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Sesquiterpene lactones derived from Saussurea lappa induce apoptosis and inhibit invasion and migration in neuroblastoma cells Q1
Keiichi Tabata a, y, Yuki Nishimura a, Taiji Takeda a, Masahiro Kurita a, Taketo Uchiyama a, Takashi Suzuki a, b, * a b
School of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, Japan School of Medicine, Nihon University, 30-1 Oyaguchikami-cho, Itabashi-ku, Tokyo 173-0032, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 September 2014 Received in revised form 7 January 2015 Accepted 12 January 2015 Available online xxx
Neuroblastoma is among the most fatal of solid tumors in the pediatric age group, even when treated aggressively. Therefore, a new effective therapeutic drug(s) for neuroblastoma is urgently needed. To clarify the anticancer effects of the sesquiterpene lactones dehydrocostus lactone and costunolide, derived from Saussurea lappa, we examined the cytotoxic and migration/invasion-inhibitory effects of these compounds against neuroblastoma cell lines. Both the compounds exerted significant cytotoxicity against the neuroblastoma cell lines IMR-32, NB-39, SK-N-SH, and LA-N-1. Evidence of cellular apoptosis, such as nuclear condensation and membrane inversion, were observed after treatment with these compounds. Both compounds induced caspase-7 activation and PARP cleavage as confirmed by Western blotting. Furthermore, the sesquiterpene lactones also suppressed invasion and migration of the neuroblastoma cells. These results suggest that dehydrocostus lactone and costunolide are promising candidates for being developed into novel anticancer drugs effective against neuroblastoma. © 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/3.0/).
Keywords: Sesquiterpenes Neuroblastoma Apoptosis Invasion Migration
1. Introduction Neuroblastoma, a tumor of the sympathetic nervous system, is the most common extracranial solid tumor in children. About 40% of patients with neuroblastoma are in stage 4 (International Neuroblastoma Staging System (INSS)) of the disease at diagnosis (1). In spite of combined-modality therapy and aggressive treatment with the available anticancer drugs, the 5-year event-free survival rate of patients with widely disseminated neuroblastoma (stage 4) remains around 35% (1). On the other hand, the prognosis is favorable in patients under one year of age with metastasis only to the skin, liver and bone marrow (stage 4S), because of spontaneous regression of this type of tumor. Although the precise mechanism of the spontaneous regression is still unclear, the contribution of apoptosis has been proposed (2). * Corresponding author. School of Medicine, Nihon University, 30-1 Oyaguchikami-cho, Itabashi-ku, Tokyo 173-0032, Japan. E-mail address: [email protected] (T. Suzuki). Peer review under responsibility of Japanese Pharmacological Society. y
Deceased.
Evasion of apoptosis is a hallmark of human cancers, including neuroblastoma. Caspase-8, a key enzyme in the execution of apoptosis via the death receptor pathway, is silenced in aggressive neuroblastoma (3). Bcl-2, an anti-apoptotic protein that suppresses mitochondrial cytochrome c release, tends to be overexpressed in neuroblastoma cell lines (4) which may be related to the inhibition of the chemotherapeutic agent-induced apoptosis (5). Therefore, an apoptosis-inducing compound may be a valid candidate for an effective therapeutic drug against neuroblastoma. Metastasis, one of the most important factors reflecting the high malignancy grade of a tumor, is a determinant of the therapeutic strategy. Matrix metalloproteinase (MMP)-2 and MMP-9 are key molecules involved in the mechanism of tumor invasion and metastasis. In many human solid tumors, including neuroblastoma, MMP-2 and MMP-9 are markedly overexpressed during the invasive and metastatic phases (6,7). Saussurea lappa (Mokko in Japanese) is a plant of the Compositae that is used for the treatment of asthma, inflammatory diseases, ulcers, and stomach problems as a component of traditional prescriptions in oriental countries (8). Costunolide and dehydrocostus lactone, major sesquiterpene lactones derived from
http://dx.doi.org/10.1016/j.jphs.2015.01.002 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/3.0/).
Please cite this article in press as: Tabata K, et al., Sesquiterpene lactones derived from Saussurea lappa induce apoptosis and inhibit invasion and migration in neuroblastoma cells, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.01.002
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S. lappa, are reported to induce cell death in some cancer cells (9e11). Dehydrocostus lactone has been shown to induce apoptosis in lung cancer (12), breast cancer (13), hepatocellular carcinoma (14), prostate cancer (15) and leukemia (16). Costunolide has also been shown to induce apoptosis in bladder cancer (17), breast cancer (18), ovarian cancer (19), and leukemia (20,21). However, the antitumor effect of these sesquiterpenes against neuroblastoma is still unknown. In this study, we investigated the apoptosis-inducing effect and metastasiseinhibitory activity of dehydrocostus lactone and costunolide (Fig. 1), derived from S. lappa, against neuroblastoma cell lines. 2. Materials and methods 2.1. Materials Dehydrocostus lactone and costunolide, used as the test substances for this study, were procured from Isukura Industry Co. Ltd. (Tokyo). Both the compounds were stored as 50 mM stock solutions in 100% dimethyl sulfoxide (DMSO). The final concentration of DMSO was 0.2%. Cisplatin was purchased from Wako (Osaka). Thiazolyl blue tetrazolium bromide (MTT) was purchased from SigmaeAldrich (St. Louis, MO, USA). 2.2. Cell lines The human neuroblastoma cell lines IMR-32, LA-N-1, SK-N-SH (purchased from RIKEN BRC, Tsukuba) and NB-39 (provided by Dr. Toshimitsu Suzuki, Fukushima Medical University, Fukushima) were cultured in RPMI-1640 medium (Invitrogen-Life Technologies, Carlsbad, CA, USA) containing 10% heat-inactivated fetal bovine serum (FBS) (HyClone, Logan, UT, USA), 100 unit/mL penicillin and 100 mg/mL streptomycin at 37 C and 5% CO2, in a humid condition. Human umbilical vein endothelial cells (HUVEC) and normal human dermal fibroblasts (NHDF) (purchased from Lonza, Walkersville, MD, USA) were maintained in EGM and FGM-2 (Lonza), respectively. The cells were maintained at 37 C/5% CO2 in a humid environment. 2.3. MTT assay IMR-32, LA-N-1, SK-N-SH and NB-39 cells (1 104 cells/well) were spread on to a 96-well culture plate in phenol red-free RPMI1640 medium (with 10% FBS) and maintained for 24 h. HUVEC and NHDF cells (2 104 cells/well) were spread on to a 96-well culture plate in each cell culture medium and maintained for 24 h. Then, dehydrocostus lactone, costunolide (final concentration 106e104 M) or vehicle was applied for 48 h. After addition of 0.5% MTT solution at 10% of the volume of the medium in the well, incubation was continued for an additional 3 h at 37 C/5% CO2. An equal volume of stop solution (0.04 M HCl in isopropanol) was
Fig. 1. Chemical structure of the sesquiterpene lactones derived from Saussurea lappa.
Fig. 2. Neuroblastoma cells (IMR-32, NB-39, SK-N-SH, LA-N-1) and normal cells (NHDF, HUVEC) were treated with dehydrocostus lactone (A) and costunolide (B) for 48 h. The vertical axis indicates the % cell survival in the treated group vs. the vehicle control group.
added to the culture medium in each well, and the absorbances at 570 nm (peak) and 655 nm (trough) were measured after thorough pipetting to disperse the generated blue formazan.
2.4. Hoechst 33342 staining assay An apoptotic nuclear morphology was observed by staining with Hoechst 33342. IMR-32 cells (1 105 cells/well) were spread on to 6-well cell culture plates containing RPMI 1640 medium (with 10% FBS) and incubated at 37 C in 5% CO2 for 24 h. Then, dehydrocostus lactone, costunolide (final concentration 106e104 M) or vehicle was added and the incubation was continued for 24 or 48 h. Hoechst 33342 solution (final concentration, 0.001% of medium) was added to the wells for 15 min, and the cells were then observed under a fluorescence microscope (IX71, Olympus, Tokyo).
2.5. Flow cytometry IMR-32 cells (1 106 cells/well) were spread on to 6-well cell culture plates containing RPMI 1640 medium (with 10% FBS) and incubated at 37 C in 5% CO2 for 24 h. Then, dehydrocostus lactone, costunolide, cisplatin (final concentration 106e104 M) or vehicle was added and the incubation was continued for 24 h. Cells were collected by trypsinization and washed with annexin-binding buffer and stained with annexin V-Alexa Fluor 488 and propidium iodide (PI) for 15 min. The cell samples were then analyzed using an FC500 flow cytometer (Beckman Coulter, Brea, CA, USA) in the FL1 and FL4 range for annexin V-Alexa Fluor 488 and PI, respectively.
Please cite this article in press as: Tabata K, et al., Sesquiterpene lactones derived from Saussurea lappa induce apoptosis and inhibit invasion and migration in neuroblastoma cells, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.01.002
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Fig. 3. Morphological features of apoptosis. IMR-32 cells were exposed to the test compounds for 48 h and stained with Hoechst 33342. Confocal image (left) and luminescence image (right) obtained from same viewpoint. White bar: 20 mm.
2.6. Western blotting Western blotting was performed as described previously (22). Briefly, IMR-32 and NB-39 cells (2 106 cells/dish) were plated on to 60-mm dishes and incubated for 24 h. They were then treated with test compounds for 0e72 h (0 h refers to untreated dishes) and protein samples were prepared. Equal amounts (10 mg) of proteins were separated by SDS-PAGE and transferred to PVDF membranes. After being blocked with 5% skim milk, the membranes were probed with the primary antibodies overnight at 4 C. The blots were detected using HRP-conjugated secondary antibody and the ECL system. The primary antibodies used were as follows: anti-PARP (611038) (BD Biosciences, San Jose, CA, USA), anti-btubulin (T 4026) (SigmaeAldrich), anti-Bax (2772), anti-Bcl-2 (2876), anti-MMP-2 (4022), anti-MMP-9 (3852), anti-caspase-7 (9494), anti-caspase-9 (9502), and anti-cleaved-caspase-3 (9661) (Cell Signaling Technology, Danvers, MA, USA). 2.7. Invasion and migration assay We selected NB-39 cells for the invasion and migration assay, because they showed higher invasion and migration rates as compared to other neuroblastoma cell lines in a preliminary study. Matrigel inserts for the invasion assay and control inserts for the migration assay (BD Biosciences) were set into the companion plate containing serum-free medium, and serum-free medium was added on to each insert. After hydration of the inserts for 2 h, NB-39
cells (5 105 cells/insert) were seeded with serum-free medium. Then, dehydrocostus lactone, costunolide (final concentration 107e105 M) or vehicle was added and the incubation was continued for 24 h. The medium in the insert was discarded and the remaining cells were removed with a cotton swab. Cells that had migrated to the opposite side of the insert were fixed and stained by Diff-Quik solution (Sysmex, Kobe), photographed under a microscope (IX71, Olympus), and the cell number was counted.
2.8. Statistics Data are expressed as means ± S.E.M. (n ¼ 3). Significance testing was performed using one-way analysis of variance (ANOVA) followed by Bonferroni's test for comparing three or more data.
3. Results To determine the cytotoxicity of the sesquiterpene lactones derived from S. lappa, the MTT assay was performed on human neuroblastoma cell lines and control human normal cells. Application of dehydrocostus lactone and costunolide for 48 h induced significant cytotoxicity against the neuroblastoma cells (Fig. 2), with IC50 values of 1.26e6.52 mM. The control HUVEC was also as sensitive to these compounds as the neruoblastoma cells. On the other hand, application of costunolide at 10 mM had no effect on the NHDF cell line (Fig. 2).
Please cite this article in press as: Tabata K, et al., Sesquiterpene lactones derived from Saussurea lappa induce apoptosis and inhibit invasion and migration in neuroblastoma cells, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.01.002
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Fig. 4. Flow-cytometric analysis of cells stained with annexin V and propidium iodide. IMR-32 cells were exposed to the test compounds for 24 h. The vertical axis indicates the cells stained by PI (FL4 Log) and the horizontal axis indicates those labeled with Annexin V-Alexa Fluor 488 (FL1 Log). (viable cells: left lower, early apoptosis: right lower, late apoptosis: right upper.)
In the Hoechst 33342 assay, dehydrocostus lactone and costunolide induced cell shrinkage and nuclear condensation of IMR-32 cells in a dose-dependent manner after 48 h of treatment (Fig. 3). In the flow-cytometric analysis, movement of the cell population from the viable to the early apoptotic region was observed after treatment with either compound at 10 mM. Moreover, almost the entire cell population was detected in the late apoptotic after treatment
with either compound at 100 mM (Fig. 4). These results indicate that these sesquiterpene lactones induced apoptotic cell death in the neuroblastoma cells. The mechanisms of apoptosis induced by these sesquiterpene lactones were revealed by Western blotting (Fig. 5). Caspase-7, but not caspase-3 or caspase-9, was significantly activated by both compounds after 24 h application. Subsequently, PARP cleavage
Please cite this article in press as: Tabata K, et al., Sesquiterpene lactones derived from Saussurea lappa induce apoptosis and inhibit invasion and migration in neuroblastoma cells, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.01.002
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Fig. 5. Western-blot analysis for apoptosis-related proteins. IMR-32 cells were exposed to 10 mM of dehydrocostus lactone and costunolide for 0e24 h b-tubulin was used as the loading control.
Fig. 6. Inhibition of migration and invasion. NB-39 cells were exposed to 0.1e10 mM of dehydrocostus lactone and costunolide for 24 h. Vertical axes indicate the number of cells showing migratory (A) or invasive (B) activity. N: normal control, V: vehicle control. (C) Western blot analysis of MMP-2 in NB-39 cells. Cells were exposed to 1 mM of the test compounds for 0e72 h.
Please cite this article in press as: Tabata K, et al., Sesquiterpene lactones derived from Saussurea lappa induce apoptosis and inhibit invasion and migration in neuroblastoma cells, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.01.002
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was detected. While costunolide did not affect the expression levels of Bcl-2 or Bax, dehydrocostus lactone decreased the Bax expression level. As shown in Fig. 6A and B, both the sesquiterpene lactones significantly inhibited cell migration and invasion. In the case of 10 mM application of both compounds, cytotoxic effect should also be considered, because these compounds were found to be moderately cytotoxic against NB-39 cells at this concentration (Fig. 2). However, 1 mM of costunolide, which showed no cytotoxicity in the MTT assay, also inhibited the invasion significantly. Dehydrocostus lactone showed a tendency to inhibit the cell invasion. Interestingly, both compounds showed more powerful migration-inhibitory effects at 0.1 mM than at 1 mM. Treatment with dehydrocostus lactone and costunolide at 1 mM resulted in decrease of MMP-2 expression (Fig. 6C). Thus, reduction of MMP-2 expression contributes, at least in part, to the invasion- and migrationinhibitory effects of both the sesquiterpene lactones. 4. Discussion In this study, we demonstrated the cytotoxicity and apoptosisinducing activity of dehydrocostus lactone and costunolide against neuroblastoma cells. Both compounds possess an a-exomethylene-g-lactone moiety which is reported to be involved in the cytotoxic potential of these agents (8). Induction of apoptosis by dehydrocostus lactone and costunolide has been reported in many types of cancer cells (12,15e18,21). However, there is no report on the anticancer effects of these compounds against neuroblastoma. Caspases play important roles in the induction of apoptosis. Neuroblastoma is known to lack caspase-8 expression because of epigenetic silencing (3). In this study, the sesquiterpene lactones activated caspase-7, but not caspase-3 or -9. These sesquiterpene lactones activated caspase-7 selectively, which induced subsequent PARP cleavage, inversion of the plasma membrane and nuclear condensation. These data imply that the sesquiterpene lactones derived from S. lappa can induce apoptosis even in apoptosisresistant cells which lack functional caspase-3, -8, and/or -9. In a previous study, dehydrocostus lactone was shown to induce apoptosis with activation of caspase-3 in leukemia (16) and prostate cancer (15). Similarly, costunolide has been shown to activate caspase-3 in ovarian cancer (19) and bladder cancer (17). The precise mechanism seems to differ depending on the cell types. Caspase-7 is activated by caspase-9 via mitochondrial pathway in general. On the other hand, caspase-7 activation requires caspase-1 inflammasomes under inflammatory conditions (23). The sesquiterpene lactones may activate these pathway related in inflammation. Migration and invasion are hallmarks of malignancy. Inhibition of migration and invasion of cancers may be expected to be associated with a reduction of the malignancy grade of cancers. In this study, both dehydrocostus lactone and costunolide inhibited cell invasion in a concentration-dependent manner. On the other hand, both the sesquiterpene lactones inhibited the migratory activity of the cells to a greater degree at 0.1 mM than at 1 mM. Clarification of this lack of concentration dependency in the migration assay needs further study. These multiple effects would seem to be of clinical benefit in neuroblastoma patients in order to avoid drug resistance developed in malignant transformation and/or recurrence. Recently, two reports showed inhibition of migration and invasion by dehydrocostus lactone and costunolide in soft-tissue sarcoma cells and breast cancer cells (24,25). MMP-2 and -9 gelatinases facilitate invasion and migration by causing degradation of collagen and the fibronectin matrix (6). MMP-2 and -9 expressions are known to be related to the clinical aggressiveness of neuroblastomas (7). Marked downregulation of
the expression of MMP-2 by dehydrocostus lactone and costunolide was observed in NB-39 cells after 48 h of application. The results indicate that the downregulation of MMP-2 expression by these compounds may be related to the inhibition of the migration and invasion characteristics of the cells, at least in the late phase of this phenomenon. On the other hand, MMP-9 expression is not detected in NB-39 cells. MMP-9 expression is reported to be specifically restricted to stromal cells surrounding the tumor cell nests, and in particular, vascular and perivascular cells (7). Anti-angiogenesis effects of sesquiterpene lactones have been reported previously. Dehydrocostus lactone suppressed angiogenesis both in vitro and in vivo (26). Costunolide has been shown to inhibit capillary-like tube formation in TR-LE cells (9). Moreover, these compounds have been shown to induce cell cycle arrest (10,13,18,27) in cancer cell lines; the phase of cell cycle arrest differed among the cell lines. Taken together, sesquiterpene lactones derived from S. lappa have anticancer effects against neuroblastoma cells via multiple mechanisms. In conclusion, dehydrocostus lactone and costunolide derived from S. lappa exert cytotoxic effects against neuroblastoma cells via inducing apoptosis related to caspase-7 activation and also antimetastatic effects via inhibition of migration and invasion. Conflicts of interest The authors have no potential conflicts of interests to declare. Acknowledgements We are grateful to Mr. Satoshi Kamo (Isukura Industry Co. Ltd., Tokyo, Japan) who kindly provided us with dehydrocostus lactone and costunolide. References 1 Cohn SL, Pearson AD, London WB, Monclair T, Ambros PF, Brodeur GM, et al. The international neuroblastoma risk group (INRG) classification system: an INRG Task Force report. J Clin Oncol. 2009;27:289e297. 2 Kocak H, Ackermann S, Hero B, Kahlert Y, Oberthuer A, Juraeva D, et al. Hox-C9 activates the intrinsic pathway of apoptosis and is associated with spontaneous regression in neuroblastoma. Cell Death Dis. 2013;4:e586. 3 Teitz T, Lahti JM, Kidd VJ. Aggressive childhood neuroblastomas do not express caspase-8: an important component of programmed cell death. J Mol Med. 2001;79:428e436. 4 Reed JC, Meister L, Tanaka S, Cuddy M, Yum S, Geyer C, et al. Differential expression of bcl2 proto-oncogene in neuroblastoma and other human tumor cell lines of neural origin. Cancer Res. 1991;51:6529e6538. ~ ez G, Merchant AK, Maybaum J, Rode CK, Bloch CA, et al. Bcl-2 5 Dole M, Nun inhibits chemotherapy-induced apoptosis in neuroblastoma. Cancer Res. 1994;54:3253e3259. 6 Ribatti D, Marimpietri D, Pastorino F, Brignole C, Nico B, Vacca A, et al. Angiogenesis in neuroblastoma. Ann N Y Acad Sci. 2004;1028:133e142. 7 Sugiura Y, Shimada H, Seeger RC, Laug WE, DeClerck YA. Matrix metalloproteinases-2 and -9 are expressed in human neuroblastoma: contribution of stromal cells to their production and correlation with metastasis. Cancer Res. 1998;58:2209e2216. 8 Pandey MM, Rastogi S, Rawat AK. Saussurea costus: botanical, chemical and pharmacological review of an ayurvedic medicinal plant. J Ethnopharmacol. 2007;110:379e390. 9 Jeong D, Watari K, Shirouzu T, Ono M, Koizumi K, Saiki I, et al. Studies on lymphangiogenesis inhibitors from Korean and Japanese crude drugs. Biol Pharm Bull. 2013;36:152e157. 10 Kretschmer N, Rinner B, Stuendl N, Kaltenegger H, Wolf E, Kunert O, et al. Effect of costunolide and dehydrocostus lactone on cell cycle, apoptosis, and ABC transporter expression in human soft tissue sarcoma cells. Planta Med. 2012;78:1749e1756. 11 Sun CM, Syu WJ, Don MJ, Lu JJ, Lee GH. Cytotoxic sesquiterpene lactones from the root of Saussurea lappa. J Nat Prod. 2003;66:1175e1180. 12 Hung JY, Hsu YL, Ni WC, Tsai YM, Yang CJ, Kuo PL, et al. Oxidative and endoplasmic reticulum stress signaling are involved in dehydrocostuslactonemediated apoptosis in human non-small cell lung cancer cells. Lung Cancer. 2010;68:355e365.
Please cite this article in press as: Tabata K, et al., Sesquiterpene lactones derived from Saussurea lappa induce apoptosis and inhibit invasion and migration in neuroblastoma cells, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.01.002
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Sesquiterpene lactones derived from Saussurea lappa induce apoptosis and inhibit invasion and migration in neuroblastoma cells Q1
Keiichi Tabata a, y, Yuki Nishimura a, Taiji Takeda a, Masahiro Kurita a, Taketo Uchiyama a, Takashi Suzuki a, b, * a b
School of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, Japan School of Medicine, Nihon University, 30-1 Oyaguchikami-cho, Itabashi-ku, Tokyo 173-0032, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 September 2014 Received in revised form 7 January 2015 Accepted 12 January 2015 Available online xxx
Neuroblastoma is among the most fatal of solid tumors in the pediatric age group, even when treated aggressively. Therefore, a new effective therapeutic drug(s) for neuroblastoma is urgently needed. To clarify the anticancer effects of the sesquiterpene lactones dehydrocostus lactone and costunolide, derived from Saussurea lappa, we examined the cytotoxic and migration/invasion-inhibitory effects of these compounds against neuroblastoma cell lines. Both the compounds exerted significant cytotoxicity against the neuroblastoma cell lines IMR-32, NB-39, SK-N-SH, and LA-N-1. Evidence of cellular apoptosis, such as nuclear condensation and membrane inversion, were observed after treatment with these compounds. Both compounds induced caspase-7 activation and PARP cleavage as confirmed by Western blotting. Furthermore, the sesquiterpene lactones also suppressed invasion and migration of the neuroblastoma cells. These results suggest that dehydrocostus lactone and costunolide are promising candidates for being developed into novel anticancer drugs effective against neuroblastoma. © 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/3.0/).
Keywords: Sesquiterpenes Neuroblastoma Apoptosis Invasion Migration
1. Introduction Neuroblastoma, a tumor of the sympathetic nervous system, is the most common extracranial solid tumor in children. About 40% of patients with neuroblastoma are in stage 4 (International Neuroblastoma Staging System (INSS)) of the disease at diagnosis (1). In spite of combined-modality therapy and aggressive treatment with the available anticancer drugs, the 5-year event-free survival rate of patients with widely disseminated neuroblastoma (stage 4) remains around 35% (1). On the other hand, the prognosis is favorable in patients under one year of age with metastasis only to the skin, liver and bone marrow (stage 4S), because of spontaneous regression of this type of tumor. Although the precise mechanism of the spontaneous regression is still unclear, the contribution of apoptosis has been proposed (2). * Corresponding author. School of Medicine, Nihon University, 30-1 Oyaguchikami-cho, Itabashi-ku, Tokyo 173-0032, Japan. E-mail address: [email protected] (T. Suzuki). Peer review under responsibility of Japanese Pharmacological Society. y
Deceased.
Evasion of apoptosis is a hallmark of human cancers, including neuroblastoma. Caspase-8, a key enzyme in the execution of apoptosis via the death receptor pathway, is silenced in aggressive neuroblastoma (3). Bcl-2, an anti-apoptotic protein that suppresses mitochondrial cytochrome c release, tends to be overexpressed in neuroblastoma cell lines (4) which may be related to the inhibition of the chemotherapeutic agent-induced apoptosis (5). Therefore, an apoptosis-inducing compound may be a valid candidate for an effective therapeutic drug against neuroblastoma. Metastasis, one of the most important factors reflecting the high malignancy grade of a tumor, is a determinant of the therapeutic strategy. Matrix metalloproteinase (MMP)-2 and MMP-9 are key molecules involved in the mechanism of tumor invasion and metastasis. In many human solid tumors, including neuroblastoma, MMP-2 and MMP-9 are markedly overexpressed during the invasive and metastatic phases (6,7). Saussurea lappa (Mokko in Japanese) is a plant of the Compositae that is used for the treatment of asthma, inflammatory diseases, ulcers, and stomach problems as a component of traditional prescriptions in oriental countries (8). Costunolide and dehydrocostus lactone, major sesquiterpene lactones derived from
http://dx.doi.org/10.1016/j.jphs.2015.01.002 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/3.0/).
Please cite this article in press as: Tabata K, et al., Sesquiterpene lactones derived from Saussurea lappa induce apoptosis and inhibit invasion and migration in neuroblastoma cells, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.01.002
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S. lappa, are reported to induce cell death in some cancer cells (9e11). Dehydrocostus lactone has been shown to induce apoptosis in lung cancer (12), breast cancer (13), hepatocellular carcinoma (14), prostate cancer (15) and leukemia (16). Costunolide has also been shown to induce apoptosis in bladder cancer (17), breast cancer (18), ovarian cancer (19), and leukemia (20,21). However, the antitumor effect of these sesquiterpenes against neuroblastoma is still unknown. In this study, we investigated the apoptosis-inducing effect and metastasiseinhibitory activity of dehydrocostus lactone and costunolide (Fig. 1), derived from S. lappa, against neuroblastoma cell lines. 2. Materials and methods 2.1. Materials Dehydrocostus lactone and costunolide, used as the test substances for this study, were procured from Isukura Industry Co. Ltd. (Tokyo). Both the compounds were stored as 50 mM stock solutions in 100% dimethyl sulfoxide (DMSO). The final concentration of DMSO was 0.2%. Cisplatin was purchased from Wako (Osaka). Thiazolyl blue tetrazolium bromide (MTT) was purchased from SigmaeAldrich (St. Louis, MO, USA). 2.2. Cell lines The human neuroblastoma cell lines IMR-32, LA-N-1, SK-N-SH (purchased from RIKEN BRC, Tsukuba) and NB-39 (provided by Dr. Toshimitsu Suzuki, Fukushima Medical University, Fukushima) were cultured in RPMI-1640 medium (Invitrogen-Life Technologies, Carlsbad, CA, USA) containing 10% heat-inactivated fetal bovine serum (FBS) (HyClone, Logan, UT, USA), 100 unit/mL penicillin and 100 mg/mL streptomycin at 37 C and 5% CO2, in a humid condition. Human umbilical vein endothelial cells (HUVEC) and normal human dermal fibroblasts (NHDF) (purchased from Lonza, Walkersville, MD, USA) were maintained in EGM and FGM-2 (Lonza), respectively. The cells were maintained at 37 C/5% CO2 in a humid environment. 2.3. MTT assay IMR-32, LA-N-1, SK-N-SH and NB-39 cells (1 104 cells/well) were spread on to a 96-well culture plate in phenol red-free RPMI1640 medium (with 10% FBS) and maintained for 24 h. HUVEC and NHDF cells (2 104 cells/well) were spread on to a 96-well culture plate in each cell culture medium and maintained for 24 h. Then, dehydrocostus lactone, costunolide (final concentration 106e104 M) or vehicle was applied for 48 h. After addition of 0.5% MTT solution at 10% of the volume of the medium in the well, incubation was continued for an additional 3 h at 37 C/5% CO2. An equal volume of stop solution (0.04 M HCl in isopropanol) was
Fig. 1. Chemical structure of the sesquiterpene lactones derived from Saussurea lappa.
Fig. 2. Neuroblastoma cells (IMR-32, NB-39, SK-N-SH, LA-N-1) and normal cells (NHDF, HUVEC) were treated with dehydrocostus lactone (A) and costunolide (B) for 48 h. The vertical axis indicates the % cell survival in the treated group vs. the vehicle control group.
added to the culture medium in each well, and the absorbances at 570 nm (peak) and 655 nm (trough) were measured after thorough pipetting to disperse the generated blue formazan.
2.4. Hoechst 33342 staining assay An apoptotic nuclear morphology was observed by staining with Hoechst 33342. IMR-32 cells (1 105 cells/well) were spread on to 6-well cell culture plates containing RPMI 1640 medium (with 10% FBS) and incubated at 37 C in 5% CO2 for 24 h. Then, dehydrocostus lactone, costunolide (final concentration 106e104 M) or vehicle was added and the incubation was continued for 24 or 48 h. Hoechst 33342 solution (final concentration, 0.001% of medium) was added to the wells for 15 min, and the cells were then observed under a fluorescence microscope (IX71, Olympus, Tokyo).
2.5. Flow cytometry IMR-32 cells (1 106 cells/well) were spread on to 6-well cell culture plates containing RPMI 1640 medium (with 10% FBS) and incubated at 37 C in 5% CO2 for 24 h. Then, dehydrocostus lactone, costunolide, cisplatin (final concentration 106e104 M) or vehicle was added and the incubation was continued for 24 h. Cells were collected by trypsinization and washed with annexin-binding buffer and stained with annexin V-Alexa Fluor 488 and propidium iodide (PI) for 15 min. The cell samples were then analyzed using an FC500 flow cytometer (Beckman Coulter, Brea, CA, USA) in the FL1 and FL4 range for annexin V-Alexa Fluor 488 and PI, respectively.
Please cite this article in press as: Tabata K, et al., Sesquiterpene lactones derived from Saussurea lappa induce apoptosis and inhibit invasion and migration in neuroblastoma cells, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.01.002
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Fig. 3. Morphological features of apoptosis. IMR-32 cells were exposed to the test compounds for 48 h and stained with Hoechst 33342. Confocal image (left) and luminescence image (right) obtained from same viewpoint. White bar: 20 mm.
2.6. Western blotting Western blotting was performed as described previously (22). Briefly, IMR-32 and NB-39 cells (2 106 cells/dish) were plated on to 60-mm dishes and incubated for 24 h. They were then treated with test compounds for 0e72 h (0 h refers to untreated dishes) and protein samples were prepared. Equal amounts (10 mg) of proteins were separated by SDS-PAGE and transferred to PVDF membranes. After being blocked with 5% skim milk, the membranes were probed with the primary antibodies overnight at 4 C. The blots were detected using HRP-conjugated secondary antibody and the ECL system. The primary antibodies used were as follows: anti-PARP (611038) (BD Biosciences, San Jose, CA, USA), anti-btubulin (T 4026) (SigmaeAldrich), anti-Bax (2772), anti-Bcl-2 (2876), anti-MMP-2 (4022), anti-MMP-9 (3852), anti-caspase-7 (9494), anti-caspase-9 (9502), and anti-cleaved-caspase-3 (9661) (Cell Signaling Technology, Danvers, MA, USA). 2.7. Invasion and migration assay We selected NB-39 cells for the invasion and migration assay, because they showed higher invasion and migration rates as compared to other neuroblastoma cell lines in a preliminary study. Matrigel inserts for the invasion assay and control inserts for the migration assay (BD Biosciences) were set into the companion plate containing serum-free medium, and serum-free medium was added on to each insert. After hydration of the inserts for 2 h, NB-39
cells (5 105 cells/insert) were seeded with serum-free medium. Then, dehydrocostus lactone, costunolide (final concentration 107e105 M) or vehicle was added and the incubation was continued for 24 h. The medium in the insert was discarded and the remaining cells were removed with a cotton swab. Cells that had migrated to the opposite side of the insert were fixed and stained by Diff-Quik solution (Sysmex, Kobe), photographed under a microscope (IX71, Olympus), and the cell number was counted.
2.8. Statistics Data are expressed as means ± S.E.M. (n ¼ 3). Significance testing was performed using one-way analysis of variance (ANOVA) followed by Bonferroni's test for comparing three or more data.
3. Results To determine the cytotoxicity of the sesquiterpene lactones derived from S. lappa, the MTT assay was performed on human neuroblastoma cell lines and control human normal cells. Application of dehydrocostus lactone and costunolide for 48 h induced significant cytotoxicity against the neuroblastoma cells (Fig. 2), with IC50 values of 1.26e6.52 mM. The control HUVEC was also as sensitive to these compounds as the neruoblastoma cells. On the other hand, application of costunolide at 10 mM had no effect on the NHDF cell line (Fig. 2).
Please cite this article in press as: Tabata K, et al., Sesquiterpene lactones derived from Saussurea lappa induce apoptosis and inhibit invasion and migration in neuroblastoma cells, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.01.002
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Fig. 4. Flow-cytometric analysis of cells stained with annexin V and propidium iodide. IMR-32 cells were exposed to the test compounds for 24 h. The vertical axis indicates the cells stained by PI (FL4 Log) and the horizontal axis indicates those labeled with Annexin V-Alexa Fluor 488 (FL1 Log). (viable cells: left lower, early apoptosis: right lower, late apoptosis: right upper.)
In the Hoechst 33342 assay, dehydrocostus lactone and costunolide induced cell shrinkage and nuclear condensation of IMR-32 cells in a dose-dependent manner after 48 h of treatment (Fig. 3). In the flow-cytometric analysis, movement of the cell population from the viable to the early apoptotic region was observed after treatment with either compound at 10 mM. Moreover, almost the entire cell population was detected in the late apoptotic after treatment
with either compound at 100 mM (Fig. 4). These results indicate that these sesquiterpene lactones induced apoptotic cell death in the neuroblastoma cells. The mechanisms of apoptosis induced by these sesquiterpene lactones were revealed by Western blotting (Fig. 5). Caspase-7, but not caspase-3 or caspase-9, was significantly activated by both compounds after 24 h application. Subsequently, PARP cleavage
Please cite this article in press as: Tabata K, et al., Sesquiterpene lactones derived from Saussurea lappa induce apoptosis and inhibit invasion and migration in neuroblastoma cells, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.01.002
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Fig. 5. Western-blot analysis for apoptosis-related proteins. IMR-32 cells were exposed to 10 mM of dehydrocostus lactone and costunolide for 0e24 h b-tubulin was used as the loading control.
Fig. 6. Inhibition of migration and invasion. NB-39 cells were exposed to 0.1e10 mM of dehydrocostus lactone and costunolide for 24 h. Vertical axes indicate the number of cells showing migratory (A) or invasive (B) activity. N: normal control, V: vehicle control. (C) Western blot analysis of MMP-2 in NB-39 cells. Cells were exposed to 1 mM of the test compounds for 0e72 h.
Please cite this article in press as: Tabata K, et al., Sesquiterpene lactones derived from Saussurea lappa induce apoptosis and inhibit invasion and migration in neuroblastoma cells, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.01.002
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was detected. While costunolide did not affect the expression levels of Bcl-2 or Bax, dehydrocostus lactone decreased the Bax expression level. As shown in Fig. 6A and B, both the sesquiterpene lactones significantly inhibited cell migration and invasion. In the case of 10 mM application of both compounds, cytotoxic effect should also be considered, because these compounds were found to be moderately cytotoxic against NB-39 cells at this concentration (Fig. 2). However, 1 mM of costunolide, which showed no cytotoxicity in the MTT assay, also inhibited the invasion significantly. Dehydrocostus lactone showed a tendency to inhibit the cell invasion. Interestingly, both compounds showed more powerful migration-inhibitory effects at 0.1 mM than at 1 mM. Treatment with dehydrocostus lactone and costunolide at 1 mM resulted in decrease of MMP-2 expression (Fig. 6C). Thus, reduction of MMP-2 expression contributes, at least in part, to the invasion- and migrationinhibitory effects of both the sesquiterpene lactones. 4. Discussion In this study, we demonstrated the cytotoxicity and apoptosisinducing activity of dehydrocostus lactone and costunolide against neuroblastoma cells. Both compounds possess an a-exomethylene-g-lactone moiety which is reported to be involved in the cytotoxic potential of these agents (8). Induction of apoptosis by dehydrocostus lactone and costunolide has been reported in many types of cancer cells (12,15e18,21). However, there is no report on the anticancer effects of these compounds against neuroblastoma. Caspases play important roles in the induction of apoptosis. Neuroblastoma is known to lack caspase-8 expression because of epigenetic silencing (3). In this study, the sesquiterpene lactones activated caspase-7, but not caspase-3 or -9. These sesquiterpene lactones activated caspase-7 selectively, which induced subsequent PARP cleavage, inversion of the plasma membrane and nuclear condensation. These data imply that the sesquiterpene lactones derived from S. lappa can induce apoptosis even in apoptosisresistant cells which lack functional caspase-3, -8, and/or -9. In a previous study, dehydrocostus lactone was shown to induce apoptosis with activation of caspase-3 in leukemia (16) and prostate cancer (15). Similarly, costunolide has been shown to activate caspase-3 in ovarian cancer (19) and bladder cancer (17). The precise mechanism seems to differ depending on the cell types. Caspase-7 is activated by caspase-9 via mitochondrial pathway in general. On the other hand, caspase-7 activation requires caspase-1 inflammasomes under inflammatory conditions (23). The sesquiterpene lactones may activate these pathway related in inflammation. Migration and invasion are hallmarks of malignancy. Inhibition of migration and invasion of cancers may be expected to be associated with a reduction of the malignancy grade of cancers. In this study, both dehydrocostus lactone and costunolide inhibited cell invasion in a concentration-dependent manner. On the other hand, both the sesquiterpene lactones inhibited the migratory activity of the cells to a greater degree at 0.1 mM than at 1 mM. Clarification of this lack of concentration dependency in the migration assay needs further study. These multiple effects would seem to be of clinical benefit in neuroblastoma patients in order to avoid drug resistance developed in malignant transformation and/or recurrence. Recently, two reports showed inhibition of migration and invasion by dehydrocostus lactone and costunolide in soft-tissue sarcoma cells and breast cancer cells (24,25). MMP-2 and -9 gelatinases facilitate invasion and migration by causing degradation of collagen and the fibronectin matrix (6). MMP-2 and -9 expressions are known to be related to the clinical aggressiveness of neuroblastomas (7). Marked downregulation of
the expression of MMP-2 by dehydrocostus lactone and costunolide was observed in NB-39 cells after 48 h of application. The results indicate that the downregulation of MMP-2 expression by these compounds may be related to the inhibition of the migration and invasion characteristics of the cells, at least in the late phase of this phenomenon. On the other hand, MMP-9 expression is not detected in NB-39 cells. MMP-9 expression is reported to be specifically restricted to stromal cells surrounding the tumor cell nests, and in particular, vascular and perivascular cells (7). Anti-angiogenesis effects of sesquiterpene lactones have been reported previously. Dehydrocostus lactone suppressed angiogenesis both in vitro and in vivo (26). Costunolide has been shown to inhibit capillary-like tube formation in TR-LE cells (9). Moreover, these compounds have been shown to induce cell cycle arrest (10,13,18,27) in cancer cell lines; the phase of cell cycle arrest differed among the cell lines. Taken together, sesquiterpene lactones derived from S. lappa have anticancer effects against neuroblastoma cells via multiple mechanisms. In conclusion, dehydrocostus lactone and costunolide derived from S. lappa exert cytotoxic effects against neuroblastoma cells via inducing apoptosis related to caspase-7 activation and also antimetastatic effects via inhibition of migration and invasion. Conflicts of interest The authors have no potential conflicts of interests to declare. Acknowledgements We are grateful to Mr. Satoshi Kamo (Isukura Industry Co. Ltd., Tokyo, Japan) who kindly provided us with dehydrocostus lactone and costunolide. References 1 Cohn SL, Pearson AD, London WB, Monclair T, Ambros PF, Brodeur GM, et al. The international neuroblastoma risk group (INRG) classification system: an INRG Task Force report. J Clin Oncol. 2009;27:289e297. 2 Kocak H, Ackermann S, Hero B, Kahlert Y, Oberthuer A, Juraeva D, et al. Hox-C9 activates the intrinsic pathway of apoptosis and is associated with spontaneous regression in neuroblastoma. Cell Death Dis. 2013;4:e586. 3 Teitz T, Lahti JM, Kidd VJ. Aggressive childhood neuroblastomas do not express caspase-8: an important component of programmed cell death. J Mol Med. 2001;79:428e436. 4 Reed JC, Meister L, Tanaka S, Cuddy M, Yum S, Geyer C, et al. Differential expression of bcl2 proto-oncogene in neuroblastoma and other human tumor cell lines of neural origin. Cancer Res. 1991;51:6529e6538. ~ ez G, Merchant AK, Maybaum J, Rode CK, Bloch CA, et al. Bcl-2 5 Dole M, Nun inhibits chemotherapy-induced apoptosis in neuroblastoma. Cancer Res. 1994;54:3253e3259. 6 Ribatti D, Marimpietri D, Pastorino F, Brignole C, Nico B, Vacca A, et al. Angiogenesis in neuroblastoma. Ann N Y Acad Sci. 2004;1028:133e142. 7 Sugiura Y, Shimada H, Seeger RC, Laug WE, DeClerck YA. Matrix metalloproteinases-2 and -9 are expressed in human neuroblastoma: contribution of stromal cells to their production and correlation with metastasis. Cancer Res. 1998;58:2209e2216. 8 Pandey MM, Rastogi S, Rawat AK. Saussurea costus: botanical, chemical and pharmacological review of an ayurvedic medicinal plant. J Ethnopharmacol. 2007;110:379e390. 9 Jeong D, Watari K, Shirouzu T, Ono M, Koizumi K, Saiki I, et al. Studies on lymphangiogenesis inhibitors from Korean and Japanese crude drugs. Biol Pharm Bull. 2013;36:152e157. 10 Kretschmer N, Rinner B, Stuendl N, Kaltenegger H, Wolf E, Kunert O, et al. Effect of costunolide and dehydrocostus lactone on cell cycle, apoptosis, and ABC transporter expression in human soft tissue sarcoma cells. Planta Med. 2012;78:1749e1756. 11 Sun CM, Syu WJ, Don MJ, Lu JJ, Lee GH. Cytotoxic sesquiterpene lactones from the root of Saussurea lappa. J Nat Prod. 2003;66:1175e1180. 12 Hung JY, Hsu YL, Ni WC, Tsai YM, Yang CJ, Kuo PL, et al. Oxidative and endoplasmic reticulum stress signaling are involved in dehydrocostuslactonemediated apoptosis in human non-small cell lung cancer cells. Lung Cancer. 2010;68:355e365.
Please cite this article in press as: Tabata K, et al., Sesquiterpene lactones derived from Saussurea lappa induce apoptosis and inhibit invasion and migration in neuroblastoma cells, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.01.002
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21 Choi JH, Ha J, Park JH, Lee JY, Lee YS, Park HJ, et al. Costunolide triggers apoptosis in human leukemia U937 cells by depleting intracellular thiols. Jpn J Cancer Res. 2002;93:1327e1333. 22 Tabata K, Hamano A, Akihisa T, Suzuki T, Kuguaglycoside C. a constituent of Momordica charantia, induces caspase-independent cell death of neuroblastoma cells. Cancer Sci. 2012;103:2153e2158. 23 Lamkanfi M, Kanneganti TD. Caspase-7: a protease involved in apoptosis and inflammation. Int J Biochem Cell Biol. 2010;42:21e24. 24 Choi YK, Cho SG, Woo SM, Yun YJ, Jo J, Kim W, et al. Saussurea lappa Clarkederived costunolide prevents TNFa-induced breast cancer cell migration and invasion by inhibiting NF-kB activity. Evid Based Complement Altern Med. 2013;2013:936257. 25 Lohberger B, Rinner B, Stuendl N, Kaltenegger H, Steinecker-Frohnwieser B, Bernhart E, et al. Sesquiterpene lactones downregulate G2/M cell cycle regulator proteins and affect the invasive potential of human soft tissue sarcoma cells. PLoS One. 2013;8:e66300. 26 Wang CY, Tsai AC, Peng CY, Chang YL, Lee KH, Teng CM, et al. Dehydrocostuslactone suppresses angiogenesis in vitro and in vivo through inhibition of Akt/GSK-3b and mTOR signaling pathways. PLoS One. 2012;7:e31195. 27 Hsu JL, Pan SL, Ho YF, Hwang TL, Kung FL, Guh JH. Costunolide induces apoptosis through nuclear calcium2þ overload and DNA damage response in human prostate cancer. J Urol. 2011;185:1967e1974.
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