Quinomycin A targets Notch signaling pathway in pancreatic cancer stem cells

Abstract

Cancer stem cells (CSCs) appear to explain many aspects of the neoplastic evolution of tumors and likely account for enhanced therapeutic resistance following treatment. Dysregulated Notch signaling, which affects CSCs plays an important role in pancreatic cancer progression. We have determined the ability of Quinomycin to inhibit CSCs and the Notch signaling pathway. Quinomycin treatment resulted in significant inhibition of proliferation and colony formation in pancreatic cancer cell lines, but not in normal pancreatic epithelial cells. Moreover, Quinomycin affected pancreatosphere formation. The compound also decreased the expression of CSC marker proteins DCLK1, CD44, CD24 and EPCAM. In addition, flow cytometry studies demonstrated that Quinomycin reduced the number of DCLK1+ cells. Furthermore, levels of Notch 1-4 receptors, their ligands Jagged1, Jagged2, DLL1, DLL3, DLL4 and the downstream target protein Hes-1 were reduced. The γ-secretase complex proteins, Presenilin 1, Nicastrin, Pen2, and APH-1, required for Notch activation also exhibited decreased expression. Ectopic expression of the Notch Intracellular Domain (NICD) partially rescued the cells from Quinomycin mediated growth suppression. To determine the effect of Quinomycin on tumor growth in vivo, nude mice carrying tumor xenografts were administered Quinomycin intraperitoneally every day for 21 days. Treatment with the compound significantly inhibited tumor xenograft growth, coupled with significant reduction in the expression of CSC markers and Notch signaling proteins. Together, these data suggest that Quinomycin is a potent inhibitor of pancreatic cancer that targets the stem cells by inhibiting Notch signaling proteins.

Keywords: DCLK1; Hes1; NICD; apoptosis; tumor xenograft.

Figure 1. Quinomycin inhibits pancreatic cancer cell proliferation

(A) Chemical structure of Quinomycin. (B) Proliferation of pancreatic ductal epithelial cells is not affected by 50 nM Quinomycin treatment for 48 h. (C) Quinomycin inhibits proliferation of pancreatic cancer cells. Cells were incubated with increasing doses of Quinomycin (0–1 μM) for up to 72 h and analyzed for cell proliferation. Quinomycin treatment resulted in a significant dose and time-dependent decrease in cell proliferation in all three cell lines when compared with untreated controls. (D) Quinomycin inhibits colony formation. Pancreatic cancer cells were incubated with 5 nM Quinomycin for 48 h and allowed to grow into colonies for 10 d. Incubation with Quinomycin inhibits colony formation. Results are representative of three independent experiments.

Figure 2. Quinomycin induces cancer cell apoptosis

(A) Cell cycle analysis of Quinomycin treated cells. MiaPaCa-2 or PanC-1 cells were treated with up to 5 nM Quinomycin for 24 h and examined by flow cytometry following propidium iodide staining for DNA content. Quinomycin treatment leads to increased number of cells in the PreG0/G1 arrest. Graphs are representative of data collected from three experiments. (B) Quinomycin induces caspase 3, an apoptosis mediator. Lysates from MiaPaCa-2 or PanC-1 cells incubated with 5 nM Quinomycin were analyzed by western blotting for caspase 3 protein levels using rabbit anti-caspase 3 antibody. Quinomycin treated cells shows cleaved (activated) caspase 3 while untreated cells have no cleaved caspase-3. (C) Lysates from MiaPaCa-2 or PanC-1 cells incubated with 5 nM Quinomycin were analyzed by western blotting for cyclin D1 and c-Myc proteins. Both cyclin D1 and c-myc were reduced following Quinomycin treatment.

Figure 3. Quinomycin affects cancer stem cell marker expression

(A and B) PanC-1 cells were grown in specific spheroid media in low adherent plates and treated with increasing concentrations of Quinomycin. After 5 days, the pancreatosphere were photographed and counted. The primary spheroids were collected and separated into single cells and replated. The Quinomycin treatment significantly was inhibited in both primary and secondary pancreatospheres formation (right and left panel)(*p < 0.05). (C) Sorting of anti-DCLK1 antibody -tagged phycoerythrin untreated MiaPaCa-2 and PanC-1 cells by flow cytometry. After 24 h, Quinomycin treatment caused significant reduction in the number of DCLK1 expressing cells. (D) Western blot analyses of lysates from Quinomycin treatment showed significant reduction in cancer stem cell marker DCLK1, CD44, CD24 and EPCAM protein levels in both MiaPaCa-2 and PanC-1 cells.

Figure 4. Quinomycin affects Notch signaling

(A) Lysates from cells treated with Quinomycin caused significant reduction in the expression of Notch receptors Notch-1, 2, 3 and 4 in both MiaPaCa-2 and PanC-1 cells. (B) Lysates from cells treated with Quinomycin caused significant reduction in the expression of Notch ligands Jagged-1, 2 and Delta like ligand 1, 3 and 4 in both MiaPaCa-2 and PanC-1 cells. (C) Lysates from cells treated with Quinomycin caused significant reduction in the expression of Notch downstream target gene Hes-1 in both MiaPaCa-2 and PanC-1 cells. (D) Quinomycin also significantly reduced expression of γ-secretase complex proteins Presenilin-1, Nicastrin, APH1 and PEN2 in both MiaPaCa-2 and PanC-1 cells.

Figure 5. Quinomycin inhibits cell growth through inactivation of the γ-secretase complex

(A) Cells were cotreated with Quinomycin in combination with γ- secretase complex inhibitor DAPT for 24 h. Lysates were analyzed by western blotting. The cotreatment of the Quinomycin in combination with γ- secretase complex inhibitor DAPT further reduced Hes-1 expression, (B) Cells were cotreated with Quinomycin in combination with γ-secretase complex inhibitor DAPT and subsequently measured for proliferation (left panel) and apoptosis (right panel). The cotreatment of the Quinomycin in combination with γ- secretase complex inhibitor DAPT further reduced proliferation (left panel) and further increased apoptosis (right panel). (C) Ectopic expression of NICD overcomes Quinomycin-mediated suppression of Hes-1 expression. Cells transiently expressing NICD was treated with Quinomycin for 24 h. Lysates were analyzed by western blotting. Hes-1 was increased in the NICD expressing cells when compared to vector transfected controls. (D) Cells expressing NICD were treated with Quinomycin and subsequently measured for proliferation (left panel) and apoptosis (right panel). Ectopic expression of NICD rescued Quinomycin mediated inhibition of cell proliferation and apoptosis (*P < 0.05). (E) Cells expressing NICD were treated with Quinomycin and subsequently performed for pancreatosphere formation (left and right panel). Ectopic expression of NICD rescued Quinomycin mediated inhibition of pancreatosphere formation (*P < 0.05).

Figure 6. Quinomycin inhibits pancreatic cancer xenografts

(A) MiaPaCa-2 cells were injected in to the flanks of nude mice and palpable tumors were allowed to develop for 7 days. Subsequently, Quinomycin (20 μg/kg bw) was injected daily intraperitoneally every day for 21 days. On day 22, tumors were excised and subject to further analyses. (B) Quinomycin treatment resulted in significantly lower tumor weight when compared to control. Tumor size was measured every week. There was a significant reduction in tumor size from Quinomycin treated animals when compared control (*P < 0.05). (C) Tumor volumes in Quinomycin treated mice were smaller when compared to control (*P < 0.05).

Figure 7. Quinomycin inhibit cancer stem cell marker proteins and Notch signaling in tumor xenografts

(A) Western blot analysis showed that tissue lysates from Quinomycin treated animals have significantly lower levels of cancer stem cell markers. (B) Immunohistochemistry shows that treatment with Quinomycin significantly reduced the expression of cancer stem cell markers. (C) Western blot analysis showed that tissue lysates from Quinomycin treated animals have significantly lower levels of Notch-1, Jagged-1, Hes-1, and γ-secretase complex proteins. (D) Immunohistochemistry shows that Quinomycin treated animals have significantly lower levels of Notch-1, Jagged-1, Hes-1, and γ-secretase complex proteins Presenilin 1 and Nicastrin in the tumor xenograft tissues. (C1-C4: Controls, Q1-Q4: Quinomycin treated).

Figure 8. Quinomycin inhibit Notch receptor-2, 3 and 4 and its ligands Jagged 2, DLL-1, 3 and 4 in tumor xenografts

(A) Western blot analysis showed that tissue lysates from Quinomycin treated animals have significantly lower levels of Notch-2, 3 and 4 receptor expression. (B) Immunohistochemistry shows that treatment with Quinomycin significantly reduced the expression of Notch-2, 3 and 4 receptors. (C) Western blot analyses showed that tissue lysates from Quinomycin treated animals have significantly lower levels of Notch ligand Jagged-2, DLL-1, 3 and 4 expressions. (D) Immunohistochemistry shows that treatment with Quinomycin significantly reduced the Notch ligand Jagged-2, DLL-1, 3 and 4 expressions. (C1–C4: Controls, Q1–Q4: Quinomycin treated).