SGLT1 和 SGLT2 之天然抑制劑篩選和構效分析

Abstract

Sodium-coupled glucose co-transporters SGLT1 and SGLT2 are active glucose transporters responsible for glucose absorption in the intestine and glucose reabsorption in the kidney, respectively. Several selective SGLT2 inhibitors have been approved for the treatment of type 2 diabetes. It is also reported that SGLT1 and SGLT2 are overexpressed in many cancers. Therefore, SGLT inhibitors might be potential anticancer agents. We have established a non-radioactive cell-based method for screening SGLT inhibitors using 1-NBDG, a fluorescent D-glucose analogue, and transiently transfected COS-7 cells overexpressing hSGLT1 or hSGLT2 to perform glucose uptake assays. In this study, we found that two kaempferol glycosides, isolated from Cinnamomum macrostemon leaves, possessed potent SGLTs inhibitory activities. Based on the kaempferol core moiety, we further evaluated the inhibitory activities of a series of related flavonoids against SGLT1 and SGLT2. The assay results led to some conclusions of structure activity relationship (SAR). First, glycosyl residues are crucial since the kaempferol aglycon shows no SGLTs inhibitory activities. In addition, the sugar inter-linkage and their substitution position to the aglycon not only affect the activities but also the inhibitory selectivity toward SGLTs. In order to search for selective SGLT2 inhibitors, 1-NBDG uptake assay in hSGLT2 transfected COS-7 cells was conducted to screen crude extracts from Lauraceae plants. ISC-1236l, ISC-1966s, and ISC-1827s showed the best inhibitory activities toward SGLT2 among the crude extracts, although they were much less than the leaf extract of Cinnamomum macrostemon. Besides, 1-NBDG uptake assay and RT-qPCR were conducted in OSCC cell lines, including SCC103, SCC104 and SCC131. The results indicated that the expression of SGLT1 and SGLT2 in SCC131 was the most abundant among three cell lines. Therefore, SCC131 was chosen for drug treatment examination and cell viability was detected by the MTT assay. Obvious difference of cell viability between cells treated with 0 and 100 M of phlorizin was observed when the assay was conducted in culture medium containing more FBS and/or less glucose. The combination of phlorizin with cisplatin, doxorubicin, paclitaxel, 5-fluorouracil, vinblastine, (R)(S)(S)-BF65, NVP-BEZ235, dihydroartemisinin, and phloretin were further examined in SCC131 cells. However, no synergistic effect was observed.