Pan-Inhibition of Protein Disulfide Isomerase Caused Cell Death through Disrupting Cellular Proteostasis in Pancreatic Ductal Adenocarcinoma Cells

Ching-Sheng Hung; Kun-Lin Lee; Wei-Jan Huang; Fang-He Su; Yu-Chih Liang

doi:10.3390/ijms242216467

Pan-Inhibition of Protein Disulfide Isomerase Caused Cell Death through Disrupting Cellular Proteostasis in Pancreatic Ductal Adenocarcinoma Cells

Int J Mol Sci. 2023 Nov 17;24(22):16467. doi: 10.3390/ijms242216467.

Authors

Ching-Sheng Hung^{1

2}, Kun-Lin Lee³, Wei-Jan Huang^{4

5}, Fang-He Su², Yu-Chih Liang^{2

3

4

6}

Affiliations

¹ Department of Laboratory Medicine, Wan Fang Hospital, Taipei Medical University, Taipei 11696, Taiwan.
² School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei 11031, Taiwan.
³ Ph.D. Program in Medical Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei 11031, Taiwan.
⁴ Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei 11031, Taiwan.
⁵ School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei 11031, Taiwan.
⁶ Traditional Herbal Medicine Research Center, Taipei Medical University Hospital, Taipei 11031, Taiwan.

Abstract

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.

Keywords: apoptosis; autophagy; ferroptosis; pancreatic ductal adenocarcinoma; protein disulfide isomerase.

MeSH terms

Apoptosis
Autophagy
Carcinoma, Pancreatic Ductal* / drug therapy
Endoplasmic Reticulum Stress
Humans
Pancreatic Neoplasms*
Protein Disulfide-Isomerases
Proteostasis

Substances

Protein Disulfide-Isomerases

Grants and funding

This research was funded by Ministry of Science and Technology of the Republic of China, grant numbers MOST 107-2320-B-038-023-MY3 and MOST 110-2320-B-038-061- and Wan Fang Hospital, grant numbers 111TMU-WFH-22 and 112TMU-WFH-13.