Doxorubicin (Dox) is a chemotherapeutic agent that causes significant cardiotoxicity. We showed previously that Dox activates p53 and induces apoptosis in mouse hearts. This study was designed to elucidate the molecular events that lead to p53 stabilization, to examine the pathways involved in Dox-induced apoptosis, and to evaluate the effectiveness of pifithrin-α (PFT-α), a p53 inhibitor, in blocking apoptosis of rat H9c2 myoblasts. H9c2 cells that were exposed to 5 μM Dox had elevated levels of p53 and phosphorylated p53 at Ser15. Dox also triggered a transient activation of p38, p42/p44ERK, and p46/p54JNK MAP kinases. Caspase activity assays and Western blot analysis showed that H9c2 cells treated with Dox for 16 h had marked increase in the levels of caspases-2, -3, -8, -9, -12, Fas, and cleaved poly(ADP ribose) polymerase (PARP). There was a concomitant increase in p53 binding activity, cytochrome c release, and apoptosis. These results suggest that Dox can trigger intrinsic, extrinsic, and endoplasmic reticulum-associated apoptotic pathways. Pretreatment of cells with PFT-α followed by Dox administration attenuated Dox-induced increases in p53 levels and p53 binding activity and partially blocked the activation of p46/p54JNK and p42/p44ERK. PFT-α also led to decreased levels of caspases-2, -3, -8, -9, -12, Fas, PARP, cytochrome c release, and apoptosis. Our results suggest that p53 stabilization is a focal point of Dox-induced apoptosis and that PFT-α interferes with multiple steps of Dox-induced apoptosis.
- phosphorylated p53
- mitogen-activated protein kinase
doxorubicin (Dox) is an anthracycline antibiotic that has been widely used for the treatment of acute leukemia, malignant lymphoma, and solid tumors (16, 34, 61). Unfortunately, its effectiveness is limited by its severe cardiotoxicity (21, 45). The prevailing hypothesis for the mechanism of Dox-induced cardiotoxicity includes free radical-mediated lipid peroxidation and alteration of membrane integrity (51, 60). Recent studies suggest that apoptosis plays an important role in Dox-induced cardiotoxicity (1, 8, 20, 54, 65).
Although there are many factors that could mediate apoptosis in Dox-induced cardiotoxicity, we showed in a previous study that Dox-induced apoptosis is at least partially mediated by p53 (32). p53 is a pivotal transcription factor that is involved in cell growth and differentiation (46). In response to stresses such as ionizing radiation, hypoxia, or DNA-damaging agents, p53 mediates cell cycle arrest, DNA repair, or apoptosis to eliminate damaged cells from the organism (11, 41, 58). Proapoptotic proteins Bax, Puma, Noxa, and Bid have been identified as p53 target genes (37, 40, 44, 55). An oligonucleotide array analysis revealed a number of p53 target genes that can be categorized as cell cycle arrest related (p21, GADD45, 14–3-3σ), apoptosis related (FAS, DR5), or oxidative stress related (superoxide dismutase, Pig 3) (71).
The stability of p53 is controlled by posttranslational modifications such as phosphorylation and acetylation. Multiple phosphorylation sites have been identified on the NH2-terminal end of p53. However, the most well-studied phosphorylation site is Ser 15, which is known to be essential for the transactivation of p53. Phosphorylation of p53 could be mediated by DNA protein kinase (26, 66), ATM (2, 5), ATR (62), p38 (17, 57), p42/p44ERK (49, 69), p46/p54JNK (4, 36), Chk1 (59), and Chk2 (15).
Pifithrin-α (PFT-α) is a reversible inhibitor of p53 that has been shown to mediate antiapoptotic effects in several in vitro and in vivo systems (13). It was first reported by Komarov et al. (22) to block p53 transactivation and protect mice from the side effects of cancer therapy. Subsequently, Culmsee et al. (7) showed that PFT-α protected neurons from excitotoxic and ischemic insults by blocking Bax expression and mitochondrial dysfunction. Most recently, our study showed that PFT-α could protect cardiac functions in mice by inhibiting apoptosis (32).
The present study was designed to examine the consequences of p53 inhibition by PFT-α on Dox-induced apoptosis in rat embryonic myocardial H9c2 cells. These cells were chosen because they have been used to study the cellular mechanisms and pathways that are involved in oxidative stress and energy deprivation. They have also been used to study the mechanism of Dox-induced cardiotoxicity (27). We hypothesized that PFT-α can attenuate Dox-induced apoptosis by inhibiting p53 phosphorylation, thus preventing the activation of p53 downstream events such as caspase activation.
MATERIALS AND METHODS
Cell culture and materials.
Rat embryonic ventricular myocardial H9c2 cells were obtained from American Type Culture Collection (Manassas, VA). Cells were cultured in DMEM (GIBCO, Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS), 2 mM l-glutamine, and 25 μg/ml gentamicin at 37°C in a humidified atmosphere of 5% CO2. Dox was purchased from Sigma Chemical (St. Louis, MO). Fetal bovine serum was obtained from Biosource International (Camarillo, CA). PFT-α, digitonin, SB-203580, PD-98059, and SP-600125 were products of Calbiochem (La Jolla, CA). Antibodies against phospho-site-specific p53 at Ser-15, active caspase-3, active caspase-9, poly(ADP ribose) polymerase (PARP), phospho-site-specific p42/p44ERK, total p38, total p42/p44ERK, and total p46/p54JNK were obtained from Cell Signaling Technology (Beverly, MA). Monoclonal caspase-8, caspase-2L, p53, Fas, phospho-site-specific p38, and phospho-site-specific p46/p54JNK antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rat monoclonal caspase-12 antibodies were from Dr. Junying Yuan of Harvard Medical School. Mouse monoclonal cytochrome c and actin antibodies were purchased from Molecular Probes (Eugene, OR) and Sigma Chemical, respectively. Secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).
Western blot analysis.
Near confluent H9c2 cells (4 × 106) in 100-mm dishes were incubated with DMEM containing 0.5% FBS for 16 h so that the cells were semiquiescent. Dox was added directly to the conditioned medium at a final concentration of 5 μM for a designated period. In experiments that involved PFT-α, cells were pretreated with 20 μM PFT-α during the serum starvation period. Cells were harvested in a buffer containing 20 mM Tris·HCl (pH 7.8); 137 mM NaCl; 15% glycerol; 1% Triton X-100; 2 μg/ml each of leupeptin, aprotinin, and pepstatin; 2 mM benzamidine; 20 mM NaF; 10 mM sodium pyrophosphate; 1 mM sodium vanadate; 25 mM β-glycerophosphate; and 1 mM phenylmethylsulfonyl fluoride. Protein concentration was determined by Bio-Rad protein assay (Bio-Rad, Hercules, CA). Aliquots of 30 μg of lysates were electrophoresed on 12% SDS-PAGE and transferred to nitrocellulose membranes. Western blot analysis was carried out with primary antibodies at 4°C overnight. Appropriate secondary antibodies conjugated to horseradish peroxidase were then added for 1.5 h. Antigen-antibody complex was detected by using enhanced chemiluminescence reagent (Amersham Biosciences, Piscataway, NJ). Band density was measured by AlphaEase Image Analysis Software (Alpha Innotech, San Leandro, CA).
Caspase activity assays.
H9c2 cells (4 × 106) in 100-mm dishes were treated with 5 μM Dox and/or 20 μM PFT-α for 16 h. Cells were scraped into ice-cold PBS, washed twice with PBS, and resuspended in a lysis buffer containing 50 mM HEPES, pH 7.4, 0.1% CHAPS, 0.1 mM EDTA, and 5 mM dithiothreitol (DTT). Protein concentration was determined by Bio-Rad protein assay. Caspase-2, -3, -8, and -9 activities were assayed using fluorogenic substrates (Ac-VDVAD-AMC for caspase-2, AcDEVD-AMC for caspase-3-like, AcIETD-AMC for caspase-8, and AcLEHD-AMC for caspase-9). Reaction mixture contained 70 μ1 of 1 × reaction buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 10 mM DTT, 1 mM EDTA, 10% glycerol), 20 μl of cell lysates (30 μg protein), and 10 μl of substrate (0.3 mM). Reaction was carried out at 37°C for 3 h. On cleavage of substrates by caspases, free AMC was read in a Tecan fluorescence plate reader (excitation 360 nm, emission 465 nm). Results were expressed as relative fluorescence units per microgram protein per hour.
Cytochrome c release.
Cells (6 × 105) grown in 35-mm dishes were treated with Dox and/or PFT-α for 16 h. Mitochondria were isolated by the digitonin permeabilization method as described by Leist et al. (28). This method avoided the disruption of mitochondrial membrane by mechanical grinding. Digitonin is a weak nonionic detergent. At low concentrations, it can permeabilize plasma membrane and release cytosolic components from cells. Briefly, H9c2 cells were permeabilized with a buffer containing 210 mM mannitol, 70 mM sucrose, 10 mM HEPES, pH 7.8, 5 mM succinate, 0.2 mM EGTA, 0.15% BSA, and 80 μg/ml digitonin, on ice for 5 min. Cells were centrifuged at 12,000 g for 10 min, and 5 μg of the supernatant was separated on 12% SDS-PAGE. Western blotting was carried out with cytochrome c antibodies. A duplicate blot was probed with actin antibodies to check for protein loading.
Electrophoretic mobility shift assays.
H9c2 cells (4 × 106) in DMEM containing 10% FBS were treated with or without 5 μM Dox and/or 20 μM PFT-α for 5 h. Nuclear extracts were prepared by a Nuclear Extraction kit (Active Motif, Carlsbad, CA), and protein concentration was determined by Bio-Rad dye-binding method. Synthetic consensus p53 binding sequence (Santa Cruz Biotechnology) was end-labeled with [γ-32P]ATP by T4 polynucleotide kinase (Promega, Madison, WI). Binding reactions were carried out in a final volume of 10 μl containing 5 μg of nuclear extract, 10 mM HEPES, pH 7.9, 4 mM Tris·HCl, 60 mM KCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, and 32P-labeled probe. Reactions were incubated at room temperature for 30 min. DNA-protein complex was separated on a 5% polyacrylamide gel in 0.5 × TBE (1 × TBE: 0.045 M Tris-borate, 1 mM EDTA). Gel was dried and exposed to Kodak BioMax films. For supershift assays, 1 μg of anti-p53 antibody (clone Ab421, Oncogene Research Products, San Diego, CA) was included in the reaction.
Quantitative analysis of apoptosis was performed with a Cell Death ELISA plus kit (Roche, Indianapolis, IN). Cells (5 × 104) were plated in 48-well dishes, changed to DMEM containing 0.5% FBS for 16 h, and treated with Dox and/or PFT-α for 7 h. Cells were lysed with 0.2 ml lysis buffer provided in the kit at room temperature for 20 min. Quantities of histone-associated DNA fragments (mononucleosomes and oligonucleosomes) were determined by absorbance at 405 nm with a Tecan microplate reader.
Statistical analyses were performed by one-way ANOVA followed by Tukey’s multiple comparison test to show differences between means. Data were represented as means ± SE. P < 0.05 was considered significant.
To explore the effects of Dox on p53 levels, serum-starved H9c2 cells were treated with 5 μM Dox for different time periods. This concentration was chosen because it reproduces the plasma peak level achieved in patients receiving standard infusions of Dox (12). In our study, Dox triggered a time-dependent increase of p53 (Fig. 1A).
Because the stability of p53 is regulated by posttranslational modifications such as phosphorylation, and because phosphorylation of p53 at Ser15 is known to be essential for the transactivation of p53, we studied the effect of Dox on the level of phosphorylated p53 at Ser15. Western blot analysis was carried out with a specific antibody against phospho-p53 at Ser15. Figure 1B shows that Dox treatment led to a time-dependent increase of phospho-p53 at Ser15. Densitometric analysis demonstrated an increase of 3.3-fold and 4.7-fold after 30 min and 4 h of Dox treatment, respectively.
To determine whether MAP kinases (MAPKs) are affected by Dox administration, Western blot analysis was carried out with antibodies against phospho-site-specific MAPKs, including p38, p42/p44ERK, and p46/p54JNK. In addition, Western blot analysis was carried out with antibodies against total p38, p42/p44ERK, and p46/p54JNK. We found that Dox induced a transient increase in phosphorylation of all three MAPKs. Phosphorylation of p38 reached a peak 10 min after Dox treatment, after which phosphorylation returned to baseline. Pretreatment of H9c2 cells with 20 μM of a specific p38 inhibitor, SB-203580, completely blocked the elevation in phospho-p38 level (Fig. 2A). This concentration of SB-203580 was chosen according to the experimental design of a previous study (6).
Phosphorylation of p42/p44ERK and p46/p54JNK reached a peak 30 min after Dox treatment, after which phosphorylation returned to baseline. The elevation in p42/p44ERK and p46/p54JNK levels was abolished by 20 μM PD-98059 and 10 μM SP-600125, which are specific inhibitors of p42/p44ERK and p46/p54JNK, respectively (Fig. 2, B and C). The concentrations of these inhibitors were chosen according to the experimental design of previous studies (3, 47). These results indicated that Dox can transiently activate all three MAPKs. It is noted that 20 μM PD-98059 by itself induces the phosphorylation of p42/p44ERK. In addition, 10 μM SP-600125 seems to have a slight effect on JNK protein content.
We next explored the effect of PFT-α, a chemical inhibitor of p53, on Dox-induced levels of p53 and phospho-p53. H9c2 cells were treated with Dox in the presence or absence of PFT-α for 8 h. The levels of p53 and phospho-p53 at Ser15 were determined by Western blot analysis. We found that Dox-induced upregulation of p53 and phospho-p53 (Ser15) could be attenuated by PFT-α (Fig. 3).
To identify which MAPK is affected by PFT-α, we treated H9c2 cells with Dox in the presence or absence of PFT-α for 10 min or 30 min. Densitometric analysis revealed that at 30 min of treatment, PFT-α partially blocked Dox-induced phosphorylation of p42/p44ERK and p46/p54JNK (Fig. 4). However, PFT-α had no effect on the phospho-p38 level (results not shown).
Electrophoretic mobility shift assay showed that p53 binding activity was induced after Dox treatment for 5 h (Fig. 5, lanes 1 and 2). PFT-α itself did not affect DNA binding activity of p53 (Fig. 5, lane 3). The addition of PFT-α attenuated Dox-induced p53 binding activity (Fig. 5, lane 4). In the presence of p53 antibody Ab421, a supershifted p53 complex was generated (Fig. 5, lane 5).
The effect of Dox on caspase activation was studied in H9c2 cells that were treated with Dox with or without PFT-α for 16 h. In these experiments, caspase-2, -3, -8, and -9 activities were measured by using specific fluorogenic substrates (Ac-VDVAD-AMC, Ac-DEVD-AMC, Ac-IETD-AMC, and Ac-LEHD-AMC). Results revealed that Dox activated caspases-2, -3, -8, and -9 by 3.77-, 2.1-, 2.2-, and 4.7-fold, respectively. The addition of PFT-α along with Dox partially suppressed these enzyme activities (Fig. 6, A–D). It is interesting to note that PFT-α by itself could significantly block the endogenous level of caspase-3 (Fig. 6B).
To investigate the expression of caspases and other apoptosis-related genes at the protein level, lysates were prepared from H9c2 cells treated with Dox and/or PFT-α for 8 h. This time point was chosen because there was some caspase activation in the control cells at 12 or 16 h as a result of low serum; this basal level of caspase activation would have interfered with the interpretation of any results derived from Dox-treated lysates prepared at 12 or 16 h. Western blot analysis was carried out with specific antibodies against caspases-2L, -3, -8, -9, -12, PARP, Fas, and actin. Caspases-2, -8, and -9 are initiator caspases (10, 38, 64) and caspase-3 is an executioner caspase (42), whereas caspase-12 is an endoplasmic reticulum (ER)-associated caspase (39). Activation of caspases-2L, -3, and -9 was detected with antibodies that recognize the 12-, 20-, and 17-kDa fragments of the respective active enzymes. Activation of caspases-2L, -3 and -9 was attenuated with the addition of PFT-α (Fig. 7). Procaspase-8 was barely detectable in untreated cells. After Dox treatment, there was an elevated level of 55-kDa procaspase and 44-kDa and 42-kDa activated fragments. The band density was decreased when PFT-α was added.
PARP is a substrate for caspase-3, and cleaved PARP has been shown to be an important marker for apoptosis (30). The pattern of PARP paralleled the pattern of active caspase-3. Caspase-12 was present as 60- and 54-kDa bands in untreated cells. On Dox treatment, the cleaved 54-kDa band was intensified. PFT-α could partially suppress the activation of caspase-12 (Fig. 7).
Cytochrome c release is a marker for mitochondria-related apoptosis (29). H9c2 cells were permeabilized with 80 μg/ml of digitonin, and cytochrome c in the supernatant was subjected to Western blot analysis. Figure 8 shows that the release of cytochrome c was elevated after Dox treatment and that PFT-α partially blocked this elevation.
Quantification of apoptosis was performed by cell death ELISA assay. As shown in Fig. 9, Dox increased oligonucleosome formation by 1.8-fold. Cell death was inhibited by the addition of PFT-α along with Dox.
In this study, we present evidence that Dox rapidly upregulates p53, phosphorylated p53, p42/p44ERK, p38, and p46/p54JNK. We also demonstrate that Dox increases the activity of caspases-2, -3, -8, -9, and -12, leading to apoptosis. Most importantly, we demonstrate for the first time that PFT-α attenuates the apoptotic process by blocking Dox-induced expression of p53, phosphorylated p53, p42/p44ERK, p46/p54JNK, and the caspases listed above.
In our study, we found that Dox has an immediate effect on cell signaling pathways. Within 5–10 min, Dox rapidly phosphorylated all three MAPKs (Fig. 2). These results are in line with a study on the effect of daunomycin on the MAPKs in cardiomyocytes by Zhu et al. (72). In addition, we also found that Dox induced an upregulation of p53 level.
Given that Dox triggered an increase in the proapoptotic p53, it is reasonable to assume that Dox has proapoptotic effects. In line with this hypothesis, our study showed that Dox induced a p53-dependent activation of caspases-2, -3, -8, -9, and -12 (Figs. 6 and 7) and triggered cytochrome c release (Fig. 8). This indicates that Dox induces both mitochondria-related and death receptor-related apoptotic pathways. Furthermore, in line with the results of Jang et al. (18), we also found that Dox activated caspase-12, indicating that Dox induces the ER-related apoptotic pathway. Caspase-12 resides in the ER and is activated on ER stress, including free radicals and disturbances of the intracellular calcium level (25, 39). Previous investigations have shown that Dox treatment leads to the generation of reactive oxygen species (27, 68) and an increase of calcium influx (24); this ER stress is hypothesized to activate caspase-12 and the ER-dependent apoptotic pathway. On activation, caspase-12 is translocated from the ER membrane to the cytosol where it may activate caspase-3 directly (52).
In our study, we also showed that Dox increased the level of caspase-2L (Figs. 6 and 7). Two isoforms of caspase-2 exist as a result of alternative splicing: caspase-2S and caspase-2L (9, 64). Recent studies indicate that these two proteins have opposite effects on apoptosis: caspase-2L induces apoptosis, whereas overexpression of caspase-2S is antiapoptotic. There is strong evidence that caspase-2L serves as a direct effector of the mitochondrial apoptotic pathway by releasing proapoptotic proteins, such as cytochrome c, or by cleaving Bid (14, 53).
Previous studies have demonstrated that p53 represses the expression of Bcl-2, PTEN (phosphatase and tensin homolog deleted on chromosome 10), and survivin and upregulates the gene expression of proapoptotic proteins such as Bax, Noxa, and Puma (50). Bax translocation induces cytochrome c release and allows the formation of apoptosomes, which contain caspase-9 and Apaf1. Caspase-9, in turn, activates caspase-3 and caspase-7, which execute the death program (35, 43).
A novel finding in our study is that PFT-α blocks the effects of Dox. First, Dox-induced elevation of p53 levels was partially blocked by PFT-α (Fig. 3). Previous studies have shown that PFT-α inhibits p53 accumulation in various cell systems subjected to ultraviolet radiation, cisplatin, or resveratrol treatment (19, 22, 31, 70). To our knowledge, this is the first study that has shown that PFT-α can block Dox-induced p53 levels in H9c2 cells.
Second, we found that PFT-α was able to suppress Dox-induced apoptosis. Specifically, PFT-α partially blocked the induction of Dox-induced Fas in H9c2 cells (Fig. 7), a result that is in agreement with a previous study in human umbilical endothelial cells (33). In addition, PFT-α suppressed the activation of caspases-2, -3, -8, -9, and -12 (Figs. 6 and 7).
A reasonable question to ask is whether PFT-α blocks Dox-induced apoptosis through a p53-dependent mechanism, a p53-independent mechanism, or both. PFT-α has been reported to suppress other p53-independent effects. For example, Komarova et al. (23) found that PFT-α can suppress heat shock and glucocorticoid receptor signaling. In addition, PFT-α affects the transcription of a number of genes involved in DNA repair, apoptosis, and cell growth (48). In our study, PFT-α partially blocked the activation of p46/p54JNK and p42/p44ERK (Fig. 4), the activation of which are both p53 independent. Furthermore, in our study, PFT-α did not completely suppress the caspase activation and the overall apoptosis induced by Dox in H9c2 cells (Figs. 6 and 9), supporting the notion that there exists a p53-independent apoptotic pathway as described by Tsang et al. (63). It is possible that a p53-independent pathway plays an important part of the antiapoptotic effect of PFT-α, although this hypothesis needs to be explored further.
In summary, our results suggest that Dox induces apoptosis by upregulating p53 and caspases-2, -3, -8, -9, and -12 in H9c2 cells. We also present evidence that PFT-α partially attenuates these proapoptotic processes, although it is unclear whether the mechanism of this attenuation occurs through a p53-dependent pathway, a p53-independent pathway, or both. In our previous study, we demonstrated that PFT-α attenuated Dox-induced cardiac apoptosis in mouse hearts and had no effect on the tumor-killing activity of Dox in human prostate PC3 cells (32). As such, it is possible that combination therapy of PFT-α and Dox may be employed to prevent Dox-induced cardiotoxicity in patients who rely on Dox chemotherapy regimens.
This work was supported by Department of Veterans Affairs Merit Review and a Grant-in-Aid from the American Heart Association-Southeast Affiliate.
We thank Dr. Junying Yuan for caspase-12 antibodies.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2006 by the American Physiological Society