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.

Enhancing APE1 detection through apurinic/apyrimidinic site inhibition of DNA polymerase: an innovative, highly sensitive approach.

This study presents an innovative method for the highly sensitive detection of apurinic/apyrimidinic endonuclease 1 (APE1), a crucial biomarker and target for cancer diagnosis and treatment. The method is predicated on our discovery that the apurinic or apyrimidinic site (AP site) can inhibit the activity of Taq DNA polymerase. Subsequent experiments further led to the development of a new amplification method based on the digestion activity of Lambda exonuclease. This approach showed potential to detect trace amounts of APE1 in biological samples with high sensitivity.

APE1 inhibition enhances ferroptotic cell death and contributes to hepatocellular carcinoma therapy.

Ferroptosis, a regulated form of cell death triggered by iron-dependent lipid peroxidation, has emerged as a promising therapeutic strategy for cancer treatment, particularly in hepatocellular carcinoma (HCC). However, the mechanisms underlying the regulation of ferroptosis in HCC remain to be unclear. In this study, we have identified a novel regulatory pathway of ferroptosis involving the inhibition of Apurinic/apyrimidinic endonuclease 1 (APE1), a key enzyme with dual functions in DNA repair and redox regulation. Our findings demonstrate that inhibition of APE1 leads to the accumulation of lipid peroxidation and enhances ferroptosis in HCC. At the molecular level, the inhibition of APE1 enhances ferroptosis which relies on the redox activity of APE1 through the regulation of the NRF2/SLC7A11/GPX4 axis. We have identified that both genetic and chemical inhibition of APE1 increases AKT oxidation, resulting in an impairment of AKT phosphorylation and activation, which leads to the dephosphorylation and activation of GSK3ß, facilitating the subsequent ubiquitin-proteasome-dependent degradation of NRF2. Consequently, the downregulation of NRF2 suppresses SLC7A11 and GPX4 expression, triggering ferroptosis in HCC cells and providing a potential therapeutic approach for ferroptosis-based therapy in HCC. Overall, our study uncovers a novel role and mechanism of APE1 in the regulation of ferroptosis and highlights the potential of targeting APE1 as a promising therapeutic strategy for HCC and other cancers.

Characterizing inhibitors of human AP endonuclease 1.

AP endonuclease 1 (APE1) processes DNA lesions including apurinic/apyrimidinic sites and 3´-blocking groups, mediating base excision repair and single strand break repair. Much effort has focused on developing specific inhibitors of APE1, which could have important applications in basic research and potentially lead to clinical anticancer agents. We used structural, biophysical, and biochemical methods to characterize several reported inhibitors, including 7-nitroindole-2-carboxylic acid (CRT0044876), given its small size, reported potency, and widespread use for studying APE1. Intriguingly, NMR chemical shift perturbation (CSP) experiments show that CRT0044876 and three similar indole-2-carboxylic acids bind a pocket distal from the APE1 active site. A crystal structure confirms these findings and defines the pose for 5-nitroindole-2-carboxylic acid. However, dynamic light scattering experiments show the indole compounds form colloidal aggregates that could bind (sequester) APE1, causing nonspecific inhibition. Endonuclease assays show the compounds lack significant APE1 inhibition under conditions (detergent) that disrupt aggregation. Thus, binding of the indole-2-carboxylic acids at the remote pocket does not inhibit APE1 repair activity. Myricetin also forms aggregates and lacks APE1 inhibition under aggregate-disrupting conditions. Two other reported compounds (MLS000552981, MLS000419194) inhibit APE1 in vitro with low micromolar IC50 and do not appear to aggregate in this concentration range. However, NMR CSP experiments indicate the compounds do not bind specifically to apo- or Mg2+-bound APE1, pointing to a non-specific mode of inhibition, possibly DNA binding. Our results highlight methods for rigorous interrogation of putative APE1 inhibitors and should facilitate future efforts to discover compounds that specifically inhibit this important repair enzyme.

Pharmacological inhibition of Ref-1 enhances the therapeutic sensitivity of papillary thyroid carcinoma to vemurafenib.

The use of the BRAF inhibitor vemurafenib exhibits drug resistance in the treatment of thyroid cancer (TC), and finding more effective multitarget combination therapies may be an important solution. In the present study, we found strong correlations between Ref-1 high expression and BRAF mutation, lymph node metastasis, and TNM stage. The oxidative stress environment induced by structural activation of BRAF upregulates the expression of Ref-1, which caused intrinsic resistance of PTC to vemurafenib. Combination inhibition of the Ref-1 redox function and BRAF could enhance the antitumor effects of vemurafenib, which was achieved by blocking the action of Ref-1 on BRAF proteins. Furthermore, combination treatment could cause an overload of autophagic flux via excessive AMPK protein activation, causing cell senescence and cell death in vitro. And combined administration of Ref-1 and vemurafenib in vivo suppressed PTC cell growth and metastasis in a cell-based lung metastatic tumor model and xenogeneic subcutaneous tumor model. Collectively, our study provides evidence that Ref-1 upregulation via constitutive activation of BRAF in PTC contributes to intrinsic resistance to vemurafenib. Combined treatment with a Ref-1 redox inhibitor and a BRAF inhibitor could make PTC more sensitive to vemurafenib and enhance the antitumor effects of vemurafenib by further inhibiting the MAPK pathway and activating the excessive autophagy and related senescence process.

Real-time monitoring and effector screening of APE1 based on rGO assisted DNA nanoprobe.

Human apurinic/pyrimidine endonuclease 1 (APE1) played a critical role in the occurrence, progress and prognosis of tumors through overexpression and subcellular localization. Thus, it has become an important target for enhancing the sensitivity of tumor cells to radiotherapy and chemotherapy. Therefore, detecting and imaging its intracellular activity is of great significance for inhibitor discovery, cancer diagnosis and therapy. In this work, using DNA-based nanoprobe, we developed a new method for monitor intracellular APE1 activity. The detecting system was consisted by single fluorophore labeled hairpin probe and reduced graphene oxide (rGO). The in vitro result showed that a liner response of the detection method ranged from 0.02 U/mL to 2 U/mL with a limit of detection of 0.02 U/mL. Furthermore, this strategy possessing high specificity was successfully applied for APE1-related inhibitor screening using intracellular fluorescence imaging. Panaxytriol, an effective inhibitor of APE1 activity, was screened from traditional Chinese medicine (TCM) and its effect on APE1 activity was monitored in real time in A549 cells. In summary, this sensitive and specific APE1 detection technology is expected to provide an assistance for APE1-related inhibitor screening and diseases diagnosis.

Inhibition of APE1/Ref-1 for Neovascular Eye Diseases: From Biology to Therapy.

Proliferative diabetic retinopathy (PDR), neovascular age-related macular degeneration (nvAMD), retinopathy of prematurity (ROP) and other eye diseases are characterized by retinal and/or choroidal neovascularization, ultimately causing vision loss in millions of people worldwide. nvAMD and PDR are associated with aging and the number of those affected is expected to increase as the global median age and life expectancy continue to rise. With this increase in prevalence, the development of novel, orally bioavailable therapies for neovascular eye diseases that target multiple pathways is critical, since current anti-vascular endothelial growth factor (VEGF) treatments, delivered by intravitreal injection, are accompanied with tachyphylaxis, a high treatment burden and risk of complications. One potential target is apurinic/apyrimidinic endonuclease 1/reduction-oxidation factor 1 (APE1/Ref-1). The multifunctional protein APE1/Ref-1 may be targeted via inhibitors of its redox-regulating transcription factor activation activity to modulate angiogenesis, inflammation, oxidative stress response and cell cycle in neovascular eye disease; these inhibitors also have neuroprotective effects in other tissues. An APE1/Ref-1 small molecule inhibitor is already in clinical trials for cancer, PDR and diabetic macular edema. Efforts to develop further inhibitors are underway. APE1/Ref-1 is a novel candidate for therapeutically targeting neovascular eye diseases and alleviating the burden associated with anti-VEGF intravitreal injections.

APE1 may influence CD4+ naïve T cells on recurrence free survival in early stage NSCLC.

BACKGROUND: It was demonstrated that multifunctional protein APE1 (Apurinic/apyrimidinic endonuclease 1) is closely related to tumor immune microenvironment in a number of investigations, Meanwhile, the abundance of tumor infiltrating lymphocytes (TILs) has been shown as a prognosis indicator in some researches. However, it remains unclear whether APE1 is involved in the process of TILs affecting the prognosis of patients. To this end, we investigated the associations between APE1 and TILs in non-small cell lung cancer (NSCLC) and explored whether APE1 would influence the associations of CD4+ T cells infiltration with the prognosis of patients. METHODS: Genome-wide expression datasets were obtained from the Gene Expression Omnibus (GEO) public database under accession number GSE68465, GSE30219, GSE31210 and GSE50081. MCPcounter and CIBERSORT analysis was conducted to evaluate the abundance of TILs in 1006 NSCLC patients of GEO database. Spearman correlation tests were used to evaluate correlations between abundance of various TILs and APE1 expression. RFS (recurrence free survival) was estimated using the Kaplan-Meier method and the Cox proportional-hazards model. The expression level of APE1 and tumor-infiltrating CD4+ T cells was evaluated by immunohistochemistry (IHC). RESULTS: The results showed that the abundance of CD4+ naïve T cells was negatively associated with the APE1 expression. CD4+ naïve T cells infiltration was a favorable prognostic factor for RFS, however, there was no effect of CD4+ T cells infiltration on RFS in patients with high APE1 expression. Subsequently, it was further confirmed that CD4+ T cells infiltration was negatively associated with the APE1 expression level in 108 NSCLC tissue samples; high CD4+ T cells infiltration was associated with longer RFS in low APE1 expression group but not in APE1 high expression group. CONCLUSION: These results suggested that APE1 may affect the relationship between CD4+ T cells infiltration and prognosis in NSCLC. This study provides new insights into predictors of outcome in patients with NSCLC, and suggests that combining immunotherapy and APE1-targeted therapy may be a promising treatment for NSCLC.

Immunotherapy for breast cancer using EpCAM aptamer tumor-targeted gene knockdown.

New strategies for cancer immunotherapy are needed since most solid tumors do not respond to current approaches. Here we used epithelial cell adhesion molecule EpCAM (a tumor-associated antigen highly expressed on common epithelial cancers and their tumor-initiating cells) aptamer-linked small-interfering RNA chimeras (AsiCs) to knock down genes selectively in EpCAM+ tumors with the goal of making cancers more visible to the immune system. Knockdown of genes that function in multiple steps of cancer immunity was evaluated in aggressive triple-negative and HER2+ orthotopic, metastatic, and genetically engineered mouse breast cancer models. Gene targets were chosen whose knockdown was predicted to promote tumor neoantigen expression (Upf2, Parp1, Apex1), phagocytosis, and antigen presentation (Cd47), reduce checkpoint inhibition (Cd274), or cause tumor cell death (Mcl1). Four of the six AsiC (Upf2, Parp1, Cd47, and Mcl1) potently inhibited tumor growth and boosted tumor-infiltrating immune cell functions. AsiC mixtures were more effective than individual AsiC and could synergize with anti-PD-1 checkpoint inhibition.

Inhibitors of APE1 redox function effectively inhibit γ-herpesvirus replication in vitro and in vivo.

APE1 is a multi-functional protein with a redox function in its N-terminal domain and an apurinic/apyrimidinic endonuclease activity in the C-terminal domain. APE1 redox function plays an important role in regulating cell proliferation and survival through activating specific transcriptional activators. APE1 redox function is also found to be associated with some cancer occurrence. In this study, we demonstrated that APE1 redox function is essential for Epstein-Barr virus (EBV) lytic replication as the silencing of APE1 expression or treatment with APE1 redox inhibitors C10 and E3330 can inhibit EBV lytic replication and virion production. Furthermore, C10 and E3330 also inhibit MHV-68 replication in vitro and in vivo. C10 and E3330 were able to significantly reduce the loss of pulmonary alveoli and thickening of alveolar septa in mice caused by MHV-68 infection. Altogether, (i) APE1 redox function is validated as a new antiviral target; (ii) APE1 redox inhibitors, especially C10, have potentials to be used for the treatment of γ-herpesvirus infection and associated diseases; (iii) MHV-68 is validated to be a surrogate for the study of the pathogenesis and therapy of EBV and KSHV infection in vivo.

The redox function of apurinic/apyrimidinic endonuclease 1 as key modulator in photodynamic therapy.

Photodynamic therapy (PDT) is an anticancer modality depicting an induced oxidative stress as the mechanism of action that ultimately culminates in cell death. The apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a key protein promoting bad prognostic in several cancer types. APE1/Ref-1 is able to regulate cell response to oxidative stress by two basic protein activities, including a reduction-oxidation-function and a DNA repair-function. Therefore, the efficacy of anticancer therapies is negatively affected by APE1-overexpression. Thus, here it was evaluated the potential of APE1-chemical inhibitors as sensitizers for PDT in two different cancer cell lines (A549 and HeLa cells). Both functions of APE1 were addressed using E3330 (redox-function) and CRT0044876 (DNA repair-function) molecules. A detailed cytotoxicity screening (cell viability, cell cycle kinetics, mitochondrial perturbation, and cell death) indicated HeLa cells as extremely sensitive (~ 3.5×) to the combination of PDT with E3330 when compared to A549 cells. The treatment using PDT with E3330 induced downregulation of APE1 as detected by Western Blot. The APE1's downregulation correlated to an increase of DNA fragmentation (17% and 66% in A549 and HeLa cells, respectively) and cell death rate (total: 24% and 74% in A549 and HeLa cells, respectively) characterized by annexin V and 7-AAD markers as well as a considerable difference in superoxide detected in mitochondria (29% and 78% in A549 and HeLa cells, respectively). This study definitively detected an increase in PDT efficacy when APE1's redox function is dysregulated by E3330.

A randomized, double-blind, placebo-controlled study of B-cell lymphoma 2 homology 3 mimetic gossypol combined with docetaxel and cisplatin for advanced non-small cell lung cancer with high expression of apurinic/apyrimidinic endonuclease 1.

Background Overexpression of apurinic/apyrimidinic endonuclease 1 (APE1) is an important cause of poor chemotherapeutic efficacy in advanced non-small cell lung cancer (NSCLC) patients. Gossypol, a new inhibitor of APE1, in combination with docetaxel and cisplatin is believed to improve the efficacy of chemotherapy for advanced NSCLC with high APE1 expression. Methods Sixty-two patients were randomly assigned to two groups. Thirty-one patients in the experimental group received 75 mg/m2 docetaxel and 75 mg/m2 cisplatin on day 1 with gossypol administered at 20 mg once daily on days 1 to 14 every 21 days. The control group received placebo with the same docetaxel and cisplatin regimen. The primary endpoint was progression-free survival (PFS); secondary endpoints included overall survival (OS), response rate, and toxicity. Results There were no significant differences in PFS and OS between the experimental group and the control group. The median PFS (mPFS) in the experimental and control groups was 7.43 and 4.9 months, respectively (HR = 0.54; p = 0.06), and the median OS (mOS) was 18.37 and 14.7 months, respectively (HR = 0.68; p = 0.27). No significant differences in response rate and serious adverse events were found between the groups. Conclusion The experimental group had a better mPFS and mOS than did the control group, though no significant difference was observed. Because the regimen of gossypol combined with docetaxel and cisplatin was well tolerated, future studies with larger sample sizes should be performed.

HMCES Maintains Replication Fork Progression and Prevents Double-Strand Breaks in Response to APOBEC Deamination and Abasic Site Formation.

5-Hydroxymethylcytosine (5hmC) binding, ES-cell-specific (HMCES) crosslinks to apurinic or apyrimidinic (AP, abasic) sites in single-strand DNA (ssDNA). To determine whether HMCES responds to the ssDNA abasic site in cells, we exploited the activity of apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3A (APOBEC3A). APOBEC3A preferentially deaminates cytosines to uracils in ssDNA, which are then converted to abasic sites by uracil DNA glycosylase. We find that HMCES-deficient cells are hypersensitive to nuclear APOBEC3A localization. HMCES relocalizes to chromatin in response to nuclear APOBEC3A and protects abasic sites from processing into double-strand breaks (DSBs). Abasic sites induced by APOBEC3A slow both leading and lagging strand synthesis, and HMCES prevents further slowing of the replication fork by translesion synthesis (TLS) polymerases zeta (Polζ) and kappa (Polκ). Thus, our study provides direct evidence that HMCES responds to ssDNA abasic sites in cells to prevent DNA cleavage and balance the engagement of TLS polymerases.

Salinomycin enhances radiotherapy sensitivity and reduces expressions of BIRC5 and NEIL2 in nasopharyngeal carcinoma.

OBJECTIVE: The aim of this study was to investigate the effects of salinomycin (Sal) on expressions of baculoviral IAP repeat-containing 5 (BIRC5) and Nei endonuclease VIII-like 2 (NEIL2) and radiotherapy sensitivity of nasopharyngeal carcinoma (NPC). MATERIALS AND METHODS: Human NPC CNE-2 cell lines were used as research objects in this study. Subsequently, the cells received intervention with Sal at different concentrations, radioactive rays at different doses and Sal combined with radioactive rays. The growth inhibition rate of CNE-2 cells was detected via methyl thiazolyl tetrazolium (MTT) assay. The dose-effect relations of Sal, radioactive rays and combination therapy with the inhibitory effect on CNE-2 cells were obtained. CNE-2 cells receiving intervention with Sal at an appropriate concentration or radioactive rays at an appropriate dose alone and Sal combined with radioactive rays were used as intervention groups (Sal group, Radiation group and Combination group). However, those added with an equal amount of DMSO were set as Control group. Next, the cycle, apoptosis and apoptotic morphology of CNE-2 cells were observed via flow cytometry and Hoechst assay, respectively. Moreover, the expressions of apoptosis-related proteins Caspase-3, B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax), as well as BIRC5 and NEIL2 proteins in CNE-2 cells were determined using Western blotting. RESULTS: Under the intervention with Sal or radioactive rays alone, the growth inhibition rate of CNE-2 cells rose in a concentration/dose-dependent manner. With the increase in Sal concentration in combination therapy, the growth inhibition rate of CNE-2 cells significantly increased (p<0.05). Compared with Control group, Sal group, Radiation group, and Combination group exhibited remarkably lower colony formation rate, higher proportion of CNE-2 cells in the G2/M phase, enhanced apoptosis of CNE-2 cells with nuclear fragmentation, increased expressions of pro-apoptotic proteins Caspase-3 and Bax, decreased expression of anti-apoptotic protein Bcl-2, and lower protein expressions of BIRC5 and NEIL2 in cells (p<0.05). Compared with Radiation group, the Combination group had significantly decreased colony formation rate, increased proportion of CNE-2 cells in the G2/M phase, enhanced apoptosis of CNE-2 cells with more nuclear fragmentation and other apoptosis characteristics, increased expressions of pro-apoptotic proteins Caspase-3 and Bax, decreased expression of anti-apoptotic protein Bcl-2, and decreased protein expressions of BIRC5 and NEIL2 in cells (p<0.05). CONCLUSIONS: Sal enhances the radiotherapy sensitivity of NPC and reduces the protein expressions of BIRC5 and NEIL2 in cells.

Inhibition of APE1-endonuclease activity affects cell metabolism in colon cancer cells via a p53-dependent pathway.

The pathogenesis of colorectal cancer (CRC) involves different mechanisms, such as genomic and microsatellite instabilities. Recently, a contribution of the base excision repair (BER) pathway in CRC pathology has been emerged. In this context, the involvement of APE1 in the BER pathway and in the transcriptional regulation of genes implicated in tumor progression strongly correlates with chemoresistance in CRC and in more aggressive cancers. In addition, the APE1 interactome is emerging as an important player in tumor progression, as demonstrated by its interaction with Nucleophosmin (NPM1). For these reasons, APE1 is becoming a promising target in cancer therapy and a powerful prognostic and predictive factor in several cancer types. Thus, specific APE1 inhibitors have been developed targeting: i) the endonuclease activity; ii) the redox function and iii) the APE1-NPM1 interaction. Furthermore, mutated p53 is a common feature of advanced CRC. The relationship between APE1 inhibition and p53 is still completely unknown. Here, we demonstrated that the inhibition of the endonuclease activity of APE1 triggers p53-mediated effects on cell metabolism in HCT-116 colon cancer cell line. In particular, the inhibition of the endonuclease activity, but not of the redox function or of the interaction with NPM1, promotes p53 activation in parallel to sensitization of p53-expressing HCT-116 cell line to genotoxic treatment. Moreover, the endonuclease inhibitor affects mitochondrial activity in a p53-dependent manner. Finally, we demonstrated that 3D organoids derived from CRC patients are susceptible to APE1-endonuclease inhibition in a p53-status correlated manner, recapitulating data obtained with HCT-116 isogenic cell lines. These findings suggest the importance of further studies aimed at testing the possibility to target the endonuclease activity of APE1 in CRC.

Antitumor Activity and Mechanistic Characterization of APE1/Ref-1 Inhibitors in Bladder Cancer.

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-κB 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.

Rewiring E2F1 with classical NHEJ via APLF suppression promotes bladder cancer invasiveness.

BACKGROUND: Bladder cancer progression has been associated with dysfunctional repair of double-strand breaks (DSB), a deleterious type of DNA lesions that fuel genomic instability. Accurate DSB repair relies on two distinct pathways, homologous recombination (HR) and classical non-homologous end-joining (c-NHEJ). The transcription factor E2F1 supports HR-mediated DSB repair and protects genomic stability. However, invasive bladder cancers (BC) display, in contrast to non-invasive stages, genomic instability despite their high E2F1 levels. Hence, E2F1 is either inefficient in controlling DSB repair in this setting, or rewires the repair apparatus towards alternative, error-prone DSB processing pathways. METHODS: RT-PCR and immunoblotting, in combination with bioinformatics tools were applied to monitor c-NHEJ factors status in high-E2F1-expressing, invasive BC versus low-E2F1-expressing, non-invasive BC. In vivo binding of E2F1 on target gene promoters was demonstrated by ChIP assays and E2F1 CRISPR-Cas9 knockdown. MIR888-dependent inhibition of APLF by E2F1 was demonstrated using overexpression and knockdown experiments, in combination with luciferase assays. Methylation status of MIR888 promoter was monitored by methylation-specific PCR. The changes in invasion potential and the DSB repair efficiency were estimated by Boyden chamber assays and pulse field electrophoresis, correspondingly. RESULTS: Herein, we show that E2F1 directly transactivates the c-NHEJ core factors Artemis, DNA-PKcs, ligase IV, NHEJ1, Ku70/Ku80 and XRCC4, but indirectly inhibits APLF, a chromatin modifier regulating c-NHEJ. Inhibition is achieved by miR-888-5p, a testis-specific, X-linked miRNA which, in normal tissues, is often silenced via promoter methylation. Upon hypomethylation in invasive BC cells, MIR888 is transactivated by E2F1 and represses APLF. Consequently, E2F1/miR-888/APLF rewiring is established, generating conditions of APLF scarcity that compromise proper c-NHEJ function. Perturbation of the E2F1/miR-888/APLF axis restores c-NHEJ and ameliorates cell invasiveness. Depletion of miR-888 can establish a 'high E2F1/APLF/DCLRE1C' signature, which was found to be particularly favorable for BC patient survival. CONCLUSION: Suppression of the 'out-of-context' activity of miR-888 improves DSB repair and impedes invasiveness by restoring APLF.

The extracellular role of Ref-1 as anti-inflammatory function in lipopolysaccharide-induced septic mice.

Apurinic/apyrimidinic endonuclease/redox factor-1 (Ref-1), a multifunctional protein secreted from stimulated cells, has been identified as a new serological biomarker. Despite recent reports on the role of Ref-1 in inflammation, the biological function of secreted Ref-1 remains unknown, especially in vivo. This study aimed to evaluate the possible roles of secreted Ref-1 in lipopolysaccharide-induced systemic inflammation in vivo. We generated a secretory Ref-1 adenoviral vector system, AdPPT-LS-Ref-1, by conjugation of preprotrypsin leading sequence (PPT-LS) with full-length Ref-1 sequences. Expression of tumor necrosis factor-α (TNF-α)-induced vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells and lipopolysaccharide (LPS)-induced cyclooxygenase-2 in Raw264.7 cells was inhibited by secretory Ref-1, and this inhibitory effect was abrogated following neutralization of Ref-1 with anti-Ref-1 antibody. Plasma Ref-1 levels following administration of AdPPT-LS-Ref-1 (2 × 109 ifu, i.p.) for 24 h were substantially higher than those recorded following administration of Adßgal (84.6 ±â€¯7.2 ng/ml vs. 4.4 ±â€¯1.5 ng/ml). Treatment with LPS (10 mg/kg, i.v. for 6 h) markedly increased VCAM-1 expression, cathepsin or myeloperoxidase activity, which were significantly suppressed by treatment with AdPPT-LS-Ref-1. Furthermore, LPS-induced cytokines, such as TNF-α, interleukin (IL)-1ß, IL-6, and monocyte chemoattractant protein 1, were significantly inhibited in AdPPT-LS-Ref-1-treated mice. However, LPS-induced myeloperoxidase activities were not suppressed by treatment with the redox mutant of secretory Ref-1, AdPPT-LS-Ref-1(C65A/C93A), or wild-type AdRef-1. Collectively, these results suggest that secreted Ref-1 has anti-inflammatory properties and that its redox cysteine residue is associated with the anti-inflammatory activity in vivo. Furthermore, our findings indicate that secretory Ref-1 may be useful as a therapeutic biomolecule against systemic inflammation.

AP endonuclease 1 (Apex1) influences brain development linking oxidative stress and DNA repair.

Brain and neurons are particularly sensitive to reactive oxygen species (ROS). Oxidative damage from ROS results in increased 8-oxoguanine in DNA followed by repair through the base excision repair (BER) pathway. We reported earlier that AP endonuclease 1 (Apex1) not only participates directly in BER but also regulates transcription factor Creb1. Here, we investigated how Apex1 affects brain to respond effectively to oxidative damage during zebrafish development. Loss of Apex1 resulted in increased ROS, 8-oxoguanine, and abasic sites as well as loss of Ogg1, which recognizes 8-oxoguanine and is required for its repair. Moreover, knock-down of Apex1 not only resulted in reduction of expression of several major proteins in the BER pathway (Polb and Ogg1), and it also resulted in maldistribution and loss of four key brain transcription factors (fezf2, otx2, egr2a, and pax2a), leading to abnormal brain development. These results were independent of p53 protein level. In contrast, exposure to exogenous H2O2 resulted in increased transcription and protein of Apex1 along with other BER components, as well as Creb1. Taken together, these results indicate that oxidative stress increased when the level of Apex1 was reduced, revealing a novel pathway of how Apex1 manages oxidative stress in developing brain.

Curcumin is an APE1 redox inhibitor and exhibits an antiviral activity against KSHV replication and pathogenesis.

Curcumin, a polyphenol, is the main bioactive compound in dietary spice turmeric curcuma longa. It possesses anti-inflammatory, anti-oxidant and anti-neoplastic properties and shows potentials in treating or preventing particular diseases such as oxidative and inflammatory conditions, metabolic syndrome, arthritis, anxiety, hyperlipidemia and cancers. The diverse range and potential health beneficial effects has generated enthusiasm leading to intensive investigation into the phytochemical. However, a concern has been also raised if curcumin has a promiscuous bioassay profile and is a Pan-Assay INterference compound (PAINS). Here we present evidence indicating that curcumin is not a PAINS, but an inhibitor to APE1 redox function that affects many genes and pathways. This discovery explains the wide range of effects of curcumin on diverse human diseases and predicts a potential application in treatment of viral infection and virus-associated cancer. As a proof-of-concept, we demonstrated that curcumin is able to efficiently block Kaposi's sarcoma-associated herpesvirus replication and inhibit the pathogenic processes of angiogenesis and cell invasion.