Increased ROS in neuroblastoma and rhabdomyosarcoma cells under hypoxia. The generation of intracellular oxidative activities was evaluated by measuring intracellular peroxide-dependent oxidation of 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA, Sigma D6883) to form the fluorescent compound, 2′,7′-dichlorofluorescein (DCF) (50). Tumor cells were cultured in 96-well culture plates. After culturing overnight under normoxic or hypoxic (1%O2) conditions, the cells were incubated with 20μM of DCFDA at 37{degree sign}C for 30min. The culture medium was then removed, and the cells were washed twice with Hank's buffered salt solution. The fluorescence intensity of the cells in each well was determined using a Spectromax plate reader with excitation at 495nm and emission at 529nm. Thereafter cell viability was determined by AlamarBlue assay and DCF fluorescence was normalized to the viability. Results are expressed as a percentage of fluorescence intensity with respect to the cells under normoxic conditions. Error bars indicate SEM from three independent experiments. *P<0.01 (t test).
Funding
Threshold Pharmaceuticals, Merck-Serono, James Birrell Neuroblastoma Research Fund and CJ Memorial Fund/Sick Kids Foundation
ARTICLE ABSTRACT
Purpose: Tumor cells residing in tumor hypoxic zones are a major cause of drug resistance and tumor relapse. In this study, we investigated the efficacy of evofosfamide, a hypoxia-activated prodrug, and its combination with topotecan in neuroblastoma and rhabdomyosarcoma preclinical models.Experimental Design: Neuroblastoma and rhabdomyosarcoma cells were tested in vitro to assess the effect of evofosfamide on cell proliferation, both as a single agent and in combination with topotecan. In vivo antitumor activity was evaluated in different xenograft models. Animal survival was studied with the neuroblastoma metastatic tumor model.Results: All tested cell lines showed response to evofosfamide under normoxic conditions, but when exposed to hypoxia overnight, a 2- to 65-fold decrease of IC50 was observed. Adding topotecan to the evofosfamide treatment significantly increased cytotoxicity in vitro. In neuroblastoma xenograft models, single-agent evofosfamide treatment delayed tumor growth. Complete tumor regression was observed in the combined topotecan/evofosfamide treatment group after 2-week treatment. Combined treatment also improved survival in a neuroblastoma metastatic model, compared to single-agent treatments. In rhabdomyosarcoma xenograft models, combined treatment was more effective than single agents. We also showed that evofosfamide mostly targeted tumor cells within hypoxic regions while topotecan was more effective to tumor cells in normoxic regions. Combined treatment induced tumor cell apoptosis in both normoxic and hypoxic regions.Conclusions: Evofosfamide shows antitumor effects in neuroblastoma and rhabdomyosarcoma xenografts. Compared with single-agent, evofosfamide/topotecan, combined therapy improves tumor response, delays tumor relapse, and enhances animal survival in preclinical tumor models. Clin Cancer Res; 22(11); 2697–708. ©2015 AACR.
