The inhibitor of the redox activity of APE1/REF-1, APX2009, reduces the malignant phenotype of breast cancer cells

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/REF-1) is a multifunctional protein acting on cellular signaling pathways, including DNA repair and redox activities. APE1/REF-1 has emerged as a target for cancer therapy, and its role in breast cancer models would reveal new strategies for cancer therapy. APX2009 is a specific APE1/REF-1 redox inhibitor whose anticancer properties have not been described in breast cancer cells. Here, we investigated the effect of the APX2009 treatment in the breast cancer cell lines MDA-MB-231 and MCF-7. Breast cancer cell lines were cultured, and WST1 and colony formation assays were performed to evaluate cell proliferation. Annexin V-FITC/7-AAD and LDH-Glo™ assays were performed to evaluate cell death. The wound healing assay and Matrigel transwell assay were performed after APX2009 treatment to evaluate the cellular migration and invasion processes, respectively. Our findings demonstrated that APX2009 treatment decreased breast cancer cell proliferative, migratory, and invasive properties. Furthermore, it induced apoptosis in both cell lines. Our study is the first to show the effects of APX2009 treatment on apoptosis in a breast cancer cell. Therefore, this study suggested that APX2009 treatment is a promising anticancer molecule for breast cancer.

Role of APE1/Ref-1 in hydrogen peroxide-induced apoptosis in human renal HK-2 cells.

BACKGROUND: Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multipotent protein that plays essential roles in cellular responses to oxidative stress. METHODS: To examine the role of APE1/Ref-1 in ischemia-reperfusion (I/R) injuries and hydrogen peroxide (H2O2)-induced renal tubular apoptosis, we studied male C57BL6 mice and human proximal tubular epithelial (HK-2) cells treated with H2O2 at different concentrations. The colocalization of APE1/Ref-1 in the proximal tubule, distal tubule, thick ascending limb, and collecting duct was observed with confocal microscopy. The overexpression of APE1/Ref-1 with knockdown cell lines using an APE1/Ref-1-specific DNA or small interfering RNA (siRNA) was used for the apoptosis assay. The promotor activity of nuclear factor kappa B (NF-κB) was assessed and electrophoretic mobility shift assay was conducted. RESULTS: APE1/Ref-1 was predominantly localized to the renal tubule nucleus. In renal I/R injuries, the levels of APE1/Ref-1 protein were increased compared with those in kidneys subjected to sham operations. The overexpression of APE1/Ref-1 in HK-2 cells enhanced the Bax/Bcl-2 ratio as a marker of apoptosis. Conversely, the suppression of APE1/Ref-1 expression by siRNA in 1-mM H2O2-treated HK-2 cells decreased the Bax/Bcl-2 ratio, the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38, c-Jun N-terminal kinase (JNK) 1/2, and NF-κB. In HK-2 cells, the promoter activity of NF-κB increased following H2O2 exposure, and this effect was further enhanced by APE1/Ref-1 transfection. CONCLUSION: The inhibition of APE1/Ref-1 with siRNA attenuated H2O2-induced apoptosis through the modulation of mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 and the nuclear activation of NF-κB and proapoptotic factors.

APE1/Ref-1 as a Therapeutic Target for Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is characterized by chronic relapsing inflammation of the gastrointestinal tract. The prevalence of IBD is increasing with approximately 4.9 million cases reported worldwide. Current therapies are limited due to the severity of side effects and long-term toxicity, therefore, the development of novel IBD treatments is necessitated. Recent findings support apurinic/apyrimidinic endonuclease 1/reduction-oxidation factor 1 (APE1/Ref-1) as a target in many pathological conditions, including inflammatory diseases, where APE1/Ref-1 regulation of crucial transcription factors impacts significant pathways. Thus, a potential target for a novel IBD therapy is the redox activity of the multifunctional protein APE1/Ref-1. This review elaborates on the status of conventional IBD treatments, the role of an APE1/Ref-1 in intestinal inflammation, and the potential of a small molecule inhibitor of APE1/Ref-1 redox activity to modulate inflammation, oxidative stress response, and enteric neuronal damage in IBD.

Hydrogen peroxide activates APE1/Ref-1 via NF-κB and Parkin: a role in liver cancer resistance to oxidative stress.

Cancer cells exhibit an altered redox balance and aberrant redox signaling due to genetic, metabolic, and microenvironment-associated reprogramming. Persistently elevated levels of reactive oxygen species (ROS) contribute to many aspects of tumor development and progression. Emerging studies demonstrated the vital role of apurinic/apyrimidinic endonuclease 1 or reduction/oxidation (redox) factor 1(APE1/Ref-1) in the oxidative stress response and survival of cancer cells. APE1/Ref-1 is a multifunctional enzyme involved in the DNA damage response and functions as a redox regulator of transcription factors. We herein demonstrated that basal hydrogen peroxide (H2O2) and APE1/Ref-1 expression levels were markedly higher in cancer cell lines than in non-cancerous cells. Elevated APE1/Ref-1 levels were associated with shorter survival in liver cancer patients. Mechanistically, we showed that H2O2 activated nuclear factor-κB (NF-κB). RelA/p65 inhibited the expression of the E3 ubiquitin ligase Parkin, possibly by interfering with ATF4 activity. Parkin was responsible for the ubiquitination and proteasomal degradation of APE1/Ref-1; therefore, the H2O2-induced suppression of Parkin expression increased APE1/Ref-1 levels. The probability of survival was lower in liver cancer patients with low Parkin and high RelA expression levels. Additionally, Parkin and RelA expression levels negatively and positively correlated with APE1/Ref-1 levels, respectively, in the TCGA liver cancer cohort. We concluded that increases in APE1/Ref-1 via the NF-κB and Parkin pathways are critical for cancer cell survival under oxidative stress. The present results show the potential of the NF-κB-Parkin-APE1/Ref-1 axis as a prognostic factor and therapeutic strategy to eradicate liver cancer.

APE1/Ref-1 Inhibits Adipogenic Transcription Factors during Adipocyte Differentiation in 3T3-L1 Cells.

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein involved in DNA repair and redox regulation. The redox activity of APE1/Ref-1 is involved in inflammatory responses and regulation of DNA binding of transcription factors related to cell survival pathways. However, the effect of APE1/Ref-1 on adipogenic transcription factor regulation remains unknown. In this study, we investigated the effect of APE1/Ref-1 on the regulation of adipocyte differentiation in 3T3-L1 cells. During adipocyte differentiation, APE1/Ref-1 expression significantly decreased with the increased expression of adipogenic transcription factors such as CCAAT/enhancer binding protein (C/EBP)-α and peroxisome proliferator-activated receptor (PPAR)-γ, and the adipocyte differentiation marker adipocyte protein 2 (aP2) in a time-dependent manner. However, APE1/Ref-1 overexpression inhibited C/EBP-α, PPAR-γ, and aP2 expression, which was upregulated during adipocyte differentiation. In contrast, silencing APE1/Ref-1 or redox inhibition of APE1/Ref-1 using E3330 increased the mRNA and protein levels of C/EBP-α, PPAR-γ, and aP2 during adipocyte differentiation. These results suggest that APE1/Ref-1 inhibits adipocyte differentiation by regulating adipogenic transcription factors, suggesting that APE1/Ref-1 is a potential therapeutic target for regulating adipocyte differentiation.

Identification of Novel Pathways Regulated by APE1/Ref-1 in Human Retinal Endothelial Cells.

APE1/Ref-1 (apurinic/apyrimidinic endonuclease 1, APE1 or APEX1; redox factor-1, Ref-1) is a dual-functional enzyme with crucial roles in DNA repair, reduction/oxidation (redox) signaling, and RNA processing and metabolism. The redox function of Ref-1 regulates several transcription factors, such as NF-κB, STAT3, HIF-1α, and others, which have been implicated in multiple human diseases, including ocular angiogenesis, inflammation, and multiple cancers. To better understand how APE1 influences these disease processes, we investigated the effects of APEX1 knockdown (KD) on gene expression in human retinal endothelial cells. This abolishes both DNA repair and redox signaling functions, as well as RNA interactions. Using RNA-seq analysis, we identified the crucial signaling pathways affected following APEX1 KD, with subsequent validation by qRT-PCR. Gene expression data revealed that multiple genes involved in DNA base excision repair, other DNA repair pathways, purine or pyrimidine metabolism signaling, and histidine/one carbon metabolism pathways were downregulated by APEX1 KD. This is in contrast with the alteration of pathways by APEX1 KD in human cancer lines, such as pancreatic ductal adenocarcinoma, lung, HeLa, and malignant peripheral nerve sheath tumors. These results highlight the unique role of APE1/Ref-1 and the clinical therapeutic potential of targeting APE1 and pathways regulated by APE1 in the eye. These findings provide novel avenues for ocular neovascularization treatment.

Molecular Targets to Alleviate Enteric Neuropathy and Gastrointestinal Dysfunction.

Enteric neuropathy underlies long-term gastrointestinal (GI) dysfunction associated with several pathological conditions. Our previous studies have demonstrated that structural and functional changes in the enteric nervous system (ENS) result in persistent alterations of intestinal functions long after the acute insult. These changes lead to aberrant immune response and chronic dysregulation of the epithelial barrier. Damage to the ENS is prognostic of disease progression and plays an important role in the recurrence of clinical manifestations. This suggests that the ENS is a viable therapeutic target to alleviate chronic intestinal dysfunction. Our recent studies in preclinical animal models have progressed into the development of novel therapeutic strategies for the treatment of enteric neuropathy in various chronic GI disorders. We have tested the anti-inflammatory and neuroprotective efficacy of novel compounds targeting specific molecular pathways. Ex vivo studies in human tissues freshly collected after resection surgeries provide an understanding of the molecular mechanisms involved in enteric neuropathy. In vivo treatments in animal models provide data on the efficacy and the mechanisms of actions of the novel compounds and their combinations with clinically used therapies. These novel findings provide avenues for the development of safe, cost-effective, and highly efficacious treatments of GI disorders.

Dual Function of Secreted APE1/Ref-1 in TNBC Tumorigenesis: An Apoptotic Initiator and a Regulator of Chronic Inflammatory Signaling.

The simultaneous regulation of cancer cells and inflammatory immune cells in the tumor microenvironment (TME) can be an effective strategy in treating aggressive breast cancer types, such as triple-negative breast cancer (TNBC). Apurinic/apyrimidinic endonuclease 1/redox effector factor 1 (APE1/Ref-1) is a multi-functional nuclear protein that can be stimulated and then secreted. The extracellular APE1/Ref-1 causes a reduction in disulfide bonds in cytokine receptors, resulting in their conformational changes, thereby inhibiting inflammatory signaling. Furthermore, the secreted APE1/Ref-1 in response to acetylation has been shown to bind to a receptor for the advanced glycation end product (RAGE), initiating the apoptotic cell death of TNBC in vitro and in vivo. This study used PPTLS-APE1/Ref-1 in an adenovirus vector (Ad-PPTLS-APE1/Ref-1) for the constant expression of extracellular APE1/Ref-1, and our results demonstrated its dual function as an apoptotic initiator and inflammation regulator. Injecting MDA-MB 231 orthotopic xenografts with the Ad-PPTLS-APE1/Ref-1 inhibited tumor growth and development in response to acetylation. Moreover, Ad-PPTLS-APE1/Ref-1 generated reactive oxygen species (ROS), and tumor tissues derived from these xenografts exhibited apoptotic bodies. Compared to normal mice, a comparable ratio of anti- and pro-inflammatory cytokines was observed in the plasma of Ad-PPTLS-APE1/Ref-1-injected mice. Mechanistically, the disturbed cytokine receptor by reducing activity of PPTLS-APE1/Ref-1 inhibited inflammatory signaling leading to the inactivation of the p21-activated kinase 1-mediated signal transducer and activator of transcription 3/nuclear factor-κB axis in tumor tissues. These results suggest that the regulation of inflammatory signaling with adenoviral-mediated PPTLS-APE1/Ref-1 in tumors modulates the secretion of pro-inflammatory cytokines in TME, thereby inhibiting aggressive cancer cell progression, and could be considered as a promising and safe therapeutic strategy for treating TNBCs.

New Horizons for the Roles and Association of APE1/Ref-1 and ABCA1 in Atherosclerosis.

Atherosclerosis is the leading cause of death worldwide. APE1/Ref-1 and ABCA1 play key roles in the progression of atherosclerosis. APE1/Ref-1 suppresses atherosclerosis via multiple mechanisms, including reducing the IL-6-, TNF-α-, and IL-1ß-mediated proinflammatory responses, suppressing ROS-mediated oxidant activity and Bax/Bcl-2-mediated vascular calcification and apoptosis, and reducing LOX-1-mediated cholesterol uptake. However, APE1/Ref-1 also promotes atherosclerosis by increasing the activity of the NK-κB and S1PR1 pathways. APE1/Ref-1 localizes to the nucleus, cytoplasm, and mitochondria and can be secreted from the cell. APE1/Ref-1 localization is dynamically regulated by the disease state and may be responsible for its proatherogenic and antiatherogenic effects. ABCA1 promotes cholesterol efflux and anti-inflammatory responses by binding to apoA-I and regulates apoptotic cell clearance and HSPC proliferation to protect against inflammatory responses. Interestingly, in addition to mediating these functions, ABCA1 promotes the secretion of acetylated APE1/Ref-1 (AcAPE1/Ref-1), a therapeutic target, which protects against atherosclerosis development. The APE1/Ref-1 inhibitor APX3330 is being evaluated in a phase II clinical trial. The LXR agonist LXR-623 (WAY-252623) is an agonist of ABCA1 and the first LXR-targeting compound to be evaluated in clinical trials. In this article, we review the roles of ABCA1 and APE1/Ref-1 in atherosclerosis and focus on new insights into the ABCA1-APE1/Ref-1 axis and its potential as a novel therapeutic target in atherosclerosis.

APE1/Ref-1 as a Novel Target for Retinal Diseases.

APE1/Ref-1 (also called Ref-1) has been extensively studied for its role in DNA repair and reduction-oxidation (redox) signaling. The review titled: "The multifunctional APE1 DNA repair-redox signaling protein as a drug target in human disease" by Caston et. al. summarizes the molecular functions of Ref-1 and the role it plays in a number of diseases, with a specific focus on various types of cancer [1]. Previous studies have demonstrated that Ref-1 plays a critical role in regulating specific transcription factors (TFs) involved in a number of pathways, not only in cancer, but other disease indications as well. Disease indications of particular therapeutic interest include retinal vascular diseases such as diabetic retinopathy (DR), diabetic macular edema (DME), and neovascular age-related macular degeneration (nvAMD). While Ref-1 controls a number of TFs that are under redox regulation, three have been found to directly link cancer studies to retinal diseases; HIF-1α, NF-κB and STAT3. HIF-1α controls the expression of VEGF for angiogenesis while NF-κB and STAT3 regulate a number of known cytokines and factors involved in inflammation. These pathways are highly implicated and validated as major players in DR, DME and AMD. Therefore, findings in cancer studies for Ref-1 and its inhibition may be translated to these ocular diseases. This report discusses the path from cancer to the potential treatment of retinal disease, the Ref-1 redox signaling function as a possible target, and the current small molecules which have been identified to block this activity. One molecule, APX3330, is in clinical trials, while the others are in preclinical development. Inhibition of Ref-1 and its effects on inflammation and angiogenesis makes it a potential new therapeutic target for the treatment of retinal vascular diseases. This commentary summarizes the retinal-relevant research that built on the results summarized in the review by Caston et. al. [1].

Clinical value of serum Ape1/Ref-1 combined with TGF-ß1 monitoring in predicting the occurrence of radiation pneumonitis (RP) in non-small cell lung cancer patients.

BACKGROUND: The aim of the present study was to explore the predictive value of serum apurinic/apyrimidinic endonuclease 1/redox factor-1 (Ape1/Ref-1) combined with transforming growth factor ß1 (TGF-ß1) levels in the occurrence of radiation pneumonitis (RP) in patients with non-small cell lung cancer (NSCLS). METHODS: Eighty-one patients with NSCLS who were admitted from August 2017 to July 2019 were enrolled in the present study. All patients were treated with concurrent radiotherapy and chemotherapy. Serum Ape1/Ref-1 and TGF-ß1 levels were measured before treatment and 12 weeks after treatment. Patients with radiation-induced lung injury were assessed and divided into the RP group (lung injury ≥2) and non-RP (NRP) group (grade <2). The levels of serum Ape1/Ref-1 and TGF-ß1 before and after treatment between the 2 groups were compared. The relationship between clinical characteristics, serum Ape1/Ref-1, TGF-ß1 levels, and the occurrence of RP were then analyzed, and the relationship between serum Ape1/Ref-1, TGF-ß1 levels, and their predictive value for the occurrence of RP was also assessed. RESULTS: The incidence of RP in 81 patients was 30.86%. After treatment, the serum Ape1/Ref-1 and TGF-ß1 levels of the 2 groups were significantly higher than those before treatment (P<0.05). Furthermore, after treatment, the levels of serum Ape1/Ref-1 and TGF-ß1 in the RP group were significantly higher than those of the NRP group (P<0.05). Multivariate logistic regression analysis showed that V20, Ape1/Ref-1, and TGF-ß1 were associated with the occurrence of RP (P<0.05). The levels of serum Ape1/Ref-1 were positively correlated with TGF-ß1 (r=0.734, P<0.05). Finally, the area under the curve of RP occurrence, which was predicted by the levels of serum Ape1/Ref-1, TGF-ß1, and the combination of both were 0.779, 0.69, and 0.842, respectively. CONCLUSIONS: The occurrence of RP in NSCLS patients is closely related to the levels of serum Ape1/Ref-1 and TGF-ß1, and the combination of both has important predictive values for the occurrence of RP.

Combined inhibition of Ref-1 and STAT3 leads to synergistic tumour inhibition in multiple cancers using 3D and in vivo tumour co-culture models.

With a plethora of molecularly targeted agents under investigation in cancer, a clear need exists to understand which pathways can be targeted simultaneously with multiple agents to elicit a maximal killing effect on the tumour. Combination therapy provides the most promise in difficult to treat cancers such as pancreatic. Ref-1 is a multifunctional protein with a role in redox signalling that activates transcription factors such as NF-κB, AP-1, HIF-1α and STAT3. Formerly, we have demonstrated that dual targeting of Ref-1 (redox factor-1) and STAT3 is synergistic and decreases cell viability in pancreatic cancer cells. Data presented here extensively expands upon this work and provides further insights into the relationship of STAT3 and Ref-1 in multiple cancer types. Using targeted small molecule inhibitors, Ref-1 redox signalling was blocked along with STAT3 activation, and tumour growth evaluated in the presence and absence of the relevant tumour microenvironment. Our study utilized qPCR, cytotoxicity and in vivo analysis of tumour and cancer-associated fibroblasts (CAF) response to determine the synergy of Ref-1 and STAT3 inhibitors. Overall, pancreatic tumours grown in the presence of CAFs were sensitized to the combination of STAT3 and Ref-1 inhibition in vivo. In vitro bladder and pancreatic cancer demonstrated the most synergistic responses. By disabling both of these important pathways, this combination therapy has the capacity to hinder crosstalk between the tumour and its microenvironment, leading to improved tumour response.

Inhibition of APE1/Ref-1 Redox Signaling Alleviates Intestinal Dysfunction and Damage to Myenteric Neurons in a Mouse Model of Spontaneous Chronic Colitis.

BACKGROUND: Inflammatory bowel disease (IBD) associates with damage to the enteric nervous system (ENS), leading to gastrointestinal (GI) dysfunction. Oxidative stress is important for the pathophysiology of inflammation-induced enteric neuropathy and GI dysfunction. Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a dual functioning protein that is an essential regulator of the cellular response to oxidative stress. In this study, we aimed to determine whether an APE1/Ref-1 redox domain inhibitor, APX3330, alleviates inflammation-induced oxidative stress that leads to enteric neuropathy in the Winnie murine model of spontaneous chronic colitis. METHODS: Winnie mice received APX3330 or vehicle via intraperitoneal injections over 2 weeks and were compared with C57BL/6 controls. In vivo disease activity and GI transit were evaluated. Ex vivo experiments were performed to assess functional parameters of colonic motility, immune cell infiltration, and changes to the ENS. RESULTS: Targeting APE1/Ref-1 redox activity with APX3330 improved disease severity, reduced immune cell infiltration, restored GI function ,and provided neuroprotective effects to the enteric nervous system. Inhibition of APE1/Ref-1 redox signaling leading to reduced mitochondrial superoxide production, oxidative DNA damage, and translocation of high mobility group box 1 protein (HMGB1) was involved in neuroprotective effects of APX3330 in enteric neurons. CONCLUSIONS: This study is the first to investigate inhibition of APE1/Ref-1's redox activity via APX3330 in an animal model of chronic intestinal inflammation. Inhibition of the redox function of APE1/Ref-1 is a novel strategy that might lead to a possible application of APX3330 for the treatment of IBD.

Plasma APE1/Ref-1 Correlates with Atherosclerotic Inflammation in ApoE-/- Mice.

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.

The Biological Role of Apurinic/Apyrimidinic Endonuclease1/Redox Factor-1 as a Therapeutic Target for Vascular Inflammation and as a Serologic Biomarker.

Endothelial dysfunction promotes vascular inflammation by inducing the production of reactive oxygen species and adhesion molecules. Vascular inflammation plays a key role in the pathogenesis of vascular diseases and atherosclerotic disorders. However, whether there is an endogenous system that can participate in circulating immune surveillance or managing a balance in homeostasis is unclear. Apurinic/apyrimidinic endonuclease 1/redox factor-1 (henceforth referred to as APE1/Ref-1) is a multifunctional protein that can be secreted from cells. It functions as an apurinic/apyrimidinic endonuclease in the DNA base repair pathway and modulates redox status and several types of transcriptional factors, in addition to its anti-inflammatory activity. Recently, it was reported that the secretion of APE1/Ref-1 into the extracellular medium of cultured cells or its presence in the plasma can act as a serological biomarker for certain disorders. In this review, we summarize the possible biological functions of APE1/Ref-1 according to its subcellular localization or its extracellular secretions, as therapeutic targets for vascular inflammation and as a serologic biomarker.

ATP Binding Cassette Transporter A1 is Involved in Extracellular Secretion of Acetylated APE1/Ref-1.

Acetylation of nuclear apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE1/Ref-1) is associated with its extracellular secretion, despite the lack of an N-terminal protein secretion signal. In this study, we investigated plasma membrane targeting and translocation of APE1/Ref-1 in HEK293T cells with enhanced acetylation. While APE1/Ref-1 targeting was not affected by inhibition of the endoplasmic reticulum/Golgi-dependent secretion, its secretion was reduced by inhibitors of ATP-binding cassette (ABC) transporters, and siRNA-mediated down-regulation of ABC transporter A1. The association between APE1/Ref-1 and ABCA1 transporter was confirmed by proximal ligation assay and immunoprecipitation experiments. An APE1/Ref-1 construct with mutated acetylation sites (K6/K7R) showed reduced co-localization with ABC transporter A1. Exposure of trichostatin A (TSA) induced the acetylation of APE1/Ref-1, which translocated into membrane fraction. Taken together, acetylation of APE1/Ref-1 is considered to be necessary for its extracellular targeting via non-classical secretory pathway using the ABCA1 transporter.

[Correlations between Ape1/Ref-1, ICAM-1 and IL-17A Levels in Serum and Radiation Pneumonitis for Local Advanced Non-small Cell Lung Cancer Patients].

BACKGROUND: The main manifestations of radiation pneumonitis are injury of alveolar epithelial and endothelial cells, abnormal expression of cytokines, abnormal proliferation of fibroblasts and synthesis of fibrous matrix. The occurrence of radiation pneumonitis is associated with multiplecytokine level abnormality. These cytokines can also be used as bio-markers to predict the occurrence of radiation pneumonitis. This study was to evaluate the correlation between the change of apurinic/apyrimidinic endonuclease 1/redox factor-1 (Ape1/Ref-1), intercellular adhesion molecules 1 (ICAM-1) and interleukin-17A (IL-17A) before and after radiotherapy and radiation pneumonitis for local advanced non-small cell lung cancer (NSCLC) patients with concurrent chemoradiotherapy. METHODS: NSCLC patients (68 cases) were treated with concurrent radiotherapy and chemotherapy, every patient's normal tissue were controlled with a same radation dose. 68 local advanced NSCLC patients with concurrent chemoradiotherapy were detected the levels of Ape1/Ref-1, ICAM-1 and IL-17A in serum by ELISA before radiotherapy and in the 14th week after radiotherapy. Acute and advanced radiation pulmonary injury was graded according to Radiation Therapy Oncology Group/European Organization For Research and Treatment (RTOG/EORTC) diagnostic and grading criteria. Grade 2 or more radiation pneumonitis was taken as the main end point. RESULTS: Eighteen cases out of 68 developed radiation pneumonitis, 50 of 68 cases have no radiation pneumonia development. There was no significant change of Ape1/Ref-1 levels before and after radiotherapy in radiation pneumonitis group (P>0.05). There was no significant change of Ape1/Ref-1 concentration in serum after radiotherapy between radiation pneumonitis group and non-radiation pneumonitis group (P>0.05). Compared with before radiotherapy, upregulation degree of ICAM-1 levels in radiation pneumonitis group was significantly higher than that in non- radiation pneumonitis group (P<0.05). There was no significant change of IL-17A concentration before and after radiotherapy in radiation pneumonitis group, but after radiotherapy IL-17A concentration in serum were remarkably higher than that in non-radiation pneumonitis group (P<0.05). Correlation analysis found that the change of ICAM-1 before and after radiotherapy has no obvious correlation with the incidence of radiation pneumonitis, and IL-17A change has obvious correlation with the incidence of radiation pneumonitis. CONCLUSIONS: On the basis of strictly controlling radiation dose on normal tissue, IL-17A in serum could be the predictive factors of radiation pneumonitis for local advanced NSCLC patients with concurrent chemoradiotherapy.

APE1/Ref-1 redox function contributes to inflammatory pain sensitization.

Inflammatory pain is a complex and multifactorial disorder. Apurinic/apyrimidinic endonuclease 1 (APE1), also called Redox Factor-1 (Ref-1), is constitutively expressed in the central nervous system and regulates various cellular functions including oxidative stress. In the present study, we investigated APE1 modulation and associated pain behavior changes in the complete Freund's adjuvant (CFA) model of inflammatory pain in rats. In addition we tested the anti-inflammatory effects of E3330, a selective inhibitor of APE1-redox activity, in CFA pain condition. We demonstrate that APE1 expression and subcellular distribution are significantly altered in rats at 4 days post CFA injection. We observed around 30% reduction in the overall APE1 mRNA and protein levels. Interestingly, our data point to an increased nuclear accumulation in the inflamed group as compared to the sham group. E3330 inhibitor injection in CFA rats normalized APE1 mRNA expression and changed its distribution toward cytosolic accumulation. Furthermore, intrathecal injection of E3330 decreased inflammation (i.e. reduced IL-6 expression) and alleviated pain, as assessed by measuring the paw withdrawal threshold with the von Frey test. In conclusion, our data indicate that changes in APE1 expression and sub-cellular distribution are implicated in inflammatory pain mechanisms mediated by APE1 redox functions. Further studies are required to elucidate the exact function of APE1 in inflammatory pain processes.

APE1/Ref-1 redox-specific inhibition decreases survivin protein levels and induces cell cycle arrest in prostate cancer cells.

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 NFĸB 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 NFĸB 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.

Correlations between Ape1/Ref-1, ICAM-1 and IL-17A Levels in Serum and Radiation Pneumonitis for Local Advanced Non-small Cell Lung Cancer Patients / 中国肺癌杂志

BACKGROUND@#The main manifestations of radiation pneumonitis are injury of alveolar epithelial and endothelial cells, abnormal expression of cytokines, abnormal proliferation of fibroblasts and synthesis of fibrous matrix. The occurrence of radiation pneumonitis is associated with multiplecytokine level abnormality. These cytokines can also be used as bio-markers to predict the occurrence of radiation pneumonitis. This study was to evaluate the correlation between the change of apurinic/apyrimidinic endonuclease 1/redox factor-1 (Ape1/Ref-1), intercellular adhesion molecules 1 (ICAM-1) and interleukin-17A (IL-17A) before and after radiotherapy and radiation pneumonitis for local advanced non-small cell lung cancer (NSCLC) patients with concurrent chemoradiotherapy.@*METHODS@#NSCLC patients (68 cases) were treated with concurrent radiotherapy and chemotherapy, every patient's normal tissue were controlled with a same radation dose. 68 local advanced NSCLC patients with concurrent chemoradiotherapy were detected the levels of Ape1/Ref-1, ICAM-1 and IL-17A in serum by ELISA before radiotherapy and in the 14th week after radiotherapy. Acute and advanced radiation pulmonary injury was graded according to Radiation Therapy Oncology Group/European Organization For Research and Treatment (RTOG/EORTC) diagnostic and grading criteria. Grade 2 or more radiation pneumonitis was taken as the main end point.@*RESULTS@#Eighteen cases out of 68 developed radiation pneumonitis, 50 of 68 cases have no radiation pneumonia development. There was no significant change of Ape1/Ref-1 levels before and after radiotherapy in radiation pneumonitis group (P>0.05). There was no significant change of Ape1/Ref-1 concentration in serum after radiotherapy between radiation pneumonitis group and non-radiation pneumonitis group (P>0.05). Compared with before radiotherapy, upregulation degree of ICAM-1 levels in radiation pneumonitis group was significantly higher than that in non- radiation pneumonitis group (P<0.05). There was no significant change of IL-17A concentration before and after radiotherapy in radiation pneumonitis group, but after radiotherapy IL-17A concentration in serum were remarkably higher than that in non-radiation pneumonitis group (P<0.05). Correlation analysis found that the change of ICAM-1 before and after radiotherapy has no obvious correlation with the incidence of radiation pneumonitis, and IL-17A change has obvious correlation with the incidence of radiation pneumonitis.@*CONCLUSIONS@#On the basis of strictly controlling radiation dose on normal tissue, IL-17A in serum could be the predictive factors of radiation pneumonitis for local advanced NSCLC patients with concurrent chemoradiotherapy.