ERKs/p53 signal transduction pathway is involved in doxorubicin-induced apoptosis in H9c2 cells and cardiomyocytes

Abstract

The cardiotoxic effects of doxorubicin, a potent chemotherapeutic agent, have been linked to DNA damage, oxidative mitochondrial damage, and nuclear translocation of p53, but the exact molecular mechanisms causing p53 transactivation and doxorubicin-induced cardiomyopathy are not clear. The present study was carried out to determine whether extracellular signal-regulated kinases (ERKs), which are known to be activated by DNA damaging agents, are responsible for doxorubicin-induced p53 activation and oxidative mitochondrial damage in H9c2 cells. Cell death was measured by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling, annexin V-fluorescein isothiocyanate, activation of caspase-9 and -3, and cleavage of poly(ADP-ribose) polymerase (PARP). We found that doxorubicin produced cell death in H9c2 cells in a time-dependent manner, beginning at 6 h, and these changes are associated decreased expression of Bcl-2, increases in Bax and p53 upregulated modulator of apoptosis-alpha expression, and collapse of mitochondria membrane potential. The changes in cell death and Bcl-2 family proteins, however, were preceded by earlier activation and nuclear translocation of ERKs, followed by increased phosphorylation at Ser15 and nuclear translocation of the phosphorylated p53. The functional importance of ERK1/2 and p53 in doxorubicin-induced toxicity was further demonstrated by the specific ERK inhibitor U-0126 and p53 inhibitor pifithrin (PFT)-alpha, which abrogated the changes in Bcl-2 family proteins and cell death produced by doxorubicin. U-0126 blocked the phosphorylation and nuclear translocation of both ERK1/2 and p53, whereas PFT-alpha blocked only the changes in p53. Doxorubicin and ERK inhibitors produced similar changes in ERK1/2-p53, PARP, and caspase-3 in neonatal rat cultured cardiomyocytes. Thus we conclude that ERK1/2 are functionally linked to p53 and that the ERK1/2-p53 cascade is the upstream signaling pathway responsible for doxorubicin-induced cardiac cell apoptosis. ERKs and p53 may be considered as novel therapeutic targets for the treatment of doxorubicin-induced cardiotoxicity.

Fig. 1.

Doxorubicin induces cell apoptosis in a time-dependent manner, beginning at 6 h of incubation. A: H9c2 cells were incubated in medium with or without 1 μM doxorubicin for the indicated times. Representative (24-h incubation) immunofluorescence images are shown. Arrows identify apoptotic cells with condensed nuclei and positive terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) staining as visualized by green fluorescence. Cell nuclei were counterstained red with propidium iodide (PI). Magnification scale, 20 μm. TUNEL-positive cells were counted as described in materials and methods. B: whole cell lysates were processed for Western blot analysis to detect active caspase-3, caspase-9, and poly(ADP-ribose) polymerase (PARP). β-actin was used as an equal loading control. Representative Western blots are shown at left. The optical density is expressed in arbitrary units normalized against a control sample. Data in histograms represent means ± SE from 6 experiments. *P < 0.05, compared with control (0 h).

Fig. 2.

Doxorubicin induces early activation of ERK1/2 (A) and p53 (B) in H9c2 cells, beginning at 1 and 3 h of incubation, respectively. Cells were treated with 1 μM doxorubicin for the indicated times. Whole cell lysates were processed for Western blot analysis to detect changes in ERK1/2, phospho (p)-ERK1/2, p53, and p-p53. β-actin was used as an equal loading control. Representative Western blots are shown at left. The optical density is expressed in arbitrary units normalized against a control sample. Data in histograms represent means ± SE from 6 experiments. *P < 0.05, compared with control (0 h).

Fig. 3.

Phosphorylation and nuclear translocation of ERK1/2 and p53 following doxorubicin treatment in H9c2 cells. Cells were maintained in medium with or without 1 μM doxorubicin for 24 h. p-ERK1/2 (A) and p-p53 (B) proteins were measured by indirect immunofluorescence using fluorescein-conjugated secondary antibodies (p-ERK1/2, green; and p-p53, red). Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). Experiments were carried out at least 3 times, and the representative pictures are shown. The merge images demonstrate nuclear translocation of p-ERK1/2 and p-p53 after phosphorylation by doxorubicin. Cytosolic and nuclear proteins were processed for Western blot analysis to detect p-ERK1/2 and p-p53. Representative Western blots are shown at top right. The optical density is expressed in arbitrary units normalized against a control nuclear sample. Data in histograms represent means ± SE from 6 experiments. *P < 0.05, compared with cytosol of the same treatment group; †P < 0.05, compared with the same cell compartment of the untreated control.

Fig. 4.

Effects of doxorubicin on Bcl-2 family proteins in H9c2 cells. Cells were treated with 1 μM doxorubicin for the indicated times. Whole cell lysates were processed for Western blot analysis to detect changes in Bax, Bcl-2, and p53 upregulated modulator of apoptosis (PUMA)-α. β-actin was used as an equal loading control. Representative Western blots are shown. The optical density is expressed in arbitrary units normalized against a control sample. Data in histograms represent means ± SE from 6 experiments. *P < 0.05, compared with control (0 h).

Fig. 5.

Collapse of mitochondrial membrane potential (ΔΨ_m) after doxorubicin treatment in H9c2 cells. Cells plated on coverslides were maintained in medium with or without 1 μM doxorubicin for 24 h. The cells were stained with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocar bocyanine iodide (JC-1) and examined under fluorescence microscopy for red JC-1 aggregates or green JC-1 monomers, as described in materials and methods. Typical images are shown. Magnification scale, 20 μm. Ratio of normal ΔΨ_m and collapsed ΔΨm is presented at bottom. Data in histograms represent means ± SE from 10 experiments in each group. *P < 0.001, compared with control.

Fig. 6.

Effects of U-0126 and pifithrin (PFT)-α on doxorubicin (Dox)-induced apoptosis pathway in H9c2 cells. Cells were pretreated with or without 20 μM U-0126 or PFT-α for 1 h and then cultured in the presence or absence of 1 μM doxorubicin for 24 h. A: cell apoptosis was measured by annexin V-fluorescein isothiocyanate (FITC) and PI using FACSCalibur flow cytometry. Histograms show the percentages of early (annexin V-FITC positive and PI negative) and late (annexin V-FITC positive and PI positive) apoptotic cells in control and doxorubicin-treated cells with and without U-0126 and PFT-α treatment. B: mitochondrial damage was measured by changes in mitochondrial membrane potentials (ΔΨm) using fluorescent JC-1 under fluorescence microscopy. Histograms show the ratios of normal mitochondrial membrane potential (ΔΨ_m) and collapsed ΔΨm in control and doxorubicin-treated cells with and without U-0126 and PFT-α pretreatment. C: whole cell lysates were used to measure p-ERK1/2 (p-ERK), p-p53, Bax, Bcl-2, PUMA-α, caspase-9, cleaved caspase-3, and PARP by Western blot analysis. β-actin was used as an equal loading control. D: nuclear translocation of p-ERK1/2 and p-p53 was studied by indirect immunofluorescence using fluorescein-conjugated secondary antibodies (p-ERK1/2, green; and p-p53, red). Cell nuclei were counterstained with DAPI. Representative images are shown from 3 experiments. E and F: nuclear proteins were processed for Western blot analysis to detect p-ERK1/2 and p-p53. The optical density is expressed in arbitrary units normalized against a control sample. Data in histograms represent means ± SE from 6 experiments. *P < 0.05, compared with control; †P < 0.05, compared with doxorubicin.

Fig. 7.

Neither doxorubicin nor U-0126 produced significant changes on JNK and p38 MAPK proteins in H9c2 cells. Cells were pretreated with or without 20 μM U-0126 for 1 h and then in the presence or absence of 1 μM doxorubicin for 24 h. Whole cell lysates were processed for Western blot analysis of p-JNK, JNK, p-p38, and p38 MAPK. Representative Western blots are shown. Data in histograms represent means ± SE from 6 experiments.

Fig. 8.

Effects of MEK inhibitors U-0126 and PD-98059 on p-ERK1/2 and p-p53 expression, PARP activation, and cleaved caspase-3 in neonatal rat cardiomyocytes. Cells were pretreated with or without 20 μM U-0126 or 50 μM PD-98059 for 1 h and then in the presence or absence of 1 μM doxorubicin for 24 h. Whole cell lysates were processed for Western blot analysis of p-ERK1/2, ERK1/2, p-p53, PARP, and active caspase-3. β-actin was used as an equal loading control. Representative Western blots are shown. Data in histograms represent means ± SE from 6 experiments. *P < 0.05, compared with control of the same treatment group; †P < 0.05, compared with doxorubicin treatment alone without MEK inhibitors.