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Induction of apoptosis by esculetin in human leukemia U937 cells: Roles of Bcl-2 and extracellular-regulated kinase signaling
Toxicology in Vitro 24 (2010) 486–494
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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Induction of apoptosis by esculetin in human leukemia U937 cells: Roles of Bcl-2 and extracellular-regulated kinase signaling Cheol Park a, Cheng-Yun Jin b, Hyun Ju Kwon a,b,c, Hye Jin Hwang a,d, Gi-Young Kim e, Il Whan Choi f, Taeg Kyu Kwon g, Byung-Woo Kim a,b,c, Wun-Jae Kim h, Yung Hyun Choi a,b,i,* a
Blue-Bio Industry Regional Innovation Center, Dongeui University, Busan 614-714, Republic of Korea Department of Biomaterial Control (BK21 Program), Dongeui University Graduate School, Busan 614-714, Republic of Korea Department of Life Science and Biotechnology, College of Natural Science, Busan 614-714, Republic of Korea d Department of Food and Nutrition, College of Human Ecology, Busan 614-714, Republic of Korea e Faculty of Applied Marine Science, Cheju National University, Jeju 690-756, Republic of Korea f Department of Microbiology, Inje University College of Medicine, Busan 614-735, Republic of Korea g Department of Immunology, School of Medicine, Keimyung University, Taegu 700-712, Republic of Korea h Department of Urology, College of Medicine, Chungbuk National University, Cheongju, Chungbuk 361-763, Republic of Korea i Department of Biochemistry, Dongeui University College of Oriental Medicine, Busan 614-052, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 10 June 2009 Accepted 22 September 2009 Available online 26 September 2009 Keywords: Esculetinl Apoptosis Bcl-2 HA14-1 ERK
a b s t r a c t In the present study, we reported that apoptosis induced by esculetin, a phenolic compound with apoptotic activity in cancer cells, was markedly blocked by Bcl-2-overexpression, but restored by HA14-1, a small-molecule Bcl-2 inhibitor, in human leukemic U937 cells. The combined use of esculetin and HA14-1 effectively induced Bid cleavage and loss of mitochondrial membrane potential (MMP, Dwm) leading to the activation of caspases and cleavage of poly(ADP-ribose) polymerase (PARP) in Bcl-2-overexpressing (U937/Bcl-2) cells. Combined treatment with esculetin and HA14-1 upregulated the expression of death receptor 4 (DR4), and activation of extracellular-regulated kinase (ERK) in a timedependent manner. In addition, esculetin and HA14-1-mediated apoptosis was reduced by ERK inhibitors through inhibition of DR4 expression, suggesting that the synergistic effect was at least partially mediated through ERK-dependent induction of DR4 expression. The results indicate that HA14-1-induced reversal of the anti-apoptotic effect of Bcl-2 confers apoptosis sensitivity to esculetin by a mitochondrial amplification step and through the ERK-dependent induction of DR4 expression in U937/Bcl-2 cells. Thus, HA14-1 reversal of Bcl-2-mediated esculetin resistance suggests a novel strategy for increasing esculetin sensitivity in Bcl-2-overexpressing leukemia cells. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Esculetin is a coumarin derivative that is found in various natural plant products and this compound could exert an inhibitory action on various biological processes including the activities of lipoxygenase (Neichi et al., 1983; Kemal et al., 1987), platelet aggregation (Okada et al., 1995) and xanthine oxidase (Ponce et al., 2000). Recent reports have demonstrated that esculetin is a scavenger of oxygen-free radicals (Lin et al., 2000) and has an inhibitory effect on vascular smooth muscle cells (Pan et al., 2003), inducing apoptosis of human leukemia HL-60 cells (Wang et al., 2002), as well as having a synergistic effect with taxol-in-
* Corresponding author. Address: Department of Biochemistry, Dongeui University College of Oriental Medicine, Busan 614-052, Republic of Korea. Tel.: +82 51 857413; fax: +82 51 853 4036. E-mail addresses: [email protected], [email protected] (Y.H. Choi). 0887-2333/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2009.09.017
duced apoptosis in human hepatoma HepG2 cells (Kuo et al., 2006). Recently, we suggested that extracellular-regulated kinase (ERK) and/or c-Jun N-terminal kinase (JNK) signaling pathways may be mediated by esculetin-induced G1 arrest and apoptosis of human leukemic U937 cells (Lee et al., 2007; Park et al., 2008). In addition, the ERK cascade was proven to be involved in the enhancement of esculetin on the Taxol-induced apoptosis in HepG2 cells (Kuo et al., 2006), suggesting its importance in esculetin alone or in combination induced apoptosis. However, our previous data indicate that Bcl-2 overexpression may confer protection against esculetin-induced apoptosis at least in leukemia cell types (Park et al., 2008), suggesting its importance in esculetin resistance. Overexpression of Bcl-2 proteins is one mechanism for malignancies to acquire resistance to cancer therapies (Bouillet et al., 2000; Cory et al., 2003; Jeong and Seol, 2008). Intense efforts, therefore, have been devoted to identifying small-molecule antag-
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onists against this protein with the hope that such antagonists would re-sensitize drug-resistant tumors to standard anticancer treatment (Wang et al., 2000; Lugovskoy et al., 2002; Tang et al., 2007). HA 14-1 is a small-molecule antagonist against Bcl-2 protein and has been shown to synergize with the anticancer activities of diverse compounds in leukemia (Milella et al., 2002; Lickliter et al., 2003; Hao et al., 2004; Wlodkowic et al., 2006). These results indicate the potential of HA 14-1 in combination therapy for cancer treatment. However, the precise mechanism by which HA14-1 contributes to ERK and death receptor (DR)-mediated apoptosis is unclear. We used HA14-1 as a sensitizer for overcoming the resistance of Bcl-2 overexpressing U937 cells to esculetin-induced apoptosis. Our data show that the synergistic effect of HA14-1 on esculetininduced apoptosis is through ERK and DR-mediated apoptotic pathways.
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was measured at a wave length of 540 nm with an ELISA reader (Molecular Devices, Sunnyvale, CA). 2.5. Flow cytometric analysis After treatment with esculetin, the cells were collected, washed with cold phosphate-buffered saline (PBS), and fixed in 75% ethanol at 4 °C for 30 min. DNA content of the cells was measured using a DNA staining kit (CycleTESTTM PLUS Kit, Becton Dickinson, San Jose, CA). Propidium iodide (PI)-stained nuclear fractions were obtained by following the kit protocol (Liao et al., 2008). The cells were then filtered through 35-mm mesh, and DNA content fluorescence was determined using a FACSCalibur (Becton Dickinson) flow cytometer within 1 h. Cellular DNA content was analyzed by CellQuest software (Becton Dickinson).
2. Materials and methods
2.6. Protein extraction and western blot analysis
2.1. Reagents
Cells were treated with esculetin and were collected with icecold PBS. The mitochondrial and cytosolic fractions were isolated using a mitochondrial and cytosolic fractionation kit (Activemotif, Carlsbad, CA) (Kim et al., 2008). For the preparation of total proteins, the cells were gently lysed 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) for 30 min. Supernatants were collected and protein concentrations 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 electro blotting. The blots were probed with the desired antibodies for 1 h, incubated with the diluted enzyme-linked secondary antibody and visualized by enhanced chemiluminescence (ECL) western blotting detection reagents (SuperSignal, Thermo scientific, Rockford, IL) according to the recommended procedure (Amersham, Arlington Heights, IL). The peroxidase-labeled donkey anti-rabbit immunoglobulin and peroxidase-labeled sheep anti-mouse immunoglobulin were purchased from Amersham (Lee et al., 2008).
Esculetin (6,7-dihydroxycoumarin, 98% purity) was purchased from Sigma–Aldrich (St. Louis, MO), dissolved in dimethyl sulfoxide (DMSO) and adjusted to final concentrations using complete RPMI1640 (Gibco BRL, Gaithersburg, MD). 3-(4,5-Dimethyl-2thiazolyl)-2,5-diphnyl-2H-tetrazolium bromide (MTT), 4,6-diamidino-2-phenylindole (DAPI) and ethyl [2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)]-4H-chromene-3-carboxylate (HA 14-1) were purchased from Sigma. PD98059 (ERK inhibitor), SP600125 (JNK inhibitor) and the specific mitochondrial dye 5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolylcarbocyanine iodide (JC1) were purchased from Calbiochem (San Diego, CA). Caspase activity assay kits were purchased from R&D Systems (Minneapolis, MN). 2.2. Antibodies Antibodies specific for Bcl-2, Bax, Bid, cytochrome c, caspase-3, caspase-8, caspase-9 and poly(ADP-ribose) polymerases (PARP) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies specific for phospho-ERK and phospho-JNK were purchased from Cell Signaling (Beverly, MA). Antibodies specific for DR4 and DR5 were from Calbiochem. Ant-actin antibody was obtained from Sigma (St. Louis, MO). 2.3. Cell lines and cell culture The human leukemia cell line U937 was 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. Bcl-2 overexpressing U937 cells were a generous gift from Dr. T. K. Kwon (Department of Immunology, Keimyung University School of Medicine, Taegu, Korea) and were maintained in a medium containing 0.7 lg/ml geneticin (G418 sulfate, Calbiochem). 2.4. Cell viability assay Cell viability was determined by MTT assays. Cells were seeded in 6-well plates and treated with esculetin for the indicated times. After treatments, MTT working solution was added to 6-well culture plates and incubated continuously at 37 °C for 3 h. The culture supernatant was removed from wells, and DMSO was added to completely dissolve formazan crystals. The absorbance of each well
2.7. Nuclear staining with DAPI For DAPI staining, the cells were washed with PBS and fixed with 3.7% paraformaldehyde (Sigma) 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 then washed twice with PBS and analyzed by fluorescence microscopy (Carl Zeiss, Germany). 2.8. DNA fragmentation assay After esculetin treatment, 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 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). Subsequently, 5 M 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. DNA was separated on 1.5% agarose gels containing 0.1 lg/ml ethidium bromide (EtBr, Sigma), and was visualized using an ultraviolet light source.
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2.9. Mitochondrial membrane potential assay The mitochondrial membrane potential (MMP, Dwm) was determined using the lipophilic cationic probe JC-1. JC-1 is a radiometric, dual-emission fluorescent dye that is absorbed and concentrated by respiring mitochondria and can reflect changes in MMP in living cells. There are two excitation wavelengths, 527 nm (green) for the monomer form and 590 nm (red) for the JC-1-aggregate form. With normal mitochondrial function, MMP is high and red fluorescence is predominant. However, when there is mitochondrial injury, the MMP is reduced, leading to an increase in green fluorescence. The relative intensity of red and green fluorescent signals indicate whether mitochondria are damaged. U937 cells were incubated with JC-1 at a final concentration of 10 lM for 20 min at 37 °C, in the dark. At the end of the incubation period, the cells were washed twice in cold PBS, resuspended in a total volume of 500 ll, and analyzed by flow cytometry.
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 caspase8 and Leu-Glu-His-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. 2.11. Statistical analysis All data are presented as mean ± SD. Significant differences between two groups were determined using the unpaired Student’s ttest. 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
2.10. Assay of caspases activity
3.1. Bcl-2 overexpression protects against esculetin-induced apoptosis
The enzymatic activity of the caspases induced by esculetin was assayed using a colorimetric assay kit (R&D Systems, Minneapolis, MN) according to manufacturer’s protocol. U937 cells were incubated in the absence and presence of esculetin 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
To determine the role of Bcl-2 in esculetin-induced apoptosis, the extent of cell viability was assessed by MTT assay and apoptosis was analyzed using flow cytometric analysis of sub-G1 population (Fig. 1A and B). In U937/vector cells, esculetin treatment suppressed the cell viability and also induced apoptosis; however, Bcl-2 overexpression (U937/Bcl-2) was found to signif-
Fig. 1. Bcl-2 overexpression protects against esculetin-induced apoptosis in U937 cells. Control (U937) or Bcl-2 transfected (U937/Bcl-2) cells were seeded at 2 105 cells/ml and then incubated with 30 l/ml of esculetin for 6 h. (A) Cell viability was measured by the metabolic-dye-based MTT assay. (B) The cells were collected and stained with CycleTEST PLUS DNA REAGENT Kit for flow cytometry analysis. The percentages of cells with hypodiploid DNA (sub-G1 phase) content represent the fractions undergoing apoptotic DNA degradation. Each point represents the mean ± SD of three independent experiments. Significance was determined using Student’s t-test (*p < 0.05 vs. untreated control). (C) Equal amounts of cytosolic and mitochondrial proteins extracted from cells grown under the same conditions 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.
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icantly protect cells from esculetin-induced apoptosis evaluated relative to U937/vector cells. To determine how Bcl-2 exerts an effect on cell survival, we examined the levels of expression of several proteins in the cytosolic and mitochondrial fractions by immunoblotting. We found that ectopic Bcl-2 overexpression completely inhibited the esculetin-induced cytosolic release of the mitochondrial pro-apoptotic protein cytochrome c relative to esculetin treated U937/vector cells (Fig. 1C). Upon treatment
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with esculetin, cytosolic Bax expression was lost in U937/vector cells consistent with its translocation to the mitochondria (Fig. 1C). In contrast, Bcl-2 overexpression blocked Bax translocation, trapping it in the cytosolic fraction. Accordingly, these results indicate that a mitochondrial amplification step is required for apoptosis induction by esculetin and that Bcl-2 overexpression may confer protection against esculetin-induced apoptosis in U937 cells.
Fig. 2. Bcl-2 overexpression protects against esculetin-induced ERK activation and DR4 expression. The cells were treated with 30 lg/ml of esculetin for the indicated times, lysed, and cellular proteins were separated by SDS–polyacrylamide gel and then transferred onto nitrocellulose membranes. The membranes were probed with the indicated antibodies. Proteins were visualized using an ECL detection system. Actin was used as an internal control.
Fig. 3. Co-treatment with esculetin and HA14-1 restored apoptosis, ERK activation and DR4 expression in Bcl-2 overexpressing U937 cells. The cells were seeded at 2 105 cells/ml and were then incubated with 30 g/ml of esculetin and 15 lM of HA14-1 for 6 h. (A) Cell viability was measured by the metabolic-dye-based MTT assay. (B) The cells were collected and stained with CycleTEST PLUS DNA REAGENT Kit for flow cytometry analysis. The percentage of cells with hypodiploid DNA (sub-G1 phase) content represent the fractions undergoing apoptotic DNA degradation. 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). (C) Nuclei stained with DAPI solution were then observed under a fluorescent microscope using a blue filter. Magnification, X400. (D) The fragmented DNAs were separated on 1.5% agarose gel electrophoresis and visualized under UV light after staining with EtBr. (E) The cells were lysed, and cellular proteins were separated by SDS–polyacrylamide gel and transferred onto nitrocellulose membranes. The membranes were probed with the indicated antibodies. Proteins were visualized using an ECL detection system. Actin was used as an internal control.
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3.2. Bcl-2 overexpression protects against esculetin-induced ERK activation and DR4 expression Our previous reports indicated that the JNK and ERK pathways were key regulators of apoptosis in response to esculetin in U937 cells. As shown in Fig. 2A, the phosphorylation of ERK and JNK increased significantly after 2 h of esculetin treatment. However, Bcl2 overexpression was found to significantly protect cells from esculetin-induced phosphorylation of ERK and JNK evaluated relative to U937/vector cells. Next, we investigated the effect of esculetin treatment on the DR4 and DR5 expression in order to determine whether the death receptor-mediated apoptosis signaling pathway is involved in mediating the observed apoptotic response. As shown in Fig. 2B, the expression of DR4, but not DR5, increased significantly after esculetin treatment in a time-dependent manner, and blocked by Bcl-2 overexpression. These results indicate that Bcl-2 overexpression attenuated apoptosis by esculetin, and this was correlated with ERK, JNK and DR4 pathways.
3.3. Co-treatment with esculetin and HA14-1 restored the activation of ERK and increase of DR4expression in Bcl-2 overexpressing U937 cells To investigate the effects of esculetin and HA14-1 on the viability and apoptosis of Bcl-2 overexpressing U937 cells, several experiments were performed. As shown in Fig. 3A, following treatment with esculetin plus HA14-1 for 6 h, U937/Bcl-2 cells exhibited much stronger growth inhibitory effects as compared with esculetin or HA14-1 alone. In order to obtain a quantitative measure of apoptosis induction, we next investigated, using flow
cytometric analysis, the proportion of cells with sub-G1 DNA content. Treatment with esculetin and HA14-1 under the indicated conditions resulted in significant accumulation of cells with sub-G1 DNA content (Fig. 3B). As shown in Fig. 3C, the cells cultured for the indicated condition of esculetin and HA14-1 showed marked changes in condensed chromatin in the nuclei following treatment with esculetin plus HA14-1. In addition, treatment with esculetin plus HA14-1 also significantly increased DNA fragmentation for up to 6 h after treatment (Fig. 3D). These results suggest that esculetin plus HA14-1 significantly induced apoptosis in U937/Bcl-2 cells. We then determined whether esculetin plus HA14-1 reverses Bcl-2-mediated suppression of apoptotic signaling. For this experiment, U937/Bcl-2 cells were treated with esculetin, HA14-1, or their combination; western blotting was performed. As shown in Fig. 3E, treatment with esculetin and/or HA14-1 did not change the expression level of Bcl-2 and DR5. However, co-administration of esculetin and HA14-1 resulted in significantly increased ERK phosphorylation and DR4 expression levels compared with controls or cells treated with esculetin or HA14-1 alone. Together, these data indicate that co-treatment of Bcl-2 overexpressing U937 cells with esculetin and HA14-1 can restore the activation of ERK and the increases in DR4 but not in JNK and DR5.
3.4. Co-treatment with esculetin and HA14-1 synergistically induced the loss of MMP and activation of caspases The regulation of apoptosis is a complex process and involves a number of mitochondrial proteins. To determine the role of the
Fig. 4. Co-treatment with esculetin and HA14-1 restored cytochrome c release, decreases in MMP and activation of caspases in Bcl-2 overexpressing U937 cells. The cells were seeded at 2 105 cells/ml and were then incubated with 30 lg/ml of esculetin and 15 lM of HA14-1 for 6 h. (A and C) The cells were lysed and cellular proteins were separated by SDS–polyacrylamide gel and transferred onto nitrocellulose membranes. The membranes were probed with the indicated antibodies. Proteins were visualized using an ECL detection system. Actin was used as an internal control. (CF, cleavage form) (B) The cells were exposed to esculetin and HA14-1 for 6 h, stained with JC-1 and incubated at 37 °C for 20 min. Mean JC-1 fluorescence intensity was detected using a flow cytometer. (D) The cell lysates from cells grown under the same conditions were assayed for in vitro caspase-3, caspase-8 and caspase-9 activity using DEVD-pNA, IETD-pNA and LEHD-pNA, respectively, as substrates. Amounts of products were measured using relative fluorescence signal intensity. Each point represents the mean ± SD of three independent experiments. Significance was determined using Student’s t-test (*p < 0.05 vs. untreated control).
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mitochondria in esculetin and HA14-induced apoptosis of U937/ Bcl-2 cells, we further investigated the levels of cytosolic and mitochondrial Bax or cytochrome c, as well as MMP (Dwm). Exposure of U937/Bcl-2 cells to esculetin plus HA14-1 led to a significant increase in the level of cytosolic cytochrome c and a decrease of Bax (Fig. 4A). Furthermore, exposure of U937/Bcl-2 cells to esculetin plus HA14-1 for 6 h led to a significant reduction in the MMP level (Fig. 4B). These results suggest a direct role of the mitochondria in esculetin plus HA14-1 induced apoptosis. 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 activation of caspase-3, caspase-8, and caspase-9 and further assessed the cleavage of several key death substrates that indicate the activation of caspases, including PARP (substrate for caspase-3 and caspase-7) and Bid (caspase-8). As shown in Fig. 4C, western blot analyses revealed that treatment with esculetin or HA14-1 alone did not affect, or only slightly affected the cleavage of caspases, PARP, and Bid. However, treatment with esculetin plus HA14-1 significantly induced the cleavage of caspases, PARP, and Bid. Next, cell lysates containing equal amounts of total protein from cells treated with esculetin and HA14-1 were assayed for in vitro caspases activity. As shown in Fig. 4D, treatment with esculetin plus HA14-1 significantly increased the activities of caspase-3, caspase-8, and caspase-9 compared with control cells or cells treated with esculetin or HA14-1 alone. These results indicate that esculetin plus HA14-1 treatment induces apoptotic death in U937/Bcl-2 cells, at least in part through a caspase-dependent pathway.
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3.5. The ERK and DR4 pathways play important roles in esculetin plus HA14-1 induced apoptosis Next, we investigated the effect of the ERK signaling pathway and the expression of DRs on the apoptotic response in Bcl-2 overexpressing U937 cells induced by esculetin and HA14-1 treatment. We evaluated the possible roles of mitogen-activated protein kinase (MAPK) pathways in esculetin plus HA14-1 treatment-induced apoptosis. As shown in Fig. 5A, pre-treatment with PD98059 (a potent inhibitor of ERK) significantly decreased the number of cells with sub-G1 DNA content. However, pre-treatment with SP600125 (a potent inhibitor of JNK) did not have a statistically significant effect on the esculetin plus HA14-1 treatment. In addition, U937/Bcl-2 cells cultured in the presence of PD98059 showed significantly decreased DNA fragmentation following 6 h of esculetin plus HA14-1 treatment (Fig. 5B). Consistent with the DNA fragmentation, pre-treatment with PD98059 elicited marked inhibition of the appearance of apoptotic bodies due to esculetin plus HA14-1 (Fig. 5C). Next, we investigated the effect of the ERK signaling pathway on the esculetin and HA14-1 induced activation of ERK and increases in DR4 expression in Bcl-2 overexpressing U937 cells. As shown in Fig. 5D, treatment with esculetin, HA141 or PD98059 alone did not change the expression levels of Bcl-2 and DR5. However, pre-treatment with PD98059 significantly decreased the activation of ERK by co-treatment with esculetin and HA14-1. Interestingly, the increase in DR4 expression induced by esculetin plus HA14-1 was also significantly decreased by pretreatment with PD98059. These data indicate that the ERK and DR4 pathways play important roles and suggest that DR4 expression is dependent on the ERK signaling pathway in esculetin plus HA14-1- induced apoptosis.
Fig. 5. ERK and DR4 pathways play important roles in esculetin plus HA14-1-induced apoptosis in Bcl-2 overexpressing U937 cells. Cells were pretreated with PD98059 (100 uM) and SP600125 (40 lM) for 1 h before treatment with 30 lg/ml of esculetin and 15 lM of HA14-1 for 6 h. (A) The cells were collected and stained with CycleTEST PLUS DNA REAGENT Kit for flow cytometry analysis. The percentages of cells with hypodiploid DNA (sub-G1 phase) contents represent the fractions undergoing apoptotic DNA degradation. Each point represents the mean ± SD of three independent experiments. Significance was determined using Student’s t-test (*p < 0.05 vs. untreated control). (B) The fragmented DNAs were separated on 1.5% agarose gel electrophoresis and visualized under UV light after staining with EtBr. (C) Nuclei stained with DAPI solution were then observed under a fluorescence microscope using a blue filter. Magnification, 400. (D) The cells were lysed, and cellular proteins were separated by SDS–polyacrylamide gel and transferred onto nitrocellulose membranes. The membranes were probed with the indicated antibodies. Proteins were visualized using an ECL detection system. Actin was used as an internal control.
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3.6. The ERK pathways play important roles in esculetin plus HA14-1induced loss of MMP and caspases activation In order to investigate the significance of the ERK pathway in response to esculetin plus HA14-1 treatment, we also investigated apoptosis-related protein expressions in U937/Bcl-2 cells. As shown in Fig. 6A, exposure of U937/Bcl-2 cells with esculetin plus HA14-1 led to a significant increase in the level of cytosolic cytochrome c and the decrease of Bax, whereas it led to marked down-regulation of cytochrome c and up-regulation of Bax in mitochondria. However, pre-treatment with PD98059 resulted in a reduction in Bax and cytochrome c expression at mitochondrial and cytosolic levels compared to that induced by treatment with esculetin plus HA14-1. We also showed that esculetin plus HA14-1 potentiated the decrease in MMP and was inhibited by pre-treatment with PD98059 (Fig. 6 B). These results clearly indicate that the esculetin plus HA14-1-induced mitochondrial amplification step is correlated with the activation of ERK pathways. We further investigated the effects of ERK activation on the levels of caspases activation in U937/Bcl-2 cells. As shown in Fig. 6 C and D, western blot analyses revealed that treatment with esculetin and HA14-1 alone did not affect, or only slightly induced the cleavage of caspases, PARP, and Bid. However, treatment with esculetin plus HA14-1 significantly induced the cleavage of caspases, PARP, Bid, and increased in vitro caspase activity. In contrast, pre-treatment with PD98059 markedly inhibited cleavage of caspases and increased caspase activation compared with esculetin plus HA14-1 treatment. These results indicate that esculetin plus HA14-1 treatment induces caspase activation in U937/Bcl-2 cells, at least in part through ERK-dependent pathways.
4. Discussion We previously reported that esculetin induces apoptosis in human leukemia U937 cells via ERK and JNK-dependent apoptotic pathways (Park et al., 2008). Based on this, in the present experiments we investigated ERK signaling in Bcl-2-mediated apoptosis. We found that Bcl-2 overexpression attenuated esculetin-induced apoptosis via inhibition of DR4 expression and ERK inactivation. In addition, we found that Bcl-2-mediated apoptosis resistance to esculetin, which was restored by HA14-1 though ERK-dependent expression of DR4, caspases activation, and loss of MMP. Many previous studies indicate that the ERK pathway is involved in the survival of B-cell chronic lymphocytic leukemia cells (Brózik et al., 2006; Rubio et al., 2007). Our study was conducted to determine the effects of ERK on esculetin plus HA14-1-induced apoptosis. Our results demonstrate that pre-treatment with PD98059 markedly inhibited esculetin plus HA14-1-induced apoptosis, whereas this inhibition was not observed upon treatment with PD98059 alone. In contrast, blocking the JNK pathway did not alter the apoptosis that occurs upon treatment with esculetin plus HA14-1 (Fig. 5A). These data strongly suggest that esculetin plus HA14-1-induced apoptosis is associated with the ERK pathway. Strategies to overcome Bcl-2-mediated resistance to apoptosis have the potential to greatly increase treatment efficacy. One approach involves the use of small-molecule inhibitors of Bcl-2 that bind to and inhibit Bcl-2 function. One such inhibitor, HA14-1, has recently been developed using molecular modeling techniques; however, very limited data are available regarding its ability to reverse Bcl-2 mediated resistance (Fernandes-Alnemri et al.,
Fig. 6. The ERK pathway plays important roles in esculetin plus HA14-1-induced cytochrome c release, decreases in MMP and activation of caspases in Bcl-2 overexpressing U937 cells. Cells were pretreated with PD98059 (100 uM) for 1 h before treatment with 30 g/ml of esculetin and 15 lM of HA14-1 for 6 h. (A and C) The cells were lysed, and cellular proteins were separated by SDS–polyacrylamide gel and transferred onto nitrocellulose membranes. The membranes were probed with the indicated antibodies. Proteins were visualized using an ECL detection system. The integrity of mitochondria and quantitative loading of the proteins was established by analyzing the levels of cytochrome oxidase subunit IV (COX IV) and actin, respectively (CF, cleavage form) (B) Cells were exposed to esculetin and HA14-1 for 6 h, after which MMPs were determined using JC-1. (D) Cell lysates from cells grown under the same conditions 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 relative fluorescence of the products was measured. Each point represents the mean ± SD of three independent experiments. Significance was determined using Student’s t-test (*p < 0.05 vs. untreated control).
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1994; Murphy et al., 1996; Yang et al., 1997; Kluck et al., 1997). Therefore, HA14-1 was evaluated to determine if it could reverse the anti-apoptotic effect of Bcl-2 and enhance esculetin-mediated apoptosis. Treatment with HA14-1 alone did not induce apoptosis, except at the highest dose (15 lM) evaluated. A prior study of hematopoietic cell lines found that a concentration of HA14-1 exceeding 25 lM resulted in a loss of selectivity for Bcl-2 and marked cytotoxicity in several cell lines tested (Jin et al., 1997). Although treatment of U937/Bcl-2 cells with HA14-1 (15 lM) alone failed to overcome the Bcl-2-mediated suppression of mitochondrial apoptotic signaling, co-administration of HA14-1 and esculetin restored apoptotic susceptibility. This occurred in association with a loss of MMP, which enabled caspases activation. The ability of Bcl-2 to confer resistance to esculetin-induced apoptosis and its reversal via HA14-1 has important therapeutic implications. Bcl-2 is expressed in multiple human tumor cell types in the absence of gene rearrangements, including leukemia cells (Adams and Cory, 1998; Pileri et al., 2003; Inoue et al., 2004; Dargent et al., 2005; Vitolo et al., 2008). Human cancer cells are type II, and most chemotherapeutic drugs mediate apoptosis by inducing mitochondrial dysfunction; therefore, these results indicate that Bcl-2 is an important contributor to multiple drug resistance (van de Donk et al., 2006; Wilson, 2006; Eberle et al., 2007). Furthermore, the results of our study indicate that the requirement for mitochondrial amplification of various stimuli that target intrinsic apoptotic pathways may overcome Bcl-2-mediated resistance, thereby increasing therapeutic efficacy. In conclusion, we have demonstrated that Bcl-2 overexpression prevents esculetin-induced activation of ERK and expression of DR4 in U937 cells. HA14-1 restored Bcl-2 mediated apoptosis via regulation of the decreases in MMP and caspase activity. This phenomenon was associated with the ERK signaling pathway. Therefore, it is thought that combination of esculetin and HA14-1 might be a promising strategy for use in cancer chemoprevention and chemotherapy. 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 work was supported by a grant from the Personalized Tumor Engineering Research Center (PTERC) and the Korea Science and Engineering Foundation (KOSEF) and BlueBio Industry RIC at Dong-Eui University as a RIC (08-06-07) program of KIAT under Ministry of Knowledge Economy, Republic of Korea. References Adams, J.M., Cory, S., 1998. The Bcl-2 protein family: arbiters of cell survival. Science 281, 1322–1326. Bouillet, P., Huang, D.C., O’Reilly, L.A., Puthalakath, H., O’Connor, L., Cory, S., Adams, J.M., Strasser, A., 2000. The role of the pro-apoptotic Bcl-2 family member bim in physiological cell death. Ann. N.Y. Acad. Sci. 926, 83–89. Brózik, A., Casey, N.P., Hegedus, C., Bors, A., Kozma, A., Andrikovics, H., Geiszt, M., Német, K., Magócsi, M., 2006. Reduction of Bcr-Abl function leads to erythroid differentiation of K562 cells via downregulation of ERK. Ann. N.Y. Acad. Sci. 1090, 344–354. Cory, S., Huang, D.C., Adams, J.M., 2003. The Bcl-2 family: roles in cell survival and oncogenesis. Oncogene 22, 8590–8607. Dargent, J.L., Lespagnard, L., Feoli, F., Debusscher, L., Greuse, M., Bron, D., 2005. De novo CD5-positive diffuse large B-cell lymphoma of the skin arising in chronic limb lymphedema. Leukocyte Lymph. 46, 775–780. Eberle, J., Kurbanov, B.M., Hossini, A.M., Trefzer, U., Fecker, L.F., 2007. Overcoming apoptosis deficiency of melanoma-hope for new therapeutic approaches. Drug Resist. Updat. 10, 218–234. Fernandes-Alnemri, T., Litwack, G., Alnemri, E.S., 1994. CPP32, a novel human apoptotic protein with homology to Caenorhabditis elegans cell death protein Ced-3 and mammalian interleukin-1 beta-converting enzyme. J. Biol. Chem. 269, 30761–30764.
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Inhibitory effect of esculetin on oxidative damage induced by t-butyl hydroperoxide in rat liver. Arch. Toxicol. 74, 467–472. Lugovskoy, A.A., Degterev, A.I., Fahmy, A.F., Zhou, P., Gross, J.D., Yuan, J., Wagner, G., 2002. A novel approach for characterizing protein ligand complexes: molecular basis for specificity of small-molecule Bcl-2 inhibitors. J. Am. Chem. Soc. 124, 1234–1240. Milella, M., Estrov, Z., Kornblau, S.M., Carter, B.Z., Konopleva, M., Tari, A., Schober, W.D., Harris, D., Leysath, C.E., Lopez-Berestein, G., Huang, Z., Andreeff, M., 2002. Synergistic induction of apoptosis by simultaneous disruption of the Bcl-2 and MEK/MAPK pathways in acute myelogenous leukemia. Blood 99, 3461–3464. Murphy, A.N., Bredesen, D.E., Cortopassi, G., Wang, E., Fiskum, G., 1996. Bcl-2 potentiates the maximal calcium uptake capacity of neural cell mitochondria. Proc. Natl. Acad. Sci. USA 93, 9893–9898. Neichi, T., Koshihara, Y., Murota, S., 1983. 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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Induction of apoptosis by esculetin in human leukemia U937 cells: Roles of Bcl-2 and extracellular-regulated kinase signaling Cheol Park a, Cheng-Yun Jin b, Hyun Ju Kwon a,b,c, Hye Jin Hwang a,d, Gi-Young Kim e, Il Whan Choi f, Taeg Kyu Kwon g, Byung-Woo Kim a,b,c, Wun-Jae Kim h, Yung Hyun Choi a,b,i,* a
Blue-Bio Industry Regional Innovation Center, Dongeui University, Busan 614-714, Republic of Korea Department of Biomaterial Control (BK21 Program), Dongeui University Graduate School, Busan 614-714, Republic of Korea Department of Life Science and Biotechnology, College of Natural Science, Busan 614-714, Republic of Korea d Department of Food and Nutrition, College of Human Ecology, Busan 614-714, Republic of Korea e Faculty of Applied Marine Science, Cheju National University, Jeju 690-756, Republic of Korea f Department of Microbiology, Inje University College of Medicine, Busan 614-735, Republic of Korea g Department of Immunology, School of Medicine, Keimyung University, Taegu 700-712, Republic of Korea h Department of Urology, College of Medicine, Chungbuk National University, Cheongju, Chungbuk 361-763, Republic of Korea i Department of Biochemistry, Dongeui University College of Oriental Medicine, Busan 614-052, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 10 June 2009 Accepted 22 September 2009 Available online 26 September 2009 Keywords: Esculetinl Apoptosis Bcl-2 HA14-1 ERK
a b s t r a c t In the present study, we reported that apoptosis induced by esculetin, a phenolic compound with apoptotic activity in cancer cells, was markedly blocked by Bcl-2-overexpression, but restored by HA14-1, a small-molecule Bcl-2 inhibitor, in human leukemic U937 cells. The combined use of esculetin and HA14-1 effectively induced Bid cleavage and loss of mitochondrial membrane potential (MMP, Dwm) leading to the activation of caspases and cleavage of poly(ADP-ribose) polymerase (PARP) in Bcl-2-overexpressing (U937/Bcl-2) cells. Combined treatment with esculetin and HA14-1 upregulated the expression of death receptor 4 (DR4), and activation of extracellular-regulated kinase (ERK) in a timedependent manner. In addition, esculetin and HA14-1-mediated apoptosis was reduced by ERK inhibitors through inhibition of DR4 expression, suggesting that the synergistic effect was at least partially mediated through ERK-dependent induction of DR4 expression. The results indicate that HA14-1-induced reversal of the anti-apoptotic effect of Bcl-2 confers apoptosis sensitivity to esculetin by a mitochondrial amplification step and through the ERK-dependent induction of DR4 expression in U937/Bcl-2 cells. Thus, HA14-1 reversal of Bcl-2-mediated esculetin resistance suggests a novel strategy for increasing esculetin sensitivity in Bcl-2-overexpressing leukemia cells. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Esculetin is a coumarin derivative that is found in various natural plant products and this compound could exert an inhibitory action on various biological processes including the activities of lipoxygenase (Neichi et al., 1983; Kemal et al., 1987), platelet aggregation (Okada et al., 1995) and xanthine oxidase (Ponce et al., 2000). Recent reports have demonstrated that esculetin is a scavenger of oxygen-free radicals (Lin et al., 2000) and has an inhibitory effect on vascular smooth muscle cells (Pan et al., 2003), inducing apoptosis of human leukemia HL-60 cells (Wang et al., 2002), as well as having a synergistic effect with taxol-in-
* Corresponding author. Address: Department of Biochemistry, Dongeui University College of Oriental Medicine, Busan 614-052, Republic of Korea. Tel.: +82 51 857413; fax: +82 51 853 4036. E-mail addresses: [email protected], [email protected] (Y.H. Choi). 0887-2333/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2009.09.017
duced apoptosis in human hepatoma HepG2 cells (Kuo et al., 2006). Recently, we suggested that extracellular-regulated kinase (ERK) and/or c-Jun N-terminal kinase (JNK) signaling pathways may be mediated by esculetin-induced G1 arrest and apoptosis of human leukemic U937 cells (Lee et al., 2007; Park et al., 2008). In addition, the ERK cascade was proven to be involved in the enhancement of esculetin on the Taxol-induced apoptosis in HepG2 cells (Kuo et al., 2006), suggesting its importance in esculetin alone or in combination induced apoptosis. However, our previous data indicate that Bcl-2 overexpression may confer protection against esculetin-induced apoptosis at least in leukemia cell types (Park et al., 2008), suggesting its importance in esculetin resistance. Overexpression of Bcl-2 proteins is one mechanism for malignancies to acquire resistance to cancer therapies (Bouillet et al., 2000; Cory et al., 2003; Jeong and Seol, 2008). Intense efforts, therefore, have been devoted to identifying small-molecule antag-
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onists against this protein with the hope that such antagonists would re-sensitize drug-resistant tumors to standard anticancer treatment (Wang et al., 2000; Lugovskoy et al., 2002; Tang et al., 2007). HA 14-1 is a small-molecule antagonist against Bcl-2 protein and has been shown to synergize with the anticancer activities of diverse compounds in leukemia (Milella et al., 2002; Lickliter et al., 2003; Hao et al., 2004; Wlodkowic et al., 2006). These results indicate the potential of HA 14-1 in combination therapy for cancer treatment. However, the precise mechanism by which HA14-1 contributes to ERK and death receptor (DR)-mediated apoptosis is unclear. We used HA14-1 as a sensitizer for overcoming the resistance of Bcl-2 overexpressing U937 cells to esculetin-induced apoptosis. Our data show that the synergistic effect of HA14-1 on esculetininduced apoptosis is through ERK and DR-mediated apoptotic pathways.
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was measured at a wave length of 540 nm with an ELISA reader (Molecular Devices, Sunnyvale, CA). 2.5. Flow cytometric analysis After treatment with esculetin, the cells were collected, washed with cold phosphate-buffered saline (PBS), and fixed in 75% ethanol at 4 °C for 30 min. DNA content of the cells was measured using a DNA staining kit (CycleTESTTM PLUS Kit, Becton Dickinson, San Jose, CA). Propidium iodide (PI)-stained nuclear fractions were obtained by following the kit protocol (Liao et al., 2008). The cells were then filtered through 35-mm mesh, and DNA content fluorescence was determined using a FACSCalibur (Becton Dickinson) flow cytometer within 1 h. Cellular DNA content was analyzed by CellQuest software (Becton Dickinson).
2. Materials and methods
2.6. Protein extraction and western blot analysis
2.1. Reagents
Cells were treated with esculetin and were collected with icecold PBS. The mitochondrial and cytosolic fractions were isolated using a mitochondrial and cytosolic fractionation kit (Activemotif, Carlsbad, CA) (Kim et al., 2008). For the preparation of total proteins, the cells were gently lysed 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) for 30 min. Supernatants were collected and protein concentrations 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 electro blotting. The blots were probed with the desired antibodies for 1 h, incubated with the diluted enzyme-linked secondary antibody and visualized by enhanced chemiluminescence (ECL) western blotting detection reagents (SuperSignal, Thermo scientific, Rockford, IL) according to the recommended procedure (Amersham, Arlington Heights, IL). The peroxidase-labeled donkey anti-rabbit immunoglobulin and peroxidase-labeled sheep anti-mouse immunoglobulin were purchased from Amersham (Lee et al., 2008).
Esculetin (6,7-dihydroxycoumarin, 98% purity) was purchased from Sigma–Aldrich (St. Louis, MO), dissolved in dimethyl sulfoxide (DMSO) and adjusted to final concentrations using complete RPMI1640 (Gibco BRL, Gaithersburg, MD). 3-(4,5-Dimethyl-2thiazolyl)-2,5-diphnyl-2H-tetrazolium bromide (MTT), 4,6-diamidino-2-phenylindole (DAPI) and ethyl [2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)]-4H-chromene-3-carboxylate (HA 14-1) were purchased from Sigma. PD98059 (ERK inhibitor), SP600125 (JNK inhibitor) and the specific mitochondrial dye 5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolylcarbocyanine iodide (JC1) were purchased from Calbiochem (San Diego, CA). Caspase activity assay kits were purchased from R&D Systems (Minneapolis, MN). 2.2. Antibodies Antibodies specific for Bcl-2, Bax, Bid, cytochrome c, caspase-3, caspase-8, caspase-9 and poly(ADP-ribose) polymerases (PARP) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies specific for phospho-ERK and phospho-JNK were purchased from Cell Signaling (Beverly, MA). Antibodies specific for DR4 and DR5 were from Calbiochem. Ant-actin antibody was obtained from Sigma (St. Louis, MO). 2.3. Cell lines and cell culture The human leukemia cell line U937 was 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. Bcl-2 overexpressing U937 cells were a generous gift from Dr. T. K. Kwon (Department of Immunology, Keimyung University School of Medicine, Taegu, Korea) and were maintained in a medium containing 0.7 lg/ml geneticin (G418 sulfate, Calbiochem). 2.4. Cell viability assay Cell viability was determined by MTT assays. Cells were seeded in 6-well plates and treated with esculetin for the indicated times. After treatments, MTT working solution was added to 6-well culture plates and incubated continuously at 37 °C for 3 h. The culture supernatant was removed from wells, and DMSO was added to completely dissolve formazan crystals. The absorbance of each well
2.7. Nuclear staining with DAPI For DAPI staining, the cells were washed with PBS and fixed with 3.7% paraformaldehyde (Sigma) 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 then washed twice with PBS and analyzed by fluorescence microscopy (Carl Zeiss, Germany). 2.8. DNA fragmentation assay After esculetin treatment, 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 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). Subsequently, 5 M 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. DNA was separated on 1.5% agarose gels containing 0.1 lg/ml ethidium bromide (EtBr, Sigma), and was visualized using an ultraviolet light source.
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2.9. Mitochondrial membrane potential assay The mitochondrial membrane potential (MMP, Dwm) was determined using the lipophilic cationic probe JC-1. JC-1 is a radiometric, dual-emission fluorescent dye that is absorbed and concentrated by respiring mitochondria and can reflect changes in MMP in living cells. There are two excitation wavelengths, 527 nm (green) for the monomer form and 590 nm (red) for the JC-1-aggregate form. With normal mitochondrial function, MMP is high and red fluorescence is predominant. However, when there is mitochondrial injury, the MMP is reduced, leading to an increase in green fluorescence. The relative intensity of red and green fluorescent signals indicate whether mitochondria are damaged. U937 cells were incubated with JC-1 at a final concentration of 10 lM for 20 min at 37 °C, in the dark. At the end of the incubation period, the cells were washed twice in cold PBS, resuspended in a total volume of 500 ll, and analyzed by flow cytometry.
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 caspase8 and Leu-Glu-His-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. 2.11. Statistical analysis All data are presented as mean ± SD. Significant differences between two groups were determined using the unpaired Student’s ttest. 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
2.10. Assay of caspases activity
3.1. Bcl-2 overexpression protects against esculetin-induced apoptosis
The enzymatic activity of the caspases induced by esculetin was assayed using a colorimetric assay kit (R&D Systems, Minneapolis, MN) according to manufacturer’s protocol. U937 cells were incubated in the absence and presence of esculetin 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
To determine the role of Bcl-2 in esculetin-induced apoptosis, the extent of cell viability was assessed by MTT assay and apoptosis was analyzed using flow cytometric analysis of sub-G1 population (Fig. 1A and B). In U937/vector cells, esculetin treatment suppressed the cell viability and also induced apoptosis; however, Bcl-2 overexpression (U937/Bcl-2) was found to signif-
Fig. 1. Bcl-2 overexpression protects against esculetin-induced apoptosis in U937 cells. Control (U937) or Bcl-2 transfected (U937/Bcl-2) cells were seeded at 2 105 cells/ml and then incubated with 30 l/ml of esculetin for 6 h. (A) Cell viability was measured by the metabolic-dye-based MTT assay. (B) The cells were collected and stained with CycleTEST PLUS DNA REAGENT Kit for flow cytometry analysis. The percentages of cells with hypodiploid DNA (sub-G1 phase) content represent the fractions undergoing apoptotic DNA degradation. Each point represents the mean ± SD of three independent experiments. Significance was determined using Student’s t-test (*p < 0.05 vs. untreated control). (C) Equal amounts of cytosolic and mitochondrial proteins extracted from cells grown under the same conditions 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.
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icantly protect cells from esculetin-induced apoptosis evaluated relative to U937/vector cells. To determine how Bcl-2 exerts an effect on cell survival, we examined the levels of expression of several proteins in the cytosolic and mitochondrial fractions by immunoblotting. We found that ectopic Bcl-2 overexpression completely inhibited the esculetin-induced cytosolic release of the mitochondrial pro-apoptotic protein cytochrome c relative to esculetin treated U937/vector cells (Fig. 1C). Upon treatment
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with esculetin, cytosolic Bax expression was lost in U937/vector cells consistent with its translocation to the mitochondria (Fig. 1C). In contrast, Bcl-2 overexpression blocked Bax translocation, trapping it in the cytosolic fraction. Accordingly, these results indicate that a mitochondrial amplification step is required for apoptosis induction by esculetin and that Bcl-2 overexpression may confer protection against esculetin-induced apoptosis in U937 cells.
Fig. 2. Bcl-2 overexpression protects against esculetin-induced ERK activation and DR4 expression. The cells were treated with 30 lg/ml of esculetin for the indicated times, lysed, and cellular proteins were separated by SDS–polyacrylamide gel and then transferred onto nitrocellulose membranes. The membranes were probed with the indicated antibodies. Proteins were visualized using an ECL detection system. Actin was used as an internal control.
Fig. 3. Co-treatment with esculetin and HA14-1 restored apoptosis, ERK activation and DR4 expression in Bcl-2 overexpressing U937 cells. The cells were seeded at 2 105 cells/ml and were then incubated with 30 g/ml of esculetin and 15 lM of HA14-1 for 6 h. (A) Cell viability was measured by the metabolic-dye-based MTT assay. (B) The cells were collected and stained with CycleTEST PLUS DNA REAGENT Kit for flow cytometry analysis. The percentage of cells with hypodiploid DNA (sub-G1 phase) content represent the fractions undergoing apoptotic DNA degradation. 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). (C) Nuclei stained with DAPI solution were then observed under a fluorescent microscope using a blue filter. Magnification, X400. (D) The fragmented DNAs were separated on 1.5% agarose gel electrophoresis and visualized under UV light after staining with EtBr. (E) The cells were lysed, and cellular proteins were separated by SDS–polyacrylamide gel and transferred onto nitrocellulose membranes. The membranes were probed with the indicated antibodies. Proteins were visualized using an ECL detection system. Actin was used as an internal control.
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3.2. Bcl-2 overexpression protects against esculetin-induced ERK activation and DR4 expression Our previous reports indicated that the JNK and ERK pathways were key regulators of apoptosis in response to esculetin in U937 cells. As shown in Fig. 2A, the phosphorylation of ERK and JNK increased significantly after 2 h of esculetin treatment. However, Bcl2 overexpression was found to significantly protect cells from esculetin-induced phosphorylation of ERK and JNK evaluated relative to U937/vector cells. Next, we investigated the effect of esculetin treatment on the DR4 and DR5 expression in order to determine whether the death receptor-mediated apoptosis signaling pathway is involved in mediating the observed apoptotic response. As shown in Fig. 2B, the expression of DR4, but not DR5, increased significantly after esculetin treatment in a time-dependent manner, and blocked by Bcl-2 overexpression. These results indicate that Bcl-2 overexpression attenuated apoptosis by esculetin, and this was correlated with ERK, JNK and DR4 pathways.
3.3. Co-treatment with esculetin and HA14-1 restored the activation of ERK and increase of DR4expression in Bcl-2 overexpressing U937 cells To investigate the effects of esculetin and HA14-1 on the viability and apoptosis of Bcl-2 overexpressing U937 cells, several experiments were performed. As shown in Fig. 3A, following treatment with esculetin plus HA14-1 for 6 h, U937/Bcl-2 cells exhibited much stronger growth inhibitory effects as compared with esculetin or HA14-1 alone. In order to obtain a quantitative measure of apoptosis induction, we next investigated, using flow
cytometric analysis, the proportion of cells with sub-G1 DNA content. Treatment with esculetin and HA14-1 under the indicated conditions resulted in significant accumulation of cells with sub-G1 DNA content (Fig. 3B). As shown in Fig. 3C, the cells cultured for the indicated condition of esculetin and HA14-1 showed marked changes in condensed chromatin in the nuclei following treatment with esculetin plus HA14-1. In addition, treatment with esculetin plus HA14-1 also significantly increased DNA fragmentation for up to 6 h after treatment (Fig. 3D). These results suggest that esculetin plus HA14-1 significantly induced apoptosis in U937/Bcl-2 cells. We then determined whether esculetin plus HA14-1 reverses Bcl-2-mediated suppression of apoptotic signaling. For this experiment, U937/Bcl-2 cells were treated with esculetin, HA14-1, or their combination; western blotting was performed. As shown in Fig. 3E, treatment with esculetin and/or HA14-1 did not change the expression level of Bcl-2 and DR5. However, co-administration of esculetin and HA14-1 resulted in significantly increased ERK phosphorylation and DR4 expression levels compared with controls or cells treated with esculetin or HA14-1 alone. Together, these data indicate that co-treatment of Bcl-2 overexpressing U937 cells with esculetin and HA14-1 can restore the activation of ERK and the increases in DR4 but not in JNK and DR5.
3.4. Co-treatment with esculetin and HA14-1 synergistically induced the loss of MMP and activation of caspases The regulation of apoptosis is a complex process and involves a number of mitochondrial proteins. To determine the role of the
Fig. 4. Co-treatment with esculetin and HA14-1 restored cytochrome c release, decreases in MMP and activation of caspases in Bcl-2 overexpressing U937 cells. The cells were seeded at 2 105 cells/ml and were then incubated with 30 lg/ml of esculetin and 15 lM of HA14-1 for 6 h. (A and C) The cells were lysed and cellular proteins were separated by SDS–polyacrylamide gel and transferred onto nitrocellulose membranes. The membranes were probed with the indicated antibodies. Proteins were visualized using an ECL detection system. Actin was used as an internal control. (CF, cleavage form) (B) The cells were exposed to esculetin and HA14-1 for 6 h, stained with JC-1 and incubated at 37 °C for 20 min. Mean JC-1 fluorescence intensity was detected using a flow cytometer. (D) The cell lysates from cells grown under the same conditions were assayed for in vitro caspase-3, caspase-8 and caspase-9 activity using DEVD-pNA, IETD-pNA and LEHD-pNA, respectively, as substrates. Amounts of products were measured using relative fluorescence signal intensity. Each point represents the mean ± SD of three independent experiments. Significance was determined using Student’s t-test (*p < 0.05 vs. untreated control).
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mitochondria in esculetin and HA14-induced apoptosis of U937/ Bcl-2 cells, we further investigated the levels of cytosolic and mitochondrial Bax or cytochrome c, as well as MMP (Dwm). Exposure of U937/Bcl-2 cells to esculetin plus HA14-1 led to a significant increase in the level of cytosolic cytochrome c and a decrease of Bax (Fig. 4A). Furthermore, exposure of U937/Bcl-2 cells to esculetin plus HA14-1 for 6 h led to a significant reduction in the MMP level (Fig. 4B). These results suggest a direct role of the mitochondria in esculetin plus HA14-1 induced apoptosis. 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 activation of caspase-3, caspase-8, and caspase-9 and further assessed the cleavage of several key death substrates that indicate the activation of caspases, including PARP (substrate for caspase-3 and caspase-7) and Bid (caspase-8). As shown in Fig. 4C, western blot analyses revealed that treatment with esculetin or HA14-1 alone did not affect, or only slightly affected the cleavage of caspases, PARP, and Bid. However, treatment with esculetin plus HA14-1 significantly induced the cleavage of caspases, PARP, and Bid. Next, cell lysates containing equal amounts of total protein from cells treated with esculetin and HA14-1 were assayed for in vitro caspases activity. As shown in Fig. 4D, treatment with esculetin plus HA14-1 significantly increased the activities of caspase-3, caspase-8, and caspase-9 compared with control cells or cells treated with esculetin or HA14-1 alone. These results indicate that esculetin plus HA14-1 treatment induces apoptotic death in U937/Bcl-2 cells, at least in part through a caspase-dependent pathway.
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3.5. The ERK and DR4 pathways play important roles in esculetin plus HA14-1 induced apoptosis Next, we investigated the effect of the ERK signaling pathway and the expression of DRs on the apoptotic response in Bcl-2 overexpressing U937 cells induced by esculetin and HA14-1 treatment. We evaluated the possible roles of mitogen-activated protein kinase (MAPK) pathways in esculetin plus HA14-1 treatment-induced apoptosis. As shown in Fig. 5A, pre-treatment with PD98059 (a potent inhibitor of ERK) significantly decreased the number of cells with sub-G1 DNA content. However, pre-treatment with SP600125 (a potent inhibitor of JNK) did not have a statistically significant effect on the esculetin plus HA14-1 treatment. In addition, U937/Bcl-2 cells cultured in the presence of PD98059 showed significantly decreased DNA fragmentation following 6 h of esculetin plus HA14-1 treatment (Fig. 5B). Consistent with the DNA fragmentation, pre-treatment with PD98059 elicited marked inhibition of the appearance of apoptotic bodies due to esculetin plus HA14-1 (Fig. 5C). Next, we investigated the effect of the ERK signaling pathway on the esculetin and HA14-1 induced activation of ERK and increases in DR4 expression in Bcl-2 overexpressing U937 cells. As shown in Fig. 5D, treatment with esculetin, HA141 or PD98059 alone did not change the expression levels of Bcl-2 and DR5. However, pre-treatment with PD98059 significantly decreased the activation of ERK by co-treatment with esculetin and HA14-1. Interestingly, the increase in DR4 expression induced by esculetin plus HA14-1 was also significantly decreased by pretreatment with PD98059. These data indicate that the ERK and DR4 pathways play important roles and suggest that DR4 expression is dependent on the ERK signaling pathway in esculetin plus HA14-1- induced apoptosis.
Fig. 5. ERK and DR4 pathways play important roles in esculetin plus HA14-1-induced apoptosis in Bcl-2 overexpressing U937 cells. Cells were pretreated with PD98059 (100 uM) and SP600125 (40 lM) for 1 h before treatment with 30 lg/ml of esculetin and 15 lM of HA14-1 for 6 h. (A) The cells were collected and stained with CycleTEST PLUS DNA REAGENT Kit for flow cytometry analysis. The percentages of cells with hypodiploid DNA (sub-G1 phase) contents represent the fractions undergoing apoptotic DNA degradation. Each point represents the mean ± SD of three independent experiments. Significance was determined using Student’s t-test (*p < 0.05 vs. untreated control). (B) The fragmented DNAs were separated on 1.5% agarose gel electrophoresis and visualized under UV light after staining with EtBr. (C) Nuclei stained with DAPI solution were then observed under a fluorescence microscope using a blue filter. Magnification, 400. (D) The cells were lysed, and cellular proteins were separated by SDS–polyacrylamide gel and transferred onto nitrocellulose membranes. The membranes were probed with the indicated antibodies. Proteins were visualized using an ECL detection system. Actin was used as an internal control.
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3.6. The ERK pathways play important roles in esculetin plus HA14-1induced loss of MMP and caspases activation In order to investigate the significance of the ERK pathway in response to esculetin plus HA14-1 treatment, we also investigated apoptosis-related protein expressions in U937/Bcl-2 cells. As shown in Fig. 6A, exposure of U937/Bcl-2 cells with esculetin plus HA14-1 led to a significant increase in the level of cytosolic cytochrome c and the decrease of Bax, whereas it led to marked down-regulation of cytochrome c and up-regulation of Bax in mitochondria. However, pre-treatment with PD98059 resulted in a reduction in Bax and cytochrome c expression at mitochondrial and cytosolic levels compared to that induced by treatment with esculetin plus HA14-1. We also showed that esculetin plus HA14-1 potentiated the decrease in MMP and was inhibited by pre-treatment with PD98059 (Fig. 6 B). These results clearly indicate that the esculetin plus HA14-1-induced mitochondrial amplification step is correlated with the activation of ERK pathways. We further investigated the effects of ERK activation on the levels of caspases activation in U937/Bcl-2 cells. As shown in Fig. 6 C and D, western blot analyses revealed that treatment with esculetin and HA14-1 alone did not affect, or only slightly induced the cleavage of caspases, PARP, and Bid. However, treatment with esculetin plus HA14-1 significantly induced the cleavage of caspases, PARP, Bid, and increased in vitro caspase activity. In contrast, pre-treatment with PD98059 markedly inhibited cleavage of caspases and increased caspase activation compared with esculetin plus HA14-1 treatment. These results indicate that esculetin plus HA14-1 treatment induces caspase activation in U937/Bcl-2 cells, at least in part through ERK-dependent pathways.
4. Discussion We previously reported that esculetin induces apoptosis in human leukemia U937 cells via ERK and JNK-dependent apoptotic pathways (Park et al., 2008). Based on this, in the present experiments we investigated ERK signaling in Bcl-2-mediated apoptosis. We found that Bcl-2 overexpression attenuated esculetin-induced apoptosis via inhibition of DR4 expression and ERK inactivation. In addition, we found that Bcl-2-mediated apoptosis resistance to esculetin, which was restored by HA14-1 though ERK-dependent expression of DR4, caspases activation, and loss of MMP. Many previous studies indicate that the ERK pathway is involved in the survival of B-cell chronic lymphocytic leukemia cells (Brózik et al., 2006; Rubio et al., 2007). Our study was conducted to determine the effects of ERK on esculetin plus HA14-1-induced apoptosis. Our results demonstrate that pre-treatment with PD98059 markedly inhibited esculetin plus HA14-1-induced apoptosis, whereas this inhibition was not observed upon treatment with PD98059 alone. In contrast, blocking the JNK pathway did not alter the apoptosis that occurs upon treatment with esculetin plus HA14-1 (Fig. 5A). These data strongly suggest that esculetin plus HA14-1-induced apoptosis is associated with the ERK pathway. Strategies to overcome Bcl-2-mediated resistance to apoptosis have the potential to greatly increase treatment efficacy. One approach involves the use of small-molecule inhibitors of Bcl-2 that bind to and inhibit Bcl-2 function. One such inhibitor, HA14-1, has recently been developed using molecular modeling techniques; however, very limited data are available regarding its ability to reverse Bcl-2 mediated resistance (Fernandes-Alnemri et al.,
Fig. 6. The ERK pathway plays important roles in esculetin plus HA14-1-induced cytochrome c release, decreases in MMP and activation of caspases in Bcl-2 overexpressing U937 cells. Cells were pretreated with PD98059 (100 uM) for 1 h before treatment with 30 g/ml of esculetin and 15 lM of HA14-1 for 6 h. (A and C) The cells were lysed, and cellular proteins were separated by SDS–polyacrylamide gel and transferred onto nitrocellulose membranes. The membranes were probed with the indicated antibodies. Proteins were visualized using an ECL detection system. The integrity of mitochondria and quantitative loading of the proteins was established by analyzing the levels of cytochrome oxidase subunit IV (COX IV) and actin, respectively (CF, cleavage form) (B) Cells were exposed to esculetin and HA14-1 for 6 h, after which MMPs were determined using JC-1. (D) Cell lysates from cells grown under the same conditions 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 relative fluorescence of the products was measured. Each point represents the mean ± SD of three independent experiments. Significance was determined using Student’s t-test (*p < 0.05 vs. untreated control).
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1994; Murphy et al., 1996; Yang et al., 1997; Kluck et al., 1997). Therefore, HA14-1 was evaluated to determine if it could reverse the anti-apoptotic effect of Bcl-2 and enhance esculetin-mediated apoptosis. Treatment with HA14-1 alone did not induce apoptosis, except at the highest dose (15 lM) evaluated. A prior study of hematopoietic cell lines found that a concentration of HA14-1 exceeding 25 lM resulted in a loss of selectivity for Bcl-2 and marked cytotoxicity in several cell lines tested (Jin et al., 1997). Although treatment of U937/Bcl-2 cells with HA14-1 (15 lM) alone failed to overcome the Bcl-2-mediated suppression of mitochondrial apoptotic signaling, co-administration of HA14-1 and esculetin restored apoptotic susceptibility. This occurred in association with a loss of MMP, which enabled caspases activation. The ability of Bcl-2 to confer resistance to esculetin-induced apoptosis and its reversal via HA14-1 has important therapeutic implications. Bcl-2 is expressed in multiple human tumor cell types in the absence of gene rearrangements, including leukemia cells (Adams and Cory, 1998; Pileri et al., 2003; Inoue et al., 2004; Dargent et al., 2005; Vitolo et al., 2008). Human cancer cells are type II, and most chemotherapeutic drugs mediate apoptosis by inducing mitochondrial dysfunction; therefore, these results indicate that Bcl-2 is an important contributor to multiple drug resistance (van de Donk et al., 2006; Wilson, 2006; Eberle et al., 2007). Furthermore, the results of our study indicate that the requirement for mitochondrial amplification of various stimuli that target intrinsic apoptotic pathways may overcome Bcl-2-mediated resistance, thereby increasing therapeutic efficacy. In conclusion, we have demonstrated that Bcl-2 overexpression prevents esculetin-induced activation of ERK and expression of DR4 in U937 cells. HA14-1 restored Bcl-2 mediated apoptosis via regulation of the decreases in MMP and caspase activity. This phenomenon was associated with the ERK signaling pathway. Therefore, it is thought that combination of esculetin and HA14-1 might be a promising strategy for use in cancer chemoprevention and chemotherapy. 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 work was supported by a grant from the Personalized Tumor Engineering Research Center (PTERC) and the Korea Science and Engineering Foundation (KOSEF) and BlueBio Industry RIC at Dong-Eui University as a RIC (08-06-07) program of KIAT under Ministry of Knowledge Economy, Republic of Korea. References Adams, J.M., Cory, S., 1998. The Bcl-2 protein family: arbiters of cell survival. Science 281, 1322–1326. Bouillet, P., Huang, D.C., O’Reilly, L.A., Puthalakath, H., O’Connor, L., Cory, S., Adams, J.M., Strasser, A., 2000. The role of the pro-apoptotic Bcl-2 family member bim in physiological cell death. Ann. N.Y. Acad. Sci. 926, 83–89. Brózik, A., Casey, N.P., Hegedus, C., Bors, A., Kozma, A., Andrikovics, H., Geiszt, M., Német, K., Magócsi, M., 2006. Reduction of Bcr-Abl function leads to erythroid differentiation of K562 cells via downregulation of ERK. Ann. N.Y. Acad. Sci. 1090, 344–354. Cory, S., Huang, D.C., Adams, J.M., 2003. The Bcl-2 family: roles in cell survival and oncogenesis. Oncogene 22, 8590–8607. Dargent, J.L., Lespagnard, L., Feoli, F., Debusscher, L., Greuse, M., Bron, D., 2005. De novo CD5-positive diffuse large B-cell lymphoma of the skin arising in chronic limb lymphedema. Leukocyte Lymph. 46, 775–780. Eberle, J., Kurbanov, B.M., Hossini, A.M., Trefzer, U., Fecker, L.F., 2007. Overcoming apoptosis deficiency of melanoma-hope for new therapeutic approaches. Drug Resist. Updat. 10, 218–234. Fernandes-Alnemri, T., Litwack, G., Alnemri, E.S., 1994. CPP32, a novel human apoptotic protein with homology to Caenorhabditis elegans cell death protein Ced-3 and mammalian interleukin-1 beta-converting enzyme. J. Biol. Chem. 269, 30761–30764.
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