 min at 4 C. The pellet was homogenized on ice in an assay buffer, and then centrifuged at 10,000 g for 15 min at 4 C. The supernatant was reacted with DTNB and NADPH at 25 C for 20 min, and the reaction intensity as a TrxR activity was measured at an absorbance of 412 nm. 2.7. Immunoprecipitation Cells were lysed in a lysis buffer [150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 1% (v/v) Triton X-100 and 20 mM Tris, pH 7.5] containing 1% (v/v) protease inhibitor cocktail and 1% (v/v) phosphatase inhibitor cocktail (Nacalai Tesque, Kyoto, Japan), and then centrifuged at 10,000 g for 15 min at 4 C. Supernatants were incubated with an anti-ASK1 antibody (Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4 C. Then, protein G sepharose 4 beads (GE Healthcare, Piscataway, NJ, USA) was added to the extracts and incubated for 2 h at 4 C. Pellets were washed 5 times with the lysis buffer and dissolved in an SDS sample buffer for Western blotting. 2.8. Western blotting Western blotting was performed by the method as previously described (29) using antibodies against p38, ERK, and phospho-ERK (p-ERK) (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA); Trx1 (GeneTex, Inc., Irvine, CA, USA); ASK1, phospho-ASK1(p-ASK1), MKK3, phospho-MKK3 (p-MKK3), MKK4, phospho-MKK4 (pMKK4), phospho-p38 (p-p38), JNK, and phospho-JNK (p-JNK) (Cell Signaling Technology); and b-actin (SIGMA, Missouri, SL, USA). 2.9. Construction and transfection of siRNA
2.3. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining TUNEL staining was performed by the method as previously described (27). 2.4. Enzymatic assay of caspase activity Caspase activity was measured by the method as previously described (29). The substrate peptides used were as follows: AcAsp-Glu-Val-Asp-MCA for a caspase-3 substrate peptide, Ac-LeuGlu-Val-Asp-AFC for a caspase-4 substrate peptide, Ac-Ile-GluThr-Asp-MCA for a caspase-8 substrate peptide, and Ac-Leu-GluHis-Asp-MCA for a caspase-9 substrate peptide. 2.5. Monitoring of ROS Cells were incubated in the culture medium containing 10 mM 6carboxy-20 ,70 -dichlorodihydrofluorescein diacetate (carboxyH2DCFDA) (Life Technologies, Tokyo, Japan) for 30 min at 37 C. ROS generation in living cells was monitored at 488-nm argon laser with a confocal scanning laser microscope (LSM 510, Carl Zeiss Co., Ltd.) before and after bath-application with DAPE and/or GSH or VAS. The fluorescent signal intensity in each cell was measured and summated as an index of ROS production. 2.6. Assay of TrxR activity TrxR activity was monitored using a TrxR activity colorimetric assay kit (Biovision, Milpitas, CA, USA). In this assay, TrxR catalyzes reduction of 5,50 -dithiobis (2-nitrobenzoic) acid (DTNB) with NADPH to 5-thio-2-nitrobenzoic acid (TNB2), and in this reaction process TNB2 generates a strong yellow color. Cells were harvested
The siRNA silencing the ASK1-targeted gene (ASK1 siRNA) and the negative control siRNA (NC siRNA) were obtained from Invitrogen (Carlsbad, CA, USA). The sequences of ASK1 siRNA used here were 50 -UUAAUAGCAAUUCUGACUUGGUUGC-30 , and 50 -GCAACCAAGUCAGAAUUGCUAUUAA-30 . The NC siRNA contained the scrambled sequence with the same GC content and nucleic acid composition. ASK1 siRNA and NC siRNA were reverse-transfected into cells using a Lipofectamine reagent (Invitrogen). Cells were used for experiments 48 h after transfection. 2.10. Statistical analysis The data presented were mean ± standard error of the mean (SEM). Statistical differences were analyzed by unpaired t-test and analysis of variance (ANOVA) followed by a Bonferroni correction. Values of p < 0.05 were considered statistically significant. 3. Results In the MTT assay, DAPE reduced NCI-H28 cell viability in a treatment time (24e48 h)- and concentration (1e100 mM)dependent manner (Fig. 1A). In the TUNEL staining, DAPE obviously increased the number of TUNEL-positive NCI-H28 cells as compared with control (Fig. 1B). In the caspase assay, no significant activation of caspase-3, -4, -8, and -9 was obtained with DAPE (Fig. 1C). Taken together, these results indicate that DAPE induces caspase-independent apoptosis of NCI-H28 cells. In the real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis, DAPE had no significant effect on expression of mRNAs for FasL, Fas, Fas-associated death domain protein (FADD), TNFa, TNFa receptor 1 (TNFR1), TNFR2, TNFR1-associated death domain protein (TRADD), TNF-related apoptosis-inducing
Please cite this article in press as: Tsuchiya A, et al., Diarachidonoylphosphoethanolamine induces apoptosis of malignant pleural mesothelioma cells through a Trx/ASK1/p38 MAPK pathway, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.10.003
A. Tsuchiya et al. / Journal of Pharmacological Sciences xxx (2015) 1e9
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Fig. 1. DAPE induces caspase-independent apoptosis of NCI-H28 cells. (A) Cells were treated with DAPE at concentrations as indicated for 24 or 48 h, followed by MTT assay. In the graph, each point represents the mean (±SEM) percentage of basal levels (MTT intensities for untreated cells) (n ¼ 4 independent experiments). (B) TUNEL staining was carried out in cells before and after treatment with DAPE (100 mM) for 6 h. DIC, differential interference contrast. Bars, 100 mm. (C) Activities of caspase-3, -4, -8, and -9 were assayed in cells before and after treatment with DAPE (100 mM) for periods of time as indicated. In the graph, each point represents the mean (±SEM) ratio against basal caspase activities (before treatment with DAPE) (n ¼ 4 independent experiments).
ligand (TRAIL), TNF-related weak inducer of apoptosis (TWEAK), death receptor 3 (DR3), DR4, DR5, and DR6 in NCI-H28 cells (Fig. S1). This suggests that DAPE-induced apoptosis of NCI-H28 cells is not mediated through death receptors bearing activation of caspase-8 followed by the effect caspase-3. Apoptosis-inducing factor (AIF) and AIF-homologous mitochondrion-associated inducer of death (AMID) induce caspaseindependent apoptosis by accumulating in the nucleus. Endonuclease G (EndoG), alternatively, induces caspase-independent apoptosis by cleaving DNA. DAPE had no effect on total expression of AIF, AMID, and EndoG (Fig. S2AeC) or nuclear localization of AIF, AMID, and EndoG in NCI-H28 cells (Fig. S2DeF). This indicates that AIF, AMID, and EndoG are not implicated in DAPE-induced caspase-independent apoptosis of NCI-H28 cells. X-linked inhibitor of apoptosis protein (XIAP) inhibits caspase-3, -7, and -9. In response to apoptotic stimuli Smac/DIABLO is released from the mitochondria and neutralizes inhibition of caspase-3, -7, and -9 due to XIAP. Expression of XIAP and Smac/DIABLO in NCIH28 cells was not affected by DAPE (Fig. S3), indicating no implication of XIAP and Smac/DIABLO in DAPE-induced caspase-independent apoptosis of NCI-H28 cells. We next examined whether oxidative stress is implicated in DAPE-induced apoptosis of NCI-H28 cells. DAPE-induced NCI-H28 cell death was significantly inhibited by glutathione (GSH), an antioxidant (Fig. 2A). In addition, the effect of DAPE was clearly prevented by N-acetyl-L-cysteine (NAC), an antioxidant that is a precursor of reduced GSH (Fig. 2B). These results suggest that DAPE induces apoptosis of NCI-H28 cells by producing oxidants. DAPEinduced NCI-H28 cell death was also attenuated by VAS-2870 (VAS), an inhibitor of NOX, or NSC23766 (NSC), an inhibitor of Rac (Fig. 2C,D). Collectively, DAPE appears to stimulate NOXmediated ROS generation, responsible for apoptosis of NCI-H28 cells. To obtain further evidence for this, we monitored ROS production using carboxy-H2DCFDA. DAPE produced ROS in NCI-H28 cells in a treatment time (1e20 min)-dependent manner, and the effect was inhibited by GSH (Fig. 3A). DAPE-induced ROS production, alternatively, was abrogated by VAS (Fig. 3B). Taken together, these results imply that DAPE stimulates NOX-mediated ROS production in NCI-H28 cells and that produced ROS is reduced by GSH. ROS, produced by DAPE, might inhibit TrxR followed by Trx reduction. To obtain evidence for this, we assayed TrxR activity in NCI-H28 cells. Expectedly, DAPE suppressed TrxR activity in NCIH28 cells in a treatment time (10e120 min)-dependent manner, reaching approximately 60% of basal levels at 120 min (Fig. 4). Trx-SH2, which is reduced by TrxR, associates with ASK1, but otherwise Trx-S2, a non-reduced from of Trx, dissociates and
activates ASK1. To address this issue, we examined an association of Trx with ASK1 in immunoprecipitants using an anti-ASK1 antibody from NCI-H28 cells. DAPE significantly decreased immunoreactive signals for Trx in a treatment time (10e120 min)-dependent manner, reaching almost half of basal levels at 120 min (Fig. 5). This indicates that DAPE decreases an association of ASK1 with Trx, i.e., DAPE increases free ASK1. Overall, these results show that DAPE promotes NOX-mediated ROS production, causing a decrease in the reductive form of Trx due to TrxR inhibition, allowing dissociation of ASK1 from a complex with Trx. Our next attempt was to see whether ASK1, dissociated from a complex with Trx, is activated. ASK1 is activated by being phosphorylated at Thr845. DAPE apparently increased phosphorylation of
Fig. 2. ROS is implicated in DAPE-induced apoptosis of NCI-H28 cells. MTT assay was carried out in cells treated with DAPE (100 mM) for 24 h in the presence and absence of GSH (1 mM) (A), NAC (1 mM) (B), VAS (1 mM) (C), or NSC (10 mM) (D), which was applied 1 h prior to DAPE treatment. In the graphs, each point represents the mean (±SEM) percentage of basal levels (MTT intensities for cells untreated with DAPE in the absence of each inhibitor) (n ¼ 4 independent experiments). P values, unpaired t-test.
Please cite this article in press as: Tsuchiya A, et al., Diarachidonoylphosphoethanolamine induces apoptosis of malignant pleural mesothelioma cells through a Trx/ASK1/p38 MAPK pathway, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.10.003
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A. Tsuchiya et al. / Journal of Pharmacological Sciences xxx (2015) 1e9
Fig. 3. DAPE promotes NOX-mediated ROS production in NCI-H28 cells. After 30-min loading of carboxy-H2DCFDA (10 mM), cells were treated DAPE (100 mM) in the presence and absence of GSH (1 mM) (A) or VAS (1 mM) (B) for periods of time as indicated, and the fluorescent signal intensities were measured. Typical fluorescent images are shown in the upper panel and time-course profiles of ROS production are shown in the lower panel. Scale bars, 50 mm. In the graphs, each point represents the mean (±SEM) ROS production (n ¼ 4 independent experiments).
ASK1 at Thr845 in NCI-H28 cells in a bell-shaped treatment time (10e120 min)-dependent manner (Fig. 6A,B). This indicates that DAPE activates ASK1 by dissociating ASK1 from a complex with Trx. ASK1 phosphorylates and activates MKK3/6, which in turn, phosphorylates and activates p38 MAP kinase. ASK1, alternatively, phosphorylates and activates MKK4/7, which phosphorylates and activates stress-activated protein kinases (SAPK)/c-Jun N-terminal kinase (JNK). DAPE did not significantly increase MKK3 phosphorylation at Ser189 (Fig. 6A,C), but a striking increase in p38 MAPK phosphorylation at Thr180/Tyr182 was obtained (Fig. 6A, E). DAPE significantly increased JNK phosphorylation at Thr183/Tyr185 only at 30-min treatment (Fig. 6A,F), while no remarkable increase in the
phosphorylation of MKK4 at Ser257/Thr261 was found (Fig. 6A,D). DAPE did not enhance phosphorylation of the other MAPK extracellular signal-regulated kinase 1/2 (ERK1/2) at Thr202/Tyr204 (Fig. 6A,G). 1,2-Dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLPE) as well as DAPE increased ASK1 phosphorylation in NCIH28 cells, while DPPE and 1,2-dioleoyl-sn-glycero-3-phospho ethanolamine (DOPE) had no effect (Fig. S4A,B). DLPE also increased p38 MAPK phosphorylation, with much lesser potential than that for DAPE, but otherwise no increase was obtained with DOPE and DPPE (Fig. S4A,C). These results indicate that the effect of DAPE on phosphorylation of ASK1 and p38 MAPK is not due to nonspecific action of phosphatidylethanolamine.
Please cite this article in press as: Tsuchiya A, et al., Diarachidonoylphosphoethanolamine induces apoptosis of malignant pleural mesothelioma cells through a Trx/ASK1/p38 MAPK pathway, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.10.003
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GSH abolished DAPE-induced phosphorylation of p38 MAPK in NCI-H28 cells (Fig. 7A,D). GSH also decreased DAPE-induced phosphorylation of ASK1, ERK, and JNK, but not significantly (Fig. 7B,C,E). These results suggest that DAPE activates p38 MAPK in a ROS-dependent manner, possibly as mediated via ASK1. To obtain further evidence for ASK1-dependent p38 MAPK activation, we knocked-down ASK1 using the siRNA. Expression of ASK1 was clearly suppressed in NCI-H28 cells transfected with the siRNA silencing the ASK1-targeted gene, but the expression levels were not affected by DAPE (Fig. 8A,B). DAPE-induced increase in p38 MAPK phosphorylation was clearly inhibited by knockingdown ASK1 (Fig. 8A,C), although DAPE-induced JNK phosphorylation was not affected (Fig. 8A,D). DAPE-induced NCI-H28 cell death was significantly prevented by knocking-down ASK1 (Fig. 8E). Overall, these results indicate that DAPE activates p38 MAPK and induces NCI-H28 cell death in an ASK1-dependent manner. 4. Discussion Fig. 4. DAPE attenuates TrxR activity in NCI-H28 cells. TrxR activity was monitored in cells before and after treatment with DAPE (100 mM) for periods of time as indicated. In the graph, each point represents the mean (±SEM) percentage of basal TrxR activities (0 min) (n ¼ 4 independent experiments). P values, ANOVA followed by a Bonferroni correction.
Fig. 5. DAPE dissociates ASK1 from a complex with Trx1 in NCI-H28 cells. Immunoprecipitation using an anti-ASK1 antibody was carried out in cells before and after treatment with DAPE (100 mM) for periods of time as indicated, followed by Western blotting. In the graph, each column represents the mean (±SEM) signal intensity for Trx1 relative to that for ASK1 (n ¼ 4 independent experiments). P values, ANOVA followed by a Bonferroni correction. NS, not significant.
In the present study, DAPE induced caspase-independent apoptosis of NCI-H28 MPM cells. DAPE-induced NCI-H28 cell death was inhibited by the antioxidants GSH and NAC or the NOX inhibitor VAS. This suggests that DAPE stimulates NOX-mediated ROS production, which triggers to induce apoptosis of NCI-H28 cells. In the NOX assay, DAPE actually increased ROS production in NCI-H28 cells and the effect was canceled by VAS. This provides evidence that DAPE stimulates NOX activity followed by ROS production. ROS, alternatively, is recognized to be produced through damaged mitochondria. DAPE perturbed mitochondrial membrane potentials in NCI-H28 cells, but ROS production preceded mitochondrial damage (28). This implies that DAPE generates ROS by activating NOX, but not due to mitochondrial damage. The NOX family includes NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1, and DUOX2, which are composed of several subunits (30). Of them NOX1 and NOX2 contain the small G-protein Rac1 in the subunits, to regulate NOX activation (30). DAPE-induced NCI-H28 cell death was significantly attenuated by the Rac1 inhibitor NSC, suggesting that DAPE may activate NOX by targeting Rac1. Trx and TrxR are recognized to play a crucial role in the regulation of cancer cell proliferation and invasion (20). TrxR reduces Trx-S2 to Trx-SH2, which associates and inhibits ASK1; conversely, Trx-S2, a non-reduced from of Trx, dissociates and activates ASK1, to induce apoptosis (31e33). We postulated that ROS produced by DAPE might suppress TrxR activity. Indeed, DAPE attenuated TrxR activity in NCI-H28 cells and significantly decreased association of ASK1 with Trx1. Moreover, DAPE enhanced phosphorylation of ASK1 at Thr845 in NCI-H28. Taken together, these results indicate that DAPE activates NOX to generate ROS, which inhibits TrxR, allowing decrease in reduced Trx and dissociation of ASKI from a complex with Trx, and then leading to activation of ASK1. ASK1 activates MKK3/6 followed by p38 MAPK or MKK4/7 followed by SAPK/JNK through sequential phosphorylation, and both of p38 MAPK and SAPK/JNK are implicated in apoptosis (34,35). DAPE markedly enhanced phosphorylation of p38 MAPK at Thr180/ Tyr182 in NCI-H28 cells, although no significant increase in the phosphorylation of MKK3 at Ser189 was not obtained. DAPE, on the other hand, enhanced phosphorylation of JNK at Thr183/Tyr185 only at 30-min treatment, but phosphorylation of MKK4 at Ser257/ Thr261 was not affected. DAPE-induced increase in the phosphorylation of p38 MAPK, but not JNK, was abolished by GSH and clearly prevented by knocking-down ASK1. In addition, DAPE-induced NCI-H28 cell death was significantly inhibited by knocking-down ASK1. Overall, these results suggest that DAPE induces apoptosis of NCI-H28 cells dominantly through an ASK1/MKK(3/6)/p38 MAPK pathway. The questions raised are as to: why in spite of significant
Please cite this article in press as: Tsuchiya A, et al., Diarachidonoylphosphoethanolamine induces apoptosis of malignant pleural mesothelioma cells through a Trx/ASK1/p38 MAPK pathway, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.10.003
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Fig. 6. DAPE enhances phosphorylation of ASK1 and p38 MAPK in NCI-H28 cells. Cells were treated with DAPE (100 mM) for periods of time as indicated, followed by Western blotting. Typical blotting images are shown in (A). In the graphs, each column represents the mean (±SEM) signal intensity for p-ASK1 (B), p-MKK3 (C), p-MKK4 (D), p-p38 (E), pJNK (F), or p-ERK (G) relative to that for each protein (n ¼ 4 independent experiments). P value, ANOVA followed by a Bonferroni correction. NS, not significant.
Please cite this article in press as: Tsuchiya A, et al., Diarachidonoylphosphoethanolamine induces apoptosis of malignant pleural mesothelioma cells through a Trx/ASK1/p38 MAPK pathway, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.10.003
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Fig. 7. DAPE-induced phosphorylation of p38 MAPK is abrogated by GSH in NCI-H28 cells. Cells were treated with DAPE (100 mM) for 30 min in the presence and absence of GSH (1 mM), which was applied 1 h prior to DAPE treatment, and then, Western blotting was carried out. Typical blotting images are shown in (A). In the graphs, each column represents the mean (±SEM) signal intensity for p-ASK1 (B), p-ERK (C), p-p38 (D), or p-JNK (E) relative to that for each protein (n ¼ 4 independent experiments). P value, unpaired t-test. NS, not significant.
activation of p38 MAPK DAPE did not activate MKK3 significantly and why in spite of significant inhibition of DAPE-induced p38 MAPK activation GSH did not inhibit DAPE-induced ASK1 activation. This may represent an additional DAPE-induced direct p38 MAPK activation pathway, regardless of MKK3/6.
In summary, the results of the present study show that DAPE stimulates NOX-mediated ROS production, which suppresses TrxR activity and decreases reduced Trx, allowing ASK1 release from an association with Trx, resulting in the sequential activation of ASK1/ MKK(3/6)/p38 MAPK, to induce caspase-independent apoptosis of
Please cite this article in press as: Tsuchiya A, et al., Diarachidonoylphosphoethanolamine induces apoptosis of malignant pleural mesothelioma cells through a Trx/ASK1/p38 MAPK pathway, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.10.003
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Fig. 8. DAPE-induced phosphorylation of p38 MAPK and cell death are dependent upon ASK1 in NCI-H28 cells. Cells, transfected with the NC siRNA or the ASK1 siRNA, were untreated and treated with DAPE (100 mM) for 30 min, followed by Western blotting. (A) Typical blotting images. (B) In the graphs, each column represents the mean (±SEM) signal intensity for ASK1 normalized by that for b-actin (n ¼ 4 independent experiments). (C) (D) The signal intensities for p-p38 (C) and p-JNK (D) were normalized by those for each unphosphorylated protein. In the graphs, each column represents the mean (±SEM) signal intensity for p-p38 (C) and p-JNK (D) relative to that for each (n ¼ 4 independent experiments). (E) Cells, transfected with the NC siRNA and the ASK1 siRNA, were treated with DAPE (100 mM) for 48 h, followed by MTT assay. In the graph, each column represents the mean (±SEM) percentage of basal levels (MTT intensities for cells before treatment with DAPE) (n ¼ 4 independent experiments). P values, unpaired t-test. NS, not significant.
Please cite this article in press as: Tsuchiya A, et al., Diarachidonoylphosphoethanolamine induces apoptosis of malignant pleural mesothelioma cells through a Trx/ASK1/p38 MAPK pathway, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.10.003
A. Tsuchiya et al. / Journal of Pharmacological Sciences xxx (2015) 1e9
NCI-H28 MPM cells. This may establish a new apoptotic pathway relevant to DAPE signaling. Conflicts of interest The authors declare that they have no conflicts of interest to disclose. Acknowledgments This study was supported by research grants from ‘The Ministry of Education, Culture, Sports, Science, and Technology-Supported Program for the Strategic Research Foundation at Private Universities, 2012e2016’. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jphs.2015.10.003. References (1) Tsujimura T, Torii I, Sato A, Song M, Fukuoka K, Hasegawa S, et al. Pathological and molecular biological approaches to early mesothelioma. Int J Clin Oncol. 2012;17:40e47. (2) Fatma N, Jain AK, Rahman Q. Frequency of sister chromatid exchange and chromosomal aberrations in asbestos cement workers. Br J Ind Med. 1991;48: 103e105. (3) Marczynski B, Czuppon AB, Marek W, Reichel G, Baur X. Increased incidence of DNA double-strand breaks and anti-ds DNA antibodies in blood of workers occupationally exposed to asbestos. Hum Exp Toxicol. 1994;13:3e9. (4) Mossman BT, Churg A. Mechanisms in the pathogenesis of asbestosis and silicosis. Am J Respir Crit Care Med. 1998;157:1666e1680. (5) Ramos-Nino ME, Heintz N, Scappoli L, Martinelli M, Land S, Nowak N, et al. Gene profiling and kinase screening in asbestos-exposed epithelial cells and lungs. Am J Respir Cell Mol Biol. 2003;29:S51eS58. (6) Yang H, Bocchetta M, Kroczynska B, Elmishad AG, Chen Y, Liu Z, et al. TNF-a inhibits asbestos-induced cytotoxicity via a NFkB-dependent pathway, a possible mechanism for asbestos-induced oncogenesis. Proc Natl Acad Sci U.S.A. 2006;103:10397e10402. (7) Kinnula VL, Pietarinen-Runtti P, Raivio K, Kahlos K, Pelin K, Mattson K, et al. Manganese superoxide dismutase in human pleural mesothelioma cell lines. Free Radic Biol Med. 1996;21:527e532. (8) Kinnula K, Linnainmaa K, Raivio KO, Kinnula VL. Endogenous antioxidant enzymes and glutathione-S-transferase in protection of mesothelioma cells against hydrogen peroxide and epirubicin toxicity. Br J Cancer. 1998;77: 1097e1102. (9) Ogretmen B, Bahadori HR, McCauley MD, Boylan A, Green MR, Safa AR. Coordinated over-expression of the MRP and g-glutamylcysteine synthetase genes, but not MDR1, correlates with doxorubicin resistance in human malignant mesothelioma cell lines. Int J Cancer. 1998;75:757e761. (10) Kahlos K, Anttila S, Asikainen T, Kinnula K, Raivio KO, Mattson K, et al. Manganese superoxide dismutase in healthy human pleural mesothelium and in malignant pleural mesothelioma. Am J Respir Cell Mol Biol. 1998;18: 570e580. €a €kko € P, Kurttila E, Soini Y, Kinnula VL. Manganese superoxide (11) Kahlos K, Pa dismutase as a diagnostic marker for malignant pleural mesothelioma. Br J Cancer. 2000;82:1022e1029. (12) Higashikubo A, Tanaka N, Noda N, Maeda I, Yagi K, Mizoguchi T, et al. Increase in thioredoxin activity of intestinal epithelial cells mediated by oxidative stress. Biol Pharm Bull. 1999;22:900e903.
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(13) Kumar S, Holmgren A. Induction of thioredoxin, thioredoxin reductase and glutaredoxin activity in mouse skin by TPA, a calcium ionophore and other tumor promoters. Carcinogenesis. 1999;9:1761e1767. (14) Sasada T, Nakamura H, Ueda S, Sato N, Kitaoka Y, Gon Y, et al. Possible involvement of thioredoxin reductase as well as thioredoxin in cellular sensitivity to cis-diamminedichloroplatinum. Free Radic Biol Med. 1999;27: 504e514. €ily M, Koistinen P, Kakko S, P€ €kko € P, et al. Up-regulation (15) Kahlos K, Soini Y, Sa aa of thioredoxin and thioredoxin reductase in human malignant pleural mesothelioma. Int J Cancer. 2001;95:198e204. (16) Maeda R, Tabata C, Tabata R, Eguchi R, Fujimori Y, Nakano T. Is serum thioredoxin-1 a useful clinical marker for malignant pleural mesothelioma? Antioxid Redox Signal. 2011;15:685e689. (17) Holmgren A, Bjornstedt M. Thioredoxin and thioredoxin reductase. Methods Enzymol. 1995;252:199e208. (18) Lee SR, Kim JR, Kwon KS, Yoon HW, Levine RL, Ginsburg A, et al. Molecular cloning and characterization of a mitochondrial selenocysteine-containing thioredoxin reductase from rat liver. J Biol Chem. 1999;274:4722e4734. (19) Miranda-Vizuete A, Damdimopoulos AE, Spyrou G. Human mitochondrial thioredoxin reductase cDNA cloning, expression and genomic organization. Eur J Biochem. 1999;261:405e412. er ESJ, Holmgren A. The thioredoxin system in cancer. Semin Cancer Biol. (20) Arn 2006;16:420e426. (21) Holmgren A. Thioredoxin and glutaredoxin systems. J Biol Chem. 1986;264: 13963e13966. er ESJ, Holmgren A. Physiological functions of thioredoxin and thioredoxin (22) Arn reductase. Eur J Biochem. 2000;267:6102e6109. (23) Matthews JR, Wakasugi N, Virelizier JL, Yodoi J, Hay RT. Thioredoxin regulates the DNA binding activity of NF-kB by reduction of a disulphide bond involving cysteine 62. Nucleic Acids Res. 1992;20:3821e3830. (24) Gasdaska P, Oblong JE, Cotgreave IA, Powis G. The predicted amino acid sequence of human thioredoxin is identical to that of the autocrine growth factor human adult T-cell derived factor (ADF): thioredoxin mRNA is elevated in some human tumors. Biochim Biophys Acta. 1994;1218:292e296. (25) Hirota K, Murata M, Sachi Y, Nakamura H, Takeuchi J, Mori K, et al. Distinct roles of thioredoxin in the cytoplasm and in the nucleus. A two-step mechanism of redox regulation of transcription factor NF-kB. J Biol Chem. 1999;274:27891e27897. (26) Ueno M, Masutani H, Arai RJ, Yamauchi A, Hirota K, Sakai T, et al. Thioredoxindependent redox regulation of p53-mediated p21 activation. J Biol Chem. 1999;274:35809e35815. (27) Kaku Y, Tsuchiya A, Kanno T, Nakano T, Nishizaki T. Dipalmitoleoyl-phosphatidylethanolamine induces apoptosis of NCI-H28 malignant mesothelioma cells. Anticancer Res. 2014;34:1759e1764. (28) Kaku Y, Tsuchiya A, Kanno T, Nakano T, Nishizaki T. Diarachidonoylphosphoethanolamine induces necrosis/necroptosis of malignant pleural mesothelioma cells. Cell Signal. 2015;27:1713e1719. (29) Kaku Y, Tsuchiya A, Kanno T, Nakao S, Shimizu T, Tanaka A, et al. The newly synthesized anticancer drug HUHS1015 is useful for treatment of human gastric cancer. Cancer Chemother Pharmacol. 2015;75:527e535. (30) Bedard K, Krause KH. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev. 2007;87:245e313. (31) Saitoh M, Nishitoh H, Fujii M, Takeda K, Tobiume K, Sawada Y, et al. Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1. EMBO J. 1998;17:2596e2606. (32) Liu H, Nishitoh H, Ichijo H, Kyriakis JM. Activation of apoptosis signalregulating kinase 1 (ASK1) by tumor necrosis factor receptor-associated factor 2 requires prior dissociation of the ASK1 inhibitor thioredoxin. Mol Cell Biol. 2000;20:2198e2208. (33) Soga M, Matsuzawa A, Ichijo H. Oxidative stress-induced diseases via the ASK1 signaling pathway. Int J Cell Biol 2012:439587. (34) Matsuzawa A, Nishitoh H, Tobiume K, Takeda K, Ichijo H. Physiological roles of ASK1-mediated signal transduction in oxidative stress- and endoplasmic reticulum stress-induced apoptosis: advanced findings from ASK1 knockout mice. Antioxid Redox Signal. 2002;4:415e425. (35) Hayakawa T, Matsuzawa A, Noguchi T, Takeda K, Ichijo H. The ASK1-MAP kinase pathways in immune and stress responses. Microbes Infect. 2006;8: 1098e1107.
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Diarachidonoylphosphoethanolamine induces apoptosis of malignant pleural mesothelioma cells through a Trx/ASK1/p38 MAPK pathway Ayako Tsuchiya a, Yoshiko Kaku a, Takashi Nakano b, Tomoyuki Nishizaki a, * a b
Division of Bioinformation, Department of Physiology, Hyogo College of Medicine, Nishinomiya 663-8501, Japan Division of Respiratory Medicine, Department of Internal Medicine, Hyogo College of Medicine, Nishinomiya 663-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 July 2015 Received in revised form 20 August 2015 Accepted 6 October 2015 Available online xxx
1,2-Diarachidonoyl-sn-glycero-3-phosphoethanolamine (DAPE) induces both necrosis/necroptosis and apoptosis of NCI-H28 malignant pleural mesothelioma (MPM) cells. The present study was conducted to understand the mechanism for DAPE-induced apoptosis of NCI-H28 cells. DAPE induced caspaseindependent apoptosis of NCI-H28 malignant pleural mesothelioma (MPM) cells, and the effect of DAPE was prevented by antioxidants or an inhibitor of NADPH oxidase (NOX). DAPE generated reactive oxygen species (ROS) and inhibited activity of thioredoxin (Trx) reductase (TrxR). DAPE decreased an association of apoptosis signal-regulating kinase 1 (ASK1) with thioredoxin (Trx), thereby releasing ASK1. DAPE activated p38 mitogen-activated protein kinase (MAPK), which was inhibited by an antioxidant or knocking-down ASK1. In addition, DAPE-induced NCI-H28 cell death was also prevented by knockingdown ASK1. Taken together, the results of the present study indicate that DAPE stimulates NOXmediated ROS production and suppresses TrxR activity, resulting in the decrease of reduced Trx and the dissociation of ASK1 from a complex with Trx, allowing sequential activation of ASK1 and p38 MAPK, to induce apoptosis of NCI-H28 MPM cells. © 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: 1,2-Diarachidonoyl-sn-glycero-3phosphoethanolamine Reactive oxygen species Thioredoxin Apoptosis signal-regulating kinase 1 p38 Mitogen-activated protein kinase
1. Introduction MPM is an aggressive malignant tumor with very poor prognosis, and exposure to asbestos fibers is the major pathogenic factor for MPM as well as fibrotic lung disease and lung cancer (1). Asbestos causes DNA double strand breaks, chromosomal aberrations, and abnormal chromosome segregation (2,3). Asbestos induces genotoxicities directly or indirectly by producing ROS (4). Asbestos affects expression of genes involved in integrin-mediated signaling pathways, MAPK pathways, and IkB/NFkB pathways (5). Asbestos stimulates secretion of tumor necrosis factor a (TNFa) and activates NFkB cascades, to prevent MPM cells from apoptosis, i.e., to promote proliferation/differentiation of MPM cells (6). MPM cells are resistant to ROS-induced oxidative stress or radiation. This may be due to the potential antioxidant mechanisms in MPM cells. Expression of mitochondrial manganese superoxide
* Corresponding author. E-mail address: [email protected] (T. Nishizaki). Peer review under responsibility of Japanese Pharmacological Society.
dismutase and enzymes associated with glutathione metabolism such as glutathione-S-transferase and g-glutamylcysteinyl synthetase is upregulated in MPM cells (7e11). In addition, Trx may be implicated in the resistance to oxidants and cytotoxic drugs. In response to a variety of factors such as oxidative stress, tumorpromoting agents, and cytotoxic drugs expression of Trx is induced (12e14). Expression of Trx and TrxR is upregulated in MPM cells (15), and higher concentrations of serum Trx are found in MPM patients (16). The Trx system, which is composed of NADPH, cytosolic and mitochondrial Trx, and TrxR, plays a crucial role in the regulation of cancer cell proliferation and invasion (17e20). TrxR reduces and activates Trx, and thioredoxin-interacting protein (TXNIP) negatively regulates Trx. Trx is a 12 kD multifunctional protein and has an active site with two redox-active cysteine residues (-Trp-CysGly-Pro-Cys-) catalyzing protein disulfide reductions. Trx keeps the cellular redox state and regulates cell proliferation by acting as an electron donor of ribonucleotide reductase, essential for DNA synthesis (21,22). Trx, alternatively, regulates p53-mediated p21 activity and modulates gene expression through thiol redox control over transcription factors such as NFkB and AP-1 (23e26).
http://dx.doi.org/10.1016/j.jphs.2015.10.003 1347-8613/© 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Tsuchiya A, et al., Diarachidonoylphosphoethanolamine induces apoptosis of malignant pleural mesothelioma cells through a Trx/ASK1/p38 MAPK pathway, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.10.003
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A. Tsuchiya et al. / Journal of Pharmacological Sciences xxx (2015) 1e9
In our previous study, the phosphatidylethanolamine 1,2dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (DPPE) induced apoptosis of NCI-H28 MPM cells by enhancing activities of protein phosphatase 2A (PP2A) and protein tyrosine phosphatase 1B (PTP1B) (27). We have lately found that another phosphatidylethanolamine DAPE induced both necrosis/necroptosis and apoptosis of NCI-H28 MPM cells (28). DAPE-induced necrosis/necroptosis was mediated by RIP1 kinase and caused by cyclophilin D-dependent mitochondrial damage in association with ROS generation, to reduce intracellular ATP levels (28). The mechanism underlying DAPE-induced apoptosis of NCI-H28 cells, however, remains to be elucidated as yet. The present study was designed to answer this issue. We show here that DAPE induces apoptosis of NCI-H28 cells through a pathway along an ASK1/MKK(3/6)/p38 MAPK axis under the regulation of Trx. 2. Materials and methods 2.1. Cell culture NCI-H28 cell line was purchased from American Type Culture Collection (Manassas, VA, USA). Cells were grown in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated FBS, 0.003% (w/v) L-glutamine, penicillin (final concentration, 100 U/ml), and streptomycin (final concentration, 0.1 mg/ml), in a humidified atmosphere of 5% CO2 and 95% air at 37 C. 2.2. Assay of cell viability 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was performed by the method as previously described (27).
before and after treatment with DAPE for periods of time, and then centrifuged at 800 g for 5 min at 4 C. The pellet was homogenized on ice in an assay buffer, and then centrifuged at 10,000 g for 15 min at 4 C. The supernatant was reacted with DTNB and NADPH at 25 C for 20 min, and the reaction intensity as a TrxR activity was measured at an absorbance of 412 nm. 2.7. Immunoprecipitation Cells were lysed in a lysis buffer [150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 1% (v/v) Triton X-100 and 20 mM Tris, pH 7.5] containing 1% (v/v) protease inhibitor cocktail and 1% (v/v) phosphatase inhibitor cocktail (Nacalai Tesque, Kyoto, Japan), and then centrifuged at 10,000 g for 15 min at 4 C. Supernatants were incubated with an anti-ASK1 antibody (Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4 C. Then, protein G sepharose 4 beads (GE Healthcare, Piscataway, NJ, USA) was added to the extracts and incubated for 2 h at 4 C. Pellets were washed 5 times with the lysis buffer and dissolved in an SDS sample buffer for Western blotting. 2.8. Western blotting Western blotting was performed by the method as previously described (29) using antibodies against p38, ERK, and phospho-ERK (p-ERK) (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA); Trx1 (GeneTex, Inc., Irvine, CA, USA); ASK1, phospho-ASK1(p-ASK1), MKK3, phospho-MKK3 (p-MKK3), MKK4, phospho-MKK4 (pMKK4), phospho-p38 (p-p38), JNK, and phospho-JNK (p-JNK) (Cell Signaling Technology); and b-actin (SIGMA, Missouri, SL, USA). 2.9. Construction and transfection of siRNA
2.3. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining TUNEL staining was performed by the method as previously described (27). 2.4. Enzymatic assay of caspase activity Caspase activity was measured by the method as previously described (29). The substrate peptides used were as follows: AcAsp-Glu-Val-Asp-MCA for a caspase-3 substrate peptide, Ac-LeuGlu-Val-Asp-AFC for a caspase-4 substrate peptide, Ac-Ile-GluThr-Asp-MCA for a caspase-8 substrate peptide, and Ac-Leu-GluHis-Asp-MCA for a caspase-9 substrate peptide. 2.5. Monitoring of ROS Cells were incubated in the culture medium containing 10 mM 6carboxy-20 ,70 -dichlorodihydrofluorescein diacetate (carboxyH2DCFDA) (Life Technologies, Tokyo, Japan) for 30 min at 37 C. ROS generation in living cells was monitored at 488-nm argon laser with a confocal scanning laser microscope (LSM 510, Carl Zeiss Co., Ltd.) before and after bath-application with DAPE and/or GSH or VAS. The fluorescent signal intensity in each cell was measured and summated as an index of ROS production. 2.6. Assay of TrxR activity TrxR activity was monitored using a TrxR activity colorimetric assay kit (Biovision, Milpitas, CA, USA). In this assay, TrxR catalyzes reduction of 5,50 -dithiobis (2-nitrobenzoic) acid (DTNB) with NADPH to 5-thio-2-nitrobenzoic acid (TNB2), and in this reaction process TNB2 generates a strong yellow color. Cells were harvested
The siRNA silencing the ASK1-targeted gene (ASK1 siRNA) and the negative control siRNA (NC siRNA) were obtained from Invitrogen (Carlsbad, CA, USA). The sequences of ASK1 siRNA used here were 50 -UUAAUAGCAAUUCUGACUUGGUUGC-30 , and 50 -GCAACCAAGUCAGAAUUGCUAUUAA-30 . The NC siRNA contained the scrambled sequence with the same GC content and nucleic acid composition. ASK1 siRNA and NC siRNA were reverse-transfected into cells using a Lipofectamine reagent (Invitrogen). Cells were used for experiments 48 h after transfection. 2.10. Statistical analysis The data presented were mean ± standard error of the mean (SEM). Statistical differences were analyzed by unpaired t-test and analysis of variance (ANOVA) followed by a Bonferroni correction. Values of p < 0.05 were considered statistically significant. 3. Results In the MTT assay, DAPE reduced NCI-H28 cell viability in a treatment time (24e48 h)- and concentration (1e100 mM)dependent manner (Fig. 1A). In the TUNEL staining, DAPE obviously increased the number of TUNEL-positive NCI-H28 cells as compared with control (Fig. 1B). In the caspase assay, no significant activation of caspase-3, -4, -8, and -9 was obtained with DAPE (Fig. 1C). Taken together, these results indicate that DAPE induces caspase-independent apoptosis of NCI-H28 cells. In the real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis, DAPE had no significant effect on expression of mRNAs for FasL, Fas, Fas-associated death domain protein (FADD), TNFa, TNFa receptor 1 (TNFR1), TNFR2, TNFR1-associated death domain protein (TRADD), TNF-related apoptosis-inducing
Please cite this article in press as: Tsuchiya A, et al., Diarachidonoylphosphoethanolamine induces apoptosis of malignant pleural mesothelioma cells through a Trx/ASK1/p38 MAPK pathway, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.10.003
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Fig. 1. DAPE induces caspase-independent apoptosis of NCI-H28 cells. (A) Cells were treated with DAPE at concentrations as indicated for 24 or 48 h, followed by MTT assay. In the graph, each point represents the mean (±SEM) percentage of basal levels (MTT intensities for untreated cells) (n ¼ 4 independent experiments). (B) TUNEL staining was carried out in cells before and after treatment with DAPE (100 mM) for 6 h. DIC, differential interference contrast. Bars, 100 mm. (C) Activities of caspase-3, -4, -8, and -9 were assayed in cells before and after treatment with DAPE (100 mM) for periods of time as indicated. In the graph, each point represents the mean (±SEM) ratio against basal caspase activities (before treatment with DAPE) (n ¼ 4 independent experiments).
ligand (TRAIL), TNF-related weak inducer of apoptosis (TWEAK), death receptor 3 (DR3), DR4, DR5, and DR6 in NCI-H28 cells (Fig. S1). This suggests that DAPE-induced apoptosis of NCI-H28 cells is not mediated through death receptors bearing activation of caspase-8 followed by the effect caspase-3. Apoptosis-inducing factor (AIF) and AIF-homologous mitochondrion-associated inducer of death (AMID) induce caspaseindependent apoptosis by accumulating in the nucleus. Endonuclease G (EndoG), alternatively, induces caspase-independent apoptosis by cleaving DNA. DAPE had no effect on total expression of AIF, AMID, and EndoG (Fig. S2AeC) or nuclear localization of AIF, AMID, and EndoG in NCI-H28 cells (Fig. S2DeF). This indicates that AIF, AMID, and EndoG are not implicated in DAPE-induced caspase-independent apoptosis of NCI-H28 cells. X-linked inhibitor of apoptosis protein (XIAP) inhibits caspase-3, -7, and -9. In response to apoptotic stimuli Smac/DIABLO is released from the mitochondria and neutralizes inhibition of caspase-3, -7, and -9 due to XIAP. Expression of XIAP and Smac/DIABLO in NCIH28 cells was not affected by DAPE (Fig. S3), indicating no implication of XIAP and Smac/DIABLO in DAPE-induced caspase-independent apoptosis of NCI-H28 cells. We next examined whether oxidative stress is implicated in DAPE-induced apoptosis of NCI-H28 cells. DAPE-induced NCI-H28 cell death was significantly inhibited by glutathione (GSH), an antioxidant (Fig. 2A). In addition, the effect of DAPE was clearly prevented by N-acetyl-L-cysteine (NAC), an antioxidant that is a precursor of reduced GSH (Fig. 2B). These results suggest that DAPE induces apoptosis of NCI-H28 cells by producing oxidants. DAPEinduced NCI-H28 cell death was also attenuated by VAS-2870 (VAS), an inhibitor of NOX, or NSC23766 (NSC), an inhibitor of Rac (Fig. 2C,D). Collectively, DAPE appears to stimulate NOXmediated ROS generation, responsible for apoptosis of NCI-H28 cells. To obtain further evidence for this, we monitored ROS production using carboxy-H2DCFDA. DAPE produced ROS in NCI-H28 cells in a treatment time (1e20 min)-dependent manner, and the effect was inhibited by GSH (Fig. 3A). DAPE-induced ROS production, alternatively, was abrogated by VAS (Fig. 3B). Taken together, these results imply that DAPE stimulates NOX-mediated ROS production in NCI-H28 cells and that produced ROS is reduced by GSH. ROS, produced by DAPE, might inhibit TrxR followed by Trx reduction. To obtain evidence for this, we assayed TrxR activity in NCI-H28 cells. Expectedly, DAPE suppressed TrxR activity in NCIH28 cells in a treatment time (10e120 min)-dependent manner, reaching approximately 60% of basal levels at 120 min (Fig. 4). Trx-SH2, which is reduced by TrxR, associates with ASK1, but otherwise Trx-S2, a non-reduced from of Trx, dissociates and
activates ASK1. To address this issue, we examined an association of Trx with ASK1 in immunoprecipitants using an anti-ASK1 antibody from NCI-H28 cells. DAPE significantly decreased immunoreactive signals for Trx in a treatment time (10e120 min)-dependent manner, reaching almost half of basal levels at 120 min (Fig. 5). This indicates that DAPE decreases an association of ASK1 with Trx, i.e., DAPE increases free ASK1. Overall, these results show that DAPE promotes NOX-mediated ROS production, causing a decrease in the reductive form of Trx due to TrxR inhibition, allowing dissociation of ASK1 from a complex with Trx. Our next attempt was to see whether ASK1, dissociated from a complex with Trx, is activated. ASK1 is activated by being phosphorylated at Thr845. DAPE apparently increased phosphorylation of
Fig. 2. ROS is implicated in DAPE-induced apoptosis of NCI-H28 cells. MTT assay was carried out in cells treated with DAPE (100 mM) for 24 h in the presence and absence of GSH (1 mM) (A), NAC (1 mM) (B), VAS (1 mM) (C), or NSC (10 mM) (D), which was applied 1 h prior to DAPE treatment. In the graphs, each point represents the mean (±SEM) percentage of basal levels (MTT intensities for cells untreated with DAPE in the absence of each inhibitor) (n ¼ 4 independent experiments). P values, unpaired t-test.
Please cite this article in press as: Tsuchiya A, et al., Diarachidonoylphosphoethanolamine induces apoptosis of malignant pleural mesothelioma cells through a Trx/ASK1/p38 MAPK pathway, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.10.003
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Fig. 3. DAPE promotes NOX-mediated ROS production in NCI-H28 cells. After 30-min loading of carboxy-H2DCFDA (10 mM), cells were treated DAPE (100 mM) in the presence and absence of GSH (1 mM) (A) or VAS (1 mM) (B) for periods of time as indicated, and the fluorescent signal intensities were measured. Typical fluorescent images are shown in the upper panel and time-course profiles of ROS production are shown in the lower panel. Scale bars, 50 mm. In the graphs, each point represents the mean (±SEM) ROS production (n ¼ 4 independent experiments).
ASK1 at Thr845 in NCI-H28 cells in a bell-shaped treatment time (10e120 min)-dependent manner (Fig. 6A,B). This indicates that DAPE activates ASK1 by dissociating ASK1 from a complex with Trx. ASK1 phosphorylates and activates MKK3/6, which in turn, phosphorylates and activates p38 MAP kinase. ASK1, alternatively, phosphorylates and activates MKK4/7, which phosphorylates and activates stress-activated protein kinases (SAPK)/c-Jun N-terminal kinase (JNK). DAPE did not significantly increase MKK3 phosphorylation at Ser189 (Fig. 6A,C), but a striking increase in p38 MAPK phosphorylation at Thr180/Tyr182 was obtained (Fig. 6A, E). DAPE significantly increased JNK phosphorylation at Thr183/Tyr185 only at 30-min treatment (Fig. 6A,F), while no remarkable increase in the
phosphorylation of MKK4 at Ser257/Thr261 was found (Fig. 6A,D). DAPE did not enhance phosphorylation of the other MAPK extracellular signal-regulated kinase 1/2 (ERK1/2) at Thr202/Tyr204 (Fig. 6A,G). 1,2-Dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLPE) as well as DAPE increased ASK1 phosphorylation in NCIH28 cells, while DPPE and 1,2-dioleoyl-sn-glycero-3-phospho ethanolamine (DOPE) had no effect (Fig. S4A,B). DLPE also increased p38 MAPK phosphorylation, with much lesser potential than that for DAPE, but otherwise no increase was obtained with DOPE and DPPE (Fig. S4A,C). These results indicate that the effect of DAPE on phosphorylation of ASK1 and p38 MAPK is not due to nonspecific action of phosphatidylethanolamine.
Please cite this article in press as: Tsuchiya A, et al., Diarachidonoylphosphoethanolamine induces apoptosis of malignant pleural mesothelioma cells through a Trx/ASK1/p38 MAPK pathway, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.10.003
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GSH abolished DAPE-induced phosphorylation of p38 MAPK in NCI-H28 cells (Fig. 7A,D). GSH also decreased DAPE-induced phosphorylation of ASK1, ERK, and JNK, but not significantly (Fig. 7B,C,E). These results suggest that DAPE activates p38 MAPK in a ROS-dependent manner, possibly as mediated via ASK1. To obtain further evidence for ASK1-dependent p38 MAPK activation, we knocked-down ASK1 using the siRNA. Expression of ASK1 was clearly suppressed in NCI-H28 cells transfected with the siRNA silencing the ASK1-targeted gene, but the expression levels were not affected by DAPE (Fig. 8A,B). DAPE-induced increase in p38 MAPK phosphorylation was clearly inhibited by knockingdown ASK1 (Fig. 8A,C), although DAPE-induced JNK phosphorylation was not affected (Fig. 8A,D). DAPE-induced NCI-H28 cell death was significantly prevented by knocking-down ASK1 (Fig. 8E). Overall, these results indicate that DAPE activates p38 MAPK and induces NCI-H28 cell death in an ASK1-dependent manner. 4. Discussion Fig. 4. DAPE attenuates TrxR activity in NCI-H28 cells. TrxR activity was monitored in cells before and after treatment with DAPE (100 mM) for periods of time as indicated. In the graph, each point represents the mean (±SEM) percentage of basal TrxR activities (0 min) (n ¼ 4 independent experiments). P values, ANOVA followed by a Bonferroni correction.
Fig. 5. DAPE dissociates ASK1 from a complex with Trx1 in NCI-H28 cells. Immunoprecipitation using an anti-ASK1 antibody was carried out in cells before and after treatment with DAPE (100 mM) for periods of time as indicated, followed by Western blotting. In the graph, each column represents the mean (±SEM) signal intensity for Trx1 relative to that for ASK1 (n ¼ 4 independent experiments). P values, ANOVA followed by a Bonferroni correction. NS, not significant.
In the present study, DAPE induced caspase-independent apoptosis of NCI-H28 MPM cells. DAPE-induced NCI-H28 cell death was inhibited by the antioxidants GSH and NAC or the NOX inhibitor VAS. This suggests that DAPE stimulates NOX-mediated ROS production, which triggers to induce apoptosis of NCI-H28 cells. In the NOX assay, DAPE actually increased ROS production in NCI-H28 cells and the effect was canceled by VAS. This provides evidence that DAPE stimulates NOX activity followed by ROS production. ROS, alternatively, is recognized to be produced through damaged mitochondria. DAPE perturbed mitochondrial membrane potentials in NCI-H28 cells, but ROS production preceded mitochondrial damage (28). This implies that DAPE generates ROS by activating NOX, but not due to mitochondrial damage. The NOX family includes NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1, and DUOX2, which are composed of several subunits (30). Of them NOX1 and NOX2 contain the small G-protein Rac1 in the subunits, to regulate NOX activation (30). DAPE-induced NCI-H28 cell death was significantly attenuated by the Rac1 inhibitor NSC, suggesting that DAPE may activate NOX by targeting Rac1. Trx and TrxR are recognized to play a crucial role in the regulation of cancer cell proliferation and invasion (20). TrxR reduces Trx-S2 to Trx-SH2, which associates and inhibits ASK1; conversely, Trx-S2, a non-reduced from of Trx, dissociates and activates ASK1, to induce apoptosis (31e33). We postulated that ROS produced by DAPE might suppress TrxR activity. Indeed, DAPE attenuated TrxR activity in NCI-H28 cells and significantly decreased association of ASK1 with Trx1. Moreover, DAPE enhanced phosphorylation of ASK1 at Thr845 in NCI-H28. Taken together, these results indicate that DAPE activates NOX to generate ROS, which inhibits TrxR, allowing decrease in reduced Trx and dissociation of ASKI from a complex with Trx, and then leading to activation of ASK1. ASK1 activates MKK3/6 followed by p38 MAPK or MKK4/7 followed by SAPK/JNK through sequential phosphorylation, and both of p38 MAPK and SAPK/JNK are implicated in apoptosis (34,35). DAPE markedly enhanced phosphorylation of p38 MAPK at Thr180/ Tyr182 in NCI-H28 cells, although no significant increase in the phosphorylation of MKK3 at Ser189 was not obtained. DAPE, on the other hand, enhanced phosphorylation of JNK at Thr183/Tyr185 only at 30-min treatment, but phosphorylation of MKK4 at Ser257/ Thr261 was not affected. DAPE-induced increase in the phosphorylation of p38 MAPK, but not JNK, was abolished by GSH and clearly prevented by knocking-down ASK1. In addition, DAPE-induced NCI-H28 cell death was significantly inhibited by knocking-down ASK1. Overall, these results suggest that DAPE induces apoptosis of NCI-H28 cells dominantly through an ASK1/MKK(3/6)/p38 MAPK pathway. The questions raised are as to: why in spite of significant
Please cite this article in press as: Tsuchiya A, et al., Diarachidonoylphosphoethanolamine induces apoptosis of malignant pleural mesothelioma cells through a Trx/ASK1/p38 MAPK pathway, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.10.003
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Fig. 6. DAPE enhances phosphorylation of ASK1 and p38 MAPK in NCI-H28 cells. Cells were treated with DAPE (100 mM) for periods of time as indicated, followed by Western blotting. Typical blotting images are shown in (A). In the graphs, each column represents the mean (±SEM) signal intensity for p-ASK1 (B), p-MKK3 (C), p-MKK4 (D), p-p38 (E), pJNK (F), or p-ERK (G) relative to that for each protein (n ¼ 4 independent experiments). P value, ANOVA followed by a Bonferroni correction. NS, not significant.
Please cite this article in press as: Tsuchiya A, et al., Diarachidonoylphosphoethanolamine induces apoptosis of malignant pleural mesothelioma cells through a Trx/ASK1/p38 MAPK pathway, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.10.003
A. Tsuchiya et al. / Journal of Pharmacological Sciences xxx (2015) 1e9
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Fig. 7. DAPE-induced phosphorylation of p38 MAPK is abrogated by GSH in NCI-H28 cells. Cells were treated with DAPE (100 mM) for 30 min in the presence and absence of GSH (1 mM), which was applied 1 h prior to DAPE treatment, and then, Western blotting was carried out. Typical blotting images are shown in (A). In the graphs, each column represents the mean (±SEM) signal intensity for p-ASK1 (B), p-ERK (C), p-p38 (D), or p-JNK (E) relative to that for each protein (n ¼ 4 independent experiments). P value, unpaired t-test. NS, not significant.
activation of p38 MAPK DAPE did not activate MKK3 significantly and why in spite of significant inhibition of DAPE-induced p38 MAPK activation GSH did not inhibit DAPE-induced ASK1 activation. This may represent an additional DAPE-induced direct p38 MAPK activation pathway, regardless of MKK3/6.
In summary, the results of the present study show that DAPE stimulates NOX-mediated ROS production, which suppresses TrxR activity and decreases reduced Trx, allowing ASK1 release from an association with Trx, resulting in the sequential activation of ASK1/ MKK(3/6)/p38 MAPK, to induce caspase-independent apoptosis of
Please cite this article in press as: Tsuchiya A, et al., Diarachidonoylphosphoethanolamine induces apoptosis of malignant pleural mesothelioma cells through a Trx/ASK1/p38 MAPK pathway, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.10.003
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A. Tsuchiya et al. / Journal of Pharmacological Sciences xxx (2015) 1e9
Fig. 8. DAPE-induced phosphorylation of p38 MAPK and cell death are dependent upon ASK1 in NCI-H28 cells. Cells, transfected with the NC siRNA or the ASK1 siRNA, were untreated and treated with DAPE (100 mM) for 30 min, followed by Western blotting. (A) Typical blotting images. (B) In the graphs, each column represents the mean (±SEM) signal intensity for ASK1 normalized by that for b-actin (n ¼ 4 independent experiments). (C) (D) The signal intensities for p-p38 (C) and p-JNK (D) were normalized by those for each unphosphorylated protein. In the graphs, each column represents the mean (±SEM) signal intensity for p-p38 (C) and p-JNK (D) relative to that for each (n ¼ 4 independent experiments). (E) Cells, transfected with the NC siRNA and the ASK1 siRNA, were treated with DAPE (100 mM) for 48 h, followed by MTT assay. In the graph, each column represents the mean (±SEM) percentage of basal levels (MTT intensities for cells before treatment with DAPE) (n ¼ 4 independent experiments). P values, unpaired t-test. NS, not significant.
Please cite this article in press as: Tsuchiya A, et al., Diarachidonoylphosphoethanolamine induces apoptosis of malignant pleural mesothelioma cells through a Trx/ASK1/p38 MAPK pathway, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.10.003
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Please cite this article in press as: Tsuchiya A, et al., Diarachidonoylphosphoethanolamine induces apoptosis of malignant pleural mesothelioma cells through a Trx/ASK1/p38 MAPK pathway, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.10.003