Department of Laboratory Medicine, Wan Fang Hospital, Taipei Medical University, Taipei 11696, Taiwan.
School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei 11031, Taiwan.
Int J Mol Sci. 2023 Nov 17;24(22):16467. doi: 10.3390/ijms242216467.
The protein disulfide isomerase (PDI) family is a group of thioredoxin endoplasmic reticulum (ER)-resident enzymes and molecular chaperones that play crucial roles in the correct folding of proteins. PDIs are upregulated in multiple cancer types and are considered a novel target for cancer therapy. In this study, we found that a potent pan-PDI inhibitor, E64FC26, significantly decreased the proliferation of pancreatic ductal adenocarcinoma (PDAC) cells. As expected, E64FC26 treatment increased ER stress and the unfolded protein response (UPR), as evidenced by upregulation of glucose-regulated protein, 78-kDa (GRP78), phosphorylated (p)-PKR-like ER kinase (PERK), and p-eukaryotic initiation factor 2α (eIF2α). Persistent ER stress was found to lead to apoptosis, ferroptosis, and autophagy, all of which are dependent on lysosomal functions. First, there was little cleaved caspase-3 in E64FC26-treated cells according to Western blotting, but a higher dose of E64FC26 was needed to induce caspase activity. Then, E64FC26-induced cell death could be reversed by adding the iron chelator, deferoxamine, and the reactive oxygen species scavengers, ferrostatin-1 and N-acetylcysteine. Furthermore, the autophagosome-specific marker, light chain 3B (LC3B)-II, increased, but the autolysosome marker, sequestosome 1 (SQSTM1)/p62, was not degraded in E64FC26-treated cells. Using the FUW mCherry-LC3 plasmid and acridine orange staining, we also discovered a lower number of acidic vesicles, such as autolysosomes and mature lysosomes, in E64FC26-treated cells. Finally, E64FC26 treatment increased the cathepsin L precursor (pre-CTSL) but decreased mature CTSL expression according to Western blotting, indicating a defective lysosome. These results suggested that the PDI inhibitor, E64FC26, might initially impede proper folding of proteins, and then induce ER stress and disrupt proteostasis, subsequently leading to lysosomal defects. Due to defective lysosomes, the extents of apoptosis and ferroptosis were limited, and fusion with autophagosomes was blocked in E64FC26-treated cells. Blockade of autolysosomal formation further led to the autophagic cell death of PDAC cells.
蛋白质二硫键异构酶（PDI）家族是一组硫氧还蛋白内质网（ER）驻留酶和分子伴侣，它们在蛋白质的正确折叠中起着至关重要的作用。PDI 在多种癌症类型中上调，被认为是癌症治疗的新靶点。在这项研究中，我们发现一种有效的泛 PDI 抑制剂 E64FC26 显著降低了胰腺导管腺癌（PDAC）细胞的增殖。正如预期的那样，E64FC26 处理增加了内质网应激和未折叠蛋白反应（UPR），这表现在葡萄糖调节蛋白 78kDa（GRP78）、磷酸化（p）PKR 样内质网激酶（PERK）和磷酸化真核起始因子 2α（eIF2α）的上调。研究发现持续的内质网应激会导致细胞凋亡、铁死亡和自噬，所有这些都依赖于溶酶体功能。首先，Western blot 结果显示，E64FC26 处理的细胞中 cleaved caspase-3 很少，但需要更高剂量的 E64FC26 才能诱导 caspase 活性。然后，通过添加铁螯合剂去铁胺、活性氧清除剂 ferrostatin-1 和 N-乙酰半胱氨酸，可以逆转 E64FC26 诱导的细胞死亡。此外，自噬体特异性标记物 LC3B-II 增加，但 E64FC26 处理的细胞中自噬溶酶体标记物 SQSTM1/p62 没有降解。使用 FUW mCherry-LC3 质粒和吖啶橙染色，我们还发现 E64FC26 处理的细胞中酸性囊泡（如自噬溶酶体和成熟溶酶体）的数量减少。最后，Western blot 结果显示，E64FC26 处理增加了组织蛋白酶 L 前体（pre-CTSL），但降低了成熟 CTSL 的表达，表明溶酶体功能缺陷。这些结果表明，PDI 抑制剂 E64FC26 可能最初会阻碍蛋白质的正确折叠，然后诱导内质网应激和破坏蛋白质稳态，进而导致溶酶体缺陷。由于溶酶体缺陷，凋亡和铁死亡的程度受到限制，并且 E64FC26 处理的细胞中与自噬体的融合被阻断。自噬溶酶体形成的阻断进一步导致 PDAC 细胞的自噬细胞死亡。
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