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.
Highlights
Doxorubicin produces genotoxic stress and p53 activation in both carcinoma and non-carcinoma cells
That AMPK-CA transfection lowered p53 protein levels and protected both wild-type and Ampk␣1Ϫ/Ϫ mouse embryonic fibroblasts (MEFs) from doxorubicin-induced apoptosis (Fig. 3, C and D). These results indicate that AMPK inactivation by doxorubicin might play a causative role in doxorubicin-induced cellular apoptosis in normal, non-carcinoma cells. p53 Half-life Is Increased in Ampk␣1Ϫ/Ϫ MEFs—Because no significant difference in p53 gene expression levels was found between Ampk␣1Ϫ/Ϫ MEFs and wild-type MEFs (Fig. 4A), we hypothesized that p53 should be stabilized in Ampk␣1Ϫ/Ϫ MEFs
Our results suggest that AMPK activation, via SIRT1 activation, protects non-carcinoma cells from doxorubicin-induced cellular apoptosis by modulation of p53 function and stability
Summary
Doxorubicin produces genotoxic stress and p53 activation in both carcinoma and non-carcinoma cells. Results: AMP-activated protein kinase (AMPK) inhibition by doxorubicin causes p53 accumulation and SIRT1 dysfunction. We report that doxorubicin inhibits AMP-activated protein kinase (AMPK) resulting in SIRT1 dysfunction and p53 accumulation. In MEFs, doxorubicin inhibited AMPK activation, increased cell death, and induced robust p53 accumulation. Beside its role in maintaining cellular or whole body energy homeostasis, AMPK is reported to play important roles in the genotoxic stress response and in the regulation of apoptosis [23]. We find that AMPK activation protects non-carcinoma cells from genotoxic stressinduced apoptosis by regulating the function and stability of p53 through direct phosphorylation and SIRT1-dependent acetylation
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