Antitumor Activity of a Novel Double-Targeted System for Folate Receptor-Mediated Delivery of Mitomycin C

[Image: see text] In this study, we designed, formulated, and investigated the potential antitumor activity of a folate receptor (FR)-mediated double-targeted drug delivery system. The system comprised of the FR ligand folic acid (FA), glycine-phenylalanine-leucine-glycine (Gly-Phe-Leu-Gly, GFLG), which can be specifically cleaved by cathepsin B, and the anticancer drug mitomycin C (MMC). The antitumor effect of FA-GFLG-MMC was compared to that of MMC. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay revealed that FA-GFLG-MMC has a significantly higher inhibitory effect on HeLa, SiHa, and PC9 cells (high FR expression) than that on 16HBE and A549 cells (low FR expression). Furthermore, FA-GFLG-MMC inhibited cancer cell proliferation in a dose-dependent manner. Free MMC was toxic to both cancer and normal cells. Apoptosis of the HeLa, SiHa, and PC9 cells was higher than that of the A549 cells; however, the apoptotic effect on 16HBE cells was minimal. Proapoptotic protein bcl-2-associated X-protein (BAX) and antiapoptotic protein BCL-2 play critical roles in cellular defense and apoptotic signal transduction. BAX/BCL-2 ratio is used to determine the intensity of an apoptotic signal and assess whether a cell will survive or undergo apoptosis. BAX and BCL-2 expression in cells treated with 5 μM FA-GFLG-MMC was studied by Western blotting. FA-GFLG-MMC increased the BAX/BCL-2 ratio in HeLa, SiHa, and PC9 cells. The results show that FA-GFLG-MMC can effectively inhibit tumor cell proliferation by inducing apoptosis. Therefore, the system developed can enhance the delivery of anticancer drugs to cancer cells and thereby reduce their toxic effects on normal cells.