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Buforin IIb induces endoplasmic reticulum stress-mediated apoptosis in HeLa cells
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Ju Hye Jang a,1 , Yu Jin Kim a,1 , Hyun Kim a , Sun Chang Kim b , Ju Hyun Cho a,∗ a
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Department of Biology, Research Institute of Life Science, Gyeongsang National University, Jinju 660-701, South Korea Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, South Korea
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Article history: Received 11 February 2015 Received in revised form 22 April 2015 Accepted 25 April 2015 Available online xxx
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Keywords: Buforin IIb Anticancer peptide Apotosis ER stress Mitochondrial membrane permeabilization
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Introduction
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Buforin IIb, a novel cell-penetrating anticancer peptide derived from histone H2A, has been reported to induce mitochondria-dependent apoptosis in tumor cells. However, increasing evidence suggests that endoplasmic reticulum and mitochondria cooperate to signal cell death. In this study, we investigated the mechanism of buforin IIb-induced apoptosis in human cervical carcinoma HeLa cells by focusing on ER stress-mediated mitochondrial membrane permeabilization. Two-dimensional PAGE coupled with MALDI-TOF and western blot analysis showed that buforin IIb treatment of HeLa cells resulted in upregulation of ER stress proteins. PBA (ER stress inhibitor) and BAPTA/AM (Ca2+ chelator) pretreatment rescued viability of buforin IIb-treated cells through abolishing phosphorylation of SAPK/JNK and p38 MAPK. SP600125 (SAPK/JNK inhibitor) and SB203580 (p38 MAPK inhibitor) attenuated down-regulation of Bcl-xL/Bcl-2, mitochondrial translocation of Bax, and cytochrome c release from mitochondria. Taken together, our data suggest that the ER stress pathway has an important role in the buforin IIb-induced apoptosis in HeLa cells. © 2015 Published by Elsevier Inc.
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Cancer treatment by conventional chemotherapies that are based on alkylating agents, antimetabolites, and hormone agonists/antagonists is limited by factors such as severe side-effects and the development of multi-drug resistance by cancer cells [18]. There is an increasing need for new anticancer therapies with a higher selectivity for cancer cells compared with conventional chemotherapy, thereby leading to less cytotoxic side-effects during treatment, as well as avoiding the problem of chemoresistance [20,30]. Recently, anticancer peptides, cationic antimicrobial peptides (AMPs) with cancer-selective cytotoxicity, have received attention as alternative chemotherapeutic agents that overcome the limits of current drugs. These peptides have several advantages over currently used anticancer therapeutics, such as low intrinsic
Abbreviations: AMP, antimicrobial peptide; ER, endoplasmic reticulum; SAPK/JNK, stress-activated protein kinase/Jun-amino-terminal kinase; p38 MAPK, p38 mitogen-activated protein kinase; CHOP, C/EBP homologous protein; GRP78, 78 kDa glucose-regulated protein; Bcl-2, B cell lymphoma 2; Bcl-xL, B-cell lymphoma-extra large; Bax, Bcl-2-associated X protein; PBA, 4-phenylbutyric acid; BAPTA/AM, 1,2-bis(o-amimophenoxy)ethane-N,N,N ,N -tetraacetic acid. ∗ Corresponding author. Tel.: +82 55 772 1347; fax: +82 55 772 1349. E-mail address: [email protected] (J.H. Cho). 1 These authors contributed equally to this work.
cytotoxicity, decreased likelihood of resistance development, and additive effects in combination therapy [9,18]. Many natural or synthetic anticancer peptides have been reported to show anticancer activity with characteristics including the ability to kill target cells rapidly, the broad spectrum of activity, and the specificity for cancer cells [4,9]. According to the AMP database (http://aps.unmc.edu/AP/main.php), over 130 such peptides are known to have anticancer properties [37]. Among the anticancer peptides, buforin IIb [RAGLQFPVG(RLLR)3 ]-a synthetic analog of buforin II that contains a proline hinge between the two ␣-helices and a model ␣-helical sequence at the C-terminus (3 × RLLR)[19]-has received attention as a potential candidate for a novel anticancer drug since it displayed selective cytotoxicity against 62 cancer cell lines. Buforin IIb selectively targets cancer cells through interaction with the cell-surface gangliosides. Buforin IIb then traverses cancer cell membranes without damaging them and induces mitochondria-dependent apoptosis, as confirmed by caspase-9/3 activation and cytochrome c release to cytosol as well as by DNA laddering and annexin V staining [10,14]. However, increasing evidence suggests that endoplasmic reticulum (ER) and mitochondria cooperate to signal cell death [15]. ER and mitochondria form close contacts with 20% of the mitochondrial surface in direct contact with the ER [13]. These contacts have pivotal roles in numerous cellular functions including Ca2+ signaling, lipid transport, energy metabolism, and the regulation
http://dx.doi.org/10.1016/j.peptides.2015.04.024 0196-9781/© 2015 Published by Elsevier Inc.
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of apoptotic signaling [12,31,34]. A consequence of the physical and functional interaction between ER and mitochondria is that mitochondria function is sensitive to pathologic insults that induce ER stress (defined by the increased accumulation of misfolded proteins within the ER lumen). ER stress can be transmitted to mitochondria by alterations in the transfer of metabolites such as Ca2+ or by stress-responsive signaling pathways, directly influencing mitochondrial functions [22]. The Ca2+ released from the ER enters the mitochondria leading to membrane depolarization and release of cytochrome c through a mechanism involving oligomerization of Bax and Bak, the central proapoptotic B cell lymphoma 2 (Bcl-2) family proteins, on the mitochondrial outer membrane [1,33]. Mitochondrial release of cytochrome c in the cytosol induces the activation of the apoptosome, resulting in caspase-9 activation, and in the subsequent activation of effector caspases, such as caspase-3/caspase-7 that mediate the cell death program. Therefore, here we investigated the mechanism of buforin IIb-induced apoptosis in human cervical carcinoma HeLa cells by focusing on ER stress-mediated mitochondrial membrane permeabilization.
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Materials and methods
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Peptide, antibodies, and reagents Buforin IIb was synthesized on a MilliGen 9050 peptide synthesizer (Peptron, Korea). Synthetic peptide was purified by reversedphase high performance liquid chromatography and characterized by mass spectroscopy and amino acid analysis. The antibodies against phosphorylated protein kinase-like endoplasmic reticulum kinase (PERK, Thr980), C/EBP homologous protein (CHOP), 78 kDa glucose-regulated protein (GRP78), -actin, stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK), phosphorylated SAPK/JNK (Thr183/Tyr185), p38 mitogen-activated protein kinase (MAPK), phosphorylated p38 MAPK (Thr180/Tyr182), B-cell lymphoma-extra large (Bcl-xL), Bcl-2, cytochrome c, Bcl-2associated X protein (Bax), and voltage-dependent anion channel (VDAC) were from Cell Signaling Technology (Beverly, MA, USA). The antibody against phosphorylated inositol-requiring protein 1 (IRE1, Ser724) was from abcam (Cambridge, UK). The kinase inhibitors SP600125 (for SAPK/JNK) and SB203580 (for p38 MAPK), the caspase inhibitors Z-LEHD-FMK (for caspase-9) and Z-DEVD-FMK (for caspase-3), and the calcium chelator 1,2-bis(oamimophenoxy)ethane-N,N,N ,N -tetraacetic acid (BAPTA/AM) were from Calbiochem (San Diego, CA, USA). The pan-caspase inhibitor Z-VAD-FMK was from Enzo (Farmingdale, NY, USA). The ER stress inhibitor 4-phenylbutyric acid (PBA) was from Sigma (St. Louis, MO, USA). Cell culture HeLa (human cervical adenocarcinoma) cells were purchased from the American Tissue Cell Culture (Manassas, VA, USA), and cultured in a complete medium (DMEM supplemented with 10% FBS and 0.1% penicillin–streptomycin) in a humidified atmosphere of 5% CO2 at 37 ◦ C. Trypsin-EDTA (0.05%) was used to detach cells in subculturing. All the cell culture media and reagents were purchased from Lonza (Basel, Switzerland). Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and mass spectrometric analysis Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was carried out according to the procedure described elsewhere with a slight modification [5,27,28,32]. Briefly, HeLa cells, treated and untreated with buforin IIb (4 M) for 24 h, were
harvested and washed with PBS. The harvested cells were resuspended in the lysis buffer [8 M urea, 4% (w/v) 3-[(3-chlormidopropyl)dimethylammonium]-1-propanesulfonate (CHAPS), 40 mM Tris–HCl pH 8.5, and 2% Pharmalyte 3–10 (Amersham Biosciences, Piscataway, NJ, USA)]. After 30 min of gentle stirring at room temperature, the samples were centrifuged at 100 000 × g for 10 min. The clear supernatant containing proteins was frozen at −70 ◦ C until used. Isoelectric focusing was carried out using IPGphor (Amersham Biosciences). Proteins were applied by overnight ingel rehydration of Immobiline DryStrip gels (IPG strips, 13 cm, pH 3–10), and focused by using a series of voltage increases at 300 V for 1 min, 300 V to 3500 V for 1.5 h, and 3500 V for 2.5 h. The second dimension was carried out in a 12.5% polyacrylamide gel (150 × 150 × 1 mm). After fixation and silver staining, the wet gel was scanned with ImageScanner and quantified with ImageMaster 2D Elite software (Amersham Biosciences). Protein spots showing over-expressed patterns were excised from the stained gel and destained by silver stain–destain protocol [Scripps center for mass spectrometry (http://masspec.scripps.edu/ services/proteomics/silver prot.php)], and subjected to gel digestion with 20 ng of sequencing-grade trypsin (Promega, Madison, WI, USA)/l in 8 l of in 20 mM ammonium bicarbonate. The resulting samples were analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (Voyager DE STR, Applied Biosystems, Inc., Waltham, MA, USA). Peptide fragment peaks (at 805.416 and 2163.056 m/z) produced by autodigestion of trypsin were used as an internal calibration. Proteins were identified by using the ProteinProspector server (http://prospector.ucsf.edu/). In vitro cytotoxicity assay HeLa cells were seeded onto 96-well plates at a density of 7000 cells/well in 0.1 ml of complete medium. After 16 h of incubation, cells were treated with buforin IIb (8 M) and incubated for another 12 h. In some experiments, cells were pretreated with PBA (10 mM), BAPTA/AM (10 M), SP600125 (20 M), SB203580 (20 M), Z-LEHD-FMK (25 M), Z-DEVD-FMK (50 M), or Z-VAD-FMK (20 M) for 1 h before buforin IIb was added. Cell viability was measured with the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT) assay using the CellTiter 96-cell proliferation assay kit (Promega). The percentage of cell viability was determined using the following equation: Viability (%) = (As − A0 )/(Ac − A0 ) × 100, where As is the absorbance of the sample, Ac is the absorbance of control, and A0 is the background absorbance. Each experiment was performed in triplicate, and repeated at least three times independently. Differences between the groups were analyzed using Student’s t test with GraphPad Prism version 5.00 (GraphPad Software, San Diego, CA, USA), and the differences were considered significant at P < 0.05. Western blot analysis HeLa cells were seeded onto 6-well plates at a density of 15 000 cells/well in 3 ml of complete medium. After 48 h of incubation, cells pretreated with or without PBA (10 mM), BAPTA/AM (10 M), SP600125 (20 M), or SB203580 (20 M) for 1 h were treated with buforin IIb (4 M) and incubated for indicated time. Cells were harvested and lysed in ice-cold PRO-PREP Protein Extraction Solution (Intron, Korea). In some experiments, mitochondrial versus cytosolic fractions of cells were prepared using the mitochondrial isolation kit for cultured cells (Pierce, Rockford, IL, USA). The protein concentration was quantified by using the BCA Protein Assay Kit (Pierce), and equal amounts of protein (30 g for total cell lysates, 20 g for cytosolic fraction, and 5 g for mitochondrial fraction) were subjected to 12% SDS–PAGE. Separated proteins were
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transferred to polyvinyldifluoride (PVDF) membranes (Millipore, Billerica, MA, USA), the membranes were blocked with 5% (w/v) BSA in TBS containing 0.1% Tween-20, and conventional immunoblot was performed using appropriate antibodies. Chemiluminescence was detected using an ECL kit (Promega).
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Protein expression in buforin IIb-treated HeLa cells
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The effect of buforin IIb on the protein expression in HeLa cells was explored by 2-D PAGE. As shown in Fig. 1, most of the proteins were found to be down-regulated in HeLa cells treated with buforin IIb for 24 h compared to control untreated cells, whereas several proteins were highly expressed. Of them, we selected 9 protein spots with significant upregulated expression, and identified them by MALDI-TOF/MS (Fig. 1 and Table 1). Of the identified 9 proteins, 7 proteins were molecular chaperones [170 kDa glucoseregulated protein precursor (GRP170), 94 kDa glucose-regulated protein precursor (GRP94), 78 kDa glucose-regulated protein precursor (GRP78), T-complex protein 1 subunit theta (TCPQ), protein disulfide isomerase A3 precursor (PDIA3), prohibitin (PHB), and calnexin precursor (CALX)]. The others were Ca2+ binding protein [calreticulin precursor (CALR)] and annexin V (ANXA5). These results point to the presence of ER stress in buforin IIb-treated HeLa cells.
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Relationship between ER stress and buforin IIb-induced apoptosis
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The results of 2-D PAGE led us to examine the regulation of two ER stress sensors, PERK and IRE1 as well as the ER chaperone GRP78, employing western blot analysis. The protein contents of p-PERK, p-IRE1, and GRP78 were significantly increased in a time-dependent manner in HeLa cells following buforin IIb treatment. In addition, the expression of CHOP, a hallmark on the ER stress-mediated apoptosis, was also increased (Fig. 2A). We next attempted to determine whether ER stress was related directly with buforin IIb-induced apoptosis. When HeLa cells were pretreated with the ER stress inhibitor PBA for 1 h, buforin IIb-induced apoptosis was significantly decreased by about 40% compared with inhibitor-nontreated control (Fig. 2B). Moreover, we found that the pretreatment of cells with Ca2+ chelator BAPTA/AM could inhibit
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Table 1 Identified protein spots in buforin IIb-treated HeLa cells by MALDI-TOF/MS. Spot no.
Protein name
Symbol
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PI
Accession no.
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170 kDa glucose-regulated protein precursor 94 kDa glucose-regulated protein precursor 78 kDa glucose-regulated protein precursor T-complex protein 1 subunit theta Protein disulfide isomerase A3 precursor Annexin V Prohibitin Calreticulin precursor Calnexin precursor
GRP170
111.34
5.16
Q9Y4L1
GRP94
92.47
4.76
P14625
GRP78
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P11021
TCPQ
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5.41
P50990
PDIA3
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P30101
ANXA5 PHB CALR
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4.93 5.57 4.29
P08758 P35232 P27797
CALX
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4.46
P27824
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the buforin IIb-induced apoptosis (Fig. 2B). These data indicated that buforin IIb does indeed induce ER stress-mediated apoptosis in HeLa cells. Involvement of MAPKs in buforin IIb-induced apoptosis The MAPK pathway plays essential roles in the regulation of cellular response, including cell survival, apoptosis, proliferation, and differentiation, and MAPKs have been implicated in the signal pathway of ER stress [16,26]. To gain further insight into the mechanisms of the ER stress-induced apoptosis of HeLa cells by buforin IIb, we assessed whether buforin IIb activates MAPKs in HeLa cells. We observed that the treatment of buforin IIb led to upregulation of the phosphorylated form of SAPK/JNK and p38 MAPK from 6 h (Fig. 3A). Their activation was effectively blocked by specific inhibitors (SAPK/JNK inhibitor SP600125 and p38 MAPK inhibitor SB203580), and these inhibitors also blocked buforin IIbinduced apoptosis (Fig. 3B). In addition, we found that both PBA and BAPTA/AM could inhibit the activation of MAPKs (Fig. 3A), indicating that the activation of MAPKs by buforin IIb might be subsequent to ER stress and the Ca2+ release from the ER. These data indicated
Fig. 1. 2-D PAGE images of HeLa cell proteins (A) untreated and (B) treated with buforin IIb. The first dimension ran using pH 3–10 IPG strips and the second dimension was a 12.5% SDS–PAGE. Gels were stained with silver. Spots selected for MALDI-TOF/MS analysis were denoted by numbers. The list of spots identified by MALDI-TOF was shown in Table 1.
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Fig. 2. Relationship between ER stress and buforin IIb-induced apoptosis. (A) HeLa cells were treated with buforin IIb (4 M) for indicated times, after which the protein contents of p-PERK, p-IRE1, GRP78, and CHOP were determined via western blot analysis. -actin was used as loading control. (B) HeLa cells pretreated with or without PBA (10 mM) or BAPTA/AM (10 M) for 1 h were treated with buforin IIb (8 M). After a 12-h incubation with buforin IIb, cell viability was measured by the MTT assay. Data represent the mean ± SEM of 3 independent experiments performed in triplicate. Asterisks indicate significant differences at P < 0.05, compared with no inhibitor by Student’s t test.
Fig. 3. Involvement of SAPK/JNK and p38 MAPK in buforin IIb-induced apoptosis. (A) HeLa cells pretreated with or without SP600125 (20 M), SB203580 (20 M), PBA (10 mM), or BAPTA/AM (10 M) for 1 h were treated with buforin IIb (4 M) for indicated times, after which phospho-active and total forms of SAPK/JNK and p38 MAPK were determined via western blot analysis. (B) HeLa cells pretreated with or without SP600125 (20 M) or SB203580 (20 M) for 1 h were treated with buforin IIb (8 M). After a 12-h incubation with buforin IIb, cell viability was measured by the MTT assay. Data represent the mean ± SEM of 3 independent experiments performed in triplicate. Asterisks indicate significant differences at P < 0.05, compared with no inhibitor by Student’s t test.
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that buforin IIb induced ER stress and Ca2+ release from the ER, which in turn activated SAPK/JNK and p38 MAPK, resulting in ER stress-mediated apoptosis in HeLa cells.
downstream of SAPK/JNK and p38 MAPK, and involved in mitochondrial membrane permeabilization and caspase-dependent apoptosis in buforin IIb-treated HeLa cells.
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Discussion
Since Bcl-2 family proteins have been known to be involved in the signal crosstalk between ER stress and mitochondrial dysfunction [15,21,24], we next questioned if buforin IIb-induced apoptosis was mediated through Bcl-2 family proteins. Fig. 4A showed that down-regulation of anti-apoptotic Bcl-xL/Bcl-2 and marked translocation of pro-apoptotic Bax to mitochondria after buforin IIb treatment. A time-dependent release of cytochrome c into cytosol was also detected relative to gradual decrease in mitochondrial cytochrome c. Moreover, pretreatment with SP600125, SB203580, or PBA markedly abolished down-regulation of BclxL/Bcl-2, mitochondrial translocation of Bax, and cytochrome c release in response to buforin IIb treatment (Fig. 4A), reflecting that ER stress-elicited SAPK/JNK and p38 MAPK activation was involved in this event. We also found that Z-LEHD-FMK (caspase-9 inhibitor), Z-DEVD-FMK (caspase-3 inhibitor), and Z-VAD-FMK (pan-caspase inhibitor) blocked buforin IIb-induced apoptosis (Fig. 4B), which is in agreement with our previous study that showed that buforin IIb induced the activation of pro-caspase-9 and pro-caspase-3 [14]. Collectively, our data indicated that Bcl-xL, Bcl-2, and Bax were
ER is the primary site for protein synthesis, folding, and trafficking as well as for Ca2+ storage. Under a variety of stressful conditions, the accumulation of unfolded or misfolded proteins, or alterations in Ca2+ homeostasis in the ER can induce ER stress and ultimately lead to apoptosis [11]. To alleviate ER stress, cells activate a signaling pathway known as the unfolded protein response (UPR). The UPR promotes an increased capacity of protein folding and clearance to reduce the amount of misfolded proteins at ER lumen [6,36]. However, if the UPR’s mechanisms of adaptation and cell survival are insufficient to decrease the unfolded protein load, the UPR initiates cell death by apoptosis [7,8]. UPR-mediated cell death is executed by the canonical mitochondrial apoptosis pathway, where the Bcl-2 family plays a crucial role [29]. Transcriptional and post-transcriptional mechanisms are activated to regulated pro-apoptotic members of the Bcl-2 family that facilitate cytochrome c release from the mitochondria and Ca2+ release from the ER to engage downstream apoptotic signaling events [23]. Recently, an explosion of novel mechanisms and components that can suppress or potentiate apoptosis during ER stress has been
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Fig. 4. Involvement of Bcl-2 family proteins, cytochrome c, and caspase-9/3 in buforin IIb-treated HeLa cells. (A) HeLa cells pretreated with or without SP600125 (20 M), SB203580 (20 M), or PBA (10 mM) for 1 h were treated with buforin IIb (4 M) for indicated times, after which Bcl-xL and Bcl-2 were determined via western blot analysis. To examine the subcellular localization of Bax and cytochrome c, cytosolic (Cyto) and mitochondrial (Mito) fractions were prepared, and the protein distributions were analyzed by western blot analysis. VDAC was used as loading control for mitochondrial fraction. (B) HeLa cells pretreated with or without Z-LEHD-FMK (25 M), Z-DEVDFMK (50 M), or Z-VAD-FMK (20 M) for 1 h were treated with buforin IIb (8 M). After a 12-h incubation with buforin IIb, cell viability was measured by the MTT assay. Data represent the mean ± SEM of 3 independent experiments performed in triplicate. Asterisks indicate significant differences at P < 0.05, compared with no inhibitor by Student’s t test.
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identified. In the present study, we reported the mechanism of buforin IIb-induced apoptosis that involved ER stress-mediated mitochondrial membrane permeabilization in HeLa cells. Our proteomic analysis revealed that buforin IIb treatment led to upregulation of typical ER stress-related proteins including molecular chaperones and Ca2+ binding protein (Fig. 1 and Table 1). In particular, elevated expression of GRP78 and CHOP occurred at early time point (6 h) of buforin IIb treatment, signifying an initial ER stress-mediated apoptosis (Fig. 2A). It is well known that GRP78 and CHOP are typical ER stress-regulated proteins involved in ER stress-induced apoptosis [17]. Moreover, pretreatment with PBA (ER stress inhibitor) or BAPTA/AM (Ca2+ chelator) dramatically blocked the buforin IIb-induced apoptosis in HeLa cells (Fig. 2B). Therefore, it seems that buforin IIb-induced apoptosis involves ER stress. In addition to GRP78 and CHOP, MAPKs have been reported to play a critical role in ER stress-mediated apoptosis. In general, extracellular signal-regulated kinase (ERK) has been implicated in growth-factor-induced cell proliferation, whereas SAPK/JNK or p38 MAPK contributes to stress-induced apoptosis [25,35]. Based on these previous data, we hypothesized that ER stress-induced apoptosis after buforin IIb treatment involved the MAPK signaling pathways. Interestingly, SAPK/JNK and p38 MAPK were all activated by buforin IIb, and the inhibitor of each MAPK significantly blocked buforin IIb-induced apoptosis (Fig. 3), suggesting that MAPK functions upstream of buforin IIb-induced apoptosis. Although the direct molecular target of buforin IIb remains to be identified, our study indicates that buforin IIb treatment triggers a signal leading to ER stress, MAPKs, and apoptosis. Bcl-2 family proteins are a common link between ER stress and mitochondrial dysfunction [3]. The Bcl-2 family, a large class of both the anti-apoptotic proteins (Bcl-xL, Bcl-2) and pro-apoptotic proteins (Bax), tightly regulates the intrinsic apoptotic pathway by controlling the integrity of the outer mitochondrial membrane [2]. To investigate the involvement of Bcl-2 family proteins in buforin IIb-induced ER stress and apoptosis, we determined the expression levels of Bcl-2 family members in buforin IIb-treated HeLa cells. As shown in Fig. 4A, the level of Bcl-xL and Bcl-2 proteins was decreased in a time-dependent manner following buforin IIb treatment, while there was a significant enhancement in translocation of Bax from cytosol to mitochondria. One of the consequences following the changes of Bcl-2 family members is the release of mitochondrial pro-apoptotic protein, cytochrome c [1]. After
treatment of HeLa cells with buforin IIb, mitochondrial release of cytochrome c in the cytosol was increased in a time-dependent manner. In addition, SP600125, SB203580, and PBA attenuated down-regulation of Bcl-xL/Bcl-2, mitochondrial translocation of Bax, and cytochrome c release from mitochondria (Fig. 4A), and caspase inhibitors (Z-LEHD-FMK, Z-DEVD-FMK, and Z-VAD-FMK) dramatically blocked the buforin IIb-induced apoptosis in HeLa cells (Fig. 4B). These results indicates that the mitochondrial disruption by buforin IIb might be linked to ER stress, activation of MAPKs, and caspase-9/3-dependent apoptosis in HeLa cells. In conclusion, we demonstrated that buforin IIb induced ER stress and subsequently provoked ER stress-mediated mitochondrial membrane permeabilization via Ca2+ release from ER, SAPK/JNK and p38 MAPK pathway activation, down-regulation of Bcl-xL/Bcl-2, mitochondrial translocation of Bax, and cytochrome c release from mitochondria in HeLa cells. Buforin IIb has a great therapeutic potential to be developed into a novel therapeutic agent for the treatment of cancers. An understanding of the mechanism of buforin IIb-induced apoptotic cell death is a basic step in clinical therapeutic approaches. Acknowledgements This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) Q4 funded by the Ministry of Science, ICT & Future Planning (NRF- Q5 2014R1A2A1A11050944) and the Intelligent Synthetic Biology Center of Global Frontier Project funded by the Ministry of Science, ICT & Future Planning (NRF-2013M3A6A8073556). References [1] Brunelle JK, Letai A. Control of mitochondrial apoptosis by the Bcl-2 family. J Cell Sci 2009;122:437–41. [2] Chipuk JE, Moldoveanu T, Llambi F, Parsons MJ, Green DR. The BCL-2 family reunion. Mol Cell 2010;37:299–310. [3] Cory S, Adams JM. The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer 2002;2:647–56. [4] Dennison SR, Whittaker M, Harris F, Phoenix DA. Anticancer alpha-helical peptides and structure/function relationships underpinning their interactions with tumour cell membranes. Curr Protein Pept Sci 2006;7:487–99. [5] Gharahdaghi F, Weinberg CR, Meagher DA, Imai BS, Mische SM. Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity. Electrophoresis 1999;20:601–5.
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Buforin IIb induces endoplasmic reticulum stress-mediated apoptosis in HeLa cells
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Ju Hye Jang a,1 , Yu Jin Kim a,1 , Hyun Kim a , Sun Chang Kim b , Ju Hyun Cho a,∗ a
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Department of Biology, Research Institute of Life Science, Gyeongsang National University, Jinju 660-701, South Korea Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, South Korea
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Article history: Received 11 February 2015 Received in revised form 22 April 2015 Accepted 25 April 2015 Available online xxx
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Keywords: Buforin IIb Anticancer peptide Apotosis ER stress Mitochondrial membrane permeabilization
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Introduction
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Buforin IIb, a novel cell-penetrating anticancer peptide derived from histone H2A, has been reported to induce mitochondria-dependent apoptosis in tumor cells. However, increasing evidence suggests that endoplasmic reticulum and mitochondria cooperate to signal cell death. In this study, we investigated the mechanism of buforin IIb-induced apoptosis in human cervical carcinoma HeLa cells by focusing on ER stress-mediated mitochondrial membrane permeabilization. Two-dimensional PAGE coupled with MALDI-TOF and western blot analysis showed that buforin IIb treatment of HeLa cells resulted in upregulation of ER stress proteins. PBA (ER stress inhibitor) and BAPTA/AM (Ca2+ chelator) pretreatment rescued viability of buforin IIb-treated cells through abolishing phosphorylation of SAPK/JNK and p38 MAPK. SP600125 (SAPK/JNK inhibitor) and SB203580 (p38 MAPK inhibitor) attenuated down-regulation of Bcl-xL/Bcl-2, mitochondrial translocation of Bax, and cytochrome c release from mitochondria. Taken together, our data suggest that the ER stress pathway has an important role in the buforin IIb-induced apoptosis in HeLa cells. © 2015 Published by Elsevier Inc.
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Cancer treatment by conventional chemotherapies that are based on alkylating agents, antimetabolites, and hormone agonists/antagonists is limited by factors such as severe side-effects and the development of multi-drug resistance by cancer cells [18]. There is an increasing need for new anticancer therapies with a higher selectivity for cancer cells compared with conventional chemotherapy, thereby leading to less cytotoxic side-effects during treatment, as well as avoiding the problem of chemoresistance [20,30]. Recently, anticancer peptides, cationic antimicrobial peptides (AMPs) with cancer-selective cytotoxicity, have received attention as alternative chemotherapeutic agents that overcome the limits of current drugs. These peptides have several advantages over currently used anticancer therapeutics, such as low intrinsic
Abbreviations: AMP, antimicrobial peptide; ER, endoplasmic reticulum; SAPK/JNK, stress-activated protein kinase/Jun-amino-terminal kinase; p38 MAPK, p38 mitogen-activated protein kinase; CHOP, C/EBP homologous protein; GRP78, 78 kDa glucose-regulated protein; Bcl-2, B cell lymphoma 2; Bcl-xL, B-cell lymphoma-extra large; Bax, Bcl-2-associated X protein; PBA, 4-phenylbutyric acid; BAPTA/AM, 1,2-bis(o-amimophenoxy)ethane-N,N,N ,N -tetraacetic acid. ∗ Corresponding author. Tel.: +82 55 772 1347; fax: +82 55 772 1349. E-mail address: [email protected] (J.H. Cho). 1 These authors contributed equally to this work.
cytotoxicity, decreased likelihood of resistance development, and additive effects in combination therapy [9,18]. Many natural or synthetic anticancer peptides have been reported to show anticancer activity with characteristics including the ability to kill target cells rapidly, the broad spectrum of activity, and the specificity for cancer cells [4,9]. According to the AMP database (http://aps.unmc.edu/AP/main.php), over 130 such peptides are known to have anticancer properties [37]. Among the anticancer peptides, buforin IIb [RAGLQFPVG(RLLR)3 ]-a synthetic analog of buforin II that contains a proline hinge between the two ␣-helices and a model ␣-helical sequence at the C-terminus (3 × RLLR)[19]-has received attention as a potential candidate for a novel anticancer drug since it displayed selective cytotoxicity against 62 cancer cell lines. Buforin IIb selectively targets cancer cells through interaction with the cell-surface gangliosides. Buforin IIb then traverses cancer cell membranes without damaging them and induces mitochondria-dependent apoptosis, as confirmed by caspase-9/3 activation and cytochrome c release to cytosol as well as by DNA laddering and annexin V staining [10,14]. However, increasing evidence suggests that endoplasmic reticulum (ER) and mitochondria cooperate to signal cell death [15]. ER and mitochondria form close contacts with 20% of the mitochondrial surface in direct contact with the ER [13]. These contacts have pivotal roles in numerous cellular functions including Ca2+ signaling, lipid transport, energy metabolism, and the regulation
http://dx.doi.org/10.1016/j.peptides.2015.04.024 0196-9781/© 2015 Published by Elsevier Inc.
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of apoptotic signaling [12,31,34]. A consequence of the physical and functional interaction between ER and mitochondria is that mitochondria function is sensitive to pathologic insults that induce ER stress (defined by the increased accumulation of misfolded proteins within the ER lumen). ER stress can be transmitted to mitochondria by alterations in the transfer of metabolites such as Ca2+ or by stress-responsive signaling pathways, directly influencing mitochondrial functions [22]. The Ca2+ released from the ER enters the mitochondria leading to membrane depolarization and release of cytochrome c through a mechanism involving oligomerization of Bax and Bak, the central proapoptotic B cell lymphoma 2 (Bcl-2) family proteins, on the mitochondrial outer membrane [1,33]. Mitochondrial release of cytochrome c in the cytosol induces the activation of the apoptosome, resulting in caspase-9 activation, and in the subsequent activation of effector caspases, such as caspase-3/caspase-7 that mediate the cell death program. Therefore, here we investigated the mechanism of buforin IIb-induced apoptosis in human cervical carcinoma HeLa cells by focusing on ER stress-mediated mitochondrial membrane permeabilization.
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Materials and methods
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Peptide, antibodies, and reagents Buforin IIb was synthesized on a MilliGen 9050 peptide synthesizer (Peptron, Korea). Synthetic peptide was purified by reversedphase high performance liquid chromatography and characterized by mass spectroscopy and amino acid analysis. The antibodies against phosphorylated protein kinase-like endoplasmic reticulum kinase (PERK, Thr980), C/EBP homologous protein (CHOP), 78 kDa glucose-regulated protein (GRP78), -actin, stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK), phosphorylated SAPK/JNK (Thr183/Tyr185), p38 mitogen-activated protein kinase (MAPK), phosphorylated p38 MAPK (Thr180/Tyr182), B-cell lymphoma-extra large (Bcl-xL), Bcl-2, cytochrome c, Bcl-2associated X protein (Bax), and voltage-dependent anion channel (VDAC) were from Cell Signaling Technology (Beverly, MA, USA). The antibody against phosphorylated inositol-requiring protein 1 (IRE1, Ser724) was from abcam (Cambridge, UK). The kinase inhibitors SP600125 (for SAPK/JNK) and SB203580 (for p38 MAPK), the caspase inhibitors Z-LEHD-FMK (for caspase-9) and Z-DEVD-FMK (for caspase-3), and the calcium chelator 1,2-bis(oamimophenoxy)ethane-N,N,N ,N -tetraacetic acid (BAPTA/AM) were from Calbiochem (San Diego, CA, USA). The pan-caspase inhibitor Z-VAD-FMK was from Enzo (Farmingdale, NY, USA). The ER stress inhibitor 4-phenylbutyric acid (PBA) was from Sigma (St. Louis, MO, USA). Cell culture HeLa (human cervical adenocarcinoma) cells were purchased from the American Tissue Cell Culture (Manassas, VA, USA), and cultured in a complete medium (DMEM supplemented with 10% FBS and 0.1% penicillin–streptomycin) in a humidified atmosphere of 5% CO2 at 37 ◦ C. Trypsin-EDTA (0.05%) was used to detach cells in subculturing. All the cell culture media and reagents were purchased from Lonza (Basel, Switzerland). Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and mass spectrometric analysis Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was carried out according to the procedure described elsewhere with a slight modification [5,27,28,32]. Briefly, HeLa cells, treated and untreated with buforin IIb (4 M) for 24 h, were
harvested and washed with PBS. The harvested cells were resuspended in the lysis buffer [8 M urea, 4% (w/v) 3-[(3-chlormidopropyl)dimethylammonium]-1-propanesulfonate (CHAPS), 40 mM Tris–HCl pH 8.5, and 2% Pharmalyte 3–10 (Amersham Biosciences, Piscataway, NJ, USA)]. After 30 min of gentle stirring at room temperature, the samples were centrifuged at 100 000 × g for 10 min. The clear supernatant containing proteins was frozen at −70 ◦ C until used. Isoelectric focusing was carried out using IPGphor (Amersham Biosciences). Proteins were applied by overnight ingel rehydration of Immobiline DryStrip gels (IPG strips, 13 cm, pH 3–10), and focused by using a series of voltage increases at 300 V for 1 min, 300 V to 3500 V for 1.5 h, and 3500 V for 2.5 h. The second dimension was carried out in a 12.5% polyacrylamide gel (150 × 150 × 1 mm). After fixation and silver staining, the wet gel was scanned with ImageScanner and quantified with ImageMaster 2D Elite software (Amersham Biosciences). Protein spots showing over-expressed patterns were excised from the stained gel and destained by silver stain–destain protocol [Scripps center for mass spectrometry (http://masspec.scripps.edu/ services/proteomics/silver prot.php)], and subjected to gel digestion with 20 ng of sequencing-grade trypsin (Promega, Madison, WI, USA)/l in 8 l of in 20 mM ammonium bicarbonate. The resulting samples were analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (Voyager DE STR, Applied Biosystems, Inc., Waltham, MA, USA). Peptide fragment peaks (at 805.416 and 2163.056 m/z) produced by autodigestion of trypsin were used as an internal calibration. Proteins were identified by using the ProteinProspector server (http://prospector.ucsf.edu/). In vitro cytotoxicity assay HeLa cells were seeded onto 96-well plates at a density of 7000 cells/well in 0.1 ml of complete medium. After 16 h of incubation, cells were treated with buforin IIb (8 M) and incubated for another 12 h. In some experiments, cells were pretreated with PBA (10 mM), BAPTA/AM (10 M), SP600125 (20 M), SB203580 (20 M), Z-LEHD-FMK (25 M), Z-DEVD-FMK (50 M), or Z-VAD-FMK (20 M) for 1 h before buforin IIb was added. Cell viability was measured with the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT) assay using the CellTiter 96-cell proliferation assay kit (Promega). The percentage of cell viability was determined using the following equation: Viability (%) = (As − A0 )/(Ac − A0 ) × 100, where As is the absorbance of the sample, Ac is the absorbance of control, and A0 is the background absorbance. Each experiment was performed in triplicate, and repeated at least three times independently. Differences between the groups were analyzed using Student’s t test with GraphPad Prism version 5.00 (GraphPad Software, San Diego, CA, USA), and the differences were considered significant at P < 0.05. Western blot analysis HeLa cells were seeded onto 6-well plates at a density of 15 000 cells/well in 3 ml of complete medium. After 48 h of incubation, cells pretreated with or without PBA (10 mM), BAPTA/AM (10 M), SP600125 (20 M), or SB203580 (20 M) for 1 h were treated with buforin IIb (4 M) and incubated for indicated time. Cells were harvested and lysed in ice-cold PRO-PREP Protein Extraction Solution (Intron, Korea). In some experiments, mitochondrial versus cytosolic fractions of cells were prepared using the mitochondrial isolation kit for cultured cells (Pierce, Rockford, IL, USA). The protein concentration was quantified by using the BCA Protein Assay Kit (Pierce), and equal amounts of protein (30 g for total cell lysates, 20 g for cytosolic fraction, and 5 g for mitochondrial fraction) were subjected to 12% SDS–PAGE. Separated proteins were
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transferred to polyvinyldifluoride (PVDF) membranes (Millipore, Billerica, MA, USA), the membranes were blocked with 5% (w/v) BSA in TBS containing 0.1% Tween-20, and conventional immunoblot was performed using appropriate antibodies. Chemiluminescence was detected using an ECL kit (Promega).
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Protein expression in buforin IIb-treated HeLa cells
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The effect of buforin IIb on the protein expression in HeLa cells was explored by 2-D PAGE. As shown in Fig. 1, most of the proteins were found to be down-regulated in HeLa cells treated with buforin IIb for 24 h compared to control untreated cells, whereas several proteins were highly expressed. Of them, we selected 9 protein spots with significant upregulated expression, and identified them by MALDI-TOF/MS (Fig. 1 and Table 1). Of the identified 9 proteins, 7 proteins were molecular chaperones [170 kDa glucoseregulated protein precursor (GRP170), 94 kDa glucose-regulated protein precursor (GRP94), 78 kDa glucose-regulated protein precursor (GRP78), T-complex protein 1 subunit theta (TCPQ), protein disulfide isomerase A3 precursor (PDIA3), prohibitin (PHB), and calnexin precursor (CALX)]. The others were Ca2+ binding protein [calreticulin precursor (CALR)] and annexin V (ANXA5). These results point to the presence of ER stress in buforin IIb-treated HeLa cells.
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Relationship between ER stress and buforin IIb-induced apoptosis
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The results of 2-D PAGE led us to examine the regulation of two ER stress sensors, PERK and IRE1 as well as the ER chaperone GRP78, employing western blot analysis. The protein contents of p-PERK, p-IRE1, and GRP78 were significantly increased in a time-dependent manner in HeLa cells following buforin IIb treatment. In addition, the expression of CHOP, a hallmark on the ER stress-mediated apoptosis, was also increased (Fig. 2A). We next attempted to determine whether ER stress was related directly with buforin IIb-induced apoptosis. When HeLa cells were pretreated with the ER stress inhibitor PBA for 1 h, buforin IIb-induced apoptosis was significantly decreased by about 40% compared with inhibitor-nontreated control (Fig. 2B). Moreover, we found that the pretreatment of cells with Ca2+ chelator BAPTA/AM could inhibit
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Table 1 Identified protein spots in buforin IIb-treated HeLa cells by MALDI-TOF/MS. Spot no.
Protein name
Symbol
MW (kDa)
PI
Accession no.
1
170 kDa glucose-regulated protein precursor 94 kDa glucose-regulated protein precursor 78 kDa glucose-regulated protein precursor T-complex protein 1 subunit theta Protein disulfide isomerase A3 precursor Annexin V Prohibitin Calreticulin precursor Calnexin precursor
GRP170
111.34
5.16
Q9Y4L1
GRP94
92.47
4.76
P14625
GRP78
72.33
5.07
P11021
TCPQ
59.62
5.41
P50990
PDIA3
56.78
5.98
P30101
ANXA5 PHB CALR
35.94 29.80 48.14
4.93 5.57 4.29
P08758 P35232 P27797
CALX
67.57
4.46
P27824
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3
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the buforin IIb-induced apoptosis (Fig. 2B). These data indicated that buforin IIb does indeed induce ER stress-mediated apoptosis in HeLa cells. Involvement of MAPKs in buforin IIb-induced apoptosis The MAPK pathway plays essential roles in the regulation of cellular response, including cell survival, apoptosis, proliferation, and differentiation, and MAPKs have been implicated in the signal pathway of ER stress [16,26]. To gain further insight into the mechanisms of the ER stress-induced apoptosis of HeLa cells by buforin IIb, we assessed whether buforin IIb activates MAPKs in HeLa cells. We observed that the treatment of buforin IIb led to upregulation of the phosphorylated form of SAPK/JNK and p38 MAPK from 6 h (Fig. 3A). Their activation was effectively blocked by specific inhibitors (SAPK/JNK inhibitor SP600125 and p38 MAPK inhibitor SB203580), and these inhibitors also blocked buforin IIbinduced apoptosis (Fig. 3B). In addition, we found that both PBA and BAPTA/AM could inhibit the activation of MAPKs (Fig. 3A), indicating that the activation of MAPKs by buforin IIb might be subsequent to ER stress and the Ca2+ release from the ER. These data indicated
Fig. 1. 2-D PAGE images of HeLa cell proteins (A) untreated and (B) treated with buforin IIb. The first dimension ran using pH 3–10 IPG strips and the second dimension was a 12.5% SDS–PAGE. Gels were stained with silver. Spots selected for MALDI-TOF/MS analysis were denoted by numbers. The list of spots identified by MALDI-TOF was shown in Table 1.
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Fig. 2. Relationship between ER stress and buforin IIb-induced apoptosis. (A) HeLa cells were treated with buforin IIb (4 M) for indicated times, after which the protein contents of p-PERK, p-IRE1, GRP78, and CHOP were determined via western blot analysis. -actin was used as loading control. (B) HeLa cells pretreated with or without PBA (10 mM) or BAPTA/AM (10 M) for 1 h were treated with buforin IIb (8 M). After a 12-h incubation with buforin IIb, cell viability was measured by the MTT assay. Data represent the mean ± SEM of 3 independent experiments performed in triplicate. Asterisks indicate significant differences at P < 0.05, compared with no inhibitor by Student’s t test.
Fig. 3. Involvement of SAPK/JNK and p38 MAPK in buforin IIb-induced apoptosis. (A) HeLa cells pretreated with or without SP600125 (20 M), SB203580 (20 M), PBA (10 mM), or BAPTA/AM (10 M) for 1 h were treated with buforin IIb (4 M) for indicated times, after which phospho-active and total forms of SAPK/JNK and p38 MAPK were determined via western blot analysis. (B) HeLa cells pretreated with or without SP600125 (20 M) or SB203580 (20 M) for 1 h were treated with buforin IIb (8 M). After a 12-h incubation with buforin IIb, cell viability was measured by the MTT assay. Data represent the mean ± SEM of 3 independent experiments performed in triplicate. Asterisks indicate significant differences at P < 0.05, compared with no inhibitor by Student’s t test.
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that buforin IIb induced ER stress and Ca2+ release from the ER, which in turn activated SAPK/JNK and p38 MAPK, resulting in ER stress-mediated apoptosis in HeLa cells.
downstream of SAPK/JNK and p38 MAPK, and involved in mitochondrial membrane permeabilization and caspase-dependent apoptosis in buforin IIb-treated HeLa cells.
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Regulation of Bcl-2 family proteins and cytochrome c release
Discussion
Since Bcl-2 family proteins have been known to be involved in the signal crosstalk between ER stress and mitochondrial dysfunction [15,21,24], we next questioned if buforin IIb-induced apoptosis was mediated through Bcl-2 family proteins. Fig. 4A showed that down-regulation of anti-apoptotic Bcl-xL/Bcl-2 and marked translocation of pro-apoptotic Bax to mitochondria after buforin IIb treatment. A time-dependent release of cytochrome c into cytosol was also detected relative to gradual decrease in mitochondrial cytochrome c. Moreover, pretreatment with SP600125, SB203580, or PBA markedly abolished down-regulation of BclxL/Bcl-2, mitochondrial translocation of Bax, and cytochrome c release in response to buforin IIb treatment (Fig. 4A), reflecting that ER stress-elicited SAPK/JNK and p38 MAPK activation was involved in this event. We also found that Z-LEHD-FMK (caspase-9 inhibitor), Z-DEVD-FMK (caspase-3 inhibitor), and Z-VAD-FMK (pan-caspase inhibitor) blocked buforin IIb-induced apoptosis (Fig. 4B), which is in agreement with our previous study that showed that buforin IIb induced the activation of pro-caspase-9 and pro-caspase-3 [14]. Collectively, our data indicated that Bcl-xL, Bcl-2, and Bax were
ER is the primary site for protein synthesis, folding, and trafficking as well as for Ca2+ storage. Under a variety of stressful conditions, the accumulation of unfolded or misfolded proteins, or alterations in Ca2+ homeostasis in the ER can induce ER stress and ultimately lead to apoptosis [11]. To alleviate ER stress, cells activate a signaling pathway known as the unfolded protein response (UPR). The UPR promotes an increased capacity of protein folding and clearance to reduce the amount of misfolded proteins at ER lumen [6,36]. However, if the UPR’s mechanisms of adaptation and cell survival are insufficient to decrease the unfolded protein load, the UPR initiates cell death by apoptosis [7,8]. UPR-mediated cell death is executed by the canonical mitochondrial apoptosis pathway, where the Bcl-2 family plays a crucial role [29]. Transcriptional and post-transcriptional mechanisms are activated to regulated pro-apoptotic members of the Bcl-2 family that facilitate cytochrome c release from the mitochondria and Ca2+ release from the ER to engage downstream apoptotic signaling events [23]. Recently, an explosion of novel mechanisms and components that can suppress or potentiate apoptosis during ER stress has been
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Fig. 4. Involvement of Bcl-2 family proteins, cytochrome c, and caspase-9/3 in buforin IIb-treated HeLa cells. (A) HeLa cells pretreated with or without SP600125 (20 M), SB203580 (20 M), or PBA (10 mM) for 1 h were treated with buforin IIb (4 M) for indicated times, after which Bcl-xL and Bcl-2 were determined via western blot analysis. To examine the subcellular localization of Bax and cytochrome c, cytosolic (Cyto) and mitochondrial (Mito) fractions were prepared, and the protein distributions were analyzed by western blot analysis. VDAC was used as loading control for mitochondrial fraction. (B) HeLa cells pretreated with or without Z-LEHD-FMK (25 M), Z-DEVDFMK (50 M), or Z-VAD-FMK (20 M) for 1 h were treated with buforin IIb (8 M). After a 12-h incubation with buforin IIb, cell viability was measured by the MTT assay. Data represent the mean ± SEM of 3 independent experiments performed in triplicate. Asterisks indicate significant differences at P < 0.05, compared with no inhibitor by Student’s t test.
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identified. In the present study, we reported the mechanism of buforin IIb-induced apoptosis that involved ER stress-mediated mitochondrial membrane permeabilization in HeLa cells. Our proteomic analysis revealed that buforin IIb treatment led to upregulation of typical ER stress-related proteins including molecular chaperones and Ca2+ binding protein (Fig. 1 and Table 1). In particular, elevated expression of GRP78 and CHOP occurred at early time point (6 h) of buforin IIb treatment, signifying an initial ER stress-mediated apoptosis (Fig. 2A). It is well known that GRP78 and CHOP are typical ER stress-regulated proteins involved in ER stress-induced apoptosis [17]. Moreover, pretreatment with PBA (ER stress inhibitor) or BAPTA/AM (Ca2+ chelator) dramatically blocked the buforin IIb-induced apoptosis in HeLa cells (Fig. 2B). Therefore, it seems that buforin IIb-induced apoptosis involves ER stress. In addition to GRP78 and CHOP, MAPKs have been reported to play a critical role in ER stress-mediated apoptosis. In general, extracellular signal-regulated kinase (ERK) has been implicated in growth-factor-induced cell proliferation, whereas SAPK/JNK or p38 MAPK contributes to stress-induced apoptosis [25,35]. Based on these previous data, we hypothesized that ER stress-induced apoptosis after buforin IIb treatment involved the MAPK signaling pathways. Interestingly, SAPK/JNK and p38 MAPK were all activated by buforin IIb, and the inhibitor of each MAPK significantly blocked buforin IIb-induced apoptosis (Fig. 3), suggesting that MAPK functions upstream of buforin IIb-induced apoptosis. Although the direct molecular target of buforin IIb remains to be identified, our study indicates that buforin IIb treatment triggers a signal leading to ER stress, MAPKs, and apoptosis. Bcl-2 family proteins are a common link between ER stress and mitochondrial dysfunction [3]. The Bcl-2 family, a large class of both the anti-apoptotic proteins (Bcl-xL, Bcl-2) and pro-apoptotic proteins (Bax), tightly regulates the intrinsic apoptotic pathway by controlling the integrity of the outer mitochondrial membrane [2]. To investigate the involvement of Bcl-2 family proteins in buforin IIb-induced ER stress and apoptosis, we determined the expression levels of Bcl-2 family members in buforin IIb-treated HeLa cells. As shown in Fig. 4A, the level of Bcl-xL and Bcl-2 proteins was decreased in a time-dependent manner following buforin IIb treatment, while there was a significant enhancement in translocation of Bax from cytosol to mitochondria. One of the consequences following the changes of Bcl-2 family members is the release of mitochondrial pro-apoptotic protein, cytochrome c [1]. After
treatment of HeLa cells with buforin IIb, mitochondrial release of cytochrome c in the cytosol was increased in a time-dependent manner. In addition, SP600125, SB203580, and PBA attenuated down-regulation of Bcl-xL/Bcl-2, mitochondrial translocation of Bax, and cytochrome c release from mitochondria (Fig. 4A), and caspase inhibitors (Z-LEHD-FMK, Z-DEVD-FMK, and Z-VAD-FMK) dramatically blocked the buforin IIb-induced apoptosis in HeLa cells (Fig. 4B). These results indicates that the mitochondrial disruption by buforin IIb might be linked to ER stress, activation of MAPKs, and caspase-9/3-dependent apoptosis in HeLa cells. In conclusion, we demonstrated that buforin IIb induced ER stress and subsequently provoked ER stress-mediated mitochondrial membrane permeabilization via Ca2+ release from ER, SAPK/JNK and p38 MAPK pathway activation, down-regulation of Bcl-xL/Bcl-2, mitochondrial translocation of Bax, and cytochrome c release from mitochondria in HeLa cells. Buforin IIb has a great therapeutic potential to be developed into a novel therapeutic agent for the treatment of cancers. An understanding of the mechanism of buforin IIb-induced apoptotic cell death is a basic step in clinical therapeutic approaches. Acknowledgements This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) Q4 funded by the Ministry of Science, ICT & Future Planning (NRF- Q5 2014R1A2A1A11050944) and the Intelligent Synthetic Biology Center of Global Frontier Project funded by the Ministry of Science, ICT & Future Planning (NRF-2013M3A6A8073556). References [1] Brunelle JK, Letai A. Control of mitochondrial apoptosis by the Bcl-2 family. J Cell Sci 2009;122:437–41. [2] Chipuk JE, Moldoveanu T, Llambi F, Parsons MJ, Green DR. The BCL-2 family reunion. Mol Cell 2010;37:299–310. [3] Cory S, Adams JM. The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer 2002;2:647–56. [4] Dennison SR, Whittaker M, Harris F, Phoenix DA. Anticancer alpha-helical peptides and structure/function relationships underpinning their interactions with tumour cell membranes. Curr Protein Pept Sci 2006;7:487–99. [5] Gharahdaghi F, Weinberg CR, Meagher DA, Imai BS, Mische SM. Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity. Electrophoresis 1999;20:601–5.
Please cite this article in press as: Jang JH, et al. Buforin IIb induces endoplasmic reticulum stress-mediated apoptosis in HeLa cells. Peptides (2015), http://dx.doi.org/10.1016/j.peptides.2015.04.024
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