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

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.

Figure 1

The mRNA expressions of protein disulfide isomerase (PDI) family members in human pancreatic ductal adenocarcinoma (PDAC) tissues. mRNA levels of human PDAC tissues and normal pancreatic tissues were obtained from the GEPIA2 database. PDI family members were selected based on a |log2(fold change)| cutoff of 0.5 and q-value cutoff of 0.01. PDAC tissues, n = 179; normal pancreatic tissues, n = 171.

Figure 2

Effects of E64FC26 on the cell viability in human pancreatic ductal adenocarcinoma (PDAC) cells. (A) AsPC-1 and (B) BxPC-3 cells were treated with different concentrations of E64FC26 for 24 and 48 h, and cell viability was determined by an MTT assay. Each data point is presented as the mean ± S.E. of three independent experiments.

Figure 3

Effects of E64FC26 on endoplasmic reticular (ER) stress- and unfolded protein response (UPR)-related protein expressions in human pancreatic ductal adenocarcinoma (PDAC) cells. (A) AsPC-1 and BxPC-3 cells were treated with E64FC26 for different times, and protein levels of ER stress-related proteins were determined by Western blotting. (B) AsPC-1 and BxPC-3 cells were treated with different concentrations of E64FC26 for 24 h, and protein levels of ER stress-related proteins were determined by Western blotting. The relative mean intensity of each band (indicated below the bands, n ≥ 3) was normalized to the unphosphorylated total protein (including PERK, eIF2α) or α-tubulin loading control.

Figure 4

Effects of E64FC26 on activation of caspases in human pancreatic ductal adenocarcinoma (PDAC) cells. (A) AsPC-1 and BxPC-3 cells were treated with different concentrations of E64FC26 for 24 h, and protein levels of the poly(ADP ribose) polymerase (PARP) and caspase-3 proteins were determined by Western blotting. (B–D) AsPC-1 and BxPC-3 cells were treated with different concentrations of E64FC26 for 24 h, and activities of (B) caspase-3, (C) caspase-8, and (D) caspase-9 were detected with a caspase colorimetric assay kit. Concentrations of 1 µM and 100 nM of staurosporine were respectively used as positive controls (PCs) in AsPC-1 and BxPC-3 cells. Each data point is presented as the mean ± S.E. of three independent experiments. * p < 0.05 vs. the control (Ctl) group.

Figure 5

Effects of ferroptosis inhibitors on E64FC26-caused cell death in human pancreatic ductal adenocarcinoma (PDAC) cells. AsPC-1 and BcPC-3 cells were pretreated with (A) deferoxamine (DFO) for 6 h, (B) ferrostatin-1 (Ferro-1) for 1 h, or (C) N-acetylcysteine (NAC) for 24 h, and then treated with E64FC26 for 24 or 48 h. Cell viability was determined by an MTT assay. Each data point is presented as the mean ± S.E. of three independent experiments. * p < 0.05 vs. the E64FC26 alone group.

Figure 5

Effects of ferroptosis inhibitors on E64FC26-caused cell death in human pancreatic ductal adenocarcinoma (PDAC) cells. AsPC-1 and BcPC-3 cells were pretreated with (A) deferoxamine (DFO) for 6 h, (B) ferrostatin-1 (Ferro-1) for 1 h, or (C) N-acetylcysteine (NAC) for 24 h, and then treated with E64FC26 for 24 or 48 h. Cell viability was determined by an MTT assay. Each data point is presented as the mean ± S.E. of three independent experiments. * p < 0.05 vs. the E64FC26 alone group.

Figure 6

Effects of E64FC26 on autophagy marker protein expressions in human pancreatic ductal adenocarcinoma (PDAC) cells. (A) AsPC-1 and BxPC-3 cells were treated with different concentrations of E64FC26 for 24 h, and protein levels of autophagy-related proteins were determined by Western blotting. The relative mean intensity of each band (indicated below the bands, n ≥ 3) was normalized to the unphosphorylated total protein (mTOR) or GAPDH loading control. (B) AsPC-1 and (C) BxPC-3 cells were respectively treated with 5 and 1 µM of E64FC26 for 24 h, and the light chain 3B (LC3B) puncta were detected by IF staining with the CF^®488A dye (green) and nucleic acid staining with DAPI (blue). Images on the right column are magnifications of the white boxed areas. Representative LC3B puncta are indicated by white arrows. Quantification of LC3 puncta per cell is presented as the mean ± S.E. of three independent experiments. * p < 0.05 vs. the control (Ctl) group.

Figure 7

Effects of E64FC26 on autolysosome formation and lysosome functions in human pancreatic ductal adenocarcinoma (PDAC) cells. (A) AsPC-1 and BxPC-3 cells were transfected with the FUW mCherry-GFP-LC3 plasmid and then treated with 5 µM and 1 µM E64FC26, respectively, for 24 h. Autophagosomes/autolysosomes were visualized by fluorescence microscopy. Representative autolysosomes and autophagosomes are indicated by red and yellow arrows, respectively. (B) AsPC-1 and BxPC-3 cells were treated with 5 µM and 1 µM E64FC26, respectively, for 24 h, and cells were stained with acridine orange for 15 min. Under acridine orange staining, cytoplasmic and nuclear fluorescence was green, and acidic vesicular organelle fluorescence was bright red or orange-red. Images on the right column are magnifications of the white boxed areas. Representative mature lysosomes or acidic autolysosomes are indicated by white arrows. (C) AsPC-1 and BxPC-3 cells were treated with different concentrations of E64FC26 for 24 h, and the precursor (pre-), intermediate (i-), and mature (m-) forms of the cathepsin L (CTSL) protein were detected by Western blotting. The relative mean intensity of each band (indicated below the bands, n ≥ 3) was normalized to the GAPDH loading control.

Figure 8

Possible mechanisms of E64FC26-induced cell death in pancreatic ductal adenocarcinoma (PDAC) cells. E64FC26 first induced endoplasmic reticular (ER) stress through impeding proper folding of proteins, and then subsequently caused lysosome defects. ER stress might have initiated ferroptosis and autophagy signals, and cells may have undergone partial apoptosis due to persistent ER stress from higher E64FC26 concentrations. However, defective lysosomes were unable to assist in the apoptosis and ferroptosis processes and hindered fusion with autophagosomes to form autolysosomes. Finally, E64FC26 only induced limited apoptosis and ferroptosis and mainly caused autophagic cell death. Solid lines are used to indicate activated pathways, and dashed lines are used to indicate inhibited pathways. LPO, lipid peroxidation.