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

BACKGROUND: Triple-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. METHODS: Experiments 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 (18)F-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 (18)F-FDG PET. Immunohistochemistry and fluorescence photography were taken in tumor samples. Pairwise comparisons were performed using Student’s t-test. RESULTS: Our results represented that overexpression of hENT1 reversed GEM resistance in GEM-R cells by showing lower IC(50) 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 (18)F-FDG PET more effectively. CONCLUSIONS: Combined 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.