Chloroquine synergizes sunitinib cytotoxicity via modulating autophagic, apoptotic and angiogenic machineries Amal Kamal Abdel-Aziz a,b , Samia Shouman c , Ebtehal El-Demerdash a , Mohamed Elgendy b , Ashraf B. Abdel-Naim a,⇑ a Pharmacology and Toxicology Department, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt b Department of Experimental Oncology, European Institute of Oncology, Milan, Italy c Cancer Biology Department, National Cancer Institute, Cairo University, Cairo, Egypt article info Article history: Received 10 November 2013 Received in revised form 16 March 2014 Accepted 4 April 2014 Available online 18 April 2014 Keywords: Sunitinib Chloroquine Autophagy Apoptosis Angiogenesis Oxidative stress abstract Tyrosine kinases play a pivotal role in oncogenesis. Although tyrosine kinase inhibitors as sunitinib malate are used in cancer therapy, emerging studies report compromised cytotoxicity when used as monotherapy and thus combinations with other anti-cancer agents is recommended. Chloroquine is a clinically available anti-malarial agent which has been shown to exhibit anti-cancer activity. In the current study, we ques- tioned whether chloroquine can modulate sunitinib cytotoxicity. We found that chloroquine synergisti- cally augmented sunitinib cytotoxicity on human breast (MCF-7 and T-47D), cervical (Hela), colorectal (Caco-2 and HCT116), hepatocellular (HepG2), laryngeal (HEp-2) and prostate (PC3) cancer cell lines as indicated by combination and concentration reduction indices. These results were also consistent with that of Ehrlich ascites carcinoma (EAC) Swiss albino mice models as confirmed by tumor volume, weight, his- topathological examination and PCNA expression. Sunitinib induced autophagy via upregulating beclin-1 expression which was blocked by chloroquine as evidenced by accumulated SQTSM1/p62 level. Further- more, chloroquine augmented sunitinib-induced apoptosis by decreasing survivin level and increasing caspase 3 activity. Chloroquine also enhanced the antiangiogenic capacity of sunitinib as indicated by decreased CD34 expression and peritoneal/skin angiogenesis. Sunitinib when combined with chloroquine also increased reactive nitrogen species production via increasing inducible nitric oxide synthase expres- sion and nitric oxide level whilst reduced reactive oxygen species production by increasing GSH level, activities of glutathione peroxidase and catalase and reducing lipid peroxides compared to sunitinib-only treated group. Taken together, these findings suggest that chloroquine enhanced sunitinib cytotoxicity in a synergistic manner via inducing apoptosis while switching off autophagic and angiogenic machineries. Nevertheless, further studies are required to elucidate the efficacy and safety profile of such combination. Ó 2014 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Sunitinib malate, a small molecule tyrosine kinase inhibitor, inhibits vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR) and stem cell tyrosine kinase receptor (c-kit) [1]. Food and drug administration (FDA) approved the use of sunitinib malate in the treatment of metastatic renal-cell carcinoma, imatinib-resistant gastrointestinal stromal tumors and pancreatic neuroendocrine tumors [1]. Although there are ongoing clinical trials testing its efficacy in treating other types of cancer, several studies have reported com- promised sunitinib cytotoxicity when used as monotherapy [2–4]. Gotink et al. (2011) showed that cancer cells resist sunitinib cytotoxicity through lysosomal sequestration and thereby recommended co-treatment with clinically available drugs – as chloroquine – which interferes with lysosomal function [4]. Target- ing apoptosis, angiogenesis and oxidative stress has also been described as efficient tools in augmenting sunitinib anticancer activity [2,3]. http://dx.doi.org/10.1016/j.cbi.2014.04.007 0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved. Abbreviations: BSA, bovine serum albumin; CI, combination index; DRI, dose- reduction index; DTNB, 5,5-dithio-bis (2-nitrobenzoic acid); DMSO, dimethyl sulfoxide; EAC, Ehrlich ascites carcinoma; FBS, fetal bovine serum; GIST, gastroin- testinal stromal tumor; GSH, glutathione; IAP, inhibitor of apoptosis protein; IC 50 , median inhibitory concentration; iNOS, inducible nitric oxide synthase; NO x , nitric oxide; PCD, Programmed Cell Death; PCNA, proliferating cell nuclear antigen; PDGFR, platelet-derived growth factor receptor; ROS, reactive oxygen species; SRB, sulphorhodamine B; TCA, trichloroacetic acid; VEGFR, vascular endothelial growth factor receptor. ⇑ Corresponding author. Tel.: +20 119998505; fax: +20 224051107. E-mail addresses: abnaim@pharma.asu.edu.eg, abnaim@yahoo.com (A.B. Abdel-Naim). Chemico-Biological Interactions 217 (2014) 28–40 Contents lists available at ScienceDirect Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint