Research paper Autophagy regulates Selumetinib (AZD6244) induced-apoptosis in colorectal cancer cells Silvina Grasso a, b , Gustavo J.S. Pereira b , Caroline Palmeira-dos-Santos b , Andrana K. Calgarotto b , Isabel Martínez-Lacaci a, c , Jose Antonio Ferragut a , Soraya S. Smaili b, ** , Claudia Bincoletto b, * a Instituto de Biología Molecular y Celular, Universidad Miguel Hern andez, 03202 Elche, Alicante, Spain b Departamento de Farmacologia, Escola Paulista de Medicina, Universidade Federal de S~ ao Paulo (UNIFESP), S~ ao Paulo, SP, Brazil c Unidad AECC de Investigaci on Traslacional en C ancer, Hospital Universitario Virgen de la Arrixaca, Instituto Murciano de Investigaci on Biosanitaria, 30120 Murcia, Spain article info Article history: Received 9 May 2016 Received in revised form 23 June 2016 Accepted 24 June 2016 Available online 9 July 2016 Keywords: Apoptosis Autophagy Selumetinib (AZD6244) CRC cells (SW480 and HT29) abstract Objective: As Selumetinib is a MEK1/2 inhibitor that has gained interest as an anti-tumor agent, the present study was designed to investigate autophagy involvement on Selumetinib-induced apoptosis in colorectal cancer (CRC) cells. Methods: CRC cells death and cycle studies were assessed by AnnexinV-FITC and PI staining, respectively. Autophagy flux was analysed by Western Blot (LC3II and p62 protein levels) and retroviral infection of SW480 cells for siBecn1 RNA interference experiments. Confocal microscopy was used to determine mCherry-EGFPeLC3 distribution. Key findings: The Selumetinib effects were concentration-dependent in SW480 cell line. Whereas 1 mM exerted an arrest in the cell cycle (G 1 phase), higher concentrations (10 mM) induced cell death, which was accompanied by autophagy blockage in its last stages. Autophagy induction by Rapamycin (RAPA) increased cell survival, whereas pharmacology autophagy inhibition by Bafilomycin A1 (BAF), Chloro- quine (CQ) or 3-Methyladenine (3-MA) increased Selumetinib-induced CRC cells death. Conclusions: Altogether, these results suggest that autophagy plays a fundamental role in CRC cells response to Selumetinib. In addition, the combination of Selumetinib with autophagy inhibitors may be a useful therapeutic strategy to enhance its activity against colorectal tumours. © 2016 Elsevier Masson SAS. All rights reserved. 1. Introduction Colorectal cancer (CRC) is the fourth most common cancer in men and the third in women worldwide [1]. Although the survival of patients with advanced CRC has increased, due to improvement in treatment and early diagnosis, CRC remains a major health problem, with approximately 1 million new cases and 500,000 deaths every year [2]. Therefore, new molecules and signalling pathways involved in cell survival and/or proliferation are of crucial interest in the search for new chemotherapeutic agents [3]. In this context, the RAS/RAF/MEK/ERK pathway has attracted attention as a target to treat colon tumours due to high frequency of mutations in KRAS (40%) and BRAF (10%) [4e6]. Moreover, CRC cell lines and primary human tumours derived from colon frequently present constitutive ERK1/2 activation [7]. MEK1/2 is one of the critical molecules in the RAS/RAF/MEK/ERK pathway which harbours a unique inhibitor-binding pocket next to its ATP binding site that allows, for its highly specific, inhibition by small molecules. MEK1/2 is a target of Selumetinib (AZD6244, ARRY-142886), a potent, orally bioavailable, allosteric MEK1/2 in- hibitor, which is currently in clinical trials [8,9]. After binding to the allosteric inhibitor-binding pocket of MEK1/2, Selumetinib impedes RAS/RAF/MEK/ERK signalling, resulting in antiproliferative and pro-apoptotic effects in various cancer cell lines or tumour * Corresponding author. Departamento de Farmacologia, Escola Paulista de Medicina, Universidade Federal de S~ ao Paulo (UNIFESP), Rua tr^ es de maio 100, S~ ao Paulo, SP, Brazil. ** Corresponding author. Departamento de Farmacologia, Escola Paulista de Medicina, Universidade Federal de S~ ao Paulo (UNIFESP), Rua tr^ es de maio 100, S~ ao Paulo, SP, Brazil. E-mail addresses: soraya.smaili23@gmail.com (S.S. Smaili), claudia.bincoletto@ unifesp.br (C. Bincoletto). Contents lists available at ScienceDirect European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech http://dx.doi.org/10.1016/j.ejmech.2016.06.043 0223-5234/© 2016 Elsevier Masson SAS. All rights reserved. European Journal of Medicinal Chemistry 122 (2016) 611e618