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A methyl jasmonate derivative, J-7, induces apoptosis in human hepatocarcinoma Hep3B cells in vitro
Toxicology in Vitro 24 (2010) 1920–1926
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A methyl jasmonate derivative, J-7, induces apoptosis in human hepatocarcinoma Hep3B cells in vitro Cheol Park a, Cheng-Yun Jin b, Gi-Young Kim c, JaeHun Cheong d, Jee H. Jung e, Young Hyun Yoo f,*, Yung Hyun Choi a,b,g,** a
Blue-Bio Industry Regional Innovation Center, Dongeui University, Busan 614-714, South Korea Department of Biomaterial Control (BK21 Program), Dongeui University Graduate School, Busan 614-714, South Korea Faculty of Applied Marine Science, Cheju National University, Jeju 690-756, South Korea d Department of Molecular Biology, College of Natural Sciences, Pusan National University, Busan 609-735, South Korea e College of Pharmacy, Pusan National University, Busan 609-735, South Korea f Department of Anatomy and Cell Biology, Dong-A University College of Medicine and Mitochondria Hub Regulation Center, Busan 602-714, South Korea g Department of Biochemistry, Dongeui University College of Oriental Medicine, Busan 614-052, South Korea b c
a r t i c l e
i n f o
Article history: Received 2 February 2010 Accepted 2 August 2010 Available online 7 August 2010 Keywords: Methyl jasmonate derivative Hep3B Apoptosis MAPK
a b s t r a c t The pro-apoptotic activity of J-7, a synthetic methyl jasmonate derivative, on the Hep3B human hepatocarcinoma cell line was investigated. Treatment of Hep3B cells with J-7 resulted in growth inhibition and the induction of apoptosis as measured by trypan blue-excluding cells, MTT assay, nuclear staining, DNA fragmentation, and flow cytometry analysis. The increased apoptotic events in Hep3B cells caused by J-7 were associated with the alteration in the ratio of Bax/Bcl-2 protein expression. J-7 treatment induced the expression of death receptor-related proteins such as death receptor 5, which triggered the activation of caspase-8 and the down-regulation of the whole Bid expression. In addition, the apoptosis induction by J7 was correlated with the activation of caspase-9 and caspase-3, down-regulation IAP family proteins such as XIAP and cIAP-1, and concomitant degradation of poly (ADP-ribose) polymerase. However, the cytotoxic and apoptotic effects induced by J-7 were significantly inhibited by z-DEVD-fmk, a caspase-3 inhibitor, which demonstrates the important role that caspase-3 plays in the process. Furthermore, blocking the extracellular signal-regulated protein kinase and c-Jun N-terminal kinase pathways showed increased apoptosis and the activation of caspases in J-7-induced apoptosis. The results indicated that J-7 induces the apoptosis of Hep3B cells through a signaling cascade of death-receptor-mediated extrinsic as well as mitochondria-mediated intrinsic caspase pathways, which are associated with the activation of the mitogen-activated protein kinases signal pathway. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Hepatocellular carcinoma is the most common malignancies and the fourth-leading cause of cancer deaths worldwide (Lau and Lai, 2008). The disease is associated with environmental exposure to the hepatitis B virus, hepatitis C virus, and Aflatoxin B1, etc. (Ince and Wands, 1999; Okuda, 2000; Coleman, 2003; Marotta et al., 2004). Surgical resection has been considered the optimal treatment approach, but only a small proportion of the patients qualified for surgery; there is a high rate of recurrence after surgery. Approaches used to prevent recurrence have included
* Corresponding author. Tel.: +82 51 240 2926; fax: +82 51 241 3767. ** Corresponding author at: Department of Biochemistry, Dongeui University College of Oriental Medicine, Busan 614-052, South Korea. Tel.: +82 51 850 7413; fax: +82 51 853 4036. E-mail addresses: [email protected] (Y.H. Yoo), [email protected] (Y.H. Choi). 0887-2333/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2010.08.001
chemoembolization before surgery and neoadjuvant therapy after surgery, neither of which has proved to be beneficial (Carr, 2004). Therefore, new therapeutic options are needed for more effective treatment of this malignancy. The jasmonate family consists of cis-jasmone, jasmonic acid, and methyl jasmonate, which are fatty-acid-derived cyclopentanones that occur ubiquitously in the plant kingdom. They serve as natural bioregulators and are involved in plant intracellular signaling and defense in response to injury (Mitler and Lam, 1996). Jasmonates are synthesized in plants from a-linolenic acid via the lipoxygenase pathway (Farmer and Ryan, 1990; Dangl et al., 1996; Weber, 2002), a process involving the transformation of linolenic acid by lipoxygenase to 13-hydroperoxylinolenic acid, and then to jasmonic acid that is finally converted to methyl jasmonate (Weber, 2002). Recently, it has been reported that methyl jasmonate exhibits anti-cancer activity in vitro and in vivo and induces growth inhibition in cancer cells, including breast and prostate
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carcinoma cells, as well as in melanoma, lymphoma, and leukemia cells, while leaving the non-transformed cells intact (Fingrut and Flescher, 2002; Rotem et al., 2003, 2005; Kniazhanski et al., 2008). Methyl jasmonate is also reported to have the ability to act against drug resistant cells (Fingrut et al., 2005; Kniazhanski et al., 2008; Ofer et al., 2008). Methyl jasmonate was also discovered to have cytotoxic effects toward metastatic melanoma both in vitro and in vivo (Reischer et al., 2007) and to significantly increase the life span of lymphoma-bearing mice (Rotem et al., 2005). Thus, methyl jasmonate is now considered as a promising lead for cancer treatment in humans (Flescher, 2005; Cohen and Flescher 2009). Apoptosis is a tightly regulated process characterized by cell shrinkage, plasma membrane blabbing, and chromatin condensation that is consistent with DNA cleavage in ladders (Makin and Dive, 2001; Ghobrial et al., 2005). Apoptosis plays an important role in developmental processes, maintenance of homeostasis, and elimination of damaged cells. In general, apoptosis can be initiated in two ways: by the death receptor pathway (extrinsic) or by the mitochondria-dependent pathway (intrinsic), leading to activation of caspases and consequent apoptosis in mammalian cells (Earnshaw et al., 1999; Han et al., 2008). In the former case, plasma membrane death receptors are involved and the apoptosis signal is provided by the interaction between the ligand and death receptor. Changes in mitochondrial integrity by a broad range of physical and chemical stimuli, however, can trigger the intrinsic pathway of apoptosis (Nagata and Golstein, 1995; Aggarwal et al., 2004; Jeong and Seol, 2008). Recent evidence has also shown that responses to numerous types of extracellular signals are mediated by mitogen-activating protein kinases (MAPKs), members of the serine/threonine kinase family (Ichijo, 1999; Chopra et al., 2008). Several studies have revealed that c-Jun N-terminal kinase (JNK)1/stress-activated protein kinase (SAPK) and/or p38 MAPK activation were involved in apoptosis induced by a variety of different stimuli (Cuevas et al., 2007; Chopra et al., 2008). However, most cancer cells can block apoptosis, which allows them to survive despite genetic and morphologic transformations. Therefore, the induction of apoptotic cell death is an important mechanism in many anti-cancer drugs. Although the induction of apoptosis by methyl jasmonate has been observed in some cancer cell lines, the molecular mechanisms of its pro-apoptotic action on malignant cell growth are not completely unknown. Recently, in an effort to develop novel anti-inflammatory agents, we employed the jasmonate skeleton as a model structure for the synthesis of new cyclopentanonebearing derivatives and reported the synthesis and anti-inflammatory activity of methyl jasmonate analogues (Dang et al., 2008). The above findings led us to investigate whether methyl jasmonate and its derivatives inhibit cancer cell growth and induce apoptosis. In the present study, we examined the anti-cancer effect of a novel methyl jasmonate derivative, J-7 [methyl 5-chloro-4,5-didehydrojasmonate (7)], using human hepatocarcinoma Hep3B cells in vitro and examined its effects on apoptosis induction and the expression of apoptosis-related genes.
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for Bcl-2, Bcl-XL, Bax, Bad, Bid, Fas, Fas ligand (FasL), XIAP, cIAP1, cIAP-2, caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerases (PARP) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies specific for death receptor 4 (DR4) and DR5 were from Calbiochem. Ant-actin antibody was obtained from Sigma–Aldrich. Peroxidase-labeled donkey anti-rabbit immunoglobulin and peroxidase-labeled sheep anti-mouse immunoglobulin were purchased from Amersham (Arlington Heights, IL). 2.2. Cell culture, a synthetic methyl jasmonate derivative (J-7), cell viability and growth study The human hepatoma Hep3B cells were purchased from the American Type Culture Collection (Rockville, MD), and maintained at 37 °C in a humidified 95% air and 5% CO2 in RPMI1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml penicillin, and 100 lg/ml streptomycin. The structure and method of synthesis of J-7 [methyl 5-chloro-4,5didehydrojasmonate (7)], an a-haloenone jasmonate, were previously described (Dang et al., 2008). J-7 was dissolved in dimethyl sulfoxide (DMSO), and dilutions were made in RPMI1640. The final concentration of DMSO in the medium was less than 0.1% (vol/vol) in the treatment concentration (50 lM) and showed no influence on cell growth. For the cell viability and growth study, the cells were grown to 70% confluence and treated with 50 lM J-7 for 0– 24 h. Control cells were supplemented with complete media containing 0.1% DMSO for 24 h time points. After treatments, cell number and viability were determined by Trypan blue exclusion and MTT assays, respectively. 2.3. Nuclear staining with DAPI After treatment with J-7, the cells were harvested, washed with phosphate-buffered saline (PBS) and fixed with 3.7% paraformaldehyde (Sigma–Aldrich) in PBS for 10 min at room temperature. The fixed cells were washed with PBS, and stained with 2.5 lg/ml DAPI solution for 10 min at room temperature. The cells were washed two more times with PBS and analysed via a fluorescent microscope (Carl Zeiss, Germany) (Jang et al., 2010). 2.4. DNA fragmentation assay Cells were lysed in a buffer containing 10 mM Tris–HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, and 0.5% Triton X-100 for 30 min at room temperature. After centrifugation for 20 min at 14,000 rpm, supernatant samples were treated with RNase and proteinase K and incubated at 50 °C for 3 h. The DNA in the supernatant was extracted using a 25:24:1 (v/v/v) equal volume of neutral phenol:chloroform:isoamyl alcohol (Sigma–Aldrich). Subsequently, 5 M of NaCl and isopropanol were added to the samples and kept at 20 °C for 6 h. Following centrifugation for 15 min at 15,000 rpm, the pellets were dissolved in TE buffer (10 mM Tris– HCl and 1 mM EDTA) with RNase A. The DNA was separated on 1.5% agarose gels containing 0.1 lg/ml ethidium bromide (EtBr, Sigma–Aldrich) and was visualized using an ultraviolet light source (Im and Kim, 2010).
2. Materials and methods 2.5. Flow cytometric analysis 2.1. Reagents and antibodies 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphnyl-2H-tetrazolium bromide (MTT) and 4,6-diamidino-2-phenylindole (DAPI) were obtained from Sigma–Aldrich (St. Louis, MO). PD98059 (ERK inhibitor) and SP600125 (JNK inhibitor) were purchased from Calbiochem (San Diego, CA). Caspase activity assay kits were purchased from R&D Systems (Minneapolis, MN). Antibodies specific
After treatment with J-7, the cells were collected, washed with cold PBS, and fixed in 75% ethanol at 4 °C for 30 min. The DNA content of the cells was measured using a DNA staining kit (CycleTEST™ PLUS Kit, Becton Dickinson, San Jose, CA). Propidium iodide (PI)-stained nuclear fractions were obtained by following the kit protocol. The cells were then filtered through 35-mm mesh, and DNA content fluorescence was determined using a FACSCalibur
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(Becton Dickinson) flow cytometer within 1 h. The cellular DNA content was analysed by CellQuest software (Becton Dickinson). 2.6. Protein extraction and Western blot analysis For the preparation of total proteins, the cells were gently lysed for 30 min with lysis buffer (20 mM sucrose, 1 mM EDTA, 20 lM Tris–Cl, pH 7.2, 1 mM DTT, 10 mM KCl, 1.5 mM MgCl2, 5 lg/ml pepstatin A, 10 lg/ml leupeptin, and 2 lg/ml aprotinin). Supernatants were collected and protein concentrations were determined using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). For Western blot analysis, an equal amount of protein was subjected to electrophoresis on SDS–polyacrylamide gel and transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH) by electroblotting. The blots were probed with the desired antibodies for 1 h, incubated with the diluted enzyme-linked secondary antibodies, and visualized by enhanced chemiluminescence (ECL) Western blotting detection reagents (SuperSignal, Thermo Scientific, Rockford, IL) according to the recommended procedure (Jung et al., 2009). 2.7. Assay of caspases activity The enzymatic activity of the caspases induced by J-7 was assayed using a colorimetric assay kit (R&D Systems, Minneapolis, MN) according to manufacturer’s protocol. The cells were incubated in the absence and presence of J-7 for the indicated times. The cells were harvested and lysed in a lysis buffer for 30 min on an ice bath. The lysed cells were centrifuged at 14,000 rpm for 20 min, and equal amounts of protein (100 lg per 50 ll) were incubated with 50 ll of a reaction buffer and 5 ll of the colorimetric tetrapeptides, Asp-Glu-Val-Asp (DEVD)-p-nitroaniline (pNA) for caspase-3, Ile-Glu-Thr-Asp (IETD)-pNA for caspase-8 and Leu-GluHis-Asp (LEHD)-pNA for caspase-9, respectively, at 37 °C for 2 h in the dark. Caspase activity was determined by measuring changes in absorbance at 405 nm using the ELISA reader. 2.8. Statistical analysis All data is presented as mean ± SD. Significant differences between two groups were determined using the unpaired Student’s t-test. A value of *p < 0.05 was accepted as statistically significant. All the figures shown represent results from at least three independent experiments. 3. Results 3.1. Inhibition of cell proliferation and Induction of apoptosis by J-7 in Hep3B cells To investigate the effects of J-7 on the cell proliferation and apoptosis of Hep3B cells, we assayed time dependencies as determined by cell numbers or MTT assay, DNA fragmentation, chromatin condensation, and flow cytometry analyses. Subsequent treatment with 50 lM of J-7 resulted in a significant reduction in cell numbers (Fig. 1A) and cell viability (Fig. 1B) in a time-dependent manner. In order to obtain a quantitative measure of apoptosis induction, we next investigated the amount of cells with sub-G1 DNA content using flow cytometric analysis. Treatment with 50 lM of J-7 for the indicated amounts of time resulted in a significant accumulation of cells with sub-G1 DNA content in a timedependent manner (Fig. 2A). As shown in Fig. 2B, treatment with 50 lM of J-7 significantly increased DNA fragmentation for up to 8 h after treatment. In addition, Hep3B cells cultured for the indicated amounts of time also showed marked changes in condensed
Fig. 1. Inhibition of cell growth and viability by J-7 in Hep3B cells. Cells were seeded at 2 105 cells/ml and then treated with 50 lM J-7 for the indicated times. Cell number and cell viability were determined by hemocytometer counts of trypan blue-excluding cells (A) and the metabolic-dye-based MTT assay (B), respectively. The results shown are from one representative experiment of two experiments that showed similar patterns. Each point represents the mean ± SD of three independent experiments. The significance was determined using Student’s t-test (*p < 0.05 vs. untreated control).
chromatin in the nuclei following treatment with 50 lM of J-7 for 8 h up to 24 h (Fig. 2C). In this experiment, the number of cells with sub-G1 DNA content was identical to that in vehicle (0.1% DMSO)treated cells and in untreated cells. These results suggest that J-7 significantly induced apoptosis in Hep3B cells. 3.2. Activation of caspase by J-7 in Hep3B cells Caspases are also known to act as important mediators of apoptosis and to contribute to the overall apoptotic morphology through the cleavage of various cellular substrates. Therefore, we investigated the cleavage of caspase-3, caspase-8, and caspase-9 and the subsequent proteolytic cleavage of PARP in cells treated with J-7. As shown in Fig. 3A, Western blot analyses revealed that treatment with 50 lM J-7 significantly induced the cleavage of caspases and PARP in a time-dependent manner. Next, cell lysates containing equal amounts of total protein from cells treated with J-7 were assayed for in vitro caspase activity. As shown in Fig. 3B, treatment with J-7 for the indicated times significantly increased the activities of caspase-3, caspase-8, and caspase-9. These results
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Fig. 2. Induction of apoptosis by J-7 in Hep3B cells. (A) Cells were harvested and centrifuged after treatment with 50 lg/ml of J-7 for various amounts of time. To quantify the degree of apoptosis induced by J-7, the cells were evaluated for the sub-G1 DNA content using a flow cytometer. Each point represents the mean ± SD of three independent experiments. The significance was determined by a Student’s t-test (*p < 0.05, compared with control). (B) After fixing with 3.7% paraformaldehyde, the cells were stained with a DAPI solution for 10 min and stained nuclei were then observed under a fluorescent microscope using a blue filter (original magnification, 400). (C) For the analysis of DNA fragmentation, genomic DNA from cells grown under the same conditions as (A) was extracted and analysed on 1.5% agarose gel containing EtBr. Marker indicates a size marker of the DNA ladder. The results shown are from one representative experiment of two experiments that showed similar patterns.
Fig. 3. Activation of caspases and degradation of PARP by J-7 treatment in Hep3B cells. (A) After incubation with J-7, the cells were lysed and the cellular proteins were separated by SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were probed with the indicated antibodies. The proteins were visualized using an ECL detection system. Actin was used as the internal control. (B) The cell lysates from the cells grown under the same conditions as (A) were assayed for in vitro caspase-3, caspase-8 and caspase-9 activity using DEVD-pNA, IETD-pNA, and LEHD-pNA, respectively, as substrates. The released fluorescent products were measured. The data represents the average ± SD of three independent experiments. The significance was determined by Student’s t-test (*p < 0.05 vs. untreated control).
indicate that J-7 treatment induces apoptotic death in Hep3B cells, at least in part through a caspase-dependent pathway.
pro-apoptotic protein, Bid, was truncated. Furthermore, 50 lM of J-7 led to a significant increase in the level of DR5 but not in other death receptors (Fig. 4).
3.3. Modulation of apoptosis-related proteins by J-7 in Hep3B cells 3.4. Inhibition of J-7-induced apoptosis by caspase-3 inhibitor The expression of the apoptosis-related proteins in J-7-induced apoptosis of Hep3B cells was further investigated by examining the effect of J-7 on Bcl-2 and IAP member proteins, as well as the expression levels of death receptors. Exposure of cells to 50 lM of J-7 for 0–24 h led to a significant reduction in the anti-apoptotic proteins XIAP, cIAP-1, and Bcl-2 and the whole Bid indicating the
Caspase-3 represents one of the key proteases responsible for the cleavage of PARP as well as subsequent apoptosis. To further evaluate the significance of caspase activation in J-7 treatment, we used a general and potent inhibitor of caspase-3: z-DEVDfmk. As shown in Fig. 5A, treatment with 50 lM of J-7 for 24 h
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resulted in a significant accumulation of cells with sub-G1 DNA content. However, cells treated with J-7 in the presence of zDEVD-fmk (50 lM) had a normal cell cycle profile and did not exhibit an increase in cells with sub-G1 DNA content, indicating that the J-7-induced apoptosis was mediated through the activation of caspase-3. We also assessed the effects of J-7 treatment on the appearance of apoptotic bodies in the presence of z-DEVD-fmk. As shown in Fig. 5B, consistent with the results of flow cytometry, analysis by fluorescent microscopy showed that z-DEVD-fmk (50 lM) treatment decreased the appearance of apoptotic bodies following J-7 treatment. Furthermore, J-7 treatment significantly enhanced the activity of caspase-3, whereas z-DEVD-fmk (50 lM) pretreatment significantly inhibited the enhancement of caspase3 activity (Fig. 5C). These results clearly indicate that J-7-induced apoptosis was correlated with the activation of caspase-3. 3.5. Involvement of ERK and JNK pathways in J-7-induced apoptosis Next, we investigated the effect of J-7 treatment on the expression and activities of MAPKs in order to determine whether this signaling pathway is involved in mediating the observed apoptotic response. As shown in Fig. 6A, pretreatment with PD98059 (a potent inhibitor of ERK) or SP600125 (a potent inhibitor of JNK) significantly increased the number of cells with sub-G1 DNA content. However, pretreatment with SB203589 (a specific inhibitor of p38 MAPK) did not have a statistically significant effect on the J-7 treatment (data not shown). Consistent with the increase in sub-G1 DNA content, pretreatment with PD98059 and SP600125 significantly increased the J-7 treatment-induced activation of caspases (Fig. 6B), indicating that J-7-induced apoptosis may be associated with the regulation of the ERK and JNK signaling pathways. Fig. 4. Effects of J-7 on the levels of apoptosis-related gene products in Hep3B cells. The cells were incubated with 50 lM of J-7 for the indicated amounts of time, they were lysed, and equal amounts of proteins were separated by SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were probed with the indicated antibodies and detected by ECL. Actin was used as the internal control.
4. Discussion In the present study, by using a human hepatocarcinoma Hep3B cell line, we demonstrated that the treatment of cells with a syn-
Fig. 5. A caspase-3 inhibitor, z-DEVD-fmk, alleviates J-7-induced apoptosis in Hep3B cells. Cells were incubated with 50 lM of J-7 for 24 h after 1 h pretreatment with zDEVD-fmk (50 lM). DNA content was analysed by flow cytometry (A) and nuclei stained with a DAPI solution were observed under a fluorescent microscope using a blue filter (B). Caspase-3 activity was determined according to the protocol of the manufacturer (C). The all data represents the average ± SD of three independent experiments. The significance was determined by Student’s t-test (*p < 0.05 vs. untreated control).
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Fig. 6. Roles of MAPKs’ signaling pathway on the J-7-induced apoptosis in Hep3B cells are shown. PD98059 (50 lM, a potent inhibitor of ERK) and SP600125 (10 lM, a potent inhibitor of JNK) were pre-treated for 1 h before treatment with 50 lM of J7 for 24 h. The DNA content was analysed by flow cytometry (A) and caspase activities were determined using caspase assay kits obtained from R&D according to the protocol of the manufacturer (B). The data is expressed as mean ± SD of the three independent experiments. The all data represents the average ± SD of three independent experiments. The significance was determined by Student’s t-test (*p < 0.05 vs. untreated control).
thetic methyl jasmonate derivative, J-7, showed significant antiproliferating activity and induction of apoptosis. The induction of apoptosis by J-7 was confirmed by characteristic morphological changes, DNA fragmentation, and an increase of sub-G1 cells. Our observations revealed that J-7 induced apoptosis was by the activation of caspase, resulting in the accumulation of cleavage products in a time-dependent manner. Furthermore, the inhibition of ERK and JNK pathways with pretreatments of PD980595 and SP60012, respectively, increases the J-7-induced accumulation of cells with sub-G1 DNA content and activation of caspase. The regulation of apoptosis is a complex process and involves a number of gene products. During the process of apoptosis, caspase are essential for the execution of cell death through the death receptor pathway (extrinsic) as well as the mitochondrial pathway (intrinsic) in response to various stimuli (Earnshaw et al., 1999; Han et al., 2008). They are synthesized initially as single polypeptide chains representing latent precursors that undergo proteolytic processing at specific residues to produce subunits that form the active heterotetrameric protease. On the other hand, cell surface Fas/FasL and tumor necrosis factor related apoptosis-inducing ligand (TRAIL)/DRs systems and IAP and Bcl-2 family proteins are also key signaling transduction pathways of the both extrinsic and intrinsic pathways of apoptosis in cells (Aggarwal et al., 2004; Jeong and Seol, 2008). Binding FasL to Fas receptors or TRAIL to DRs leads to receptor oligomerization and the formation of the death-inducing signaling complex, followed by the activation of caspase-8, and then cleavage of Bid (tBid), which further activates a series of caspase cascades resulting in apoptotic cell death (Nag-
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ata and Golstein, 1995; Aggarwal et al., 2004; Jeong and Seol, 2008). The present data demonstrated that J-7 increased the enzymatic activity of extrinsic and intrinsic caspase cascades such as caspase-8 and caspase-9 (Fig. 3). In addition to the results that showed increased levels of death receptor-related proteins such as DR5, activation of caspase-8 and decreased whole Bid expression in Hep3B cells treated with J-7 (Fig. 4) suggest that a change in the extrinsic pathway may have partially contributed to the J7-induced apoptosis of Hep3B cells. The present study also shows that there was a time-dependent decrease of IAP family proteins such as XIAP and cIAP-1 in cells treated with J-7 and the levels of anti-apoptotic Bcl-2 dramatically decreased, resulting in an increase in the ratio of Bax/Bcl-2 (Fig. 4). Among the caspase family proteins, capase-3 is known to be one of the key executioners of apoptosis because it is either partially or totally responsible for the proteolytic cleavage of many key proteins including PARP, which is important for cell viability but also serves as a marker of apoptosis when cleaved (Lazebnik et al., 1994). Further studies have shown that exposure of Hep3B cells to J-7 caused a concomitant cleavage of PARP (Fig. 3A). The results also suggest that J-7 may increase mitochondrial dysfunction, which, in turn, results in the activation of caspase-9, leading to the activation of caspase-3, which is also associated with the degradation of caspase-3 target protein, PARP. Therefore, the present data indicates that the apoptotic effects of J-7 on Hep3B cells are associated with the activation of both intrinsic and extrinsic pathways. Additionally, treatment with J-7 in the presence of a caspase-3 inhibitor was found to prevent apoptosis by blocking the proteolytic activation of caspase-3 and apoptotic body formation induced by J-7 (Fig. 5). This data suggests that J-7-induced apoptosis is caused by caspase-3-dependent cell death and that caspase-3 plays an important role in J-7-induced apoptosis in Hep3B cells. Among three major MAPKs, JNK and p38 MAPK are preferentially activated by cytotoxic stressors (Cuevas et al., 2007; Chopra et al., 2008), while the ERK cascade is activated by growth factors and is critical for cell proliferation (Hill and Treisman, 1995). Sustained activation of JNK has been associated with the concurrent inhibition of the ERKs, suggesting that a dynamic balance between ERK and JNK activation may be important in whether a cell survives or undergoes apoptotic cell death (Xia et al., 1995). In the study, the combination of J-7 with a potent inhibitor of ERK (PD98059) or J-7 with a potent inhibitor of JNK (SP600125) markedly increased comparative J-7-induced apoptosis (Fig. 6). In contrast, blocking the p38 MAPK pathway by SB203580 does not alter the apoptosis that occurs upon treatment with J-7 (data not shown). This data strongly suggest that J-7-induced apoptosis is associated with the ERK and JNK pathways. Although the detailed molecular mechanism of induction of apoptosis by the methyl jasmonate derivatives is beyond the scope of this study, the present results demonstrated that a methyl jasmonate derivative, J-7, was capable of inhibiting cell proliferation and inducing apoptosis of Hep3B cells through activation of the intrinsic caspase pathway along with the death receptor-mediated extrinsic pathway. The apoptotic effects of J-7 were also associated with a modulation of MAPKs signaling pathway. Taken together, these novel phenomena have not been previously described and provide important new insights into the possible biological effects of J-7. Acknowledgments Cheng-Yun Jin is the recipient of postdoctoral fellowship from the Ministry of Education, Science and Technology through the Brain Korea 21 Project. This research was supported by National Research Foundation of Korea grant funded by the Korea Government (2009 0093193) and Blue-Bio Industry RIC at Dong-Eui Uni-
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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
A methyl jasmonate derivative, J-7, induces apoptosis in human hepatocarcinoma Hep3B cells in vitro Cheol Park a, Cheng-Yun Jin b, Gi-Young Kim c, JaeHun Cheong d, Jee H. Jung e, Young Hyun Yoo f,*, Yung Hyun Choi a,b,g,** a
Blue-Bio Industry Regional Innovation Center, Dongeui University, Busan 614-714, South Korea Department of Biomaterial Control (BK21 Program), Dongeui University Graduate School, Busan 614-714, South Korea Faculty of Applied Marine Science, Cheju National University, Jeju 690-756, South Korea d Department of Molecular Biology, College of Natural Sciences, Pusan National University, Busan 609-735, South Korea e College of Pharmacy, Pusan National University, Busan 609-735, South Korea f Department of Anatomy and Cell Biology, Dong-A University College of Medicine and Mitochondria Hub Regulation Center, Busan 602-714, South Korea g Department of Biochemistry, Dongeui University College of Oriental Medicine, Busan 614-052, South Korea b c
a r t i c l e
i n f o
Article history: Received 2 February 2010 Accepted 2 August 2010 Available online 7 August 2010 Keywords: Methyl jasmonate derivative Hep3B Apoptosis MAPK
a b s t r a c t The pro-apoptotic activity of J-7, a synthetic methyl jasmonate derivative, on the Hep3B human hepatocarcinoma cell line was investigated. Treatment of Hep3B cells with J-7 resulted in growth inhibition and the induction of apoptosis as measured by trypan blue-excluding cells, MTT assay, nuclear staining, DNA fragmentation, and flow cytometry analysis. The increased apoptotic events in Hep3B cells caused by J-7 were associated with the alteration in the ratio of Bax/Bcl-2 protein expression. J-7 treatment induced the expression of death receptor-related proteins such as death receptor 5, which triggered the activation of caspase-8 and the down-regulation of the whole Bid expression. In addition, the apoptosis induction by J7 was correlated with the activation of caspase-9 and caspase-3, down-regulation IAP family proteins such as XIAP and cIAP-1, and concomitant degradation of poly (ADP-ribose) polymerase. However, the cytotoxic and apoptotic effects induced by J-7 were significantly inhibited by z-DEVD-fmk, a caspase-3 inhibitor, which demonstrates the important role that caspase-3 plays in the process. Furthermore, blocking the extracellular signal-regulated protein kinase and c-Jun N-terminal kinase pathways showed increased apoptosis and the activation of caspases in J-7-induced apoptosis. The results indicated that J-7 induces the apoptosis of Hep3B cells through a signaling cascade of death-receptor-mediated extrinsic as well as mitochondria-mediated intrinsic caspase pathways, which are associated with the activation of the mitogen-activated protein kinases signal pathway. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Hepatocellular carcinoma is the most common malignancies and the fourth-leading cause of cancer deaths worldwide (Lau and Lai, 2008). The disease is associated with environmental exposure to the hepatitis B virus, hepatitis C virus, and Aflatoxin B1, etc. (Ince and Wands, 1999; Okuda, 2000; Coleman, 2003; Marotta et al., 2004). Surgical resection has been considered the optimal treatment approach, but only a small proportion of the patients qualified for surgery; there is a high rate of recurrence after surgery. Approaches used to prevent recurrence have included
* Corresponding author. Tel.: +82 51 240 2926; fax: +82 51 241 3767. ** Corresponding author at: Department of Biochemistry, Dongeui University College of Oriental Medicine, Busan 614-052, South Korea. Tel.: +82 51 850 7413; fax: +82 51 853 4036. E-mail addresses: [email protected] (Y.H. Yoo), [email protected] (Y.H. Choi). 0887-2333/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2010.08.001
chemoembolization before surgery and neoadjuvant therapy after surgery, neither of which has proved to be beneficial (Carr, 2004). Therefore, new therapeutic options are needed for more effective treatment of this malignancy. The jasmonate family consists of cis-jasmone, jasmonic acid, and methyl jasmonate, which are fatty-acid-derived cyclopentanones that occur ubiquitously in the plant kingdom. They serve as natural bioregulators and are involved in plant intracellular signaling and defense in response to injury (Mitler and Lam, 1996). Jasmonates are synthesized in plants from a-linolenic acid via the lipoxygenase pathway (Farmer and Ryan, 1990; Dangl et al., 1996; Weber, 2002), a process involving the transformation of linolenic acid by lipoxygenase to 13-hydroperoxylinolenic acid, and then to jasmonic acid that is finally converted to methyl jasmonate (Weber, 2002). Recently, it has been reported that methyl jasmonate exhibits anti-cancer activity in vitro and in vivo and induces growth inhibition in cancer cells, including breast and prostate
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carcinoma cells, as well as in melanoma, lymphoma, and leukemia cells, while leaving the non-transformed cells intact (Fingrut and Flescher, 2002; Rotem et al., 2003, 2005; Kniazhanski et al., 2008). Methyl jasmonate is also reported to have the ability to act against drug resistant cells (Fingrut et al., 2005; Kniazhanski et al., 2008; Ofer et al., 2008). Methyl jasmonate was also discovered to have cytotoxic effects toward metastatic melanoma both in vitro and in vivo (Reischer et al., 2007) and to significantly increase the life span of lymphoma-bearing mice (Rotem et al., 2005). Thus, methyl jasmonate is now considered as a promising lead for cancer treatment in humans (Flescher, 2005; Cohen and Flescher 2009). Apoptosis is a tightly regulated process characterized by cell shrinkage, plasma membrane blabbing, and chromatin condensation that is consistent with DNA cleavage in ladders (Makin and Dive, 2001; Ghobrial et al., 2005). Apoptosis plays an important role in developmental processes, maintenance of homeostasis, and elimination of damaged cells. In general, apoptosis can be initiated in two ways: by the death receptor pathway (extrinsic) or by the mitochondria-dependent pathway (intrinsic), leading to activation of caspases and consequent apoptosis in mammalian cells (Earnshaw et al., 1999; Han et al., 2008). In the former case, plasma membrane death receptors are involved and the apoptosis signal is provided by the interaction between the ligand and death receptor. Changes in mitochondrial integrity by a broad range of physical and chemical stimuli, however, can trigger the intrinsic pathway of apoptosis (Nagata and Golstein, 1995; Aggarwal et al., 2004; Jeong and Seol, 2008). Recent evidence has also shown that responses to numerous types of extracellular signals are mediated by mitogen-activating protein kinases (MAPKs), members of the serine/threonine kinase family (Ichijo, 1999; Chopra et al., 2008). Several studies have revealed that c-Jun N-terminal kinase (JNK)1/stress-activated protein kinase (SAPK) and/or p38 MAPK activation were involved in apoptosis induced by a variety of different stimuli (Cuevas et al., 2007; Chopra et al., 2008). However, most cancer cells can block apoptosis, which allows them to survive despite genetic and morphologic transformations. Therefore, the induction of apoptotic cell death is an important mechanism in many anti-cancer drugs. Although the induction of apoptosis by methyl jasmonate has been observed in some cancer cell lines, the molecular mechanisms of its pro-apoptotic action on malignant cell growth are not completely unknown. Recently, in an effort to develop novel anti-inflammatory agents, we employed the jasmonate skeleton as a model structure for the synthesis of new cyclopentanonebearing derivatives and reported the synthesis and anti-inflammatory activity of methyl jasmonate analogues (Dang et al., 2008). The above findings led us to investigate whether methyl jasmonate and its derivatives inhibit cancer cell growth and induce apoptosis. In the present study, we examined the anti-cancer effect of a novel methyl jasmonate derivative, J-7 [methyl 5-chloro-4,5-didehydrojasmonate (7)], using human hepatocarcinoma Hep3B cells in vitro and examined its effects on apoptosis induction and the expression of apoptosis-related genes.
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for Bcl-2, Bcl-XL, Bax, Bad, Bid, Fas, Fas ligand (FasL), XIAP, cIAP1, cIAP-2, caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerases (PARP) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies specific for death receptor 4 (DR4) and DR5 were from Calbiochem. Ant-actin antibody was obtained from Sigma–Aldrich. Peroxidase-labeled donkey anti-rabbit immunoglobulin and peroxidase-labeled sheep anti-mouse immunoglobulin were purchased from Amersham (Arlington Heights, IL). 2.2. Cell culture, a synthetic methyl jasmonate derivative (J-7), cell viability and growth study The human hepatoma Hep3B cells were purchased from the American Type Culture Collection (Rockville, MD), and maintained at 37 °C in a humidified 95% air and 5% CO2 in RPMI1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml penicillin, and 100 lg/ml streptomycin. The structure and method of synthesis of J-7 [methyl 5-chloro-4,5didehydrojasmonate (7)], an a-haloenone jasmonate, were previously described (Dang et al., 2008). J-7 was dissolved in dimethyl sulfoxide (DMSO), and dilutions were made in RPMI1640. The final concentration of DMSO in the medium was less than 0.1% (vol/vol) in the treatment concentration (50 lM) and showed no influence on cell growth. For the cell viability and growth study, the cells were grown to 70% confluence and treated with 50 lM J-7 for 0– 24 h. Control cells were supplemented with complete media containing 0.1% DMSO for 24 h time points. After treatments, cell number and viability were determined by Trypan blue exclusion and MTT assays, respectively. 2.3. Nuclear staining with DAPI After treatment with J-7, the cells were harvested, washed with phosphate-buffered saline (PBS) and fixed with 3.7% paraformaldehyde (Sigma–Aldrich) in PBS for 10 min at room temperature. The fixed cells were washed with PBS, and stained with 2.5 lg/ml DAPI solution for 10 min at room temperature. The cells were washed two more times with PBS and analysed via a fluorescent microscope (Carl Zeiss, Germany) (Jang et al., 2010). 2.4. DNA fragmentation assay Cells were lysed in a buffer containing 10 mM Tris–HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, and 0.5% Triton X-100 for 30 min at room temperature. After centrifugation for 20 min at 14,000 rpm, supernatant samples were treated with RNase and proteinase K and incubated at 50 °C for 3 h. The DNA in the supernatant was extracted using a 25:24:1 (v/v/v) equal volume of neutral phenol:chloroform:isoamyl alcohol (Sigma–Aldrich). Subsequently, 5 M of NaCl and isopropanol were added to the samples and kept at 20 °C for 6 h. Following centrifugation for 15 min at 15,000 rpm, the pellets were dissolved in TE buffer (10 mM Tris– HCl and 1 mM EDTA) with RNase A. The DNA was separated on 1.5% agarose gels containing 0.1 lg/ml ethidium bromide (EtBr, Sigma–Aldrich) and was visualized using an ultraviolet light source (Im and Kim, 2010).
2. Materials and methods 2.5. Flow cytometric analysis 2.1. Reagents and antibodies 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphnyl-2H-tetrazolium bromide (MTT) and 4,6-diamidino-2-phenylindole (DAPI) were obtained from Sigma–Aldrich (St. Louis, MO). PD98059 (ERK inhibitor) and SP600125 (JNK inhibitor) were purchased from Calbiochem (San Diego, CA). Caspase activity assay kits were purchased from R&D Systems (Minneapolis, MN). Antibodies specific
After treatment with J-7, the cells were collected, washed with cold PBS, and fixed in 75% ethanol at 4 °C for 30 min. The DNA content of the cells was measured using a DNA staining kit (CycleTEST™ PLUS Kit, Becton Dickinson, San Jose, CA). Propidium iodide (PI)-stained nuclear fractions were obtained by following the kit protocol. The cells were then filtered through 35-mm mesh, and DNA content fluorescence was determined using a FACSCalibur
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(Becton Dickinson) flow cytometer within 1 h. The cellular DNA content was analysed by CellQuest software (Becton Dickinson). 2.6. Protein extraction and Western blot analysis For the preparation of total proteins, the cells were gently lysed for 30 min with lysis buffer (20 mM sucrose, 1 mM EDTA, 20 lM Tris–Cl, pH 7.2, 1 mM DTT, 10 mM KCl, 1.5 mM MgCl2, 5 lg/ml pepstatin A, 10 lg/ml leupeptin, and 2 lg/ml aprotinin). Supernatants were collected and protein concentrations were determined using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). For Western blot analysis, an equal amount of protein was subjected to electrophoresis on SDS–polyacrylamide gel and transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH) by electroblotting. The blots were probed with the desired antibodies for 1 h, incubated with the diluted enzyme-linked secondary antibodies, and visualized by enhanced chemiluminescence (ECL) Western blotting detection reagents (SuperSignal, Thermo Scientific, Rockford, IL) according to the recommended procedure (Jung et al., 2009). 2.7. Assay of caspases activity The enzymatic activity of the caspases induced by J-7 was assayed using a colorimetric assay kit (R&D Systems, Minneapolis, MN) according to manufacturer’s protocol. The cells were incubated in the absence and presence of J-7 for the indicated times. The cells were harvested and lysed in a lysis buffer for 30 min on an ice bath. The lysed cells were centrifuged at 14,000 rpm for 20 min, and equal amounts of protein (100 lg per 50 ll) were incubated with 50 ll of a reaction buffer and 5 ll of the colorimetric tetrapeptides, Asp-Glu-Val-Asp (DEVD)-p-nitroaniline (pNA) for caspase-3, Ile-Glu-Thr-Asp (IETD)-pNA for caspase-8 and Leu-GluHis-Asp (LEHD)-pNA for caspase-9, respectively, at 37 °C for 2 h in the dark. Caspase activity was determined by measuring changes in absorbance at 405 nm using the ELISA reader. 2.8. Statistical analysis All data is presented as mean ± SD. Significant differences between two groups were determined using the unpaired Student’s t-test. A value of *p < 0.05 was accepted as statistically significant. All the figures shown represent results from at least three independent experiments. 3. Results 3.1. Inhibition of cell proliferation and Induction of apoptosis by J-7 in Hep3B cells To investigate the effects of J-7 on the cell proliferation and apoptosis of Hep3B cells, we assayed time dependencies as determined by cell numbers or MTT assay, DNA fragmentation, chromatin condensation, and flow cytometry analyses. Subsequent treatment with 50 lM of J-7 resulted in a significant reduction in cell numbers (Fig. 1A) and cell viability (Fig. 1B) in a time-dependent manner. In order to obtain a quantitative measure of apoptosis induction, we next investigated the amount of cells with sub-G1 DNA content using flow cytometric analysis. Treatment with 50 lM of J-7 for the indicated amounts of time resulted in a significant accumulation of cells with sub-G1 DNA content in a timedependent manner (Fig. 2A). As shown in Fig. 2B, treatment with 50 lM of J-7 significantly increased DNA fragmentation for up to 8 h after treatment. In addition, Hep3B cells cultured for the indicated amounts of time also showed marked changes in condensed
Fig. 1. Inhibition of cell growth and viability by J-7 in Hep3B cells. Cells were seeded at 2 105 cells/ml and then treated with 50 lM J-7 for the indicated times. Cell number and cell viability were determined by hemocytometer counts of trypan blue-excluding cells (A) and the metabolic-dye-based MTT assay (B), respectively. The results shown are from one representative experiment of two experiments that showed similar patterns. Each point represents the mean ± SD of three independent experiments. The significance was determined using Student’s t-test (*p < 0.05 vs. untreated control).
chromatin in the nuclei following treatment with 50 lM of J-7 for 8 h up to 24 h (Fig. 2C). In this experiment, the number of cells with sub-G1 DNA content was identical to that in vehicle (0.1% DMSO)treated cells and in untreated cells. These results suggest that J-7 significantly induced apoptosis in Hep3B cells. 3.2. Activation of caspase by J-7 in Hep3B cells Caspases are also known to act as important mediators of apoptosis and to contribute to the overall apoptotic morphology through the cleavage of various cellular substrates. Therefore, we investigated the cleavage of caspase-3, caspase-8, and caspase-9 and the subsequent proteolytic cleavage of PARP in cells treated with J-7. As shown in Fig. 3A, Western blot analyses revealed that treatment with 50 lM J-7 significantly induced the cleavage of caspases and PARP in a time-dependent manner. Next, cell lysates containing equal amounts of total protein from cells treated with J-7 were assayed for in vitro caspase activity. As shown in Fig. 3B, treatment with J-7 for the indicated times significantly increased the activities of caspase-3, caspase-8, and caspase-9. These results
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Fig. 2. Induction of apoptosis by J-7 in Hep3B cells. (A) Cells were harvested and centrifuged after treatment with 50 lg/ml of J-7 for various amounts of time. To quantify the degree of apoptosis induced by J-7, the cells were evaluated for the sub-G1 DNA content using a flow cytometer. Each point represents the mean ± SD of three independent experiments. The significance was determined by a Student’s t-test (*p < 0.05, compared with control). (B) After fixing with 3.7% paraformaldehyde, the cells were stained with a DAPI solution for 10 min and stained nuclei were then observed under a fluorescent microscope using a blue filter (original magnification, 400). (C) For the analysis of DNA fragmentation, genomic DNA from cells grown under the same conditions as (A) was extracted and analysed on 1.5% agarose gel containing EtBr. Marker indicates a size marker of the DNA ladder. The results shown are from one representative experiment of two experiments that showed similar patterns.
Fig. 3. Activation of caspases and degradation of PARP by J-7 treatment in Hep3B cells. (A) After incubation with J-7, the cells were lysed and the cellular proteins were separated by SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were probed with the indicated antibodies. The proteins were visualized using an ECL detection system. Actin was used as the internal control. (B) The cell lysates from the cells grown under the same conditions as (A) were assayed for in vitro caspase-3, caspase-8 and caspase-9 activity using DEVD-pNA, IETD-pNA, and LEHD-pNA, respectively, as substrates. The released fluorescent products were measured. The data represents the average ± SD of three independent experiments. The significance was determined by Student’s t-test (*p < 0.05 vs. untreated control).
indicate that J-7 treatment induces apoptotic death in Hep3B cells, at least in part through a caspase-dependent pathway.
pro-apoptotic protein, Bid, was truncated. Furthermore, 50 lM of J-7 led to a significant increase in the level of DR5 but not in other death receptors (Fig. 4).
3.3. Modulation of apoptosis-related proteins by J-7 in Hep3B cells 3.4. Inhibition of J-7-induced apoptosis by caspase-3 inhibitor The expression of the apoptosis-related proteins in J-7-induced apoptosis of Hep3B cells was further investigated by examining the effect of J-7 on Bcl-2 and IAP member proteins, as well as the expression levels of death receptors. Exposure of cells to 50 lM of J-7 for 0–24 h led to a significant reduction in the anti-apoptotic proteins XIAP, cIAP-1, and Bcl-2 and the whole Bid indicating the
Caspase-3 represents one of the key proteases responsible for the cleavage of PARP as well as subsequent apoptosis. To further evaluate the significance of caspase activation in J-7 treatment, we used a general and potent inhibitor of caspase-3: z-DEVDfmk. As shown in Fig. 5A, treatment with 50 lM of J-7 for 24 h
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resulted in a significant accumulation of cells with sub-G1 DNA content. However, cells treated with J-7 in the presence of zDEVD-fmk (50 lM) had a normal cell cycle profile and did not exhibit an increase in cells with sub-G1 DNA content, indicating that the J-7-induced apoptosis was mediated through the activation of caspase-3. We also assessed the effects of J-7 treatment on the appearance of apoptotic bodies in the presence of z-DEVD-fmk. As shown in Fig. 5B, consistent with the results of flow cytometry, analysis by fluorescent microscopy showed that z-DEVD-fmk (50 lM) treatment decreased the appearance of apoptotic bodies following J-7 treatment. Furthermore, J-7 treatment significantly enhanced the activity of caspase-3, whereas z-DEVD-fmk (50 lM) pretreatment significantly inhibited the enhancement of caspase3 activity (Fig. 5C). These results clearly indicate that J-7-induced apoptosis was correlated with the activation of caspase-3. 3.5. Involvement of ERK and JNK pathways in J-7-induced apoptosis Next, we investigated the effect of J-7 treatment on the expression and activities of MAPKs in order to determine whether this signaling pathway is involved in mediating the observed apoptotic response. As shown in Fig. 6A, pretreatment with PD98059 (a potent inhibitor of ERK) or SP600125 (a potent inhibitor of JNK) significantly increased the number of cells with sub-G1 DNA content. However, pretreatment with SB203589 (a specific inhibitor of p38 MAPK) did not have a statistically significant effect on the J-7 treatment (data not shown). Consistent with the increase in sub-G1 DNA content, pretreatment with PD98059 and SP600125 significantly increased the J-7 treatment-induced activation of caspases (Fig. 6B), indicating that J-7-induced apoptosis may be associated with the regulation of the ERK and JNK signaling pathways. Fig. 4. Effects of J-7 on the levels of apoptosis-related gene products in Hep3B cells. The cells were incubated with 50 lM of J-7 for the indicated amounts of time, they were lysed, and equal amounts of proteins were separated by SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were probed with the indicated antibodies and detected by ECL. Actin was used as the internal control.
4. Discussion In the present study, by using a human hepatocarcinoma Hep3B cell line, we demonstrated that the treatment of cells with a syn-
Fig. 5. A caspase-3 inhibitor, z-DEVD-fmk, alleviates J-7-induced apoptosis in Hep3B cells. Cells were incubated with 50 lM of J-7 for 24 h after 1 h pretreatment with zDEVD-fmk (50 lM). DNA content was analysed by flow cytometry (A) and nuclei stained with a DAPI solution were observed under a fluorescent microscope using a blue filter (B). Caspase-3 activity was determined according to the protocol of the manufacturer (C). The all data represents the average ± SD of three independent experiments. The significance was determined by Student’s t-test (*p < 0.05 vs. untreated control).
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Fig. 6. Roles of MAPKs’ signaling pathway on the J-7-induced apoptosis in Hep3B cells are shown. PD98059 (50 lM, a potent inhibitor of ERK) and SP600125 (10 lM, a potent inhibitor of JNK) were pre-treated for 1 h before treatment with 50 lM of J7 for 24 h. The DNA content was analysed by flow cytometry (A) and caspase activities were determined using caspase assay kits obtained from R&D according to the protocol of the manufacturer (B). The data is expressed as mean ± SD of the three independent experiments. The all data represents the average ± SD of three independent experiments. The significance was determined by Student’s t-test (*p < 0.05 vs. untreated control).
thetic methyl jasmonate derivative, J-7, showed significant antiproliferating activity and induction of apoptosis. The induction of apoptosis by J-7 was confirmed by characteristic morphological changes, DNA fragmentation, and an increase of sub-G1 cells. Our observations revealed that J-7 induced apoptosis was by the activation of caspase, resulting in the accumulation of cleavage products in a time-dependent manner. Furthermore, the inhibition of ERK and JNK pathways with pretreatments of PD980595 and SP60012, respectively, increases the J-7-induced accumulation of cells with sub-G1 DNA content and activation of caspase. The regulation of apoptosis is a complex process and involves a number of gene products. During the process of apoptosis, caspase are essential for the execution of cell death through the death receptor pathway (extrinsic) as well as the mitochondrial pathway (intrinsic) in response to various stimuli (Earnshaw et al., 1999; Han et al., 2008). They are synthesized initially as single polypeptide chains representing latent precursors that undergo proteolytic processing at specific residues to produce subunits that form the active heterotetrameric protease. On the other hand, cell surface Fas/FasL and tumor necrosis factor related apoptosis-inducing ligand (TRAIL)/DRs systems and IAP and Bcl-2 family proteins are also key signaling transduction pathways of the both extrinsic and intrinsic pathways of apoptosis in cells (Aggarwal et al., 2004; Jeong and Seol, 2008). Binding FasL to Fas receptors or TRAIL to DRs leads to receptor oligomerization and the formation of the death-inducing signaling complex, followed by the activation of caspase-8, and then cleavage of Bid (tBid), which further activates a series of caspase cascades resulting in apoptotic cell death (Nag-
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ata and Golstein, 1995; Aggarwal et al., 2004; Jeong and Seol, 2008). The present data demonstrated that J-7 increased the enzymatic activity of extrinsic and intrinsic caspase cascades such as caspase-8 and caspase-9 (Fig. 3). In addition to the results that showed increased levels of death receptor-related proteins such as DR5, activation of caspase-8 and decreased whole Bid expression in Hep3B cells treated with J-7 (Fig. 4) suggest that a change in the extrinsic pathway may have partially contributed to the J7-induced apoptosis of Hep3B cells. The present study also shows that there was a time-dependent decrease of IAP family proteins such as XIAP and cIAP-1 in cells treated with J-7 and the levels of anti-apoptotic Bcl-2 dramatically decreased, resulting in an increase in the ratio of Bax/Bcl-2 (Fig. 4). Among the caspase family proteins, capase-3 is known to be one of the key executioners of apoptosis because it is either partially or totally responsible for the proteolytic cleavage of many key proteins including PARP, which is important for cell viability but also serves as a marker of apoptosis when cleaved (Lazebnik et al., 1994). Further studies have shown that exposure of Hep3B cells to J-7 caused a concomitant cleavage of PARP (Fig. 3A). The results also suggest that J-7 may increase mitochondrial dysfunction, which, in turn, results in the activation of caspase-9, leading to the activation of caspase-3, which is also associated with the degradation of caspase-3 target protein, PARP. Therefore, the present data indicates that the apoptotic effects of J-7 on Hep3B cells are associated with the activation of both intrinsic and extrinsic pathways. Additionally, treatment with J-7 in the presence of a caspase-3 inhibitor was found to prevent apoptosis by blocking the proteolytic activation of caspase-3 and apoptotic body formation induced by J-7 (Fig. 5). This data suggests that J-7-induced apoptosis is caused by caspase-3-dependent cell death and that caspase-3 plays an important role in J-7-induced apoptosis in Hep3B cells. Among three major MAPKs, JNK and p38 MAPK are preferentially activated by cytotoxic stressors (Cuevas et al., 2007; Chopra et al., 2008), while the ERK cascade is activated by growth factors and is critical for cell proliferation (Hill and Treisman, 1995). Sustained activation of JNK has been associated with the concurrent inhibition of the ERKs, suggesting that a dynamic balance between ERK and JNK activation may be important in whether a cell survives or undergoes apoptotic cell death (Xia et al., 1995). In the study, the combination of J-7 with a potent inhibitor of ERK (PD98059) or J-7 with a potent inhibitor of JNK (SP600125) markedly increased comparative J-7-induced apoptosis (Fig. 6). In contrast, blocking the p38 MAPK pathway by SB203580 does not alter the apoptosis that occurs upon treatment with J-7 (data not shown). This data strongly suggest that J-7-induced apoptosis is associated with the ERK and JNK pathways. Although the detailed molecular mechanism of induction of apoptosis by the methyl jasmonate derivatives is beyond the scope of this study, the present results demonstrated that a methyl jasmonate derivative, J-7, was capable of inhibiting cell proliferation and inducing apoptosis of Hep3B cells through activation of the intrinsic caspase pathway along with the death receptor-mediated extrinsic pathway. The apoptotic effects of J-7 were also associated with a modulation of MAPKs signaling pathway. Taken together, these novel phenomena have not been previously described and provide important new insights into the possible biological effects of J-7. Acknowledgments Cheng-Yun Jin is the recipient of postdoctoral fellowship from the Ministry of Education, Science and Technology through the Brain Korea 21 Project. This research was supported by National Research Foundation of Korea grant funded by the Korea Government (2009 0093193) and Blue-Bio Industry RIC at Dong-Eui Uni-
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