Synthesis, characterization and induction of ferroptosis of iridium(III) complexes against B16 melanoma cells

The synthesis of the ligand 2-(2-methyl-4-hydroxyl)phenyl-1H-imidazo[4,5-f][1,10]phenanthroline (MHIP) and its corresponding new iridium(III) complexes [Ir(ppy)₂(MHIP)]PF₆ (ppy = 2-phenylpyridine, 9a), [Ir(bzq)₂(MHIP)]PF₆ (bzq = benzo[h]quinolone, 9b) and [Ir(piq)₂(MHIP)]PF₆ (piq = 1-phenylisoquinoline, 9c) was reported. The antiproliferative activity of compounds 9a-9c on HepG2, B16, and A549 cancer cells as well as on normal NIH 3T3 cells was tested using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. It was found that the three complexes showed moderate cytotoxicity in A549 and B16 cells. However, after further irradiation, the cytotoxicity was greatly enhanced; especially, 9a, 9b and 9c displayed significant cytotoxicity toward B16 cells with a low IC₅₀ value of 3.1 ± 0.3 μM for 9a, 4.9 ± 0.8 μM for 9b, and 0.4 ± 0.1 μM for 9c. The effects of 9a-9c on the invasive ability of B16 cells were explored via colony formation and scratch experiments. Results demonstrated that the complexes could efficiently block cell proliferation and migration. The co-localization assay found that 9a-9c accumulated in the mitochondria and led to the apoptosis of B16 cells by decreasing mitochondrial membrane potential, altering the structure of microtubule proteins, damaging the structure of cellular DNA, and changing the expression of related proteins. The decrease in glutathione (GSH) concentration, the increase in malondialdehyde (MDA), the downregulation of GPX4, and C11-BODIPY staining results confirmed that 9a, 9b and 9c led to ferroptosis. In addition, we explored the relevant signaling pathways through an RNA sequencing assay and speculated on the possible anticancer mechanisms. Together, the results of this study indicate that the synthesized new iridium(III) complexes 9a-9c can induce cell death via ROS-mediated mitochondrial dysfunction, apoptosis and ferroptosis.