Inhibition of AMP-activated protein kinase α (AMPKα) by doxorubicin accentuates genotoxic stress and cell death in mouse embryonic fibroblasts and cardiomyocytes: role of p53 and SIRT1

Abstract

Doxorubicin, an anthracycline antibiotic, is widely used in cancer treatment. Doxorubicin produces genotoxic stress and p53 activation in both carcinoma and non-carcinoma cells. Although its side effects in non-carcinoma cells, especially in heart tissue, are well known, the molecular targets of doxorubicin are poorly characterized. Here, we report that doxorubicin inhibits AMP-activated protein kinase (AMPK) resulting in SIRT1 dysfunction and p53 accumulation. Spontaneously immortalized mouse embryonic fibroblasts (MEFs) or H9C2 cardiomyocyte were exposed to doxorubicin at different doses and durations. Cell death and p53, SIRT1, and AMPK levels were examined by Western blot. In MEFs, doxorubicin inhibited AMPK activation, increased cell death, and induced robust p53 accumulation. Genetic deletion of AMPKα1 reduced NAD(+) levels and SIRT1 activity and significantly increased the levels of p53 and cell death. Pre-activation of AMPK by 5-aminoimidazole-4-carboxamide ribonucleoside or transfection with an adenovirus encoding a constitutively active AMPK (AMPK-CA) markedly reduced the effects of doxorubicin in MEFs from Ampkα1 knock-out mice. Conversely, pre-inhibition of Ampk further sensitized MEFs to doxorubicin-induced cell death. Genetic knockdown of p53 protected both wild-type and Ampkα1(-/-) MEFs from doxorubicin-induced cell death. p53 accumulation in Ampkα1(-/-) MEFs was reversed by SIRT1 activation by resveratrol. Taken together, these data suggest that AMPK inhibition by doxorubicin causes p53 accumulation and SIRT1 dysfunction in MEFs and further suggest that pharmacological activation of AMPK might alleviate the side effects of doxorubicin.

FIGURE 1.

AMPKα1 is the predominant isoform of AMPK expressed in mouse embryonic fibroblasts, and long term treatment of doxorubicin inhibits AMPK activation. A, AMPKα1, AMPKα2, and total AMPKα protein expression in MEFs from wild-type, Ampkα1^−/−, and Ampkα2^−/− mice were detected by immunoblotting. B, qualification of the expression profile of AMPKα1 and AMPKα2 in MEFs. C and D, doxorubicin treated at 0.5, 1.0 μm for 16 h inhibits AMPK function in wild-type MEFs (#, *, p < 0.05 versus control; n = 3). E, 2 mm 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) inhibited and 1 μm compound C sensitized wild-type MEFs from doxorubicin (1.0 μm for 16 h)-induced cellular apoptosis. DMSO, dimethyl sulfoxide; Dox, doxorubicin; Ctrl, control; p-ACC, phospho-acetyl-CoA carboxylase.

FIGURE 2.

Genetic deletion of AMPKα1 sensitizes MEFs to doxorubicin-induced apoptosis. A, Ampkα1-deficient MEFs show increased DNA damage and apoptosis compared with wild-type MEFs. B, genetic deletion of Ampkα1 increased doxorubicin-induced apoptosis in MEFs. C, morphology changes in wild-type and Ampkα1^−/− MEFs after doxorubicin treatment. PARP, poly (ADP-ribose) polymerase.

FIGURE 3.

AMPK deletion leads to defects in p53 function and increased p53 accumulation. A, low p53 levels in wild-type MEFs under normal conditions and doxorubicin-induced robust accumulation of p53 and p21 induction. B, Ampkα1^−/− MEFs showed stronger DNA damage signals but lower Ser¹⁵ phosphorylation levels of p53 compared with wild-type MEFs under doxorubicin treatment. C and D, AMPK-CA increased p53 phosphorylation levels and rescued Ampkα1^−/− MEFs from doxorubicin-induced apoptosis. Ctrl, control.

FIGURE 4.

Elongated half-life of p53 protein in Ampkα1-deficient MEFs. A, PCR results of mRNA expression levels of p53 in wild-type and Ampkα1^−/− MEFs. B, p53 levels in wild-type MEFs treated with cycloheximide (CHX, 50 μg/ml) for the indicated times. C, Ampkα1^−/− MEFs were treated with cycloheximide (50 μg/ml) for the indicated times, and p53 levels were detected with Western blot. D and E, quantitative analysis of the half-life of p53 in wild-type and Ampkα1^−/− MEFs. Ctrl, control.

FIGURE 5.

Increased acetylation and decreased degradation of p53 in Ampkα1^−/− MEFs due to defective SIRT1 activation. A, increased acetylation of p53 (Ace-p53) and total p53 levels in Ampkα1^−/− MEFs compared with wild-type MEFs. B, inhibition of AMPK by compound C (CmpC) increased the doxorubicin (Dox)-induced acetylation of p53 in wild-type MEFs. C, intracellular NAD⁺ content (**, p < 0.01; n = 6 in each group). D, NADH levels in MEFs. E, calculated NAD⁺/NADH ratio (**, p < 0.01; n = 6 in each group). F, p53 protein levels after resveratrol treatment in Ampkα1-deficient MEFs for 16 h at the indicated dose. G, p53 protein levels after resveratrol treatment in Ampkα1^−/− MEFs at 100 μm for indicated time points. H, MG132 blocks the effect of resveratrol in decreasing p53 levels in Ampkα1^−/− MEFs. Ctrl, control; p-ACC, phospho-acetyl-CoA carboxylase; AICAR, 5-aminoimidazole-4-carboxamide ribonucleoside.

FIGURE 6.

Cytoplasmic localization of p53 in Ampkα1-deficient MEFs and genetic silencing of p53 partially protect Ampkα1^−/− MEFs from doxorubicin-induced apoptosis. A, Ampkα1^−/− MEFs were labeled with mito-RFP (red) and then stained with anti-p53 (green) and DAPI (blue). The yellow shading in the merged image indicates mitochondria-localized p53 (phase contrast image; magnification, 40×). B, an obvious increase in cytoplasmic (Cyto) localization of p53 is seen in Ampkα1^−/− MEFs compared with wild-type controls. C and D, transient silencing of p53 by siRNA in wild-type (C) and Ampkα1-deficient (D) MEFs protects cells against doxorubicin (Dox)-induced apoptosis. E and F, stable knockdown of p53 by shRNA lentiviral particles protect MEFs from doxorubicin-induced apoptosis. G, representative phase contrast images of MEFs after doxorubicin (1 μm) treatment in wild-type and Ampkα1^−/− MEFs. Ctrl, control; Nucl, nucleus; PARP, poly (ADP-ribose) polymerase; LDH, Lactate dehydrogenase.

FIGURE 7.

Doxorubicin inhibits the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC), increases the levels of p53, and promotes apoptosis in cardiomyocytes. A, H9C2 cardiomyocytes were treated with doxorubicin at 1 μm for the indicated times. Western blots were performed for indicated target proteins. B, H9C2 cardiomyocytes were treated with doxorubicin at indicated doses for 16 h. Proteins were detected in Western blots by using the specific antibodies (* and #, p < 0.05 compared with control group, respectively). Ctrl, control; PARP, poly (ADP-ribose) polymerase.

FIGURE 8.

Proposed mechanisms of AMPK regulation of the genotoxic stress response in mouse embryonic fibroblast cells. Genotoxic stress inducers, such as doxorubicin, induce DNA damage and inhibit AMPK activation. AMPK inactivation by doxorubicin leads to p53 dysfunction and an altered NAD⁺/NADH ratio, resulting in decreased SIRT1 activation, which, in turn, leads to the accumulation of p53 and cellular apoptosis.