Abstract
Drug repurposing is an effective approach to identify active drugs with known toxicity profiles for rare cancers such as ACC. The objective of this study was to determine the anticancer activity of combination treatment for ACC from previously identified candidate agents using quantitative high-throughput screening (qHTS). In this study, we evaluated the anticancer activity of flavopiridol and carfilzomib in three ACC cell lines in vitro and in vivo. Human ACC samples were analyzed for drug-target analysis, and cancer-related pathway arrays were used to identify biomarkers of treatment response. Because flavopiridol is a potent cyclin-dependent kinase (CDK) inhibitor, we found significantly higher CDK1 and CDK2 mRNA expression in three independent cohorts human ACC (p<0.01) and CDK1 protein by immunohistochemistry (p<0.01) in human ACC samples. In vitro treatment with flavopiridol and carfilzomib in all three ACC cell lines resulted in a dose-dependent, anti-proliferative effect, and the combination had synergistic activity as well as in three-dimensional tumor spheroids. We observed increased G2M cell-cycle arrest and apoptosis with combination treatment compared to other groups in vitro. The combination treatment decreased XIAP protein expression in ACC cell lines. Mice with human ACC xenografts treated with flavopiridol and carfilzomib had significantly lower tumor burden, compared to other groups (p<0.05). We observed increased cleaved-caspase expression and decreased XIAP in tumor xenografts of mice treated with combined agents. Our preclinical data supports the evaluation of combination therapy with flavopiridol and carfilzomib in patients with advanced ACC.
Highlights
NCI-H295R (6×103), SW-13 (4×103), and BD140A (4×103) cells were plated into 96-well clear bottom, black plate (Costar®, Corning, NY)
At the beginning of the third week of culture, multicellular aggregates (MCAs) were treated with selected combinations and their corresponding single drug and vehicle controls using concentrations based on IC50 from the monolayer proliferation assay below maximum tolerable serum levels
MCAs were photographed with a Nikon D5100 (Nikon, Inc., Melville, NY) under a 12.5× magnification microscope (Olympus SZX9 microscope with DF PLAPO 1X-2 lens, Olympus America, Inc., Center Valley, PA)
Summary
NCI-H295R and SW-13 cells were grown and maintained in DMEM supplemented with 2.5% NuSerum (BD Biosciences, San Jose, CA), and 0.1% ITS premix (BD Biosciences, San Jose, CA). Both cell lines were purchased from American Type Culture CollectionTM (Manassas, VA). BD140A cells, kindly provided by Drs Kimberly Bussey and Michael Demeure (TGen, Phoenix, AZ), were cultured in RPMI supplemented with 1% L-glutamine (Gibco), 1% penicillin-streptomycin (Gibco), and 10% FBS (Invitrogen). The cell lines were authenticated using short tandem repeat profiling. Cells were routinely subcultured every three to four days and maintained in a 5% CO2 atmosphere at 37 C. NCIH295R cells used to generate human ACC xenograft were transfected with a linearized pGL4.51[luc2/CMV/Neo] vector (Promega, Madison, WI) encoding the luciferase reporter gene luc (Photinus pyralis) as previously described (17)
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