The role of APE/Ref-1 signaling pathway in hepatocellular carcinoma progression.

Hepatocellular carcinoma (HCC) is responsible for a third of the estimated cancer-caused deaths worldwide. To deeply understand the mechanisms controlling HCC progression is of primary importance to develop new approaches for treatment. Apurinic/apyrimidinic endonuclease-1/redox effector factor 1 (APE/Ref-1) has been uncovered elevated in various types of cancer, including HCC. Additionally, HCC progression is always correlated with elevated copper (Cu). Our previous data demonstrated that Cu treatment initiated APE/Ref-1 expression and its downstream targets. Therefore, we hypothesized that APE/Ref-1 may be involved in HCC progression through mediating the effect of Cu to its signaling cascades. Following different treatments, human HCC cell line (Hep3B) and immortalized non-malignant hepatocyte cell line (THLE3) were analyzed to explore the role of APE/Ref-1 signaling pathway. Unstained human tissue microarrays (TMA) were subjected to IHC analysis to study the relationship between APE/Ref-1 expression and clinic features. APE/Ref-1 was upregulated in HCC cells consistent with the strong expression of APE/Ref-1 in HCC tissue microarray. Greater cytoplasmic accumulation of APE/Ref-1 was found in poorly differentiated and more aggressive tumors. Also we provide evidence to show that APE/Ref-1 signaling pathway stimulates cellular proliferation, enhances anti-apoptosis, and facilitates metastasis through experimental knockdown of APE/Ref-1 using siRNA in Hep3B cells or overexpressing APE/Ref-1 in THLE3 cells. These results define a novel role of APE/Ref-1 in HCC progression as being an important mediating and potentiating molecule, and also provide a basis for further investigations utilizing appropriate APE/Ref-1 inhibitors in combination with chemo-drugs for HCC treatment.

Nitric oxide initiates progression of human melanoma via a feedback loop mediated by apurinic/apyrimidinic endonuclease-1/redox factor-1, which is inhibited by resveratrol.

It is well recognized that nitric oxide (NO) is involved in tumor progression, including melanoma. Measurement of proliferative and metastatic capacity by MTS and Matrigel invasion assays, respectively, was done and showed that NO-treated melanoma cells exhibited a higher capacity compared with control, especially metastatic Lu1205 cells. Apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE/Ref-1) is a multifunctional protein and its role in tumor biology has attracted considerable attention. To determine whether APE/Ref-1 plays a role in mediating NO stimulation of melanoma progression, we investigated the effect of DETA/NO on levels of APE/Ref-1 and related downstream targets [activator protein-1 (AP-1)/JunD, matrix metalloproteinase-1 (MMP-1), Bcl-2, and inducible nitric oxide synthase (iNOS)] by Western blot and reverse transcription-PCR analysis. Following DETA/NO treatment, APE/Ref-1 and other downstream molecules were induced. Knockdown of APE/Ref-1 or AP-1/JunD by specific small interfering RNA markedly reversed the induction by NO stress of target proteins. These results present evidence for the existence of a functional feedback loop contributing to progression and metastasis of melanoma cells. Resveratrol has been shown to be an APE/Ref-1 inhibitor and significant decreases in AP-1/JunD, MMP-1, Bcl-2, and iNOS protein levels occurred after exposure to resveratrol. This phenolic antioxidant may be an appropriate choice for combining with other compounds that develop resistance by up-regulation of these molecules.

Redox effector factor-1, combined with reactive oxygen species, plays an important role in the transformation of JB6 cells.

Apurinic/apyrimidinic endonuclease/redox effector factor-1 (APE/Ref-1) is a multifunctional protein involved both in DNA base excision repair and redox regulation. Studies have suggested that abnormal Ref-1 levels and/or activities are associated with tumor progression and sensitivities to treatment, but no direct evidence has yet been published regarding the role of Ref-1 in malignant transformation. We utilized the well-documented tumor promotor-sensitive JB6 mouse epithelial cell model as well as new transformants [by ultraviolet light B (UVB), H2O2 or Cd] to study this phenomenon. Significant increases of reactive oxygen species (ROS) were observed in JB6P+ and all the transformants compared with promotor-resistant JB6P- cells. These increases were paralleled by a sustained elevation of Ref-1 expression. Further analysis exhibited a strong inverse correlation between oxidative DNA lesions [8-oxodeoxyguanosine (8-oxo-dG)] and Ref-1 levels in all JB6 cells. Notably, apoptosis occurred after knock-down of Ref-1 by small interfering RNA (siRNA)] demonstrated by a approximately 2-fold increase of Annexin V-positive JB6P+ cells. Ref-1 depletion also inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced anchorage-independent growth of JB6P+ by 40% and reduced the colony numbers of JB6P+/H2O2 and JB6P+/Cd cells. Mechanistic studies revealed that Ref-1 reduction was associated with an increase of intracellular ROS levels and a marked decrease of activator protein-1 (AP-1) transcription activities in JB6P+/H2O2 cells. This is the first report of the novel role of Ref-1 in cellular transformation. Based on the data presented here, we propose that induction of Ref-1, serving as an adaptive response to elevated ROS, plays a critical role in transformation and protects cells from excess ROS stresses through both DNA repair and activation of transcription factors such as activator protein-1.