Combination treatment with hENT1 and miR-143 reverses gemcitabine resistance in triple-negative breast cancer

Abstract

BackgroundTriple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer and is susceptible to develop gemcitabine (GEM) resistance. Decreased expression of human equilibrative nucleoside transporter 1 (hENT1) accompanied by compensatory increase of glycolysis is strongly associated with GEM resistance in TNBC. In this study, we investigated the treatment feasibility of combined hENT1 upregulation and miR-143-mediated inhibition of glycolysis for reversing GEM resistance in TNBC.MethodsExperiments were performed in vitro and in vivo to compare the efficacy of GEM therapies. In this study, we established stable drug-resistant cell line, GEM-R cells, from parental cells (MDA-MB-231) through exposure to GEM following a stepwise incremental dosing strategy. Then GEM-R cells were transfected by lentiviral plasmids and GEM-R cells overexpressing hENT1 (GEM-R-hENT1) were established. The viability and apoptosis of wild-type (MDA-MB-231), GEM-R, and GEM-R-hENT1 cells treated with GEM or GEM + miR-143 were analyzed by CCK8 assay and flow cytometry. The RNA expression and protein expression were measured by RT-PCR and western blotting respectively. GEM uptake was determined by multiple reaction monitoring (MRM) analysis. Glycolysis was measured by glucose assay and 18F-FDG uptake. The antitumor effect was assessed in vivo in a tumor xenograft model by evaluating toxicity, tumor volume, and maximum standardized uptake value in 18F-FDG PET. Immunohistochemistry and fluorescence photography were taken in tumor samples. Pairwise comparisons were performed using Student’s t-test.ResultsOur results represented that overexpression of hENT1 reversed GEM resistance in GEM-R cells by showing lower IC50 and higher rate of apoptosis. MiR-143 suppressed glycolysis in GEM-R cells and enhanced the effect of reversing GEM resistance in GEM-R-hENT1 cells. The therapeutic efficacy was validated using a xenograft mouse model. Combination treatment decreased tumor growth rate and maximum standardized uptake value in 18F-FDG PET more effectively.ConclusionsCombined therapy of exogenous upregulation of hENT1 expression and miR-143 mimic administration was effective in reversing GEM resistance, providing a promising strategy for treating GEM-resistant TNBC.