RESEARCH ARTICLE Extracellular NAMPT/visfatin causes p53 deacetylation via NAD production and SIRT1 activation in breast cancer cells Kiarash Behrouzfar 1 | Mohammad Alaee 1 | Mitra Nourbakhsh 2 | Zafar Gholinejad 1 | Abolfazl Golestani 1 1 Department of Biochemistry, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran 2 Department of Biochemistry, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran Correspondence Mitra Nourbakhsh, Department of Biochemistry, Faculty of Medicine, Iran University of Medical Sciences, 1449614535, Tehran, Iran. Email: nourbakhsh.m@iums.ac.ir Funding information Tehran University of Medical Sciences and Health Services, Grant/Award Number: 92‐ 03‐30‐23794 Visfatin, which is secreted as an adipokine and cytokine, has been implicated in cancer development and progression. In this study, we investigated the NAD‐producing ability of visfatin and its relationship with SIRT1 (silent information regulator 2) and p53 to clarify the role of visfatin in breast cancer. MCF‐7 breast cancer cells were cultured and treated with visfatin. SIRT1 activity was assessed by measuring fluorescence intensity from fluoro‐substrate peptide. To investigate the effect of visfatin on p53 acetylation, SDS‐PAGE followed by western blotting was performed using specific antibodies against p53 and its acetylated form. Total NAD was measured both in cell lysate and the extracellular medium by colorimetric method. Visfatin increased both extracellular and intracellular NAD concentrations. It also induced proliferation of breast cancer cells, an effect that was abolished by inhibition of its enzymatic activity. Visfatin significantly increased SIRT1 activity, accompanied by induction of p53 deacetylation. In conclusion, the results show that extracellular visfatin produces NAD that causes upregulation of SIRT1 activity and p53 deacetylation. These findings explain the relationship between visfatin and breast cancer progression. KEYWORDS acetylation, breast cancer, p53, SIRT1, visfatin 1 | INTRODUCTION Breast cancer is the most prevalent cancer in women and the second cause of cancer mortality, comprising 25% of all cancers and 15% of cancer deaths. 1 Obesity is one of the main risk factors for breast cancer. 2 Adipose tissue secretes adipokines that have been shown to be associated with an increased risk of breast cancer. 3 Visfatin, also known as PBEF (pre‐B cell enhancing factor), is a novel adipokine that is secreted from adipocytes as well as macrophages in adipose and inflamed endothelial tissues. 4-6 Visfatin is often increased in breast tumours and is associated with more malignant cancer behaviour and poor survival. 7 Circulating visfatin is elevated in patients with breast cancer and is correlated with lymph node invasion and thus is suggested as a risk factor for post‐menopausal breast cancer. 8 Visfatin has various functions and is involved in inflammation, cell proliferation, apoptosis, cellular migration, and metastasis of cancer cells. 9 However, the cellular mechanism of visfatin and its contribution to breast cancer progression is not clearly understood, and there are controversial views on enzymatic activity of extracellular form of visfatin. 10,11 Intracellular form of visfatin, which is called nicotinamide phosphoribosyltransferase (NAMPT), is the rate limiting enzyme in nicotinamide adenine dinucleotide (NAD) biosynthesis. 12 Not only NAD plays a crucial role as a co‐factor in metabolic pathways but also it participates in deacetylation reactions performed by sirtuins. Therefore, cellular NAD is kept stable via equilibrium between NAD degradation and NAD synthesis, and increasing NAD levels in cancer cells can influence deacetylation reactions. 13 Sirtuins belong to class III histone deacetylases, which can deacetylate both chromatin and nonhistone proteins. SIRT1 is the principal member of this class of enzymes and participates in various cellular processes. SIRT1 is implicated in tumour initiation and progression as well as drug resistance by blocking senescence and apoptosis, and by promoting cell growth and angiogenesis. 14 Studies show that SIRT1 is also involved in oestrogen‐dependent proliferation of breast cancer cells. 15 p53, which is one of the most common tumour suppressors and causes apoptosis and cell cycle arrest, is one of the Received: 21 March 2017 Revised: 9 June 2017 Accepted: 5 July 2017 DOI: 10.1002/cbf.3279 Cell Biochem Funct. 2017;35:327–333. Copyright © 2017 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/cbf 327