| 摘要: | 根據美國癌症協會 (American Cancer Society, ACS)於2023年所發表的統計圖表中，胰臟癌位列癌症致死率第4名，高致死率的主因是早期轉移和預後不良，其中最常見的是胰臟導管腺癌 (Pancreatic Ductal Adenocarcinoma, PDAC)。蛋白質雙硫鍵異構? (protein disulfide isomerase, PDI)被視為新穎的抗癌標的(target)，PDI在內質網中透過雙硫鍵氧化還原的方式將蛋白質進行正確折疊，穩定蛋白質的結構與功能，並在癌細胞當中過度表達以應付快速增殖所產生的大量蛋白質。當抑制PDI或是下調PDI會導致未折疊以及錯誤折疊蛋白質的積累而活化內質網壓力以及未折疊蛋白反應，進而誘導細胞凋亡。先前研究指出胰臟導管腺癌細胞具有高表達的自噬基礎，以攝取外來胺基酸維持細胞生長並增強對於化、放療的抵抗性。然而PDI在PDAC中對自噬作用的潛在分子機制仍不清楚。先前文獻指出不同PDI抑制劑會透過鐵死亡、細胞凋亡以及自噬作用等機制造成細胞死亡，在這項研究中探討PDI廣泛型抑制劑E64FC26造成細胞死亡之分子機制。首先分析E64FC26對PDAC細胞的毒性，結果顯示E64FC26顯著誘導PDAC細胞的死亡，並透過給予鐵死亡抑制劑確定E64FC26與鐵死亡的相關性。此外以西方墨點法分析PDAC中關於內質網壓力路徑以及細胞凋亡路徑相關的蛋白質表現，結果顯示E64FC26會增加內質網壓力相關蛋白質phospho-PERK、GRP78和phospho-eIF2α表達，以及細胞凋亡相關蛋白質clv-caspase-3和clv-PARP表達，並增強caspases 酵素活性。此外E64FC26上調自噬囊泡蛋白質LC3-Ⅱ的表達，而降解指標蛋白質SQSTM1/p62的積累說明了自噬作用最終的降解步驟受到影響。透過轉染FUW mCherry-LC3質體以及?啶橙染色並使用共軛焦顯微鏡確認自噬溶小體的形成，並透過西方墨點法得知溶?體中m-CTSL表現量下降。以上結果揭示PDI會透過內質網壓力誘導細胞凋亡以及阻斷自噬小體和溶?體的融合和溶?體的功能影響自噬作用等分子機制，進而引起人類胰臟導管腺癌細胞的死亡，達到最終的抗癌效果。
According to the statistical charts published by the American Cancer Society (ACS) in 2023, pancreatic cancer ranks fourth in cancer mortality, indicating a high fatality rate. The main reasons for this high mortality rate are early metastasis and poor prognosis. The most common type of pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). Protein disulfide isomerase (PDI) is considered a novel target for cancer treatment. PDI is responsible for correct protein folding through disulfide bond oxidation and reduction in the endoplasmic reticulum, stabilizing protein structure and function. PDI is overexpressed in cancer cells to cope with the excessive production of proteins caused by rapid proliferation. Inhibiting or downregulating PDI leads to the accumulation of unfolded or misfolded proteins, activating endoplasmic reticulum stress (ER stress) and the unfolded protein response (UPR), eventually inducing apoptosis. Previous studies have shown that pancreatic ductal adenocarcinoma cells have a high basal level of autophagy, which enables them to uptake external amino acids for cell growth and enhances resistance to chemotherapy and radiation therapy. However, the potential molecular mechanisms of PDI to regulate autophagy in PDAC are still unclear. In this study, the molecular mechanisms of cell death induced by the potent pan-inhibitor of the PDI family, E64FC26 were investigated. First, we initially treated PDAC cells with E64FC26 to examine its toxicity. The results showed that E64FC26 significantly induced cell death in PDAC cells. The correlation between E64FC26 and ferroptosis was confirmed by pre-treated different ferroptosis inhibitors. Additionally, we analyzed the expression of proteins related to endoplasmic reticulum stress pathways and apoptosis pathways in PDAC using western blot. The results showed that E64FC26 increased the expression of endoplasmic reticulum stress-related proteins, such as phospho-PERK, GRP78, and phospho-eIF2α, as well as apoptosis-related proteins, including clv-caspase-3 and clv-PARP, and increased caspase enzyme activity. Furthermore, E64FC26 upregulated the expression of autophagosome protein LC3-Ⅱ while accumulating the degradation marker protein SQSTM1/p62, indicating that the final degradation step of autophagy was affected. To observe the formation of autolysosomes, PDAC cells were transfected with the FUW mCherry-LC3 plasmid and stained with acridine orange, followed by confocal microscopy. Additionally, we observed that E64FC26 decreased the expression of mature-CTSL through western blot. These findings suggested that PDI induced apoptosis through endoplasmic reticulum stress and blocked the fusion of autophagosomes and lysosomes, as well as impaired lysosomal function, affecting the molecular mechanisms of autophagy. Consequently, it led to the death of human pancreatic ductal adenocarcinoma cells, achieving the ultimate anti-cancer effect. |
