Reduced expression of DNA repair and redox signaling protein APE1/Ref-1 impairs human pancreatic cancer cell survival, proliferation, and cell cycle progression

Abstract

Pancreatic cancer is a deadly disease that is virtually never cured. Understanding the chemoresistance intrinsic to this cancer will aid in developing new regimens. High expression of APE1/Ref-1, a DNA repair and redox signaling protein, is associated with resistance, poor outcome, and angiogenesis; little is known in pancreatic cancer. Immunostaining of adenocarcinoma shows greater APE1/Ref-1 expression than in normal pancreas tissue. A decrease in APE1/Ref-1 protein levels results in pancreatic cancer cell growth inhibition, increased apoptosis, and altered cell cycle progression. Endogenous cell cycle inhibitors increase when APE1/ Ref-1 is reduced, demonstrating its importance to proliferation and growth of pancreatic cancer.

Figure 1

APE1/Ref-1 levels are elevated in pancreatic adenocarcinoma. (A) Normal pancreatic tissue (20×). (B) Primary pancreatic adenocarcinoma (20×). (C) Primary pancreatic adenocarcinoma (200×). (D) Pancreatic metastasis into the regional lymph node (20×).

Figure 2

APE1/Ref-1 knockdown via siRNA results in a dose- and time-dependent decrease in APE1/Ref-1 protein in the mitochondria and nuclei. (A) Western blot of Panc-1 and PaCa-2 cells after transfection with APE1/Ref-1 siRNA. siRNA dose course on Day 3; oligofectamine (OF), scrambled (SC) and APE1/Ref-1 siRNA (Si). (B) Time course with siRNA 25 nM. (C) Representative Western blot probed for APE1/Ref-1 from mitochondria of PaCa-2 cells with quantitation (right). WCE = whole cell extract, Nuc/Cyto = Nuclei + cytoplasm, Mito = purified mitochondria.

Figure 3

A reduction in APE1/Ref-1 protein reduces the growth rate and colony-forming ability of pancreatic cancer cell lines and increases the amount of apoptosis. (A) Growth rate of cells after exposure to 50 nM APE1/Ref-1 siRNA. (B) Colony-formation assays with each data point representing the mean ± SD from at least three separate experiments, with each experiment representing six replicate dishes per treatment. Solid squares depict siAPE/Ref-1-treated pancreatic cancer cells. *p < .005 using Student's t-test, comparing SC versus SiAPE1/Ref-1 at corresponding dose. (C) Bar graphs show average percentage of Annexin-positive cells (n = 4) ± SE. Middle panel: Representative Western blot probed with PARP-1 antibody on Day 3 after transfection with scrambled (SC) or APE1/Ref-1 siRNA (Si) at 50 nM.

Figure 4

Reduced levels of APE1/Ref-1 alter cell cycle profile and levels of endogenous cell cycle proteins in asynchronous cells. Representative dot plots of BrdU incorporation assays in (A) asynchronous Panc-1 cells treated with SC and SiAPE1/Ref-1 (25 nM). Bar graphs demonstrate quantitation of ≥3 independent experiments with average ± SD. *p < .05 using Student's t-test, comparing SC vs SiAPE1/Ref-1. (B) Representative Western blot of cell cycle proteins including p21, p27, total cdc-2, and phosphorylated cdc-2. Tubulin is used as a loading control. (C) Quantitation of Western blot data for proteins shown in Panel B. Bar graphs demonstrate quantitation of ≥4 independent experiments with average ± SE. *p < .05 using Student's t-test, comparing SC versus SiAPE1/Ref-1.

Figure 5

APE1/Ref-1 knockdown delays the reentry of cells into the cell cycle following synchronization. (A) Representative dot plots of synchronized PaCa-2 cells treated with SC and SiAPE1/Ref-1 (25 nM) stained with BrdU-FITC antibody and 7-AAD on Day 3 following transfection. (B) Bar graphs demonstrate quantitation of at least three independent experiments with average ± SD of SC control in black bars and SiAPE1/Ref-1 in gray bars. *p < .05 using student's t-test, comparing SC versus SiAPE1/Ref-1.

Figure 6

Knocking down APE1/Ref-1 increases DNA damage in pancreatic cancer cells but not the levels of ROS. (A) Knocking down of APE1/Ref-1 did not increase ROS generation in Panc-1 and PaCa-1 cell lines, but treatment with tert-butyl hydrogen peroxide (TBHP) did. The MFI (mean fluorescence intensity) of the oxidized form of dihydrorhodamine123 was analyzed by flow cytometry in APE1/Ref-1 siRNA (50 nM) transfected Panc-1 and PaCa-2 cells and analyzed on Day 3. These data are representative of three individual experiments and the averages are quantitated in Panel B. (C) Whole cell protein extracts from PaCa-2 cells were collected on Day 3 following transfection and analyzed for the presence of H2AX-γ by Western blot. A representative Western blot is presented, and the bar graph summarizes data from three experiments, with Tubulin used to normalize the amount of protein per lane. The amount of H2AX-γ is expressed relative to the respective scrambled controls in D. *p < .05 using student's t-test, comparing SC vs SiAPE1/Ref-1.