Expression of Notch receptors and their ligands in pancreatic ductal adenocarcinoma

Abstract

Pancreatic cancer is the fourth leading cause of cancer-associated mortality in developed countries. Pancreatic ductal adenocarcinoma (PDAC) accounts for ~90% of all pancreatic cancer cases. The Notch signaling pathway serves a crucial role in embryonic development, as well as during the tumorigenesis of different types of cancer. However, Notch signaling serves either oncogenic or tumor suppressor roles depending on the tissue type. There are four Notch receptors (Notch1-4) and five ligands [Jagged1, Jagged2, δ-like ligand protein (DLL)1, DLL3 and DLL4]; therefore, it has been suggested that the different Notch receptors serve distinct roles in the same type of tissue. To determine whether this is the case, the present study measured the expression of all Notch receptors and their ligands in PDAC tissue samples and cells. Immunohistochemistry was performed to measure the expression of Notch receptors and their ligands in paraffin-embedded PDAC tissue samples. Immunofluorescence was used to detect the expression of Notch receptors in the pancreatic cancer cell lines human pancreatic adenocarcinoma (HPAC) and PANC-1. In addition, levels of Notch receptors and ligands in HPAC and PANC-1 cells were analyzed by western blot analysis. The results revealed that levels of Notch1 and Notch3 were increased in PDAC tissues, whereas levels of Notch2 and Notch3 were not. The expression of Notch receptors in the pancreatic cancer cell lines HPAC and PANC-1 was consistent with their expression in PDAC tissues. Additionally, levels of the ligands DLL1, DLL3 and DLL4 were increased in HPAC and PANC-1 cells, as well as PDAC tissue samples. However, the expression of Jagged1 and 2 remained low. These results indicate that Notch1, Notch3, DLL1, DLL3 and DLL4 are upregulated in PDAC, a positive correlation was observed between the expression of Notch1 and Notch3, and between Notch1 and the ligands DLL1, DLL3 and DLL4. whereas Notch2, Notch4, Jagged1 and Jagged2 are not. The interaction of Notch1 and Notch3 with Notch ligands DLL1, DLL3 and DLL4 may be important in maintaining the tumor phenotype of pancreatic cancer.

Keywords: Notch ligands; Notch receptors; cancer biomarkers; expression; pancreatic cancer.

Figure 1.

Representative examples of immunohistochemistical staining for Notch receptors in PDAC tissues, indicating that Notch1 and 3 expression levels are increased whereas Notch 2 and 4 levels are decreased in PDAC tissue. Rabbit polyclonal anti-Notch antibodies were used to determine the expression of (A) Notch1, (B) Notch2, (C) Notch3 and (D) Notch4 in PDAC tissues. Scale bar=20 µm; magnification ×40. PDAC, pancreatic ductal adenocarcinoma.

Figure 2.

Representative examples of immunohistochemistry staining for Notch ligands in pancreatic ductal adenocarcinoma tissues, indicating that DLL1, DLL3 and DLL4 levels are increased whereas Jagged 1 and 2 levels remain low in PDAC. Rabbit polyclonal antibodies were used to measure the expression of (A) Jagged1, (B) Jagged2, (C) DLL1, (D) DLL3 and (E) DLL4 in PDAC tissues. (F) negative control. Scale bar=50 µm. PDAC, pancreatic ductal adenocarcinoma; DLL, δ-like ligand.

Figure 3.

Representative examples of immunofluorescent staining for Notch receptors in HPAC pancreatic cancer cells. DAPI staining is indicated in blue and Notch staining is indicated in red. Scale bar=50 µm. HPAC, human pancreatic adenocarcinoma.

Figure 4.

Representative examples of immunofluorescent staining for Notch receptors in PANC-1 pancreatic cancer cells. DAPI staining is indicated in blue and Notch is indicated in red. PBS was used for the negative control. Scale bar=10 µm. PDAC, pancreatic ductal adenocarcinoma.

Figure 5.

Western blot analysis measuring the expression of Notch receptors in pancreatic cancer cells. (A) HPAC cells; (B) PANC-1 cells. HPAC, human pancreatic adenocarcinoma; PDAC, pancreatic ductal adenocarcinoma.

Figure 6.

Western blot analysis measuring the expression of Notch ligands in pancreatic cancer cells. (A) HPAC cells; (B) PANC-1 cells. HPAC, human pancreatic adenocarcinoma; PDAC, pancreatic ductal adenocarcinoma; DLL, δ-like ligand.