Apurinic/apyrimidinic endonuclease/redox factor-1 (Ape1/Ref-1) activates Notch signaling through Jagged1 expression and promotes colon cancer progression
ABSTRACT: To investigate the possible role of Ape1/Ref-1 in tumorigenicity of colon cancer and explored the oncogenic mechanism of Ape1/Ref-1 in colon cancer cells. Control and Ref-1 overexpressing SW480cells, control and Ref-1-deficient SW480 cells. Duplicate
Project description:Pancreatic cancer is especially a deadly form of cancer with a survival rate less than 2%. Pancreatic cancers respond poorly to existing chemotherapeutic agents and radiation, and progress for the treatment of pancreatic cancer remains elusive. To address this unmet medical need, a better understanding of critical pathways and molecular mechanisms involved in pancreatic tumor development, progression, and resistance to traditional therapy is therefore critical. Reduction-oxidation (redox) signaling systems are emerging as important targets in pancreatic cancer. AP endonuclease1/Redox effector factor 1 (APE1/Ref-1) is upregulated in human pancreatic cancer cells and modulation of its redox activity blocks the proliferation and migration of pancreatic cancer cells and pancreatic cancer-associated endothelial cells in vitro. Modulation of APE1/Ref-1 using a specific inhibitor of APE1/Ref-1's redox function, E3330, leads to a decrease in transcription factor activity for NF?B, AP-1, and HIF1? in vitro. This study aims to further establish the redox signaling protein APE1/Ref-1 as a molecular target in pancreatic cancer. Here, we show that inhibition of APE1/Ref-1 via E3330 results in tumor growth inhibition in cell lines and pancreatic cancer xenograft models in mice. Pharmacokinetic studies also show that E3330 attains more than10 ?mol/L blood concentrations and is detectable in tumor xenografts. Through inhibition of APE1/Ref-1, the activity of NF?B, AP-1, and HIF1? that are key transcriptional regulators involved in survival, invasion, and metastasis is blocked. These data indicate that E3330, inhibitor of APE1/Ref-1, has potential in pancreatic cancer and clinical investigation of APE1/Ref-1 molecular target is warranted.
Project description:Reduction-oxidation factor 1-apurinic/apyrimidinic endonuclease (Ref-1/APE1) is a critical node in tumor cells, both as a redox regulator of transcription factor activation and as part of the DNA damage response. As a redox signaling protein, Ref-1/APE1 enhances the transcriptional activity of STAT3, HIF-1?, nuclear factor kappa B, and other transcription factors to promote growth, migration, and survival in tumor cells as well as inflammation and angiogenesis in the tumor microenvironment. Ref-1/APE1 is activated in a variety of cancers, including prostate, colon, pancreatic, ovarian, lung and leukemias, leading to increased aggressiveness. Transcription factors downstream of Ref-1/APE1 are key contributors to many cancers, and Ref-1/APE1 redox signaling inhibition slows growth and progression in a number of tumor types. Ref-1/APE1 inhibition is also highly effective when paired with other drugs, including standard-of-care therapies and therapies targeting pathways affected by Ref-1/APE1 redox signaling. Additionally, Ref-1/APE1 plays a role in a variety of other indications, such as retinopathy, inflammation, and neuropathy. In this review, we discuss the functional consequences of activation of the Ref-1/APE1 node in cancer and other diseases, as well as potential therapies targeting Ref-1/APE1 and related pathways in relevant diseases. APX3330, a novel oral anticancer agent and the first drug to target Ref-1/APE1 for cancer is entering clinical trials and will be explored in various cancers and other diseases bringing bench discoveries to the clinic.
Project description:Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein possessing both DNA repair and redox regulatory activities. It has been shown that blocking redox function leads to genotoxic, antiangiogenic, cytostatic, and proapoptotic effects in cells. Therefore, the selective inhibitors against APE1's redox function can be served as potential pharmaceutical candidates in cancer therapeutics. In the present study, we identified the biological specificity of the Chinese herbal compound tanshinone IIA (T2A) in blocking the redox function of APE1. Using dual polarization interferometry, the direct interaction between APE1 and T2A was observed with a KD value at subnanomolar level. In addition, we showed that T2A significantly compromised the growth of human cervical cancer and colon cancer cells. Furthermore, the growth-inhibitory or proapoptotic effect of T2A was diminished in APE1 knockdown or redox-deficient cells, suggesting that the cytostatic effect of T2A might be specifically through inhibiting the redox function of APE1. Finally, T2A pretreatment enhanced the cytotoxicity of ionizing radiation or other chemotherapeutic agents in human cervical cancer and colon cancer cell lines. The data presented herein suggest T2A as a promising bioactive inhibitor of APE1 redox activity.
Project description:A key feature of prostate cancer progression is the induction and activation of survival proteins, including the Inhibitor of Apoptosis (IAP) family member survivin. Apurinic/apyrimidinic endonuclease 1/redox effector factor 1 (APE1/Ref-1) is a multifunctional protein that is essential in activating oncogenic transcription factors. Because APE1/Ref-1 is expressed and elevated in prostate cancer, we sought to characterize APE1/Ref-1 expression and activity in human prostate cancer cell lines and determine the effect of selective reduction-oxidation (redox) function inhibition on prostate cancer cells in vitro and in vivo. Due to the role of oncogenic transcriptional activators NF?B and STAT3 in survivin protein expression, and APE1/Ref-1 redox activity regulating their transcriptional activity, we assessed selective inhibition of APE1/Ref-1's redox function as a novel method to halt prostate cancer cell growth and survival. Our study demonstrates that survivin and APE1/Ref-1 are significantly higher in human prostate cancer specimens compared to noncancerous controls and that APE1/Ref-1 redox-specific inhibition with small molecule inhibitor, APX3330 and a second-generation inhibitor, APX2009, decreases prostate cancer cell proliferation and induces cell cycle arrest. Inhibition of APE1/Ref-1 redox function significantly reduced NF?B transcriptional activity, survivin mRNA and survivin protein levels. These data indicate that APE1/Ref-1 is a key regulator of survivin and a potentially viable target in prostate cancer.
Project description:Apurinic/Apyrmidinic Endonuclease 1/Redox Factor-1 (APE1/Ref-1) is a reductant which is important for vascular homeostasis. SIRTUIN1 (SIRT1) is a lysine deacetylase that also promotes endothelium-dependent vasorelaxation. We asked if APE1/Ref-1 governs the redox state and activity of SIRT1, and whether SIRT1 mediates the effect of APE1/Ref-1 on endothelium-dependent vascular function. APE1/Ref-1 maintains sulfhydryl (thiol) groups of cysteine residues in SIRT1 in the reduced form and promotes endothelial SIRT1 activity. APE1/Ref-1 stimulates SIRT1 activity by targeting highly conserved vicinal thiols 371 and 374 which form a zinc tetra-thiolate motif in the deacetylase domain of SIRT1. Cysteine residues in the N-terminal redox domain of APE1/Ref-1 are essential for reducing SIRT1 and stimulating its activity. APE1/Ref-1 protects endothelial SIRT1 from hydrogen peroxide-induced oxidation of sulfhydryls and from inactivation. APE1/Ref-1 also promotes lysine deacetylation of the SIRT1 target endothelial nitric oxide synthase (eNOS). SIRT1 mutated at cysteines 371 and 374, which renders it non-reducible by APE1/Ref-1, prevents lysine deacetylation of eNOS by APE1/Ref-1. SIRT1 free thiol (reduced sulfhydryl) content and deacetylase activity are diminished in all examined tissues of APE1/Ref-1(+/-) mice, including the vasculature. Overexpression of SIRT1 in aortas of APE1/Ref-1(+/-) mice restores endothelium-dependent vasorelaxation and bioavailable nitric oxide (NO) to levels similar to those observed in wild-type mice. Thus, APE1/Ref-1, by maintaining functionally important cysteine sulfhydryls in SIRT1 in the reduced form, promotes endothelial SIRT1 activity. This reductive activation of endothelial SIRT1 by APE1/Ref-1 mediates the effect of APE1/Ref-1 on eNOS acetylation, promoting endothelium-derived NO and endothelium-dependent vasorelaxation.
Project description:Pancreatic cancer is a largely incurable disease, and increasing evidence supports strategies targeting multiple molecular mediators of critical functions of pancreatic ductal adenocarcinoma cells. Intracellular redox state modulates the activity of various signal transduction pathways and biological processes, including cell survival, drug resistance and responsiveness to microenvironmental factors. Recently, it has been shown that the transcription factor STAT3 is under redox control, but the mechanisms involved in its regulation are unknown. Here, we demonstrate for the first time that STAT3 DNA binding and transcriptional activity is directly regulated by the redox function of the APE1/Ref-1 endonuclease, using overexpression and redox-specific mutational strategies, and gene knockdown. Also, pharmacological blockade of APE1/Ref-1 by the redox-selective inhibitor E3330 abrogates STAT3 DNA binding. Since APE1/Ref-1 also exerts redox control on other cancer-associated transcription factors, we assessed the impact of dual-targeting of STAT3 signaling and APE1/Ref-1 redox on pancreatic cancer cell functions. We observed that disruption of APE1/Ref-1 redox activity synergizes with STAT3 blockade to potently inhibit the proliferation and viability of human PDAC cells. Mechanistically, we show that STAT3-APE1/Ref-1 dual targeting promotes marked tumor cell apoptosis, with engagement of caspase-3 signaling, which are significantly increased in comparison to the effects triggered by single target blockade. Also, we show that STAT3-APE1/Ref-1 dual blockade results in significant inhibition of tumor cell migration. Overall, this work demonstrates that the transcriptional activity of STAT3 is directly regulated by the redox function of APE1/Ref-1, and that concurrent blockade of STAT3 and APE1/Ref-1 redox synergize effectively inhibit critical PDAC cell functions.
Project description:Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is involved in DNA base repair and reducing activity. However, the role of APE1/Ref-1 in atherosclerosis is unclear. Herein, we investigated the role of APE1/Ref-1 in atherosclerotic apolipoprotein E (ApoE-/-) mice fed with a Western-type diet. We found that serologic APE1/Ref-1 was strongly correlated with vascular inflammation in these mice. Neutrophil/lymphocyte ratio (NLR), endothelial cell/macrophage activation, and atherosclerotic plaque formation, reflected by atherosclerotic inflammation, were increased in the ApoE-/- mice fed with a Western-type diet. APE1/Ref-1 expression was upregulated in aortic tissues of these mice, and was co-localized with cells positive for cluster of differentiation 31 (CD31) and galectin-3, suggesting endothelial cell/macrophage expression of APE1/Ref-1. Interestingly, APE1/Ref-1 plasma levels of ApoE-/- mice fed with a Western-type diet were significantly increased compared with those of the mice fed with normal diet (15.76 ± 3.19 ng/mL vs. 3.51 ± 0.50 ng/mL, p < 0.05), and were suppressed by atorvastatin administration. Correlation analysis showed high correlation between plasma APE1/Ref-1 levels and NLR, a marker of systemic inflammation. The cut-off value for APE1/Ref-1 for predicting atherosclerotic inflammation at 4.903 ng/mL showed sensitivity of 100% and specificity of 91%. We conclude that APE1/Ref-1 expression is upregulated in aortic endothelial cells/macrophages of atherosclerotic mice, and that plasma APE1/Ref-1 levels could predict atherosclerotic inflammation.
Project description:Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein that plays a central role in the cellular response to DNA damage and redox regulation against oxidative stress. APE1/Ref-1 functions in the DNA base excision repair pathway, the redox regulation of several transcription factors, and the control of intracellular redox status through the inhibition of reactive oxygen species (ROS) production. APE1/Ref-1 is predominantly localized in the nucleus; however, its subcellular localization is dynamically regulated and it may be found in the mitochondria or elsewhere in the cytoplasm. Studies have identified a nuclear localization signal and a mitochondrial target sequence in APE1/Ref-1, as well as the involvement of the nuclear export system, as determinants of APE1/Ref-1 subcellular distribution. Recently, it was shown that APE1/Ref-1 is secreted in response to hyperacetylation at specific lysine residues. Additionally, post-translational modifications such as phosphorylation, S-nitrosation, and ubiquitination appear to play a role in fine-tuning the activities and subcellular localization of APE1/Ref-1. In this review, we will introduce the multifunctional role of APE1/Ref-1 and its potential usefulness as a therapeutic target in cancer and cardiovascular disease.
Project description:Bladder cancer is the ninth most common cause of cancer-related deaths worldwide. Although cisplatin is used routinely in treating bladder cancer, refractory disease remains lethal for many patients. The recent addition of immunotherapy has improved patient outcomes; however, a large cohort of patients does not respond to these treatments. Therefore, identification of innovative molecular targets for bladder cancer is crucial. Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein involved in both DNA repair and activation of transcription factors through reduction-oxidation (redox) regulation. High APE1/Ref-1 expression is associated with shorter patient survival time in many cancer types. In this study, we found high APE1/Ref-1 expression in human bladder cancer tissue relative to benign urothelium. Inhibition of APE1/Ref-1 redox signaling using APE1/Ref-1-specific inhibitors attenuates bladder cancer cell proliferation in monolayer, in three-dimensional cultures, and in vivo. This inhibition corresponds with an increase in apoptosis and decreased transcriptional activity of NF-?B and STAT3, transcription factors known to be regulated by APE1/Ref-1, resulting in decreased expression of downstream effectors survivin and Cyclin D1 in vitro and in vivo. We also demonstrate that in vitro treatment of bladder cancer cells with APE1/Ref-1 redox inhibitors in combination with standard-of-care chemotherapy cisplatin is more effective than cisplatin alone at inhibiting cell proliferation. Collectively, our data demonstrate that APE1/Ref-1 is a viable drug target for the treatment of bladder cancer, provide a mechanism of APE1/Ref-1 action in bladder cancer cells, and support the use of novel redox-selective APE1/Ref-1 inhibitors in clinical studies. SIGNIFICANCE: This work identifies a critical mechanism for APE1/Ref-1 in bladder cancer growth and provides compelling preclinical data using selective redox activity inhibitors of APE1/Ref-1 in vitro and in vivo.
Project description:Apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1) dysregulation has been identified in several human tumors and in patients with a variety of neurodegenerative diseases. However, the function of Ape1/Ref-1 is unclear. We show here that Ape1/Ref-1 increases the expression of glial cell-derived neurotropic factor (GDNF) receptor alpha1 (GFRalpha1), a key receptor for GDNF. Expression of Ape1/Ref-1 led to an increase in the GDNF responsiveness in human fibroblast. Ape1/Ref-1 induced GFRalpha1 transcription through enhanced binding of NF-kappaB complexes to the GFRalpha1 promoter. GFRalpha1 levels correlate proportionally with Ape1/Ref-1 in cancer cells. The knockdown of endogenous Ape1/Ref-1 in pancreatic cancer cells markedly suppressed GFRalpha1 expression and invasion in response to GNDF, while overexpression of GFRalpha1 restored invasion. In neuronal cells, the Ape1/Ref-1-mediated increase in GDNF responsiveness not only stimulated neurite outgrowth but also protected the cells from beta-amyloid peptide and oxidative stress. Our results show that Ape1/Ref-1 is a novel physiological regulator of GDNF responsiveness, and they also suggest that Ape1/Ref-1-induced GFRalpha1 expression may play important roles in pancreatic cancer progression and neuronal cell survival.