RESUMO
Equilibrative nucleoside transporters (ENTs) play a vital role in nucleotide synthesis, regulation of adenosine function and chemotherapy. Current inhibitors of ENTs are mostly ENT1-selective. Our previous study has demonstrated that 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) is a novel inhibitor of ENTs, which is more selective to ENT2 than to ENT1. The present study aimed to screen a series of FPMINT analogues and study their structure-activity relationship. Nucleoside transporter-deficient cells transfected with cloned human ENT1 and ENT2 were used as in vitro models. The results of the [3H]uridine uptake study showed that the replacement of the naphthalene moiety with the benzene moiety could abolish the inhibitory effects on ENT1 and ENT2. The addition of chloride to the meta position of this benzene moiety could restore only the inhibitory effect on ENT1 but had no effect on ENT2. However, the addition of the methyl group to the meta position or the ethyl or oxymethyl group to the para position of this benzene moiety could regain the inhibitory activity on both ENT1 and ENT2. The presence of a halogen substitute, regardless of the position, in the fluorophenyl moiety next to the piperazine ring was essential for the inhibitory effects on ENT1 and ENT2. Among the analogues tested, compound 3c was the most potent inhibitor. Compound 3c reduced V max of [3H]uridine uptake in ENT1 and ENT2 without affecting K m. The inhibitory effect of compound 3c could not be washed out. Compound 3c did not affect cell viability, protein expression and internalization of ENT1 and ENT2. Therefore, similar to FPMINT, compound 3c was an irreversible and non-competitive inhibitor. Molecular docking analysis also showed that the binding site of compound 3c in ENT1 may be different from that of other conventional inhibitors. It is expected that structural modification may further improve its potency and selectivity and lead to the development of useful pharmacological agents.
RESUMO
BACKGROUND: Therapeutic regimens of breast cancer treatment are increasingly inclined to adopt combination strategy based on the broad spectrum antitumor effect of doxorubicin (Dox). Currently, combination therapy comprises of conventional anti-cancer drugs and angiogenesis inhibitors have been corroborated as an effective approach in cancer treatment. PURPOSE: We explored the ability of a natural anti-angiogenic compound glycyrrhetinic acid (GA), derived from an edible-medicinal herb licorice, to enhance the breast cancer suppression effect of Dox. STUDY DESIGN: The drug ratio of GA and Dox with synergistic anticancer effect against MCF-7 cells was optimized by combination index (CI) value in vitro, followed by evaluation of the improved anticancer effects and reduced side-effects of this combination in vitro and in vivo. METHODS: Cell viability was measured by MTT assay. Analyses of mitochondrial membrane potential and cell apoptosis on MCF-7 cells were performed by JC-1 dye and Annexin V-FITC/PI assays. The cellular accumulation of Dox when combined with GA was evaluated. Levels of apoptosis-related proteins in MCF-7 cells were measured by Western blot analysis. Synergistic anti-angiogenic effects on HUVECs were evaluated. A breast cancer mouse model was established to investigate the anti-tumor effects in vivo. RESULTS: Based on the optimization by CI value, Dox and GA at 1:20 molar ratio was chosen as the optimal combination drug ratio that exhibited synergistic effect against MCF-7 breast cancer cells. In addition, the combination of GA and Dox exhibited significantly enhanced cytotoxicity, apoptosis, and loss of mitochondrial membrane potential via the upregulation of a mitochondrial-dependent apoptosis pathway against MCF-7 cells. Interestingly, the addition of GA increased the intracellular accumulation of Dox in MCF-7 cells. Moreover, VEGF-induced HUVECs proliferation, migration, and tube formation were strongly inhibited by Dox when used with GA via the significant down-regulation of VEGFR2-mediated pathway, indicating that the combination of Dox and GA could exhibit ideal synergistic anti-angiogenesis effect. Expectedly, the enhanced anti-tumor efficacy of Dox and reduced Dox-induced cardiotoxicity when used in combination with GA were evident in a mouse breast tumor model. CONCLUSIONS: These findings support that the combination of Dox with GA is a novel and promising therapeutic strategy for the treatment of breast cancer.
