Melatonin down-regulates MDM2 gene expression and enhances p53 acetylation in MCF-7 cells Abstract: Compelling evidence demonstrated that melatonin increases p53 activity in cancer cells. p53 undergoes acetylation to be stabilized and activated for driving cells destined for apoptosis/growth inhibition. Over- expression of p300 induces p53 acetylation, leading to cell growth arrest by increasing p21 expression. In turn, p53 activation is mainly regulated in the nucleus by MDM2. MDM2 also acts as E3 ubiquitin ligase, promoting the proteasome-dependent p53 degradation. MDM2 entry into the nucleus is finely tuned by two different modulations: the ribosomal protein L11, acts by sequestering MDM2 in the cytosol, whereas the PI3K-AkT-dependent MDM2 phosphorylation is mandatory for MDM2 translocation across the nuclear membrane. In addition, MDM2-dependent targeting of p53 is regulated in a nonlinear fashion by MDM2/MDMX interplay. Melatonin induces both cell growth inhibition and apoptosis in MCF7 breast cancer cells. We previously reported that this effect is associated with reduced MDM2 levels and increased p53 activity. Herein, we demonstrated that melatonin drastically down- regulates MDM2 gene expression and inhibits MDM2 shuttling into the nucleus, given that melatonin increases L11 and inhibits Akt-PI3K-dependent MDM2 phosphorylation. Melatonin induces a 3-fold increase in both MDMX and p300 levels, decreasing simultaneously Sirt1, a specific inhibitor of p300 activity. Consequently, melatonin-treated cells display significantly higher values of both p53 and acetylated p53. Thus, a 15-fold increase in p21 levels was observed in melatonin-treated cancer cells. Our results provide evidence that melatonin enhances p53 acetylation by modulating the MDM2/MDMX/ p300 pathway, disclosing new insights for understanding its anticancer effect. Sara Proietti 1 , Alessandra Cucina 1 , Gabriella Dobrowolny 2,3 , Fabrizio D’Anselmi 1 , Simona Dinicola 1 , Maria Grazia Masiello 1,4 , Alessia Pasqualato 1 , Alessandro Palombo 1,5 , Veronica Morini 1 , Russel J. Reiter 6 and Mariano Bizzarri 7 1 Department of Surgery “P. Valdoni”, “Sapienza” University of Rome, Rome, Italy; 2 IIT, IIT viale Regina Elena 291, Rome, Italy; 3 Section of Histology and Medical Embryology, Department of Anatomy, Histology, Forensic Medicine and Orthopedics, Sapienza University of Rome, Rome, Italy; 4 Department of Clinical and Molecular Medicine, “Sapienza” University of Rome, Rome, Italy; 5 University of Rome “Tor Vergata”, Rome, Italy; 6 Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, TX, USA; 7 Department of Experimental Medicine, University “La Sapienza”, Rome, Italy Key words: AkT, MDM2, MDMX, melatonin, p300, p53-acetylation, Sirt1 Address reprint requests to Mariano Bizzarri, 14-16, Via Antonio Scarpa, Roma 00161, Italy. E-mail: mariano.bizzarri@uniroma1.it Received April 28, 2014; Accepted June 6, 2014. Introduction Melatonin exerts a variety of anticancer effects, both in vitro and in vivo [1, 2]. Moreover, preliminary clinical data have shown a significant usefulness of melatonin in onco- logical trials [3]. Melatonin’s anticancer effects are mediated by both receptor-dependent as well as receptor-independent mechanisms [4], through which the indoleamine interferes with several biochemical pathways, eventually leading to growth inhibition and increased apoptosis. Melatonin reduces the Akt activation, interferes with estrogen-depen- dent pathways, and up-regulates the expression of growth- inhibitory (p21) and pro-apoptotic genes (Bax, PARP) [5]. From the early pioneering studies, melatonin was shown to reactivate p53-dependent pathway and enhancing p53 levels, particularly in breast cancer cells [6]. The effect of melatonin and p53 is of outmost relevance, as the p53 protein plays a critical role in orchestrating the cell response to stressful events. In unstressed cells, p53 remains dormant at low levels, whereas in cells exposed to a various stress signals, p53 becomes activated and induces proliferation arrest and/or apoptosis [7]. Such processes are of importance especially during development and in tumor surveillance, given that eliminating damaged or trans- formed cells represents a critical issue in carcinogenesis. The switch between the activated and the dormant sta- tus of p53 is mainly provided by its negative regulator MDM2 (murine double minute 2) [8]. The ability of MDM2 to inactivate p53 relies on a direct physical inter- action between the two proteins in the nucleus, given that the N-terminal portion of MDM2 interacts with a specific p53-domain. As MDM2 is an E3 ligase ubiquitylating a defined set of lysine residues at the C-terminus of p53, p53 ubiquitylation by MDM2 is followed by subsequent rapid proteasome-based degradation of p53, both in the nucleus and in the cytoplasm after nuclear export [9]. Yet, MDM2-dependent degradation of p53 is not the sole mechanism through which MDM2 controls p53 activation. Indeed, MDM2 suppresses p53 transcriptional activity and mediates translocation of p53 to the cytoplasm [10], thus removing it from its site of actions, and, in associa- tion with HDAC1, deacetylases key lysine residues of p53, thereby making them available for ubiquitination [10]. P53 stabilization requires specific posttranslational modification events. Although phosphorylation of p53 at multiple serine residues occurs in response to different 120 J. Pineal Res. 2014; 57:120–129 Doi:10.1111/jpi.12150 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Journal of Pineal Research Molecular, Biological, Physiological and Clinical Aspects of Melatonin