NF-κB; APE1(Ref-1)

E3330 (300 mg/kg, p.o.) attenuates the elevation of plasma tumor necrosis factor activity and shields mice against liver damage in endotoxin-mediated hepatitis.[4] E3330 (100 mg/kg, p.o.) also shields rats against severe liver damage caused by galactosamine and endotoxin in a rat model.[5]

Trypan Blue Exclusion Assay [2]
Refer to our previous report. In summary, 2 × 105 PANC1 cells were placed in one well of a 12-well plate and treated with 5 to 30 μmol/L E3330. After a 24, 48, and 72 h of culture, the cells were washed with PBS and stained with trypan blue, and cell viability was examined by counting the live cell numbers.

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Detection of Reactive Oxygen Species by Flow Cytometry [2]
Flow cytometric detection of reactive oxygen species (ROS) was done as described. Briefly, PANC1 cells treated with 20 μmol/L E3330for 2 days and subsequently washed with PBS and resuspended in complete medium followed by incubation with 0.5 μmol/L dihydrorhodamine 123 for 30 min at 37°C. ROS fluorescence intensity was determined by flow cytometry with excitation at 490 nm and emission at 520 nm.

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SHP-2 Oxidation Assay by “Positive Labeling” of Oxidized Thiols with PEO-Iodoacetyl biotin [2]
SHP-2 oxidation was measured by “positive labeling” of oxidized thiols as described previously. In summary, PANC1 cells were serum starved and subsequently stimulated with E3330 for 2 h. Then, the E3330-treated cells were lysated for 1 h at 25°C in an anaerobic chamber. All free thiols were masked with 10 mmol/L NEM and 10 mmol/L iodoacetamide. Reversibly oxidized thiols were reduced with 4 mmol/L DTT in the anaerobic chamber and then were labeled with 0.5 mmol/L PEO-iodoacetyl biotin. Biotin incorporation into immunoprecipitated SHP-2 was detected by Western blotting with horseradish peroxidase–conjugated streptavidin and enhanced chemiluminescence.

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HIF-1α DNA-Binding ELISA Assay [2]
HIF-1α DNA binding was assessed by using HIF-1α DNA-binding ELISA assay kit purchased from Active Motif. HIF-1α DNA binding was assessed as described. PANC1 or XPA1 cells were treated with E3330 at various doses for 6 h. Then, the cells were harvested and whole-cell proteins were isolated. The procedures of ELISA assay were done according to the manufacturer’s protocol.

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Ape1 is a molecule with dual functions in DNA repair and redox regulation of transcription factors. In Ape1-deficient mice, embryos do not survive beyond embryonic day 9, indicating that this molecule is required for normal embryo development. Currently, direct evidence of the role of Ape1 in regulating hematopoiesis is lacking. We used the embryonic stem (ES) cell differentiation system and an siRNA approach to knockdown Ape1 gene expression to test the role of Ape1 in hematopoiesis. Hemangioblast development from ES cells was reduced 2- to 3-fold when Ape1 gene expression was knocked down by Ape1-specific siRNA, as was primitive and definitive hematopoiesis. Impaired hematopoiesis was not associated with increased apoptosis in siRNA-treated cells. To begin to explore the mechanism whereby Ape1 regulates hematopoiesis, we found that inhibition of the redox activity of Ape1 with E3330, a specific Ape1 redox inhibitor, but not Ape1 DNA repair activity, which was blocked using the small molecule methoxyamine, affected cytokine-mediated hemangioblast development in vitro. In summary, these data indicate Ape1 is required in normal embryonic hematopoiesis and that the redox function, but not the repair endonuclease activity, of Ape1 is critical in normal embryonic hematopoietic development [1].

Cell Cycle Analysis by Flow Cytometry [2]
Cell cycle analysis was done on PANC1 cells incubated for 48 h with E3330 (10, 20, or 30 μmol/L). The cells were fixed in chilled methanol overnight before staining with propidium iodide (50 Ag/mL) in the presence of 20 μg/mL RNase and 0.1% NP-40. Analysis was done immediately after staining using a FACScan.

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NF-κB Reporter Assay [2]
PANC1 or XPA1 cells were plated at 1 × 105 per well in 24-well plates on the day before transfection. The cells were transiently transfected by Fugene 6 Transfection Reagent with 950 ng of a NF-κB reporter plasmid (pNF-κB-Luc) and 50 ng of a construct directing expression of Renilla luciferase under the control of a constitutively active thymidine kinase promoter (pRL-TK). Twenty-four hours after transfection, the cells were treated by E3330 at various doses for 6 h, and luciferase activity was measured using a Dual-Luciferase Reporter Assay System according to the manufacturer’s instructions.

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In vitro Migration Assays [2]
Migration of three APE1 expressing pancreatic cancer cells (PANC1, XPA1, and BxPC3) was assayed using 6.5-mm-diameter chambers with 8-μm pore filters (Trans-well, 24-well cell culture) as described. Pancreatic cancer cells were suspended in serum-free medium at a concentration of 2 × 105/mL. Thereafter, 0.2 mL of the cell suspension was added to the upper chamber, and 0.5 mL serum-free medium with 100 ng/mL stromal cell–derived factor-1 was added to the lower chamber. Various concentrations of E3330 were added to the upper chamber. The chambers were incubated for 12 h at 37°C in a humid atmosphere of 5% CO2/95% air. After incubation, the filters were fixed and stained with Diff-Quick reagent. Cell viability (MTT) assays were done on the upper chamber lysates, to normalize any effects of E3330-induced growth inhibition (reduced cell numbers) on the migration readout. The upper surface of the filters was scraped twice with cotton swabs to remove nonmigrating cells. The experiments were repeated in triplicate wells, and the number of migrating cells in five high-power fields per filter was counted microscopically at ×400 magnification.

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3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide Assay [2]
E3330 powder was dissolved with pure ethanol. PANC1, XPA1, or HPNE cells were incubated with various concentrations of either E3330 (10-30 μmol/L) or methoxyamine (1 μmol/L-10 mmol/L) for 72 h in 96-well plates. At the culmination of the study, cell viability was evaluated by measuring the mitochondrial-dependent conversion of the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), to a colored formazan product as described previously. A similar analysis was done to assess growth of ESA-positive primary pancreatic cancer cells in the presence of E3330, with the exception that MTT assays were culminated at 48 h.

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PANC1 cells are treated with 5 to 30 μM E3330 after being inserted into one well of a 12-well plate. Following cultures for 24, 48, and 72 hours, the cells are rinsed with PBS, stained with trypan blue, and their viability is assessed by counting the number of living cells.

E3330 [(2E)-3-[5-(2,3-dimethoxy-6-methyl-1,4-benzoquinoyl)]-2-nonyl-2- propenoic acid] is a newly synthesized hepatoprotective quinone derivative. We examined the protective effects and possible mechanism of action of E3330 in three different endotoxin (lipopolysaccharide)-induced murine hepatitis models, in which tumor necrosis factor is suggested to play a critical role in the pathogenesis. One of these models was induced by i.v. injection of lipopolysaccharide in combination with D-galactosamine to mice. Oral pretreatment with E3330 improved the survival rate and attenuated the increase in plasma aminotransferase activities of the survivors. The other two models were induced by i.v. injection of lipopolysaccharide or a mixture of D-galactosamine and lipopolysaccharide in Propionibacterium acnes-primed mice. In both of these models, tumor necrosis factor was detected in the plasma within 3 hr of the injection. Oral pretreatment with E3330 attenuated the elevation of plasma tumor necrosis factor activity and protected mice from liver injury. Furthermore, E3330 inhibited the production of tumor necrosis factor from cultured Propionibacterium acnes-elicited murine peritoneal macrophages on stimulation with lipopolysaccharide in vitro. These findings suggest that the inhibition by E3330 of tumor necrosis factor production is the major mechanism of the protective effect of E3330 in these endotoxin-mediated hepatitis models in mice. [4]

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The effect of E3330 ((2E)-3-[5-(2,3-dimethoxy-6-methyl-1,4-benzoquinoyl)]-2-nonyl-2-++ +propenoic acid), a novel quinone derivative, was studied in the galactosamine-induced hepatitis model in F344 rats, in which endogenous endotoxin is believed to play a critical pathogenetic role. Subcutaneous injection of 300 mg/kg of galactosamine into rats resulted in liver injury. Oral treatment with E3330 (10-100 mg/kg) 1 hr after galactosamine challenge attenuated the liver injury. E3330 was also effective when administered p.o. 6 or 12 hr after galactosamine challenge. Subcutaneous injection of 1000 mg/kg of galactosamine into rats resulted in more severe liver injury with endotoxemia. The plasma endotoxin was detected 24 to 48 hr after the galactosamine challenge. The time course of increase in plasma endotoxin level was in good agreement with that in plasma aminotransferase activity. E3330 (100 mg/kg) significantly attenuated the liver injury, but did not affect the endotoxin level. Exogenous administration of endotoxin enhanced the hepatotoxicity of galactosamine. Pretreatment with E3330 also protected rats from severe liver injury induced with endotoxin plus galactosamine. These results suggest that E3330 may exert its hepatoprotective effects through inhibition of an effect of endotoxin in galactosamine-induced hepatitis in rats. [5]

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Suspended in 0.5% methylcellulose solution; 300 mg/kg; p.o.

Mice with endotoxin-mediated hepatitis

[1]. Blood . 2007 Mar 1;109(5):1917-22.

[2]. Mol Cancer Ther . 2008 Jul;7(7):2012-21.

[3]. PLoS One . 2013 Aug 15;8(8):e70909.

[4]. J Pharmacol Exp Ther . 1992 Jul;262(1):145-50.

[5]. J Pharmacol Exp Ther . 1993 Jan;264(1):496-500.

APE1/Ref-1 Redox Inhibitor APX3330 is an orally bioavailable inhibitor of apurinic/apyrimidinic endonuclease 1/reduction-oxidation (redox) effector factor-1 (APE1/Ref-1; APEX1), with potential anti-angiogenic and antineoplastic activities. Upon administration, the APE1/Ref-1 Inhibitor APX3330 selectively targets and binds to APE1/Ref-1. This inhibits the redox-dependent signaling activity of APE1/Ref-1, by preventing the reduction and activation of numerous APE1/Ref-1-dependent oncogenic transcription factors (TFs), such as nuclear factor kappa B (NF-kB), AP-1, STAT3, p53, NRF2 and HIF-1alpha, that are involved in signaling, cell proliferation, tumor progression and survival of cancer cells. Therefore, this agent inhibits the activation of multiple TF-mediated signaling pathways and inhibits tumor cell proliferation and survival. APE1/Ref-1, a multifunctional protein overexpressed in many cancer cell types, plays a key role as a redox regulator of transcription factor activation and in base excision repair upon DNA damage. It drives cancer cell proliferation, migration, drug resistance, angiogenesis and inflammation and its expression level correlates with increased tumor aggressiveness and decreased patient survival. APX3330 specifically blocks the redox activity of APE1/Ref-1 and does not affect its ability to act as a DNA repair endonuclease.

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AP endonuclease 1 (APE1; also known as REF-1) contains a DNA repair domain and a redox regulation domain. APE1 is overexpressed in several human cancers, and disruption of APE1 function has detrimental effects on cancer cell viability. However, the selective contribution of the redox and the DNA repair domains to maintenance of cellular homeostasis in cancer has not been elucidated. In the present study, we used E3330, a small-molecule inhibitor of APE1 redox domain function, to interrogate the functional relevance of sustained redox function in pancreatic cancer. We show that E3330 significantly reduces the growth of human pancreatic cancer cells in vitro. This phenomenon was further confirmed by a small interfering RNA experiment to knockdown APE1 expression in pancreatic cancer cells. Further, the growth-inhibitory effects of E3330 are accentuated by hypoxia, and this is accompanied by striking inhibition in the DNA-binding ability of hypoxia-inducible factor-1alpha, a hypoxia-induced transcription factor. E3330 exposure promotes endogenous reactive oxygen species formation in pancreatic cancer cells, and the resulting oxidative stress is associated with higher levels of oxidized, and hence inactive, SHP-2, an essential protein tyrosine phosphatase that promotes cancer cell proliferation in its active state. Finally, E3330 treatment inhibits pancreatic cancer cell migration as assessed by in vitro chemokine assays. E3330 shows anticancer properties at multiple functional levels in pancreatic cancer, such as inhibition of cancer cell growth and migration. Inhibition of the APE1 redox function through pharmacologic means has the potential to become a promising therapeutic strategy in this disease. [2]

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APE1/Ref-1 is a main regulator of cellular response to oxidative stress via DNA-repair function and co-activating activity on the NF-κB transcription factor. APE1 is central in controlling the oxidative stress-based inflammatory processes through modulation of cytokines expression and its overexpression is responsible for the onset of chemoresistance in different tumors including hepatic cancer. We examined the functional role of APE1 overexpression during hepatic cell damage related to fatty acid accumulation and the role of the redox function of APE1 in the inflammatory process. HepG2 cells were stably transfected with functional and non-functional APE1 encoding plasmids and the protective effect of APE1 overexpression toward genotoxic compounds or FAs accumulation, was tested. JHH6 cells were stimulated with TNF-α in the presence or absence of E3330, an APE1 redox inhibitor. IL-8 promoter activity was assessed by a luciferase reporter assay, gene expression by Real-Time PCR and cytokines (IL-6, IL-8, IL-12) levels measured by ELISA. APE1 over-expression did not prevent cytotoxicity induced by lipid accumulation. E3330 treatment prevented the functional activation of NF-κB via the alteration of APE1 subcellular trafficking and reduced IL-6 and IL-8 expression induced by TNF-α and FAs accumulation through blockage of the redox-mediated activation of NF-κB. APE1 overexpression observed in hepatic cancer cells may reflect an adaptive response to cell damage and may be responsible for further cell resistance to chemotherapy and for the onset of inflammatory response. The efficacy of the inhibition of APE1 redox activity in blocking TNF-α and FAs induced inflammatory response opens new perspectives for treatment of inflammatory-based liver diseases.[3]

C21H30O6

378.46

378.204

C, 66.65; H, 7.99; O, 25.36

136164-66-4

6439397

Yellow to orange solid powder

1.1±0.1 g/cm3

542.2±50.0 °C at 760 mmHg

183.2±23.6 °C

0.0±3.1 mmHg at 25°C

1.517

4.5

1

6

12

27

666

0

CCCCCCCCC/C(C(O)=O)=C\C1=C(C)C(C(OC)=C(OC)C1=O)=O

AALSSIXXBDPENJ-FYWRMAATSA-N

InChI=1S/C21H30O6/c1-5-6-7-8-9-10-11-12-15(21(24)25)13-16-14(2)17(22)19(26-3)20(27-4)18(16)23/h13H,5-12H2,1-4H3,(H,24,25)/b15-13+

(2E)-2-[(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)methylidene]undecanoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.50 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.50 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (5.50 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)