u-0126 and pifithrin

Nutlin-3 is a small-molecule antagonist of murine double minute 2 (MDM2) that blocks its binding to p53, leading to an increase in p53 protein levels. The tumor suppressor p53 induces growth arrest or apoptosis in response to genotoxic stress. Along with its growth-suppressive effect, it has been reported that p53 stimulates the mitogen-activated protein kinase (MAPK) pathway via the upregulation of heparin- binding epidermal growth factor-like growth factor (HB-EGF), an epidermal growth factor receptor (EGFR) ligand, and discoidin domain receptor 1 (DDR1), a tyrosine kinase receptor, at the transcription level. In this study, we propose a novel mechanism involved in the p53-induced MAPK activation. Nutlin-3 induced the phosphorylation of EGFR, MAPK/ERK kinase (MEK)1/2 and extracellular signal-regulated kinase (ERK)1/2 in U2OS human osteosarcoma cells harboring wild-type p53. This phosphorylation was completely inhibited by p53 siRNA, but not by pifithrin (PFT)-α, an inhibitor of the trans-criptional activity of p53. While the nutlin-3-induced EGFR phosphorylation was prevented by the inactivation of ERK1/2, the nutlin-3-induced MEK1/2-ERK1/2 phosphorylation was still observed in the cells in which EGFR phosphorylation was inhibited using EGFR siRNA and AG1478, an inhibitor of EGFR tyrosine kinase. Of note, nutlin-3 caused the accumulation of mitochondrial reactive oxygen species (ROS) and this correlated with the mitochondrial translocation of p53. 2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO), a ROS scavenger, prevented the phosphorylation of ERK1/2. PFT-μ, which prevented the mitochondrial localization of p53, suppressed ERK1/2 phosphorylation, as well as ROS accumulation. Finally, we analyzed the effect of ERK1/2 activation on nutlin-3-induced apoptosis. The knockdown of MEK1/2 and ERK1/2 activity using U0126, a MEK inhibitor, or siRNAs, resulted in the enhancement of nutlin-3-induced apoptosis. In addition, TEMPO and PFT-μ also promoted nutlin-3-induced apoptosis. Taking the above findings into account, it can be concluded that nutlin-3 activates ERK1/2 prior to EGFR phosphorylation via ROS generation following the mitochondrial translocation of p53 and that nutlin-3-induced ERK1/2 activation may play a role in protecting U2OS cells from p53-dependent apoptosis, constituting a negative feedback loop for p53-induced apoptosis.

Topics: Antioxidants; Apoptosis; Benzothiazoles; Butadienes; Cell Line, Tumor; Cyclic N-Oxides; Enzyme Activation; Enzyme Inhibitors; ErbB Receptors; Humans; Imidazoles; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Mitochondria; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Osteosarcoma; Phosphorylation; Piperazines; Protein Transport; Quinazolines; Reactive Oxygen Species; RNA Interference; RNA, Small Interfering; Toluene; Tumor Suppressor Protein p53; Tyrphostins

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.

Topics: Active Transport, Cell Nucleus; Animals; Animals, Newborn; Antibiotics, Antineoplastic; Apoptosis; bcl-2-Associated X Protein; Benzothiazoles; Butadienes; Caspases; Cells, Cultured; Doxorubicin; Membrane Potential, Mitochondrial; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myocytes, Cardiac; Nitriles; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Signal Transduction; Time Factors; Toluene; Tumor Suppressor Protein p53