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Capsanthin is the main carotenoid compound in red paprika (Capsicum annuum L.). However, little is known about the beneficial effects of capsanthin in nonalcoholic fatty liver disease (NAFLD). In t...

Arterial thrombosis and its associated diseases are considered to constitute a major healthcare problem. Arterial thrombosis, defined as blood clot formation in an artery that interrupts blood circulation, is associated with many cardiovascular diseases. Oxidative stress is one of many important factors that aggravates the pathophysiological process of arterial thrombosis. Apurinic/apyrimidinic endonuclease 1/redox factor-1 (Ref-1) has a multifunctional role in cells that includes the regulation of oxidative stress and anti-inflammatory function. The aim of this study was to investigate the therapeutic effect of adenovirus-mediated Ref-1 overexpression on arterial thrombosis induced by 60% FeCl3 solution in rats. Blood flow was measured to detect the time to occlusion, thrombus formation was detected by hematoxylin and eosin staining, reactive oxygen species (ROS) levels were detected by high-performance liquid chromatography, and the expression of tissue factor and other proteins was detected by Western blot. FeCl3 aggravated thrombus formation in carotid arteries and reduced the time to artery occlusion. Ref-1 significantly delayed arterial obstruction via the inhibition of thrombus formation, especially by downregulating tissue factor expression through the Akt-GSK3β-NF-κB signaling pathway. Ref-1 also reduced the expression of vascular inflammation markers ICAM-1 and VCAM-1, and reduced the level of ROS that contributed to thrombus formation. The results showed that adenovirus-mediated Ref-1 overexpression reduced thrombus formation in the rat carotid artery. In summary, Ref-1 overexpression had anti-thrombotic effects in a carotid artery thrombosis model and could be a target for the treatment of arterial thrombosis.

Downregulation of CR6 interacting factor 1 (CRIF1) has been reported to induce mitochondrial dysfunction, resulting in reduced activity of endothelial nitric oxide synthase (eNOS) and NO production in endothelial cells. Tetrahydrobiopterin (BH4) is an important cofactor in regulating the balance between NO (eNOS coupling) and superoxide production (eNOS uncoupling). However, whether the decreased eNOS and NO production in CRIF1-deficient cells is associated with relative BH4 deficiency-induced eNOS uncoupling remains completely unknown. Our results showed that CRIF1 deficiency increased eNOS uncoupling and depleted levels of total biopterin and BH4 by reducing the enzymes of BH4 biosynthesis (GCH-1, PTS, SPR, and DHFR) in vivo and vitro, respectively. Supplementation of CRIF1-deficient cells with BH4 significantly increased the recovery of Akt and eNOS phosphorylation and NO synthesis. In addition, scavenging ROS with MitoTEMPO treatment replenished BH4 levels by elevating levels of GCH-1, PTS, and SPR, but with no effect on the level of DHFR. Downregulation of DHFR synthesis regulators p16 or p21 in CRIF1-deficient cells partially recovered the DHFR expression. In summary, CRIF1 deficiency inhibited BH4 biosynthesis and exacerbated eNOS uncoupling. This resulted in reduced NO production and increased oxidative stress, which contributes to endothelial dysfunction and is involved in the pathogenesis of cardiovascular diseases.

There is growing evidence that apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) regulates inflammatory responses. Rheumatoid arthritis (RA) is an autoimmune disease, which is characterized with synovitis and joint destruction. Therefore, this study was planned to investigate the relationship between APE1/Ref-1 and RA. Serum and synovial fluid (SF) were collected from 46 patients with RA, 45 patients with osteoarthritis (OA), and 30 healthy control (HC) patients. The concentration of APE1/Ref-1 in serum or SF was measured using the sandwich enzyme-linked immunosorbent assay (ELISA). The disease activity in RA patients was measured using the 28-joint disease activity score (DAS28). The serum APE1/Ref-1 levels in RA patients were significantly increased compared to HC and OA patients (0.44 ± 0.39 ng/mL for RA group vs. 0.19 ± 0.14 ng/mL for HC group, p &lt, 0.05 and vs. 0.19 ± 0.11 ng/mL for OA group, p &lt, 0.05). Likewise, the APE1/Ref-1 levels of SF in RA patients were also significantly increased compared to OA patients (0.68 ± 0.30 ng/mL for RA group vs. 0.31 ± 0.12 ng/mL for OA group, p &lt, 0.001). The APE1/Ref-1 concentration in SF of RA patients was positively correlated with DAS28. Thus, APE1/Ref-1 may reflect the joint inflammation and be associated with disease activity in RA.

Elevated plasma homocysteine levels can induce vascular endothelial dysfunction, however, the mechanisms regulating homocysteine metabolism in impaired endothelial cells are currently unclear. In this study, we deleted the essential mitoribosomal gene CR6 interacting factor 1 (CRIF1) in human umbilical vein endothelial cells (HUVECs) and mice to induce endothelial cell dysfunction, then, we monitored homocysteine accumulation. We found that CRIF1 downregulation caused significant increases in intracellular and plasma concentrations of homocysteine, which were associated with decreased levels of folate cycle intermediates such as 5-methyltetrahydrofolate (MTHF) and tetrahydrofolate (THF). Moreover, dihydrofolate reductase (DHFR), a key enzyme in folate-mediated metabolism, exhibited impaired activity and decreased protein expression in CRIF1 knockdown endothelial cells. Supplementation with folic acid did not restore DHFR expression levels or MTHF and homocysteine concentrations in endothelial cells with a CRIF1 deletion or DHFR knockdown. However, the overexpression of DHFR in CRIF1 knockdown endothelial cells resulted in decreased accumulation of homocysteine. Taken together, our findings suggest that CRIF1-deleted endothelial cells accumulated more homocysteine, compared with control cells, this was primarily mediated by the disruption of DHFR expression.

The vascular endothelium is an active tissue that plays a crucial role in the maintenance of vascular homeostasis [...]

Keloids are a type of aberrant skin scarring characterized by excessive accumulation of collagen and extracellular matrix (ECM), arising from uncontrolled wound healing responses. While typically non-pathogenic, keloids are occasionally regarded as a form of benign tumor. CR6-interacting factor 1 (CRIF1) is a well-known CR6/GADD45-interacting protein, that has both nuclear and mitochondrial functions, and also exerts regulatory effects on cell growth and apoptosis. In this study, cell proliferation, cell migration, collagen production and TGF-β signaling was compared between normal fibroblasts (NFs) and keloid fibroblasts (KFs). Subsequently, the effects of CRIF1 deficiency were investigated in both NFs and KFs. Cell proliferation, cell migration, collagen production and protein expressions of TGF-β, phosphorylation of Smad2 and Smad3 were all found to be higher in KFs compared to NFs. CRIF1 deficiency in NFs and KFs inhibited cell proliferation, migration, and collagen production. In addition, phosphorylation of Smad2 and Smad3, which are transcription factors of collagen, was decreased. In contrast, mRNA expression levels of Smad7 and SMURF2, two important inhibitory proteins of Smad2/3, were increased, suggesting that CRIF1 may regulate collagen production. CRIF1 deficiency decreases the proliferation and migration of KFs, thereby inhibiting their overgrowth via the transforming growth factor-β (TGF-β)/Smad pathway. CRIF1 may therefore represent a potential therapeutic target in keloid pathogenesis.

The CR6-interacting factor1 (CRIF1) mitochondrial protein is indispensable for peptide synthesis and oxidative phosphorylation. Cardiomyocyte-specific deletion of CRIF1 showed impaired mitochondrial function and cardiomyopathy. We developed an endothelial cell-specific CRIF1 deletion mouse to ascertain whether dysfunctional endothelial CRIF1 influences cardiac function and is mediated by the antioxidant protein sirtuin 1 (SIRT1). We also examined the effect of the potent SIRT1 activator SRT1720 on cardiac dysfunction. Mice with endothelial cell-specific CRIF1 deletion showed an increased heart-to-body weight ratio, increased lethality, and markedly reduced fractional shortening of the left ventricle, resulting in severe cardiac dysfunction. Moreover, endothelial cell-specific CRIF1 deletion resulted in mitochondrial dysfunction, reduced ATP levels, inflammation, and excessive oxidative stress in heart tissues, associated with decreased SIRT1 expression. Intraperitoneal injection of SRT1720 ameliorated cardiac dysfunction by activating endothelial nitric oxide synthase, reducing oxidative stress, and inhibiting inflammation. Furthermore, the decreased endothelial junction-associated protein zonula occludens-1 in CRIF1-deleted mice was significantly recovered after SRT1720 treatment. Our results suggest that endothelial CRIF1 plays an important role in maintaining cardiac function, and that SIRT1 induction could be a therapeutic strategy for endothelial dysfunction-induced cardiac dysfunction.

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.

Background and Objectives: Propofol-based total intravenous anesthesia (TIVA) is presumed to have more favorable effects on the prognosis of patients with cancer compared with volatile inhaled anesthesia (VIA). We hypothesized that these anesthetics target plasma apurinic apyrimidinic endonuclease/redox effector factor-1 (APE1/Ref-1) as a possible mechanism of action. Materials and Methods: The plasma APE1/Ref-1 level was evaluated three times during surgery for cancer, i.e., before anesthesia, immediately after cancer resection, and finally, in the recovery room. Blood (3 cc) was drawn from the radial artery catheter, and plasma APE1/Ref-1 levels were compared according to measurement time and between the two groups. Spearman’s Rho correlation analysis was performed to determine relationships among body mass index, American Society of Anesthesiologists classification, age, sex, cancer type, and tumor-node-metastasis (TNM) stage. A total of 166 patients (VIA: 129; TIVA: 37) were enrolled. Results: Plasma APE1/Ref-1 level increased significantly (p = 0.028) after cancer resection compared with before surgery, but no significant difference was observed between anesthetics (p = 0.134). The post-resection plasma APE1/Ref-1 level showed a positive correlation with the NM stages, but not the T stage. Conclusions: The plasma APE1/Ref-1 level increased during surgery with more severe lymph node invasion, but there were no significant differences according to the anesthetics used.

Background: Paraplegia is a devastating complication following operations on the thoracoabdominal aorta. We investigated whether histidine-tryptophan-ketoglutarate (HTK) solution could reduce the extent of ischemia/reperfusion (IR) spinal cord injuries in a rat model using a direct delivery method. Methods: Twenty-four Sprague-Dawley male rats were randomly divided into four groups. The sham group (n=6) underwent a sham operation, the IR group (n=6) underwent only an aortic occlusion, the saline infusion group (saline group, n=6) underwent an aortic occlusion and direct infusion of cold saline into the occluded aortic segment, and the HTK infusion group (HTK group, n=6) underwent an aortic occlusion and direct infusion of cold HTK solution into the occluded aortic segment. An IR spinal cord injury was induced by transabdominal clamping of the aorta distally to the left renal artery and proximally to the aortic bifurcation for 60 minutes. A neurological evaluation of locomotor function was performed using the modified Tarlov score after 48 hours of reperfusion. The spinal cord was harvested for histopathological and immunohistochemical examinations. Results: The spinal cord IR model using direct drug delivery in rats was highly reproducible. The Tarlov score was 4.0 in the sham group, 1.17±0.75 in the IR group, 1.33±1.03 in the saline group, and 2.67±0.81 in the HTK group (p=0.04). The histopathological analysis of the HTK group showed reduced neuronal cell death. Conclusion: Direct infusion of cold HTK solution into the occluded aortic segment may reduce the extent of spinal cord injuries in an IR model in rats.

Nafamostat mesilate (NM), a serine protease inhibitor, has a broad range of clinical applications that include use as an anticoagulant during hemodialysis in cerebral hemorrhage patients, as a hemoperfusion anticoagulant for patients with intravascular coagulation, hemorrhagic lesions, and hemorrhagic tendencies, and for the improvement of acute pancreatitis. However, the effects of NM on acute cerebral ischemia have yet to be investigated. Thus, the present study utilized a rat model in which transient middle cerebral artery occlusion (MCAO) was used to induce ischemic injury to investigate the effects of NM on infarct volume and histological and biological changes. NM (1mg/kg) was intravenously administered prior to and after the MCAO procedure. Compared to control rats, the administration of NM significantly decreased infarct size and the extent of brain edema after the induction of focal ischemia via MCAO. Additionally, NM treatment attenuated MCAO-induced neuronal degeneration and activation of microglia and astrocytes. NM treatment also inhibited the MCAO-induced expression levels of glucose-regulated protein 78 (GRP78), CATT/EBP homologous protein (CHOP), and p-eukaryotic initiation factor 2α (eIF2α), which are endoplasmic reticulum (ER) stress markers, in the cerebral cortex. The present findings demonstrate that NM exerts neuroprotective effects in the brain following focal ischemia via, at least in part, the inhibition of ER stress.

Bladder cancer has a complex and multifactorial pathophysiology. Numerous environmental, molecular, or genetic factors are involved in the development of urothelial cell cancers. There is a growing interest in understanding the kinds of genetic alterations or its cellular signaling pathways that contribute to the pathophysiology for progression or recurrence of bladder cancer. Genetic or epigenetic alterations of tumor suppressor genes, growth factor receptors, and DNA repair genes are all thought to be responsible. Established molecular biomarkers and expression profiles of bladder cancer are useful candidates in the early prediction or recurrence of bladder cancers.

Myocarditis is an inflammatory disease of the myocardium that causes cardiogenic shock and death. However, endomyocardial biopsy that is, the gold standard for a diagnosis is limited. Apurinic/apyrimidinic endonuclease 1/redox effector factor-1 (APE1/Ref-1) is a multifunctional protein, which is involved in DNA-based excision repair pathway, and in redox signaling, its changes are observed in various cardiovascular diseases including hypertension and coronary artery disease. We analyzed serum APE1/Ref-1 in experimental murine myocarditis. To induce myocarditis, coxsackievirus B3 was injected intraperitoneally to BALB/c mice. The serum APE1/Ref-1, N-terminal pro-B-type natriuretic peptide (NT-proBNP) and troponin I were measured. The histology and virus titers measurements were performed. The troponin I and inflammation were significantly elevated at day 3, peaked to day 7 and decreased at day 10. The NT-proBNP and virus titers were significantly peaked at day 3, and dropped at day 7 and 10. The serum APE1/Ref-1 was gradually raised and its elevation is still maintained until a later time, namely day 10. Also, its level was positively correlated with myocardial inflammation, reflecting severity of myocardial injury. We suggest that serum APE1/Ref-1 can be used to assess for myocardial injury in viral myocarditis without endomyocardial biopsy.

Elevated endothelial arginase activity decreases nitric oxide (NO) production by competing with the substrate l-arginine, previously reported, and reciprocally regulating endothelial nitric oxide synthase (eNOS) activity. Thus, arginase inhibitors may help treat vascular diseases associated with endothelial dysfunction. A screening of metabolites from medicinal plants revealed that (2S)-5,2',5'-trihydroxy-7,8-dimethoxy flavanone (TDF) was a noncompetitive inhibitor of arginase. We investigated whether TDF reciprocally regulated endothelial NO production and its possible mechanism. TDF noncompetitively inhibited arginase I and II activity in a dose-dependent manner. TDF incubation decreased arginase activity and increased NO production in human umbilical vein endothelial cells and isolated mouse aortic vessels and reduced reactive oxygen species (ROS) generation in the endothelium of the latter. These TDF-mediated effects were associated with increased eNOS phosphorylation and dimerization but not with changes in protein content. Endothelium-dependent vasorelaxant responses to acetylcholine (Ach) were significantly increased in TDF-incubated aortic rings and attenuated by incubation with soluble guanylyl cyclase inhibitor. Phenylephrine-induced vasoconstrictor responses were markedly attenuated in TDF-treated vessels from wild-type mice. In atherogenic-prone ApoE(-/-) mice, TDF attenuated the high-cholesterol diet (HCD)-induced increase in arginase activity, which was accompanied by restoration of NO production and reduction of ROS generation. TDF incubation induced eNOS dimerization and phosphorylation at Ser1177. In addition, TDF improved Ach-dependent vasorelaxation responses and attenuated U46619-dependent contractile responses but did not change sodium nitroprusside-induced vasorelaxation or N-NAME-induced vasoconstriction. The findings suggest that TDF may help treat cardiovascular diseases by reducing pathophysiology derived from HCD-mediated endothelial dysfunction.

Background and Objectives Apurinic/apyrimidinic endonuclease 1/redox effector factor-1 (APE1/Ref-1) is a multifunctional protein involved in the DNA base excision repair pathway, inflammation, angiogenesis, and survival pathways. We investigated serum APE1/Ref-1 in patients with coronary artery disease (CAD). Subjects and Methods Serum APE1/Ref-1 was measured with a sandwich enzyme-linked immunosorbent assay from 360 patients who received coronary angiograms. They were divided into two groups; a control (n=57) and a CAD group (n=303), the latter included angina (n=128) and myocardial infarction (MI, n=175). Results The levels of APE1/Ref-1 were higher in the CAD than the control (0.63±0.07 vs. 0.12±0.07 ng/100 µL, respectively; p

Purpose Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein that shows elevated expression in a number of cancers. We attempted to determine whether serum APE1/Ref-1 is elevated in patients with bladder cancer. Materials and methods Serum APE1/Ref-1 levels were determined using enzyme-linked immunosorbent assay in serum from patients with bladder cancer who had not received chemotherapy or radiotherapy (n=51) and non-tumor controls (n=55). The area under the receiver operating characteristic area under the curve was applied to determine the correlation between clinical factors and the serum levels of APE1/Ref-1. Results Serum levels of APE1/Ref-1 in bladder cancer patients were significantly elevated compared to those of the control group (3.548 ± 0.333 ng/100 μL [n=51] for bladder cancer vs. 1.547 ± 0.319 ng/100 μL [n=55] for the control group), with a sensitivity and specificity of 93% and 59%, respectively. Serum APE1/Ref-1 levels are associated with tumor stage, grade, muscle invasion, and recurrence. Conclusion Serum APE1/Ref-1 might be useful as a potential serologic biomarker for bladder cancer.

Vascular calcification plays a role in the pathogenesis of atherosclerosis, diabetes, and chronic kidney disease; however, the role of apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) in inorganic phosphate (Pi)-induced vascular smooth muscle cell (VSMC) calcification remains unknown. In this study, we investigated the possible role of APE1/Ref-1 in Pi-induced VSMC calcification. We observed that Pi decreased endogenous APE1/Ref-1 expression and promoter activity in VSMCs, and that adenoviral overexpression of APE1/Ref-1 inhibited Pi-induced calcification in VSMCs and in an ex vivo organ culture of a rat aorta. However, a redox mutant of APE1/Ref-1(C65A/C93A) did not reduce Pi-induced calcification in VSMCs, suggesting APE1/Ref-1-mediated redox function against vascular calcification. Additionally, APE1/Ref-1 overexpression inhibited Pi-induced intracellular and mitochondrial reactive oxygen species production, and APE1/Ref-1 overexpression resulted in decreased Pi-induced lactate dehydrogenase activity, pro-apoptotic Bax levels, and increased anti-apoptotic Bcl-2 protein levels. Furthermore, APE1/Ref-1 inhibited Pi-induced osteoblastic differentiation associated with alkaline phosphatase activity and inhibited Pi-exposure-induced loss of the smooth muscle phenotype. Our findings provided valuable insights into the redox function of APE1/Ref-1 in preventing Pi-induced VSMC calcification by inhibiting oxidative stress and osteoblastic differentiation, resulting in prevention of altered osteoblastic phenotypes in VSMCs.

Mitochondrial dysfunction has emerged as a major contributing factor to endothelial dysfunction and vascular disease, but the key mechanisms underlying mitochondrial dysfunction-induced endothelial dysfunction remain to be elucidated. In this study, we aim at determining whether mitochondrial dysfunction in endothelial cells plays a key role in vascular disease, by examining the phenotype of endothelial-specific CR6-interacting factor 1 (CRIF1) knockout mice. We also used siRNA-mediated downregulation of CRIF1 gene in the endothelial cells to study about the in vitro pathophysiological underlying mechanisms.Downregulation of CRIF1 in endothelial cells caused disturbances of mitochondrial oxidative phosphorylation complexes and membrane potential, leading to enhanced mitochondrial reactive oxygen species production. Gene silencing of CRIF1 results in decreased SIRT1 expression along with increased endothelial nitric oxide synthase (eNOS) acetylation, leading to reduced nitric oxide production both in vitro and in vivo. Endothelium-dependent vasorelaxation of aortic rings from CRIF1 knockout (KO) mice was considerably less than in wild-type mice, and it was partially recovered by Sirt1 overexpression in CRIF1 KO mice.Our results show for the first time a relationship between mitochondrial dysfunction and impaired vascular function induced in CRIF1 deficiency conditions and also the possible underlying pathway involved.These findings indicate that CRIF1 plays an important role in maintaining mitochondrial and endothelial function through its effects on the SIRT1-eNOS pathway. Antioxid. Redox Signal. 27, 234-249.

Naematoloma sublateritium (Fr.) P. Karst is a basidiomycete that has been used as traditional medicine. N. sublateritium produces a triterpenoid antitumor compound, clavaric acid, but, in general, the effects of N. sublateritium constituents against tumor invasion and metastasis have been poorly studied. To investigate the inhibitory effect of N. sublateritium constituents on highly invasive and metastatic tumor cells, the TNF-α-stimulated human breast cancer cell line, MDA-MB‑231 was treated with the hexane fraction of an N. sublateritium extract (HFNS). Non-cytotoxic concentrations of HFNS markedly inhibited the invasion and migration of the MDA-MB‑231 cells in the Matrigel invasion assay and wound-healing analysis, respectively. Gelatin zymography showed that HFNS suppressed the activity of MMP-9, but not of MMP-2. Immunoblotting demonstrated that treatment with HFNS had decreased the level of MMP-9 and urokinase plasminogen activator-1 (uPA-1), but had upregulated expression of the endogenous inhibitor proteins, including TIMP-1,-2, and PAI-1, in a dose-dependent manner. Furthermore, HFNS suppressed the phosphorylation of p38 and JNK1/2, but not that of ERK1/2. This was confirmed by pretreatment of cells with specific inhibitors prior to stimulation with TNF-α. HFNS treatment also led to a dose-dependent inhibition of the DNA-binding activities of AP-1 and NFκB, which are downstream targets of JNK and p38. These data suggested that HFNS inhibits the metastatic potential of MDA-MB‑231 cells by inhibiting the phosphorylation of JNK/p38 and reducing AP-1 and NFκB DNA-binding activities. Therefore, HFNS may be a potential therapeutic agent against metastasis of breast cancer.

In persistent inflammatory state involving the chronic activation of the immune system, the interplay between breast cancer and stromal cells is closely associated. We have previously shown that APE1/Ref-1 which could inhibit inflammatory signaling by reductive conformational change of cytokines receptors. In response to hyperacetylation, stimulation by secreted Ac-APE1/Ref-1 binding to RAGE initiated apoptotic cell death in TNBC models, in vitro and in vivo. In the present study, we used Ad-APE1/Ref-1 adenovirus, which can be overexpressed and be secreted into the blood of xenografts. We investigated how the Ad-APE1/Ref-1 influences growth retardation and apoptotic cell death of inflammation associated TNBC in vivo. The Ad-APE1/Ref-1-injection in MDA-MB 231 orthotopic xenografts caused a decrease in the volume and weight of tumors as shown in IVIS images. The treatment of Ad-APE1/Ref-1 also induced an inhibition of tumor growth and development, leading to apoptotic cell death, accompanied by generation of reactive oxygen species. Tumor tissues derived from Ad-APE1/Ref-1 exhibited apoptotic bodies compared with tumors of control or Adβ-galactosidase-injected mice. Moreover, level of inflammatory cytokines was evaluated in plasma of the xenografts; ratio of anti- to pro inflammatory cytokines was comparable with one of normal mice. The PAK1-STAT-3-NFkB axis in inflammatory signaling of tumor tissues derived from AdAPE1/Ref-1 injected mice was significantly inhibited compared to those of control or Adβ-galactosidase-injected mice. These results suggest that regulation of inflammatory signaling with adenoviral mediated APE1/Ref-1 in tumors of MDA-MB-231 xenografts modulates cytokine secretion, thereby inhibiting cancer cell growth. Note: This abstract was not presented at the meeting. Citation Format: Sunga Choi, Yu Ran Lee, Yeon Hyang Kim, Ki Mo Kim, Myoung Soo Park, Byeong Hwa Jeon. Anti-tumor effect of adenovirus encoding APE1/Ref-1 in breast cancer xenografts: anti-inflammation and apoptotic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4777.

Lung cancer, the leading cause of cancer-related death, still lacks reliable biomarkers. Apurinic/apyrimidinic endonuclease 1/Ref-1 is a multifunctional protein involved in the base excision repair of DNA damaged by oxidative stress or alkylating compounds, as well as in the regulation of multiple transcription factors. To validate apurinic/apyrimidinic endonuclease 1/Ref-1 as a biomarker for prediction of lung cancer recurrence, we studied 42 patients who received curative resection and mediastinal lymph node dissection for stage I non-small-cell lung cancer. They were divided into 2 groups based on recurrence, and compared by immunohistochemistry staining of paraffin-embedded tissues and Western blot analysis. Immunohistochemical staining showed a significant difference between the cytoplasm and nucleus in patients who had a recurrence compared to those with nonrecurrent adenocarcinoma. In Western blot analysis, the recurrent adenocarcinoma group showed increased expression of apurinic/apyrimidinic endonuclease 1/Ref-1 in cytoplasm, nucleus, and in total. This indicates that apurinic/apyrimidinic endonuclease 1/Ref-1 is unregulated in recurrent stage I adenocarcinoma. For clinical application as a prognostic marker for non-small-cell lung cancer, further investigation into the role of apurinic/apyrimidinic endonuclease 1/Ref-1 in carcinogenesis is needed in an expanded prospective study.

Mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDH2) plays an essential role protecting cells against oxidative stress-induced damage. A deficiency in IDH2 leads to mitochondrial dysfunction and the production of reactive oxygen species (ROS) in cardiomyocytes and cancer cells. However, the function of IDH2 in vascular endothelial cells is mostly unknown. In this study the effects of IDH2 deficiency on mitochondrial and vascular function were investigated in endothelial cells. IDH2 knockdown decreased the expression of mitochondrial oxidative phosphorylation (OXPHOS) complexes I, II and III, which lead to increased mitochondrial superoxide. In addition, the levels of fission and fusion proteins (Mfn-1, OPA-1, and Drp-1) were significantly altered and MnSOD expression also was decreased by IDH2 knockdown. Furthermore, knockdown of IDH2 decreased eNOS phosphorylation and nitric oxide (NO) concentration in endothelial cells. Interestingly, treatment with Mito-TEMPO, a mitochondrial-specific superoxide scavenger, recovered mitochondrial fission-fusion imbalance and blunted mitochondrial superoxide production, and reduced the IDH2 knockdown-induced decrease in MnSOD expression, eNOS phosphorylation and NO production in endothelial cells. Endothelium-dependent vasorelaxation was impaired, and the concentration of bioavailable NO decreased in the aortic ring in IDH2 knockout mice. These findings suggest that IDH2 deficiency induces endothelial dysfunction through the induction of dynamic mitochondrial changes and impairment in vascular function.

Aims Hyperhomocysteinaemia is an independent risk factor for atherosclerotic vascular disease and is associated with vascular endothelial dysfunction. Homocysteine modulates cellular methylation reactions. P66shc is a protein that promotes oxidative stress whose expression is governed by promoter methylation. We asked if homocysteine induces endothelial p66shc expression via hypomethylation of CpG dinucleotides in the p66shc promoter, and whether p66shc mediates homocysteine-stimulated endothelial cell dysfunction. Methods and results Homocysteine stimulates p66shc transcription in human endothelial cells and hypomethylates specific CpG dinucleotides in the human p66shc promoter. Knockdown of p66shc inhibits the increase in reactive oxygen species, and decrease in nitric oxide, elicited by homocysteine in endothelial cells and prevents homocysteine-induced up-regulation of endothelial intercellular adhesion molecule-1. In addition, knockdown of p66shc mitigates homocysteine-induced adhesion of monocytes to endothelial cells. Inhibition of DNA methyltransferase activity or knockdown of DNA methyltransferase 3b abrogates homocysteine-induced up-regulation of p66shc. Comparison of plasma homocysteine in humans with coronary artery disease shows a significant difference between those with highest and lowest p66shc promoter CpG methylation in peripheral blood leucocytes. Conclusion Homocysteine up-regulates human p66shc expression via hypomethylation of specific CpG dinucleotides in the p66shc promoter, and this mechanism is important in homocysteine-induced endothelial cell dysfunction.

SummaryTwo vascular growth factor families, VEGF and the angiopoietins, play critical and coordinate roles in tumor progression and metastasis. A single inhibitor targeting both VEGF and angiopoietins is not available. Here, we developed a chimeric decoy receptor, namely double anti-angiogenic protein (DAAP), which can simultaneously bind VEGF-A and angiopoietins, blocking their actions. Compared to VEGF-Trap or Tie2-Fc, which block either VEGF-A or angiopoietins alone, we believe DAAP is a highly effective molecule for regressing tumor angiogenesis and metastasis in implanted and spontaneous solid tumors; it can also effectively reduce ascites formation and vascular leakage in an ovarian carcinoma model. Thus, simultaneous blockade of VEGF-A and angiopoietins with DAAP is an effective therapeutic strategy for blocking tumor angiogenesis, metastasis, and vascular leakage.

Lead (Pb) is a metal that is generally considered to be toxic to the cardiovascular system. Pb-exposed animals display the evidence of increased oxidative stress and hypertension. The current study was designed to examine whether Korean red ginseng (KRG) has protective effects against Pb-induced hypertension and oxidative stress in Pb-exposed rats. Male Sprague-Dawley rats were randomly assigned to Pb exposure or control groups. KRG was administered in drinking water at a concentration of 100 ㎎/㎏/day; the control group received plain drinking water. Animals in the Pb-exposed groups were provided with drinking water containing 100 ppm Pb acetate for 12 weeks. Blood pressure, plasma glutathione, blood Pb concentration, and hematologic data, such as red blood cell quantity, were determined. Pb poisoning was assessed by measuring the blood Pb concentration. Pb exposure (100 ppm) for 12 weeks resulted in a marked rise in systolic blood pressure and blood Pb concentration, as well as a significant reduction in plasma glutathione levels and red blood cell quantity. Other measurements, such as heart rate, body weight, and white blood cell quantity, were unchanged. Treatment with KRG significantly lowered blood pressure, raised plasma glutathione and increased red blood cell numbers in Pb-exposed animals; it also had no effect on heart rate, body weight, or white blood cell quantity. However, the elevated blood Pb concentration was not reduced by treatment with KRG (100 ㎎/㎏). Taken together, these data indicate that treatment with KRG in Pb-exposed animals can reduce oxidative stress and lower blood pressure, suggesting that KRG might be protective against Pb-exposed hypertension and oxidative stress.

Chemotherapy is often hindered by abnormal tumor vascularity, which causes impaired delivery of drugs into the tumor. Angiopoietin-1 has potent roles in angiogenesis and vessel maturation. We report here that an angiopoietin-1 variant, COMP-Ang1, promotes vascular remodeling and pericyte coverage and thereby promotes the efficient delivery of a chemotherapeutic drug into tumors of murine Lewis lung carcinoma. The combination of COMP-Ang1 with the cytotoxic drug 5-fluorouracil potentiated the effect of 5-fluorouracil on tumor growth without increasing animal toxicity. Moreover, COMP-Ang1 increased perfusion into the tumor. Although COMP-Ang1 increased the functional vasculature in the tumor, COMP-Ang1 alone did not promote tumor growth, possibly due to its promotion of increased pericyte coverage. This study suggests that COMP-Ang1 may improve the microcirculation within a tumor by increasing functional vasculature and tissue perfusion and that the combination of chemotherapy together with COMP-Ang1 might be an advantageous therapeutic approach. (Mol Cancer Res 2009;7(12):1920–7)

Endotoxemia is characterized by multiple dysfunctions of the micro-vascular endothelium. Of these, vascular leakage is an initial event that aggravates vascular dysfunction can lead to systemic vascular collapse and organ failure. Thus, prevention of vascular leakage may ameliorate endotoxin-induced dysfunctions of blood vessels. Here we examine the effect of an angiopoietin-1 variant, COMP-Ang1, on endotoxin-induced vascular leakage in mice. COMP-Ang1 significantly reduced endotoxin-induced vascular leakage in the lung, heart, and kidney, but not in liver or intestine. Interestingly, COMP-Ang1 attenuated endotoxin-induced lung damage, presumably due to reduced infiltration of macrophages. Moreover, COMP-Ang1 restored the level of PECAM-1 expression, which is significantly reduced by endotoxin challenge. This study suggests that COMP-Ang1 reduces endotoxin-induced vascular leakage by restoration of cellular junctions and subsequent attenuation of leukocyte infiltration.

The transcription factor, p53, and the adaptor protein, p66shc, both play essential roles in promoting oxidative stress in the vascular system. However, the relationship between the two in the context of endothelium-dependent vascular tone is unknown. Here, we report a novel, evolutionarily conserved, p53-mediated transcriptional mechanism that regulates p66shc expression and identify p53 as an important determinant of endothelium-dependent vasomotor function. We provide evidence of a p53 response element in the promoter of p66shc and show that angiotensin II-induced upregulation of p66shc in endothelial cells is dependent on p53. In addition, we demonstrate that downregulation of p66shc expression, as well as inhibition of p53 function in mice, mitigates angiotensin II-induced impairment of endothelium-dependent vasorelaxation, decrease in bioavailable nitric oxide, and hypertension. These findings reveal a novel p53-dependent transcriptional mechanism for the regulation of p66shc expression that is operative in the vascular endothelium and suggest that this mechanism is important in impairing endothelium-dependent vascular relaxation.

Vascular inflammation is an important step in the development of cardiovascular disorder. Since it has not been known whether Korean red ginseng has a role to play on the vascular inflammation, we investigated the effects of Korean red ginseng extract (KRGE) on monocyte adhesion and its underlying signaling mechanism. Monocyte adhesion assay and Western blot were conducted on the human umbilical vein endothelial cells to study monocyte adhesion and the expression of adhesion molecules. Intracellular calcium was measured with Fura-2 fluorescent staining, and superoxide production was measured with lucigenin chemiluminescence in the endothelial cells. KRGE inhibits tumor necrosis factor (TNF)-alpha-induced monocyte adhesion on the endothelial cells at the range of 0.03~1 ㎎/㎖. TNF-alpha-induced vascular cell adhesion molecule-1 and intercellular cell adhesion molecule-1 expression were inhibited by the pretreatment of KRGE in the endothelial cells. KRGE also inhibits TNF-alpha-induced intracellular calcium and the superoxide production in the endothelial cells. This study first demonstrated that KRGE inhibits TNF-alpha-induced monocyte adhesion by inhibiting the adhesion molecule expression, intracellular calcium and superoxide production in the endothelial cells. Therefore, the anti-inflammatory function of KRGE may be contributed to protecting the endothelial dysfunction in the vascular inflammatory disorders.

Summary Purpose Apurinic/apyrimidinic endonuclease1/ref-1(APE1/ref-1) is a key enzyme in the base excision repair and in transcriptional modulation against oxidative stress. We investigated the altered expression of APE1/ref-1 and antioxidant systems in lung cancer. Patients and methods Tumor specimens from 48 patients with operable non-small cell lung cancer were obtained from 2004 to 2006. Immunohistochemistry, Western blot, lipid peroxidation and superoxide production were performed on the tumor samples and a cultured H460 cell line. Results APE1/ref-1 was mainly localized to the nucleus in the non-tumor regions of the lung cancer tissue specimens. However, nuclear and cytoplasmic expressions of APE1/ref-1 in the lung cancers were markedly up-regulated in the non-small cell lung cancer (NSCLC) specimens including squamous cell and adenocarcinoma specimens. Extracellular superoxide dismutase (ECSOD) and catalase were down-regulated and manganese superoxide dismutase (MnSOD) was up-regulated in the tumor regions of the NSCLC. Tumor regions of the NSCLC showed higher superoxide production and lipid peroxidation levels than non-tumor regions. In the lung adenocarcinoma cell line, H460, treatment with hydrogen peroxide in the presence of a catalase inhibitor, aminotriazole, increased APE1/ref-1 expression, suggesting oxidative stress might have contributed to the induction of APE1/ref-1. Conclusion The results of this study suggest that APE1/ref-1 is up-regulated in the tumor regions of NSCLC. Altered expression of antioxidant systems lead to enhanced production of superoxide production and lipid peroxidation, which can induce APE1/ref-1 in the tumor regions of NSCLS.

Bladder cancer (BCa) is one of the most common urothelial cancers with still noticeable incidence rate. Early detection of BCa is highly correlated with successful therapeutic outcomes. We previously showed that apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) was expressed at an increased level in the serum of BCa patients when compared to the level in healthy controls. In this study, we investigated whether urinary APE1/Ref-1 was also elevated in patients with BCa. In this case-control study, voided urine was collected from 277 subjects including 169 BCa patients and 108 non-BCa controls. Urinary APE1/Ref-1 level was assessed by enzyme-linked immunosorbent assay (ELISA). APE1/Ref-1 levels were significantly elevated in BCa patients relative to levels in non-BCa controls and were correlated with tumor grade and stage. Urinary APE1/Ref-1 levels were also higher in patients with recurrence history of BCa. The receiver operating characteristics (ROC) curve of APE1/Ref-1 showed an area under the curve of 0.83, indicating the reliability and validity of this biomarker. The optimal combination of sensitivity and specificity was determined to be 82% and 80% at a cut-off value of 0.376 ng/100 μL for detection of APE1/Ref-1 in urine. In conclusion, urinary APE1/Ref-1 levels measured from noninvasively obtained body fluids would be clinically applicable for diagnosis of BCa.

γ-Aminobutyric acid (GABA) generates persistent tonic inhibitory currents ( Itonic) and conventional inhibitory postsynaptic currents in the hypothalamic paraventricular nucleus (PVN) via activation of GABAA receptors (GABAARs). We investigated the pathophysiological significance of astroglial GABA uptake in the regulation of Itonic in the PVN neurons projecting to the rostral ventrolateral medulla (PVN-RVLM). The Itonic of PVN-RVLM neurons were significantly reduced in heart failure (HF) compared with sham-operated (SHAM) rats. Reduced Itonic sensitivity to THIP argued for the decreased function of GABAAR δ subunits in HF, whereas similar Itonic sensitivity to benzodiazepines argued against the difference of γ2 subunit-containing GABAARs in SHAM and HF rats. HF Itonic attenuation was reversed by a nonselective GABA transporter (GAT) blocker (nipecotic acid, NPA) and a GAT-3 selective blocker, but not by a GAT-1 blocker, suggesting that astroglial GABA clearance increased in HF. Similar and minimal Itonic responses to bestrophin-1 blockade in SHAM and HF neurons further argued against a role for astroglial GABA release in HF Itonic attenuation. Finally, the NPA-induced inhibition of spontaneous firing was greater in HF than in SHAM PVN-RVLM neurons, whereas diazepam induced less inhibition of spontaneous firing in HF than in SHAM neurons. Overall, our results showed that combined with reduced GABAARs function, the enhanced astroglial GABA uptake-induced attenuation of Itonic in HF PVN-RVLM neurons explains the deficit in tonic GABAergic inhibition and increased sympathetic outflow from the PVN during heart failure.

Background Midazolam, a benzodiazepine, has a hypnotic effect and is widely used as a sedative. The role of midazolam in activation of macrophages during sepsis is not known. The aim of this study was to evaluate the antiinflammatory actions of midazolam in cultured macrophages. Methods Using a macrophage cell line, RAW264.7 cells, the effect of midazolam on proinflammatory mediators and activation of mitogen-activated protein kinase was measured by Western blot. Nuclear factor-kappaB (NF-kappaB) activation and translocation of p65 subunit of NF-kappaB was measured using luciferase assay and immunocytochemistry. Superoxide production was measured by lucigenin chemiluminescence. Results Midazolam significantly inhibited lipopolysaccharide-induced up-regulation of both cyclooxygenase 2 and inducible nitric oxide synthase in a dose-dependent manner (approximately 3-30 microm). IkappaB-alpha degradation and NF-kappaB transcriptional activity induced by lipopolysaccharide were also suppressed by the midazolam. Nuclear translocation of the p65 subunit of NF-kappaB was inhibited by midazolam. Furthermore, midazolam suppressed phosphorylation of p38 mitogen-activated protein kinase and also inhibited lipopolysaccharide-induced superoxide production in macrophages. Conclusions These results suggest that midazolam has an antiinflammatory action by inhibiting inducible nitric oxide synthase and cyclooxygenase-2 expression, possibly through suppression of NF-kappaB and p38 mitogen-activated protein kinase activation.

Fasciola hepatica, a parasitic flatworm belonging to the Class Trematoda, is one of the first metazoan groups to possess a centralized nervous system. However, the electrophysiological properties of neurones in F. hepatica are largely unknown. In the present study, we acutely isolated viable neurones from F. hepatica and characterized their electrophysiological properties. A hyperpolarization-activated cation current was recorded in the cells using the whole-cell patch-clamp. The current was found to be activated slowly at membrane potentials negative to 0 mV and did not display any time-dependent inactivation. This current was reduced by 1 mM Gd3+ to the level of the leak current, while 3 mM of Cs+ had no effect. However, the current was inhibited by extracellular acidosis in the pH range 7.0–7.8, and the membrane potentials of these cells were depolarized by extracellular alkalosis in the pH range of 5.8 to 8.2. Gd3+ (1 mM), which inhibited the pH-sensitive hyperpolarization-activated cation current, also hyperpolarized the cells. In summary, we isolated single neurones from F. hepatica, and these were found to express a pH-sensitive hyperpolarization-activated cation current. This current may participate in the membrane depolarization of F. hepatica neurones during alkaline challenge.

Activation of protein kinase C (PKC) is closely linked with endothelial dysfunction. However, the effect of PKCβII on endothelial dysfunction has not been characterized in cultured endothelial cells. Here, using adenoviral PKCβII gene transfer and pharmacological inhibitors, the role of PKCβII on endothelial dysfucntion was investigated in cultured endothelial cells. Phorbol 12-myristate 13-acetate (PMA) increased reactive oxygen species (ROS), p66shc phosphorylation, intracellular adhesion molecule-1, and monocyte adhesion, which were inhibited by PKCβi (10 nM), a selective inhibitor of PKCβII. PMA increased the phosphorylation of CREB and manganese superoxide dismutase (MnSOD), which were also inhibited by PKCβi. Gene silencing of CREB inhibited PMA-induced MnSOD expression, suggesting that CREB plays a key role in MnSOD expression. Gene silencing of PKCβII inhibited PMA-induced mitochondrial ROS, MnSOD, and ICAM-1 expression. In contrast, overexpression of PKCβII using adenoviral PKCβII increased mitochondrial ROS, MnSOD, ICAM-1, and p66shc phosphorylation in cultured endothelial cells. Finally, PKCβII-induced ICAM-1 expression was inhibited by Mito-TEMPO, a mitochondrial ROS scavenger, suggesting the involvement of mitochondrial ROS in PKC-induced vascular inflammation. Taken together, the results suggest that PKCβII plays an important role in PMA-induced endothelial dysfunction, and that the inhibition of PKCβII-dependent p66shc signaling acts as a therapeutic target for vascular inflammatory diseases.

To investigate whether alpha1-adrenoceptors are involved in pain behaviors in streptozotocin (STZ)-induced diabetic rats, we measured the effects of phenylephrine or prazosin on allodynia in the diabetic rats. Phenylephrine aggravated allodynia, while prazosin alleviated allodynia in the diabetic rats. We also measured alpha1-adrenoceptors gene expression or density of [3H]-prazosin binding sites in the dorsal root ganglia (DRG) and spinal cord in painful diabetic rats. Alpha1-adrenoceptors mRNA and density of [3H]prazosin binding sites were increased in the DRG of the diabetic rats, however there were no significant differences in alpha1-adrenoceptors expression in the spinal cord between the control and diabetic rats. These results suggest increased alpha1-adrenoceptors in the DRG may play a role in the pathogenesis of painful diabetic neuropathy.

It has been known that many immediately early genes are expressed during ischemia/reperfusion (I/R) injury. Here, employing a model of hepatic I/R, we show that inducible nitric oxide synthase (iNOS) is induced via the activation of nuclear factor kappaB (NF-kappaB) after I/R in rat liver. When liver was subjected to ischemia followed by reperfusion, but not ischemia alone, an NF-kappaB complex composed of p50/p65 heterodimer and p50 homodimer was rapidly activated within 1 h and remained elevated for up to 3 h, and then tended to decline after 5 h of reperfusion. Also, the expression of iNOS mRNA was initiated after 1 h and continued to increase after 5 h of reperfusion during the time course studied. This upregulated iNOS mRNA expression coincides with increased iNOS enzyme activity and NF-kappaB binding activity after hepatic I/R. Administration of N-acetylcysteine (NAC, 20 mg/kg i.v. 10 min before reperfusion), an antioxidant, not only significantly inhibited the expression of iNOS mRNA but also blocked upregulated NF-kappaB binding activity after reperfused liver. These results suggest that NF-kappaB is activated by oxidative stress during hepatic I/R and may play a significant role in the induction of the iNOS gene.

n-3-Polyunsaturated fatty acids (PUFAs) protect against myocardial infarction, arteriosclerosis and high blood pressure by stimulating endothelial nitric oxide synthase (eNOS) to increase nitric oxide (NO) production. However, the mechanism remains to be elucidated. This study investigated the role of SIRT1 in the protective effects of docosahexaenoic acid (DHA) in vascular endothelial cells. Exposure of human umbilical vein endothelial cells (HUVECs) to 0.3–30 μM DHA did not affect cell viability, and DHA treatment dose-dependently increased SIRT1 expression. The DHA-mediated increase in SIRT1 expression induced eNOS deacetylation, increasing endothelial NO. However, inhibition of SIRT1 inhibited DHA-mediated increases in NO production. This effect was mediated via deacetylation of lysines 496 and 506 in the eNOS calmodulin-binding domain. The effects of DHA were also demonstrated in rat aortic rings, in which DHA treatment increased SIRT1 expression and bioavailable NO. Our results demonstrate that SIRT1 plays an important role in DHA-mediated increases in bioavailable NO via decreased eNOS acetylation.

BACKGROUND The effect of paricalcitol on renal ischemia-reperfusion injury (IRI) has not been investigated. We examined whether paricalcitol is effective in preventing inflammation in a mouse model of IRI, and evaluated the cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) pathways as a protective mechanism of paricalcitol. METHODS Paricalcitol (0.3 μg/kg) was administered to male C57BL/6 mice 24 h before IRI. Bilateral kidneys were subjected to 23 min of ischemia, and mice were killed 72 h after IRI. The effects of paricalcitol on renal IRI were evaluated in terms of renal function, tubular necrosis, apoptotic cell death, inflammatory cell infiltration and inflammatory cytokines. The effects of paricalcitol on COX-2, PGE2 and its receptors were investigated. RESULTS Paricalcitol pretreatment improved renal function (decreased blood urea nitrogen and serum creatinine levels), tubular necrosis and apoptotic cell death in IRI-mice kidneys. The infiltration of inflammatory cells (T cells and macrophages), and the production of proinflammatory cytokines (RANTES, tumor necrosis factor-α, interleukin-1β and interferon-γ) were reduced in paricalcitol-treated mice with IRI. Paricalcitol up-regulated COX-2 expression, PGE2 synthesis and mRNA expression of receptor subtype EP4 in post-ischemic renal tissue. The cotreatment of a selective COX-2 inhibitor with paricalcitol restored functional injury and tubular necrosis in paricalcitol-treated mice with IRI. CONCLUSIONS Our study demonstrates that paricalcitol pretreatment prevents renal IRI via the inhibition of renal inflammation, and the up-regulation of COX-2 and PGE2 is one of the protective mechanisms of paricalcitol in renal IRI.

The present study evaluated the mechanism of apoptosis caused by posttranslational modification, hyperacetylation in triple-negative breast cancer (TNBC) cells. We previously showed that trichostatin A (TSA) induced secretion of acetylated apurinic apyrimidinic endonuclease 1/redox factor-1 (Ac-APE1/Ref-1). This is the first report showing that Ac-APE1/Ref-1 initiates apoptosis in TNBC cells by binding to the receptor for advanced glycation end products (RAGE). The functional significance of secreted Ac-APE1/Ref-1 was studied by induction of intracellular hyperacetylation through co-treatment with acetylsalicylic acid and TSA in MDA-MB-231 cells. In response to hyperacetylation, secretion of Ac-APE1/Ref-1 in vesicles was observed, resulting in significantly decreased cell viability and induction of apoptosis with increased expression of RAGE. The hyperacetylation-induced apoptosis was similar in two other TNBC cell lines: BT-459 and MDA-MB-468. Therefore, hyperacetylation may be a therapeutic target for treatment of TNBCs. This study introduces a novel paradigm whereby posttranslational modification induces apoptotic cell death in breast cancer cells resistant to standard chemotherapeutic agents through secretion of auto- or paracrine molecules such as Ac-APE1/Ref-1. Citation Format: Yu Ran Lee, Hee Kyoung Joo, Ki Mo Kim, Byeong Hwa Jeon, Sunga Choi. Extracellularly secreted APE1/Ref-1 triggers apoptosis in triple-negative breast cancer cells via RAGE binding, which is mediated through acetylation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4422.

PURPOSE Peroxynitrite plays a critical role in vascular pathophysiology by increasing arginase activity and decreasing endothelial nitric oxide synthase (eNOS) activity. Therefore, the aims of this study were to investigate whether arginase inhibition and L-arginine supplement could restore peroxynitrite-induced endothelial dysfunction and determine the involved mechanism. MATERIALS AND METHODS Human umbilical vein endothelial cells (HUVECs) were treated with SIN-1, a peroxynitrite generator, and arginase activity, nitrite/nitrate production, and expression levels of proteins were measured. eNOS activation was evaluated via Western blot and dimer blot analysis. We also tested nitric oxide (NO) and reactive oxygen species (ROS) production and performed a vascular tension assay. RESULTS SIN-1 treatment increased arginase activity in a time- and dose-dependent manner and reciprocally decreased nitrite/nitrate production that was prevented by peroxynitrite scavenger in HUVECs. Furthermore, SIN-1 induced an increase in the expression level of arginase I and II, though not in eNOS protein. The decreased eNOS phosphorylation at Ser1177 and the increased at Thr495 by SIN-1 were restored with arginase inhibitor and L-arginine. The changed eNOS phosphorylation was consistent in the stability of eNOS dimers. SIN-1 decreased NO production and increased ROS generation in the aortic endothelium, all of which was reversed by arginase inhibitor or L-arginine. N(G)-Nitro-L-arginine methyl ester (L-NAME) prevented SIN-1-induced ROS generation. In the vascular tension assay, SIN-1 enhanced vasoconstrictor responses to U46619 and attenuated vasorelaxant responses to acetylcholine that were reversed by arginase inhibition. CONCLUSION These findings may explain the beneficial effect of arginase inhibition and L-arginine supplement on endothelial dysfunction under redox imbalance-dependent pathophysiological conditions.

Cordycepin (3-deoxyadenosine), found in Cordyceps spp., has been known to have many therapeutic effects including immunomodulatory, anti-inflammatory, antimicrobial, and anti-aging effects. Moreover, anti-tumor and anti-metastatic effects of cordycepin have been reported, but the mechanism causing cancer cell death is poorly characterized. The present study was designed to investigate whether the mechanisms of cordycepin-induced cell death were associated with estrogen receptor in breast cancer cells. Exposure of both MDA-MB-231 and MCF-7 human breast cancer cells to cordycepin resulted in dose-responsive inhibition of cell growth and reduction in cell viability. The cordycepin-induced cell death in MDA-MB-231 cells was associated with several specific features of the mitochondria-mediated apoptotic pathway, which was confirmed by DNA fragmentation, TUNEL, and biochemical assays. Cordycepin also caused a dose-dependent increase in mitochondrial translocation of Bax, triggering cytosolic release of cytochrome c and activation of caspases-9 and -3. Interestingly, MCF-7 cells showed autophagy-associated cell death, as observed by the detection of an autophagosome-specific protein and large membranous vacuole ultrastructure morphology in the cytoplasm. Cordycepin-induced autophagic cell death has applications in treating MCF-7 cells with apoptotic defects, irrespective of the ER response. Although autophagy has a survival function in tumorigenesis of some cancer cells, autophagy may be important for cordycepin-induced MCF-7 cell death. In conclusion, the results of our study demonstrate that cordycepin effectively kills MDA-MB-231 and MCF-7 human breast cancer cell lines in culture. Hence, further studies should be conducted to determine whether cordycepin will be a clinically useful, ER-independent, chemotherapeutic agent for human breast cancer.

Nafamostat mesilate (NM) is a serine protease inhibitor with anticoagulant and anti-inflammatory effects. NM has been used in Asia for anticoagulation during extracorporeal circulation in patients undergoing continuous renal replacement therapy and extra corporeal membrane oxygenation. Oxidative stress is an independent risk factor for atherosclerotic vascular disease and is associated with vascular endothelial function. We investigated whether NM could inhibit endothelial dysfunction induced by tumor necrosis factor-α (TNF-α). Human umbilical vein endothelial cells (HUVECs) were treated with TNF-α for 24 h. The effects of NM on monocyte adhesion, vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) protein expression, p38 mitogen-activated protein kinase (MAPK) activation, and intracellular superoxide production were then examined. NM (0.01~100 µg/mL) did not affect HUVEC viability; however, it inhibited the increases in reactive oxygen species (ROS) production and p66shc expression elicited by TNF-α (3 ng/mL), and it dose dependently prevented the TNF-α-induced upregulation of endothelial VCAM-1 and ICAM-1. In addition, it mitigated TNF-α-induced p38 MAPK phosphorylation and the adhesion of U937 monocytes. These data suggest that NM mitigates TNF-α-induced monocyte adhesion and the expression of endothelial cell adhesion molecules, and that the anti-adhesive effect of NM is mediated through the inhibition of p66shc, ROS production, and p38 MAPK activation.

The p66shc adaptor protein mediates age-associated oxidative stress. We examined the role of p66shc in endothelial nitric oxide synthase (eNOS) signaling. Overexpression of p66shc inhibited eNOS-dependent NO production. RNAi-mediated down-regulation of endogenous p66shc led to activation of the proto-oncogene ras, and Akt kinase, with a corresponding increase in phosphorylation of eNOS at S1177 (S1179 on bovine eNOS). In rat aortic rings, down-regulation of p66shc suppressed the vasoconstrictor response to phenyephrine that was abrogated by treatment with the NOS inhibitor l-NAME, and enhanced vasodilation induced by sub-maximal doses of acetylcholine. These findings highlight a pivotal role for p66shc in inhibiting endothelial NO production, and endothelium-dependent vasorelaxation, that may provide important mechanistic information about endothelial dysfunction seen with aging.

Apurinic/apyrimidinic endonuclease1/redox factor-1 (APE1/Ref-1) is a DNA repair enzyme whose expression is increased in several forms of cancerous cells. We investigated whether serum APE1/Ref-1 is elevated in patients with bladder cancer. Tumor tissue and serum from patients with bladder cancer who had not received chemotherapy or radiotherapy (n=51) and non-tumor healthy controls (n=55) was used. In immunohistochemistry, and immunoblot analysis, the APE1/Ref-1 protein level of bladder tissues was increased in patients with bladder cancer. The serum APE1/Ref-1 levels were significantly elevated in bladder cancer patients than in those of non-tumor healthy controls and its levels were correlated with tumor staging and grading. The ROC curve of APE1/Ref-1 showed an excellent area under the ROC curve of 0.824. In this study, the optimal combination of sensitivity and specificity were determined as 93% and 59% for a cut-off value of serum APE1/Ref-1 of 2.83 ng/100 μl, respectively. The serum APE1/Ref-1 levels are increased in patients with bladder cancer with recurrence. These data indicate serum APE1/Ref-1 may help to identify patients with bladder cancer and it can be used as a serological marker of bladder cancer. Citation Format: Sunga Choi, Ju Hyun Shin, Yu Ran Lee, Myoung Soo Park, Chang Nam Yim, Young Gil Na, Jae Sung Lim, Byeong Hwa Jeon. APE1/Ref-1 as a serological biomarker for the detection of bladder cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 872. doi:10.1158/1538-7445.AM2014-872

Anti-Pv200 antibody levels were assessed in samples from endemic areas of Plasmodium vivax malaria in the Republic of Korea (ROK), using an indirect enzyme-linked immunosorbent assay (ELISA) method. Asymptomatic carriers of P. vivax were detected using nested polymerase chain reaction (PCR) of blood samples. Anti-Pv200 antibody levels in 20 vivax malaria patients (optical density +/- standard deviation [OD +/- SD] values 1.85 +/- 0.29 of IgG isotype and 1.33 +/- 1.33 of IgM isotype) were markedly higher than those of uninfected, malaria-naive controls (0.08 +/- 0.16 of IgG isotype and 0.04 +/- 0.04 of IgM isotype). Antibody levels for 7 out of 8 soldiers with a recent malaria infection were sustained above the cut-off values for 4 months after successful treatment. Analysis of serum collected from 40 healthy, asymptomatic soldiers who had a P. vivax malaria attack within 3 months after our sampling, revealed 11 antibody-positive samples (27.5%), compared to 5 positive samples (12.5%) collected from a random selection of 40 soldiers. Among a larger pool of 1,713 soldiers who had served in high-risk areas for P. vivax transmission, 15% were antibody positive. Among 1,000 blood samples from asymptomatic soldiers who had served in the high-risk areas, 4 samples (0.4%) were parasite positive, as determined by nested PCR. Our results show that anti-Pv200 antibody levels can provide useful information in the late diagnosis of P. vivax malaria infection in a previously naive population and also in large seroepidemiologic studies. Furthermore, our results suggest that asymptomatic P. vivax carriers could be important in the current outbreak of malaria in Korea.

Abdominal aortic coarctation above renal artery is known to cause hypertension proximal to the level of constriction as a consequence of reduced renal perfusion and subsequent activation of the renin– angiotensin system. A differential expression of p66shc protein in the change of arterial pressure such as abdominal aortic coarctationwas not known. We investigated the endothelium-dependent relaxation, expressions of p66shc, nitric oxide synthase (eNOS) and superoxide production in the aortic coarctation rats. In the present study, we used the abdominal aortic coarctation rat model which has shown hypertension in proximal part and hypotension in distal part of aortic coarctation to see the effects of different levels of blood pressure on the vascular tissue in the same animals. Endothelium-dependent relaxation in response to acetylcholine (Ach) was impaired only in the aortic segments above aortic coarctation (35.0±7.1% at 1muM Ach), compared to sham controls (86.6±6.0% at 1muMAch). The aortic segments exposed to increased blood pressure showed the decreased phosphorylation of eNOS and the increased phosphorylation of p66shc and increased superoxideproduction. In contrast, phosphorylation of p66shc was reduced in the aortic segments below coarctation. Angiotensin II elicited significantly increased phosphorylation of p66shc in the endothelial cells. This study demonstrates that the phosphorylation of p66shc acts as an important signal to affect the endothelial dysfunction using the abdominal aortic coarctation rat model.

Despite years of therapeutic development and research, metastasis is still the major driver of mortality in cancer patients. Breast cancer is a second leading cause of cancer deaths in woman world-wide. The prevalence of breast cancer continues to increase by approximately 2% each year. Breast cancer also presents as metastatic disease, which has spread beyond the original organ, breast to bone, liver, lung and brain. Recently, many studies have reported that natural products- dietary phytochemicals including mushroom are known to exhibit a variety of anti-cancer, anti-invasive and anti-metastatic activities. Naematolma spp. is a basidiomycete of the fungus that is known to produce anti-tumor compound, clavaric acid. In our previous studies, HFNS has been shown to significantly inhibit cell proliferation and induce apoptosis of human estrogen-nonresponsive MDA-MB-231 breast cancer cells. In this present study, we report for the first time that hexane fraction of Naematoloma sublateritium (HFNS) regulates TNF-alpha induced invasion and migration in MDA-MB-231 cells. We observed that non-cytotoxic concentrations (10-50 μg/mL) of HFNS markedly inhibited the invasion and migration of highly metastatic MDA-MB-231 cells as shown by a Matrigel invasion assay and wound healing analysis, respectively. The results of a gelatin zymography showed that HFNS suppressed the activity of matrix metalloproteinase (MMP)-9 and urokinase plasminogen activator (uPA). Western blot results demonstrated that treatment with HFNS had decreased level of MMP-9, and uPA, while the expression of the endogenous inhibitor proteins including tissue inhibitors of MMP (TIMP-1 and TIMP-2), and plasminogen activator inhibitor (PAI)-1, was up-regulated in a dose-dependent manner. Furthermore, HFNS suppressed the phosphorylation of p38, and JNK1/2 in MDA-MB-231 cells, where extracellular signal-regulated kinase (ERK) 1/2 were not affected. It was confirmed by pretreatment with SB203580 (p38 inhibitor), SP600125 (JNK inhibitor), and PD98059 (ERK inhibitor), respectively, before stimulation with TNF-alpha. Interestingly, the HFNS treatment also led to a dose-dependent inhibition on NF-κB and activator protein-1(AP-1) binding activity which are downstream targets of JNK and p38. The gel-shift assay results supported that HFNS treatment caused down-regulation of MMP-9 and uPA gene in mRNA level. Taken together, these data suggest the inhibitory effect of HFNS on the metastatic potential of MDA-MB-231 cells through inhibiting the phosphorylation of JNK/p38 and reducing NFκB and AP-1 DNA binding activities. In conclusion, we propose that HFNS may be a safe and potential therapeutic agent against metastasis of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2568. doi:1538-7445.AM2012-2568

Caesalpinia sappan (C. sappan) is a medicinal plant used for promoting blood circulation and removing stasis. During a screening procedure on medicinal plants, the ethylacetate extract of the lignum of C. sappan (CLE) showed inhibitory activity on arginase which has recently been reported as a novel therapeutic target for the treatment of cardiovascular diseases such as atherosclerosis. CLE inhibited arginase II activity prepared from kidney lysate in a dose-dependent manner. In HUVECs, inhibition of arginase activity by CLE reciprocally increased NOx production through enhancement of eNOS dimer stability without any significant changes in the protein levels of eNOS and arginase II expression. Furthermore, CLE-dependent arginase inhibition resulted in increase of NO generation and decrease of superoxide production on endothelium of isolated mice aorta. These results indicate that CLE augments NO production on endothelium through inhibition of arginase activity, and may imply their usefulness for the treatment of cardiovascular diseases associated with endothelial dysfunction.