1521-0103/362/2/219–229$25.00 https://doi.org/10.1124/jpet.117.240986 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 362:219–229, August 2017 Copyright ª 2017 by The American Society for Pharmacology and Experimental Therapeutics Carboxyamidotriazole Synergizes with Sorafenib to Combat Non–Small Cell Lung Cancer through Inhibition of NANOG and Aggravation of Apoptosis Chen Chen, Rui Ju, Jing Shi, Wei Chen, Fangrui Sun, Lei Zhu, Juan Li, Dechang Zhang, Caiying Ye, and Lei Guo Department of Pharmacology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing, China Received February 25, 2017; accepted May 16, 2017 ABSTRACT Lung cancer is currently the leading cause of cancer-related deaths worldwide. In this study, we investigated the combination of carboxyamidotriazole (CAI) and sorafenib in non–small cell lung cancer (NSCLC) in vitro and in vivo to test whether CAI enhances the antitumor effects of sorafenib and reduces its side effects. The combination index (CI) showed that coadministra- tion of CAI and sorafenib synergistically inhibited the proliferation of NSCLC cells (Lewis lung carcinoma, A549, and NCI-H1975 cells). Cell death as a result of the combination treatment was attributed to apoptosis, which was accompanied by activation of caspase-3 and poly(ADP-ribose) polymerase. In addition, com- bination therapy induced the accumulation of mitochondrial- associated reactive oxygen species, as well as depolarization of mitochondrial and reduced NANOG (homeobox protein NANOG) mRNA and protein expression. Basic fibroblast growth factor, a stimulator of NANOG, was applied to identify the possible mechanism. The addition of basic fibroblast growth factor followed by combined treatment may stimulate NANOG ex- pression and synchronously rescue the accumulation of re- active oxygen species. C57BL/6J mice bearing Lewis lung carcinoma were randomized to receive vehicle (polyethylene glycol 400), CAI (30 mg/kg), low-dose sorafenib (SFB-L; 10 mg/kg), high-dose sorafenib (SFB-H; 30 mg/kg), or a CAI and SFB-L combination. Tumor growth was significantly suppressed in the combination group, and the efficacy of combination treatment was equivalent to that of the SFB-H monotherapy group. Furthermore, the combination group had reduced side effects compared with the SFB-H group, as indicated by weight preservation in mice. Our study illustrates that CAI enhances the antitumor activity of sorafenib in NSCLC and provides a novel strategy for NSCLC treatment. Introduction Lung cancer is currently the leading cause of cancer-related death worldwide, and the 5-year survival of patients with lung cancer is approximately 4%–17%, with variations in disease stage and region (Hirsch et al., 2017). Non–small cell lung cancer (NSCLC) accounts for 80% of all lung cancers worldwide (Jemal et al., 2011). The most frequently used treatments for NSCLC are surgery, chemotherapy, and radiotherapy. Unfortunately, ap- proximately two-thirds of patients have advanced or inoperable disease at diagnosis. Many platinum-based regimens are first- line chemotherapies; single-agent docetaxel, pemetrexed, and erlotinib are considered second-line therapies. However, if patients fail to respond to conventional chemotherapies, treat- ment options are limited (Langer et al., 2013). The currently available therapies are shown to be inadequate, and novel strategies are urgently required. Sorafenib is a multikinase inhibitor that blocks the Raf kinase, platelet-derived growth factor receptors, and vascular endothe- lial growth factor receptors (EGFRs). The U.S. Food and Drug Administration approved sorafenib for the treatment of ad- vanced renal cell carcinoma and unresectable hepatocellular carcinoma (Wilhelm et al., 2006). Sorafenib has confirmed activity in preclinical models of NSCLC (Wilhelm et al., 2004; Gridelli et al., 2007) and has also been evaluated in several clinical trials (phases I–III) in patients with advanced NSCLC (Clark et al., 2005; Moore et al., 2005; Blumenschein et al., 2009; Wakelee et al., 2012; Paz-Ares et al., 2015). One limitation that hampers the use of sorafenib as a monotherapy is its side effects (e.g., hand-foot syndrome, rash, diarrhea, hypertension, and fatigue), which affect approximately 80% of patients receiving This research was supported by the Ministry of Science and Technology of China [Major Scientific and Technological Special Project 2014ZX09507003- 003], the National Science Foundation of China [Grant 81402943], the Chinese Academy of Medical Sciences Major Collaborative Innovation Project [Grant 2016-I2M-1-011], and the Peking Union Medical College Youth Fund [Grant 3332015168]. https://doi.org/10.1124/jpet.117.240986. ABBREVIATIONS: bFGF, basic fibroblast growth factor; CAI, carboxyamidotriazole; CI, combination index; CSC, cancer stem cell; DCFH-DA, 29,79-dichlorofluorescein diacetate; DMSO, dimethylsulfoxide; EGFR, endothelial growth factor receptor; FCM, flow cytometry; GSH, glutathione; LLC, Lewis lung carcinoma; MDA, malondialdehyde; NSCLC, non–small cell lung cancer; OXFOS, oxidative phosphorylation; PARP, poly(ADP- ribose) polymerase; PBS, phosphate-buffered saline; PEG400, polyethylene glycol 400; PI, propidium iodide; ROS, reactive oxygen species; SFB-H, high-dose sorafenib; SFB-L, low-dose sorafenib; SRB, sulforhodamine B; Z-VAD-FMK, Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone. 219 at ASPET Journals on October 25, 2017 jpet.aspetjournals.org Downloaded from