Cisplatin Induces Ferroptosis In Cancer Cells And Its Mechanism

Part1Cisplatin Induces Ferroptosis in Cancer Cell and Its MechanismObjectives To study the ferroptosis in cancer cells induced by cisplatin and its mechanism.Methods Human cancer cell lines including A549, H460, Calu-1, HCT116and HT1080were respectively treated with cisplatin, adriamycin,5-Fu, paclitaxel, sulfasalazine respectively, and MTT assay was performed to observe the protection of the specific inhibitor of ferroptosis ferrostatin-1to the tumor cells. Then we observed whether the cytotoxicity of cisplatin could be reversed by apoptosis inhibitor(z-vad-fmk), necrosis inhibitor(necrostatin-1), autophagy inhibitor (chloroquine) or iron chelator (deferoxamine) respectively by MTT assay. Transmission electron microscopy was performed to observe the specific morphological changes of cells after the utilization of cisplatin. Further, flow cytometry was performed to detect the ROS level of cells treated with cisplatin. RNA interference technology was used to inhibit the expression of ferroptosis related gene (IREB2) in HCT116to observe the variety of its susceptivity to cisplatin. We also detected the level of glutathione and glutathione perxidases in each group by biochemical methods.Results The cytotoxic effect of cisplatin on A549and HCT116can be partially reversed by the ferroptosis specific inhibitor fer-1(46.79±2.84%VS.34.70±2.06%in HCT116,65.23±3.37%VS.57.99±3.07%in A549, P<0.05). Deferoxamine and z-vad-fmk can protect HCT116cells from cytotoxicity of cisplatin, and no similar effect was found in necrostatin-1and chloroquine. We observed obvious mitochondrial shrinkage in HCT116being treated with cisplatin by transmission electron microscopy. When treated with cisplatin, the intracellular ROS levels will increase and β-ME could inhibit the increasing of ROS level caused by cisplatin. Downregulation IREB2by RNAi could inhibit cisplatin cytotoxicity greatly. The intracellular GSH level would be depressed when treated with cisplatin, so was the GPX activity.Conclusion Cisplatin will induce specific ultrastructural changes and action of pathway of ferroptosis in cancer cells, which can be inhibited by the inhibitor of ferroptosis or the interference of the pathway could inhibit the cytotoxicity of cisplatin, confirmed the occurrence of ferroptosis in cancer cells induced by cisplatin. Synergistic Effect of Antitumor Activity by Combining Cisplatin with Erastin or Inhibition of GPX4in Cancer CellsObjectives Being distinct from apoptosis, necrosis, autophagy and other cell death forms, ferroptosis may provide a new idea for cancer therapy. In this case, this study was designed to observe the synergistic effect of antitumor activity and investigate its preliminary mechanism by combining cisplatin with erastin or inhibition of GPX4in cancer cells.Methods Treated with the specific inducer of ferroptosis(Erastin) combined with cisplatin at different concentration in HCT116and A549cells to observe the fluctuation of cisplatin cytotoxicity. Flow cytometry was used to detect the intracellular level of ROS in different group and biochemical methods were used to detect the changes of GPXs activity. Further, we detected the difference of cell viability, intracellular GSH levels, GPXs activity and intracellular ROS levels after downregulation of GPX4gene by RNA interference combined with cisplatin.Result Erastin can visibly increase the cytotoxicity of cisplatin at different concentration in HCT116and A549cells, and the difference was statistically significant. Compared with z-vad-fmk, β-ME and ferrostatin-1could be more effective to reverse the cytotoxicity. Erastin combined with cisplatin could elevate the level of intracellular ROS, and decrease the level of GSH and the activity of GPXs. Inhibiting GPX4by RNAi could result in higher level of intracellular ROS, but the level of GSH and the activity of GPXs were not statistical different compared with the siNeg group, which implied great increase in cisplatin cytotoxicity.Conclusion Erastin and targeted inhibition of GPX4can significantly increase cisplatin cytotoxicity of cancer cells by the reduction of ferroptosis. Ferroptosis is a cell death form which is different from apoptosis, which may provide a new approach to reverse drug resistance and increase cisplatin cytotoxicity in tumor therapy.