Meihua Luo

Role of the Multifunctional DNA Repair and Redox Signaling Protein Ape1/Ref-1 in Cancer and Endothelial Cells: Small-Molecule Inhibition of the Redox Function of Ape1

The DNA base excision-repair pathway is responsible for the repair of DNA damage caused by oxidation/alkylation and protects cells against the effects of endogenous and exogenous agents. Removal of the damaged base creates a baseless (AP) site. AP endonuclease1 (Ape1) acts on this site to continue the BER-pathway repair. Failure to repair baseless sites leads to DNA strand breaks and cytotoxicity. In addition to the repair role of Ape1, it also functions as a major redox-signaling factor to reduce and activate transcription factors such as AP1, p53, HIF-1alpha, and others that control the expression of genes important for cell survival and cancer promotion and progression. Thus, the Ape1 protein interacts with proteins involved in DNA repair, growth-signaling pathways, and pathways involved in tumor promotion and progression. Although knockdown studies with siRNA have been informative in studying the role of Ape1 in both normal and cancer cells, knocking down Ape1 does not reveal the individual role of the redox or repair functions of Ape1. The identification of small-molecule inhibitors of specific Ape1 functions is critical for mechanistic studies and translational applications. Here we discuss small-molecule inhibition of Ape1 redox and its effect on both cancer and endothelial cells.

Redox regulation of DNA repair: implications for human health and cancer therapeutic development.

Redox reactions are known to regulate many important cellular processes. In this review, we focus on the role of redox regulation in DNA repair both in direct regulation of specific DNA repair proteins as well as indirect transcriptional regulation. A key player in the redox regulation of DNA repair is the base excision repair enzyme apurinic/apyrimidinic endonuclease 1 (APE1) in its role as a redox factor. APE1 is reduced by the general redox factor thioredoxin, and in turn reduces several important transcription factors that regulate expression of DNA repair proteins. Finally, we consider the potential for chemotherapeutic development through the modulation of APE1s redox activity and its impact on DNA repair.

Evolution of the redox function in mammalian Apurinic/ apyrimidinic endonuclease

Human apurinic/apyrimidinic endonuclease (hApe1) encodes two important functional activities: an essential base excision repair (BER) activity and a redox activity that regulates expression of a number of genes through reduction of their transcription factors, AP-1, NFkappaB, HIF-1alpha, CREB, p53 and others. The BER function is highly conserved from prokaryotes (E. coli exonuclease III) to humans (hApe1). Here, we provide evidence supporting a redox function unique to mammalian Apes. An evolutionary analysis of Ape sequences reveals that, of the 7 Cys residues, Cys 93, 99, 208, 296, and 310 are conserved in both mammalian and non-mammalian vertebrate Apes, while Cys 65 is unique to mammalian Apes. In the zebrafish Ape (zApe), selected as the vertebrate sequence most distant from human, the residue equivalent to Cys 65 is Thr 58. The wild-type zApe enzyme was tested for redox activity in both in vitro EMSA and transactivation assays and found to be inactive, similar to C65A hApe1. Substitution of Thr 58 with Cys in zApe, however, resulted in a redox active enzyme, suggesting that a Cys residue in this position is indeed critical for redox function. In order to further probe differences between redox active and inactive enzymes, we have determined the crystal structures of vertebrate redox inactive enzymes, the C65A human Ape1 enzyme and the zApe enzyme at 1.9 and 2.3A, respectively. Our results provide new insights on the redox function and highlight a dramatic gain-of-function activity for Ape1 in mammals not found in non-mammalian vertebrates or lower organisms.

Novel Small-Molecule Inhibitor of Apurinic/Apyrimidinic Endonuclease 1 Blocks Proliferation and Reduces Viability of Glioblastoma Cells

Apurinic/apyrimidinic (AP) endonuclease 1 (Ape1) is an essential DNA repair protein that plays a critical role in repair of AP sites via base excision repair. Ape1 has received attention as a druggable oncotherapeutic target, especially for treating intractable cancers such as glioblastoma. The goal of this study was to identify small-molecule inhibitors of Ape1 AP endonuclease. For this purpose, a fluorescence-based high-throughput assay was used to screen a library of 60,000 small-molecule compounds for ability to inhibit Ape1 AP endonuclease activity. Four compounds with IC50 values less than 10 μM were identified, validated, and characterized. One of the most promising compounds, designated Ape1 repair inhibitor 03 [2,4,9-trimethylbenzo[b][1,8]-naphthyridin-5-amine; AR03), inhibited cleavage of AP sites in vivo in SF767 glioblastoma cells and in vitro in whole cell extracts and inhibited purified human Ape1 in vitro. AR03 has low affinity for double-stranded DNA and weakly inhibits the Escherichia coli endonuclease IV, requiring a 20-fold higher concentration than for inhibition of Ape1. AR03 also potentiates the cytotoxicity of methyl methanesulfonate and temozolomide in SF767 cells. AR03 is chemically distinct from the previously reported small-molecule inhibitors of Ape1. AR03 is a novel small-molecule inhibitor of Ape1, which may have potential as an oncotherapeutic drug for treating glioblastoma and other cancers.

Design and Synthesis of Novel Quinone Inhibitors Targeted to the Redox Function of Apurinic/Apyrimidinic Endonuclease 1/Redox Enhancing Factor-1 (Ape1/Ref-1)

The multifunctional enzyme apurinic endonuclease 1/redox enhancing factor 1 (Ape1/ref-1) maintains genetic fidelity through the repair of apurinic sites and regulates transcription through redox-dependent activation of transcription factors. Ape1 can therefore serve as a therapeutic target in either a DNA repair or transcriptional context. Inhibitors of the redox function can be used as either therapeutics or novel tools for separating the two functions for in vitro study. Presently there exist only a few compounds that have been reported to inhibit Ape1 redox activity; here we describe a series of quinones that exhibit micromolar inhibition of the redox function of Ape1. Benzoquinone and naphthoquinone analogues of the Ape1-inhibitor E3330 were designed and synthesized to explore structural effects on redox function and inhibition of cell growth. Most of the naphthoquinones were low micromolar inhibitors of Ape1 redox activity, and the most potent analogues inhibited tumor cell growth with IC(50) values in the 10-20 microM range.

Functional analysis of novel analogues of E3330 that block the redox signaling activity of the multifunctional AP endonuclease/redox signaling enzyme APE1/Ref-1

APE1 is a multifunctional protein possessing DNA repair and redox activation of transcription factors. Blocking these functions leads to apoptosis, antiangiogenesis, cell-growth inhibition, and other effects, depending on which function is blocked. Because a selective inhibitor of the APE redox function has potential as a novel anticancer therapeutic, new analogues of E3330 were synthesized. Mass spectrometry was used to characterize the interactions of the analogues (RN8-51, 10-52, and 7-60) with APE1. RN10-52 and RN7-60 were found to react rapidly with APE1, forming covalent adducts, whereas RN8-51 reacted reversibly. Median inhibitory concentration (IC(50) values of all three compounds were significantly lower than that of E3330. EMSA, transactivation assays, and endothelial tube growth-inhibition analysis demonstrated the specificity of E3330 and its analogues in blocking the APE1 redox function and demonstrated that the analogues had up to a sixfold greater effect than did E3330. Studies using cancer cell lines demonstrated that E3330 and one analogue, RN8-51, decreased the cell line growth with little apoptosis, whereas the third, RN7-60, caused a dramatic effect. RN8-51 shows particular promise for further anticancer therapeutic development. This progress in synthesizing and isolating biologically active novel E3330 analogues that effectively inhibit the APE1 redox function validates the utility of further translational anticancer therapeutic development.

APE1/Ref-1 Regulates STAT3 Transcriptional Activity and APE1/Ref-1-STAT3 Dual-Targeting Effectively Inhibits Pancreatic Cancer Cell Survival

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.

Characterization of the redox activity and disulfide bond formation in Apurinic/apyrimidinic endonuclease

Apurinic/apyrimidinic endonuclease (APE1) is an unusual nuclear redox factor in which the redox-active cysteines identified to date, C65 and C93, are surface inaccessible residues whose activities may be influenced by partial unfolding of APE1. To assess the role of the five remaining cysteines in APE1s redox activity, double-cysteine mutants were analyzed, excluding C65A, which is redox-inactive as a single mutant. C93A/C99A APE1 was found to be redox-inactive, whereas other double-cysteine mutants retained the same redox activity as that observed for C93A APE1. To determine whether these three cysteines, C65, C93, and C99, were sufficient for redox activity, all other cysteines were substituted with alanine, and this protein was shown to be fully redox-active. Mutants with impaired redox activity failed to stimulate cell proliferation, establishing an important role for APE1s redox activity in cell growth. Disulfide bond formation upon oxidation of APE1 was analyzed by proteolysis of the protein followed by mass spectrometry analysis. Within 5 min of exposure to hydrogen peroxide, a single disulfide bond formed between C65 and C138 followed by the formation of three additional disulfide bonds within 15 min; 10 total disulfide bonds formed within 1 h. A single mixed-disulfide bond involving C99 of APE1 was observed for the reaction of oxidized APE1 with thioredoxin (TRX). Disulfide-bonded APE1 or APE1-TRX species were further characterized by size exclusion chromatography and found to form large complexes. Taken together, our data suggest that APE1 is a unique redox factor with properties distinct from those of other redox factors.

Inhibition of apurinic/apyrimidinic endonuclease I's redox activity revisited.

The essential base excision repair protein, apurinic/apyrimidinic endonuclease 1 (APE1), plays an important role in redox regulation in cells and is currently targeted for the development of cancer therapeutics. One compound that binds APE1 directly is (E)-3-[2-(5,6-dimethoxy-3-methyl-1,4-benzoquinonyl)]-2-nonylpropenoic acid (E3330). Here, we revisit the mechanism by which this negatively charged compound interacts with APE1 and inhibits its redox activity. At high concentrations (millimolar), E3330 interacts with two regions in the endonuclease active site of APE1, as mapped by hydrogen-deuterium exchange mass spectrometry. However, this interaction lowers the melting temperature of APE1, which is consistent with a loss of structure in APE1, as measured by both differential scanning fluorimetry and circular dichroism. These results are consistent with other findings that E3330 concentrations of >100 μM are required to inhibit APE1s endonuclease activity. To determine the role of E3330s negatively charged carboxylate in redox inhibition, we converted the carboxylate to an amide by synthesizing (E)-2-[(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)methylene]-N-methoxy-undecanamide (E3330-amide), a novel uncharged derivative. E3330-amide has no effect on the melting temperature of APE1, suggesting that it does not interact with the fully folded protein. However, E3330-amide inhibits APE1s redox activity in in vitro electrophoretic mobility shift redox and cell-based transactivation assays, producing IC(50) values (8.5 and 7 μM) lower than those produced with E3330 (20 and 55 μM, respectively). Thus, E3330s negatively charged carboxylate is not required for redox inhibition. Collectively, our results provide additional support for a mechanism of redox inhibition involving interaction of E3330 or E3330-amide with partially unfolded APE1.

Inhibition of the redox function of APE1/Ref-1 in myeloid leukemia cell lines results in a hypersensitive response to retinoic acid-induced differentiation and apoptosis

OBJECTIVE The standard of care for promyelocytic leukemia includes use of the differentiating agent all-trans retinoic acid (RA) and chemotherapy. RA induces cell differentiation through retinoic acid receptor (RAR) transcription factors. Because redox mechanisms influence how readily transcription factors bind to DNA response elements (RARE), the impact of small molecule (E3330) inhibition of the redox regulatory protein, apurinic-apyrimidinic endonuclease/redox effector factor (APE1/Ref-1) on RAR DNA binding and function in RA-induced myeloid leukemia cell differentiation and apoptosis was investigated. MATERIALS AND METHODS The redox function of APE1 was studied using the small molecule inhibitor E3330 in HL-60 and PLB acute myeloid leukemia cells. Electrophoretic mobility shift assays were employed to determine effect of inhibitor on APE1/Ref-1 redox signaling function. Trypan blue assays, Annexin-V/propidium iodide and CD11b staining, and real-time polymerase chain reaction analyses were employed to determine survival, apoptosis, and differentiation status of cells in culture. RESULTS RARα binds to its RARE in a redox-dependent manner mediated by APE1/Ref-1 redox regulation. Redox-dependent RAR-RARE binding is blocked by E3330, a small molecule redox inhibitor of APE1/Ref-1. Combination treatment of RA + E3330 results in a profound hypersensitivity of myeloid leukemia cells to RA-induced differentiation and apoptosis. Additionally, redox inhibition by E3330 results in enhanced RAR target gene, BLR-1, expression in myeloid leukemia cells. CONCLUSIONS The redox function of APE1/Ref-1 regulates RAR binding to its DNA RAREs influencing the response of myeloid leukemia cells to RA-induced differentiation. Targeting of APE1/Ref-1 redox function may allow manipulation of the retinoid response with therapeutic implications.