Interplay of ferroptotic and apoptotic cell death and its modulation by BH3-mimetics

Abstract

Ferroptosis and apoptosis are typically recognized as distinct forms of cellular death. Ferroptosis occurs primarily through excessive lipid peroxidation when cellular antioxidant systems are overwhelmed and fail to effectively convert lipid-hydroperoxides into alcohols. In contrast, apoptosis is triggered by both external and internal stressors and is characterized by caspase activation. Contrary to the belief that apoptosis and ferroptosis are independent signaling pathways, this study found that cell death triggered by the ferroptosis inducer RSL3 in HT-1080 cells was only partially reduced by the ferroptosis inhibitor ferrostatin-1 (FER). Complete prevention of RSL3-induced cell death required the addition of the apoptosis inhibitor Q-VD-OPh (QVD). Treatment with RSL3 resulted in lipid peroxidation, which was inhibited by FER but not by QVD, as measured by BODIPY 581/591 C11 staining. In addition to this hallmark of ferroptosis, the release of cytochrome c from the mitochondria and the processing of caspase-3, markers of apoptosis, were also observed. This further emphasizes the simultaneous occurrence of apoptosis and ferroptosis following the inhibition of the antioxidant enzyme GPX4 by RSL3. Beside their central role in apoptosis integration, mitochondria also play a critical role in redox signaling and are a primary source of oxidative stress that could lead to ferroptotic cell death. In apoptosis, proteins of the BCL-2 family regulate the permeabilization of the outer mitochondrial membrane, initiating caspase activation. These proteins are classified into pro-survival and pro-apoptotic groups, with pro-apoptotic proteins further divided into those with multiple BH domains and BH3-only proteins. BH3 mimetics, which mimic BH3-only proteins, bind to the hydrophobic groove of antiapoptotic BCL-2 proteins to induce apoptosis. Since this study showed that RSL3 impacts mitochondria causing the release of cytochrome c, it was hypothesized that combining RSL3 with BH3 mimetics would enhance cell death in cancer cells such as HT-1080. In experiments, targeting anti-apoptotic proteins like BCL-2, MCL-1, or BCL-XL with BH3 mimetics significantly enhanced RSL3-induced cell death in HT-1080 cells. This intervention shifted the mode of cell death from mixed to purely apoptotic in some cases. Additionally, the MCL-1 inhibitor S63845 increased RSL3-induced cell death in Pfa1 mouse embryonic fibroblasts, but surprisingly protected U87 glioblastoma cells. Moreover, the BCL-XL inhibitor WEHI-539 protected Pfa1 and U87 cells from RSL3-induced cell death. To confirm that WEHI-539 can mitigate the effects of GPX4 inhibition, Pfa1 cells were stimulated with alternative GPX4 inhibitors, ML-162 or ML-210, followed by WEHI-539 treatment. Additionally, WEHI-539 was applied to prevent cell death caused by GPX4 depletion. In each case, WEHI-539 successfully prevented cell death, demonstrating its protective role against GPX4 inhibition and deletion in Pfa1 cells. Interestingly, while WEHI-539 protected cells from RSL3-induced death, it did not do so by targeting GPX4 activity or through its known pharmacological target BCL-XL. Further investigation into the mechanism of WEHI-539’s action revealed its role in combating lipid peroxidation, a hallmark of ferroptosis. To determine whether WEHI-539 could inhibit RSL3-induced lipid peroxidation and thus protect cells, assays including DPPH, FENIX, and lipidomic studies were conducted. Results from all assays demonstrated that WEHI-539 possesses antioxidant activity and effectively reduces lipid peroxidation induced by RSL3, thereby protecting cells. In summary, the findings revealed an interaction between ferroptosis and apoptosis, with BH3 mimetics modulating cell death outcomes in repsonse to RSL3-induced ferroptotic stress. In some cases, the addition of BH3 mimetics synergistically enhanced RSL3-mediated cell death while in others it rescued cells from the effects of GPX4 inhibition. This highlights the critical need to predict cell fate in response to the combined effects of GPX4 inhibition and BH3 mimetics, which will be essential for optimizing potential cancer therapies.