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HO-1 modulates ferroptosis via ROS-mediated mitochondrial dysfunction in cyclophosphamide -induced ovarian granulosa cells

ABSTRACT: Abnormal granulosa cells (GCs) death contributes to cyclophosphamide (CTX) -induced primary ovarian insufficiency (POI). To investigate the contribution of GCs in POI, gene profiles of GCs exposed to CTX were assessed using RNA-Seq and bioinformatics analysis. The results showed the differentially expressed genes (DEGs) are enriched in ferroptosis-related pathway, which is correlated with the upregulated Heme oxygenase 1 (HO-1) and downregulated glutathione peroxidase-4 (GPX4). Using CTX-induced cell culture (COV434 and KGN cells), the levels of iron, reactive oxygen species (ROS), lipid peroxide, mitochondrial superoxide, mitochondrial morphology and mitochondrial membrane potential (MMP) were detected by DCFDA, MitoSOX, C11-BODIPY, MitoTracker, Nonylacridine Orange (NAO), JC-1 and transmission electron microscopy respectively. The results showed that iron overload and disrupting ROS, including cytoROS, mtROS and lipROS homeostasis by HO-1 upregultion could induce ferroptosis via mitochondrial dysfunction in CTX-induced GCs. Moreover, HO-1 inhibition could suppress ferroptosis induced GPX4 depletion. This implies the role of ROS in CTX-induced ferroptosis and highlighted the effect of HO-1 modulators of improving CTX-induced ovarian damage, which may provide a theoretical basis for preventing or restoring GC and ovarian function in patients with POI.

ORGANISM(S): Homo sapiens

PROVIDER: GSE243720 | GEO | 2023/10/30

REPOSITORIES: GEO

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Project description:Ferroptosis is a form of regulated cell death characterized by iron-dependent overaccumulation of lipid peroxides. It can be prevented by cellular mechanisms such as GPX4-mediated elimination of the lipid peroxides at the cost of glutathione (GSH). Since originally being discovered as a property of RAS-mutant cancer cells, ferroptosis has been shown to be regulated by several important oncogenes and tumor suppressors. In particular, LIFR, the receptor of the pleiotropic cytokine LIF, has recently been shown to be required for ferroptosis, as loss of Lifr in mouse liver tumor confers the cells resistance to ferroptosis. In this study, however, we found that the LIFR-targeting drugs, EC330 and EC359, are potent inducers of ferroptosis, thus reflecting an interesting “gain-of-function” effect of EC330/EC359 on LIFR-associated ferroptosis. Treatment of various types of cells with EC330/EC359 causes a rapid nonapoptotic cell death with characteristic morphology of ferroptosis, which can be inhibited by iron chelator (DFO) and lipid ROS scavenger (Fer-1), but not pan-caspase inhibitor (z-VAD-fmk). Consistent with previous observations, the efficiency of EC330/EC359 appears to be correlated with the expression levels of LIF and LIFR. RNA-seq analysis showed that the EC330/EC359 treatment leads to (i) a gene expression profile highly resembling that of RSL3, the GPX4-targeting ferroptosis inducer, and (ii) a specific decrease of expression of a few gene encoding membrane-located proteins, especially those located on the mitochondria (e.g., TOMM6 and COX14), implying defects in cellular/mitochondrial membrane functions. Indeed, transmission electron microscopy imaging of the EC330/EC359-treated cells shows shrunken mitochondria and loss of mitochondrial cristae. EC330/EC359 also decreases the activity of GPX4 in the cells to the level comparable with RSL3; meanwhile, the intracellular level of GSH is also decreased, and thus GPX4 inactivation and GSH depletion may jointly disable the cells to eliminate the toxic lipid peroxides. Lastly, although the STAT3 and AKT signaling pathways downstream of LIFR are inhibited by EC330/EC359, inhibition of these pathways per se can only induce non-ferroptotic cell death, suggesting that the EC330/EC359-induced ferroptosis is independent of these downstream pathways; the LIFR-NFkB-LCN2 axis discovered recently in mouse liver tumor cells might not explain the EC330/EC359-induced ferroptosis either, because LCN2 is not detectable in the herein used cell lines. Taken together, these results reveal an unexpected role of the LIFR-targeting drugs EC330/EC359 as potent inducer of ferroptosis and, in combination with previous studies, suggest that there could be either unknown mechanism for the LIFR-associated ferroptosis or unknown target for EC330/EC359 to effect the ferroptosis induction.

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HO-1 modulates ferroptosis via ROS-mediated mitochondrial dysfunction in cyclophosphamide -induced ovarian granulosa cells

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