Labile iron, ROS, and cell death are prominently induced by haemin, but not by non-transferrin-bound iron.

BACKGROUND: In transfusion-related iron overload, haem-derived iron accumulation in monocytes/macrophages is the initial event. When iron loading exceeds the ferritin storage capacity, iron is released into the plasma. When iron loading exceeds transferrin binding capacity, labile, non-transferrin-bound iron (NTBI) appears and causes organ injury. Haemin-induced cell death has already been investigated; however, whether NTBI induces cell death in monocytes/macrophages remains unclear. MATERIAL AND METHODS: Human monocytic THP-1 cells were treated with haemin or NTBI, particularly ferric ammonium citrate (FAC) or ferrous ammonium sulfate (FAS). The intracellular labile iron pool (LIP) was measured using an iron-sensitive fluorescent probe. Ferritin expression was measured by western blotting. RESULTS: LIP was elevated after haemin treatment but not after FAC or FAS treatment. Reactive oxygen species (ROS) generation and cell death induction were remarkable after haemin treatment but not after FAC or FAS treatment. Ferritin expression was not different between the FAC and haemin treatments. The combination of an iron chelator and a ferroptosis inhibitor significantly augmented the suppression of haemin cytotoxicity (p = 0.011). DISCUSSION: The difference in LIP suggests the different iron traffic mechanisms for haem-derived iron and NTBI. The Combination of iron chelators and antioxidants is beneficial for iron overload therapy.

After haemin treatment intracellular non-haem iron increases prior to haem oxygenase-1 induction: A study in human monocytic cell line THP-1.

BACKGROUND: Iron overload is a major health concern for transfusion-dependent patients. Repeated transfusions result in the loading of large amounts of haem-derived iron on macrophages, in turn, inducing cell death. We previously demonstrated that haemin-induced cell death in human monocytic THP-1 cells is consistent with ferroptosis, an iron-dependent cell death regulation mechanism. However, direct measurement of iron after haemin treatment has not yet been conducted. In this study, we measured intracellular non-haem iron concentration and haem oxygenase levels after haemin treatment. MATERIAL AND METHODS: Human monocytic THP-1 cells were treated with haemin, and the cell lysate was prepared. Non-haem iron concentration of the cell lysate was measured using the Nitroso-PSAP method. Expression of haem oxygenase-1 (HO-1) and haem oxygenase-2 (HO-2) was quantified by western blotting. RESULTS: We measured intracellular non-haem iron and the expression of haem oxygenases post-haemin treatment. Concentration of non-haem iron post-haemin treatment increased dependently with time and dose. HO-1 expression was detected 4 h after haemin treatment, whereas HO-2 expression was constitutive. DISCUSSION: Increase in non-haem iron prior to induction of HO-1 expression suggests the involvement of HO-2 in haem-induced cytotoxicity. (184 words).

Haemin-induced cell death in human monocytic cells is consistent with ferroptosis.

BACKGROUND: Iron overload is a major issue for transfusion-dependent patients. Repeated transfusions result in the loading of large amounts of haem-derived iron on macrophages, and the haemin in turn induces cell death and the generation of reactive oxygen species (ROS) in both murine macrophages and human monocytic THP-1 cells. This haemin-induced cell death process has been shown to be iron-dependent. Thus, we hypothesized that haemin-induced THP-1 cell death is a result of ferroptosis, an iron-dependent mechanism of cell death regulation. MATERIAL AND METHODS: Human monocytic THP-1 cells were treated with haemin, and haemin-induced cell death and ROS generation were assessed using flow cytometry. RESULTS: Haemin-induced THP-1 cell death showed a necrosis pattern, and treatment with iron chelators suppressed both haemin-induced cell death and ROS generation. Treatment with ferrostatin-1, a ferroptosis inhibitor, suppressed haemin-induced cell death without affecting ROS generation, whereas erastin, a ferroptosis inducer, enhanced both haemin-induced cell death and ROS generation. DISCUSSION: Our findings support haemin-induced cell death as an example of ferroptosis. Therefore, ferroptosis inhibitors may be useful for the treatment or prevention of transfusion iron overload.