Induction of ferroptosis by new pyrrole tubulin assembly inhibitors

Ferroptosis is an iron-dependent cell death, which is different from apoptosis, necrosis, autophagy, and other forms of cell death. We synthesized new aroyl diheterocyclic pyrroles (ARDHEPs) as tubulin polymerization inhibitors. Among these compounds, derivative (4-(furan-2-yl)-1-(pyrimidin-2-yl)-1H-pyrrol-3-yl)(3,4,5-trimethoxyphenyl)methanone exhibited the hallmarks of ferroptosis. The latter compound strongly inhibited U-87 MG, OVCAR-3, and MCF-7 cancer cells, induced an increase of cleaved PARP, but was not toxic for normal human primary T lymphocytes at 0.1 microM concentration. Analysis of the levels of lactoperoxidase, malondialdehyde, lactic acid, total glutathione, and ATP suggested that the in vivo inhibition of cancer cell proliferation by the new compound went through stimulation of oxidative stress injury and Fe2+ accumulation. Quantitative polymerase chain reaction analysis of the mRNA expression in U-87 MG and SKOV-3 tumor tissues from new ARDHEP-treated mice showed the presence of Ptgs2/Nfe2l2/Sat1/Akr1c1/Gpx4 genes correlated with ferroptosis in both groups. Immunofluorescence staining revealed significantly lower expressions of proteins Ki67, CD31, and ferroptosis negative regulation proteins glutathione peroxidase 4 (GPX4) and FTH1. Finally, new ARDHEP was found to be metabolically stable when incubated with human liver microsomes