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. 2008 Jul;7(7):2012-21.
doi: 10.1158/1535-7163.MCT-08-0113.

Small-molecule inhibitor of the AP endonuclease 1/REF-1 E3330 inhibits pancreatic cancer cell growth and migration

Gang-Ming Zou  1 , Anirban Maitra
Affiliations

Small-molecule inhibitor of the AP endonuclease 1/REF-1 E3330 inhibits pancreatic cancer cell growth and migration

Gang-Ming Zou et al. Mol Cancer Ther. 2008 Jul.

Abstract

AP endonuclease 1 (APE1; also known as REF-1) contains a DNA repair domain and a redox regulation domain. APE1 is overexpressed in several human cancers, and disruption of APE1 function has detrimental effects on cancer cell viability. However, the selective contribution of the redox and the DNA repair domains to maintenance of cellular homeostasis in cancer has not been elucidated. In the present study, we used E3330, a small-molecule inhibitor of APE1 redox domain function, to interrogate the functional relevance of sustained redox function in pancreatic cancer. We show that E3330 significantly reduces the growth of human pancreatic cancer cells in vitro. This phenomenon was further confirmed by a small interfering RNA experiment to knockdown APE1 expression in pancreatic cancer cells. Further, the growth-inhibitory effects of E3330 are accentuated by hypoxia, and this is accompanied by striking inhibition in the DNA-binding ability of hypoxia-inducible factor-1alpha, a hypoxia-induced transcription factor. E3330 exposure promotes endogenous reactive oxygen species formation in pancreatic cancer cells, and the resulting oxidative stress is associated with higher levels of oxidized, and hence inactive, SHP-2, an essential protein tyrosine phosphatase that promotes cancer cell proliferation in its active state. Finally, E3330 treatment inhibits pancreatic cancer cell migration as assessed by in vitro chemokine assays. E3330 shows anticancer properties at multiple functional levels in pancreatic cancer, such as inhibition of cancer cell growth and migration. Inhibition of the APE1 redox function through pharmacologic means has the potential to become a promising therapeutic strategy in this disease.

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Figures

Figure 1
Figure 1
A, APE1 expression in pancreatic cancer cells (Western blot). The total cell lysates are analyzed by Western blot. All six tested pancreatic cancer cell lines PANC1, PK9, BxPC3, CAPAN-1, ASPC-1, and XPA1 express a dominant level of APE1. Non-tumorigenic human pancreatic cell line HPNE and HPDE express lower level of APE1 compared with these malignant cell lines. B, dose-dependent inhibition of growth of pancreatic cancer cells (PANC1 and XPA1) but not the nonneoplastic human pancreatic epithelial cells HPNE by E3330. PANC1, XPA1, or HPNE cells were treated with various dose of E3330 for 72 h. Then, MTT assays were done to analyze cell viability. C, trypan blue staining and cell count for E3330-treated PANC1 cells. D, methoxyamine, the APE1 DNA repair domain inhibitor, has no growth-inhibitory effects on pancreatic cancer cells. Both PANC1 and XPA1 cells were treated with methoxyamine for 72 h followed by a MTT assay. E, difference of spontaneous growth rate between low APE1-expressing pancreatic cell (HPNE) and high APE1-expressing pancreatic cells (PANC1). Transduction of exogenous wild-type APE1 into HPNE cells enhances their growth rate.
Figure 2
Figure 2
APE1 is essential for pancreatic cancer cell growth in vitro. A, knockdown of APE1 expression affects cell growth in trypan blue exclusion assay. PANC1, XPA1, or HPNE cells were transfected with scramble (control) siRNA 25 or 50 nmol/L or APE1 siRNA 25 or 50 nmol/L. After transfection, the cells were cultivated for 3 d. The live cell numbers were counted after trypan blue staining. The MTT assays were carried out to examine the cell viability. Mean of three independent experiments; bars, SD. B, APE1 redox domain inhibitor E3330 inhibits the in vitro growth of primary pancreatic cancer cells. ESA-positive primary pancreatic cancer cells were isolated from three independent freshly resected pancreatic cancer tissue specimens using ESA-positive MACS bead. The isolated pancreatic cancer cells were then treated with various doses of E3330. After 48-h incubation, the MTT assay was done to examine cell viability. Columns, mean of three independent experiments; bars, SD. C, DNA contents of PANC1 cells treated with ethanol (as control cells) or with E3330 at various doses (10, 20, and 30 μmol/L) for 48 h. Representative result from three independent experiments. D, percentage of Ki-67-positive cells in E3330-treated PANC1 cells in flow cytometry assay. PANC1 cells were treated with either with ethanol (as control cells) or with various doses of E3330 (1, 5, 10, 20, or 30 μmol/L) for 48 h. The cells were then stained with FITC-conjugated anti-Ki-67 antibody and read on flow cytometry. Columns, mean of three independent experiments; bars, SD. *, P < 0.05; **, P < 0.01.
Figure 3
Figure 3
Hypoxia enhances E3330-mediated growth inhibition in pancreatic cancer cells. A, PANC1 cells. B, XPA1 cells. In normoxia or hypoxia conditions (cells were treated with 100 μmol/L CoCl2), the cells were treated with E3330 at various dose for 48 h. The MTT assay was done. Columns, mean of three independent experiments; bars, SD. *, P < 0.05; **, P < 0.01. C, E3330 inhibits HIF-1α activity. HIF-1α DNA-binding activity was analyzed by ELISA. Columns, mean of three independent experiments; bars, SD. *, P < 0.05. D, NF-κB activity assay in pancreatic cancer cells treated with E3330. Pancreatic cancer cells were transfected with NF-κB reporter plasmid pNF-κB-Luc. The cells were treated with E3330 at various doses for 6 h, and the luciferase activities were measured using a Dual-Luciferase Reporter Assay System.
Figure 4
Figure 4
E3330 enhances ROS production in PANC1 cells. A, ROS was detected by flow cytometry using dihydrorhodamine 123 as a probe. The cells were treated with ethanol (as control cells) or with E3330 at the indicated dose for 2 d. The cells were then harvested, stained with 0.5 μmol/L dihydrorhodamine 123 for 30 min, and analyzed on flow cytometry. Representative results from three independent experiments. B, knockdown of APE1 expression enhance endogenous ROS level in PANC1 cells. PANC1 cells were transfected with control siRNA or APE1 siRNA. After 72 h, the intracellular ROS level was analyzed by labeling cells with dihydrorhodamine 123 and assessed by flow cytometry. MFI, mean fluorescence intensity. C, SHP-2 expression in mitochondria of PANC1 cells in Western blot assay: 50 μg whole-cell lysate of PANC1 cells or 50 μg purified mitochondria of PANC1 cells were assayed in their SHP-2 protein level. D, inhibition of APE1 redox function with E3330 increases SHP-2 oxidation and inactivation in pancreatic carcinoma. PANC1 cells were treated with ethanol (as control cells) or indicated dose of E3330 for 48 h. The cells were lysated in an anaerobic chamber with 1 mL O2 free lysis buffer. The level of oxidation of SHP-2 is examined by immunoprecipitation and blot assay. Representative results from three independent experiments.
Figure 5
Figure 5
E3330 inhibits stromal cell – derived factor-1 – mediated migration of pancreatic cancer cells. A, pancreatic cancer cell lines PANC1, XPA1, and BxPC3 were stimulated by stromal cell – derived factor-1 at 100 ng/mL and exposed to varying concentrations of E3330. Stromal cell – derived factor-1 – stimulated migration was inhibited by E3330 at dosages ≥5 μmol/L. Columns, mean of three independent experiments in triplicate wells and normalized for any growth-inhibitory effects of E3330. *, P < 0.05. B, fluorescence-activated cell sorting analysis of CD44 expression in PANC1 cells. Dotted line, cells stained with isotype control antibody; solid line, cells stained with anti-CD44 antibody. C, E3330 inhibits CD44 expression in PANC1 cells. PANC1 cells were treated with E3330 at the indicated dose for 48 h. The cells were then harvested, stained with fluorescence-conjugated anti-CD44 antibody, and analyzed on flow cytometry. Columns, mean of three independent experiments; bars, SD. *, P < 0.05; **, P < 0.01.
Figure 6
Figure 6
Proposed model about the role of APE1 regulating SHP-2 activity in mitochondria and HIF-1α activity in nucleus through redox mechanism in pancreatic cancer cells.
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