Lee Young-Sun, Vafaeinik Farzaneh, Mouakkad Lila, Kim Dong-Hyun, Song Xinxin, Zhang Lin, Lee Yong J
Department of Surgery, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California, USA.
J Cell Biochem. 2025 Jan;126(1):e30681. doi: 10.1002/jcb.30681.
We previously reported that ferroptosis interplays with apoptosis through the integration of two independent pathways: the endoplasmic reticulum (ER) stress signaling pathway and the mitochondria-dependent apoptotic signaling pathway. In this study, we investigated a potential gatekeeper molecule, Mcl-1, between the two signal transduction pathways. Morphology studies and cell death analyses confirmed that a combination treatment of ferroptotic agent erastin (ERA) and apoptotic agent TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) synergistically enhances TRAIL-induced apoptosis in human pancreatic adenocarcinoma BxPC3 and human colorectal carcinoma HCT116 cells. We further observed that ERA upregulated the proapoptotic proteins PUMA (p53 upregulated modulator of apoptosis) and NOXA, as well as the anti-apoptotic protein Mcl-1 (myeloid cell leukemia sequence 1). These results suggest that ERA upregulates these molecules which results in maintenance of the balance between them. Interestingly, this balance was offset when BxPC3 cells and HCT116 cells were treated with ERA in combination with TRAIL. Our studies suggest that the imbalance between PUMA and NOXA and Mcl-1 during the combined treatment is responsible for ERA-enhanced TRAIL-induced apoptosis. This hypothesis was tested by employing a HCT116 Mcl-1 knock-in of phosphorylation site mutant (S121A/E125A/S159A/T163A) and investigated the synergistic interaction between the ERA and TRAIL. Along with morphology and cell death studies, immunoblotting analyses revealed that HCT116 Mcl-1 knock-in mutant cells effectively inhibited reduction of Mcl-1 and apoptosis promoted by the combination treatment. Moreover, ERA enhanced Mcl-1 inhibitor-induced apoptosis. Collectively, our studies suggest that Mcl-1 is a gatekeeper molecule between the ER stress pathway and the mitochondria-dependent apoptotic pathway.
我们之前报道过，铁死亡通过整合两条独立的信号通路与凋亡相互作用：内质网（ER）应激信号通路和线粒体依赖性凋亡信号通路。在本研究中，我们探究了这两条信号转导通路之间一种潜在的守门分子——髓样细胞白血病序列1（Mcl-1）。形态学研究和细胞死亡分析证实，铁死亡诱导剂埃拉斯汀（ERA）和凋亡诱导剂肿瘤坏死因子相关凋亡诱导配体（TRAIL）联合处理可协同增强TRAIL诱导的人胰腺腺癌BxPC3细胞和人结肠直肠癌HCT116细胞凋亡。我们进一步观察到，ERA上调了促凋亡蛋白p53上调凋亡调节因子（PUMA）和NOXA以及抗凋亡蛋白Mcl-1。这些结果表明，ERA上调这些分子从而维持它们之间的平衡。有趣的是，当BxPC3细胞和HCT116细胞用ERA与TRAIL联合处理时，这种平衡被打破。我们的研究表明，联合处理期间PUMA、NOXA与Mcl-1之间的失衡是ERA增强TRAIL诱导凋亡的原因。通过构建磷酸化位点突变体（S121A/E125A/S159A/T163A）的HCT116 Mcl-1基因敲入细胞系来验证这一假说，并研究ERA与TRAIL之间的协同相互作用。连同形态学和细胞死亡研究，免疫印迹分析显示，HCT116 Mcl-1基因敲入突变细胞有效抑制了联合处理导致的Mcl-1减少和凋亡。此外，ERA增强了Mcl-1抑制剂诱导的凋亡。总的来说，我们的研究表明，Mcl-1是内质网应激通路和线粒体依赖性凋亡通路之间的守门分子。
