Iron overload induces apoptosis of murine preosteoblast cells via ROS and inhibition of AKT pathway

Abstract

Objective

This study investigates the inhibitory effect of iron overload on MC3T3-E1 cells and its molecular mechanism.

Methods

MC3T3-E1 cells were grown under different concentrations of FAC (ferric ammonium citrate), the WST-8 assay was used to investigate the proliferation of cells following FAC with or without deferasirox. DCFH-DA fluorescence probe was applied to detect the cellular reactive oxygen species (ROS) level. The apoptotic cells were analyzed with Annexin V-FITC/PI and the Hoechst 33258 nuclear staining assay. The JC-1 staining assay was applied to observe the change in the mitochondrial transmembrane potential. The expression levels of caspase-3, PARP, Bcl-2 family proteins and AKT kinase were detected with the western blot assay.

Results

Iron overload had a cytotoxic effect on MC3T3-E1 cells in a dosage-dependent way and resulted in increasing level of intracellular ROS. Iron overload induced apoptosis in MC3T3-E1 cells via a caspase-dependent mechanism that is accompanied by mitochondria dysfunction and decreased expression of anti-apoptotic proteins. The expression levels of cleaved-caspase-3 and cleaved-PARP were upregulated, while the expression levels of caspase-9, caspase-7, caspase-3 and PARP were downregulated. Phosphorylation of AKT kinase decreased.

Conclusion

Iron overload can generate ROS in cells, inhibit AKT kinase and its downstream proteins activity and subsequently initiate apoptotic events.

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