(S)-Erypoegin K, an isoflavone isolated from Erythrina poeppigiana, is a novel inhibitor of topoisomerase IIα: Induction of G2 phase arrest in human gastric cancer cells.

Erypoegin K, an isoflavone isolated from the stem bark of Erythrina poeppigiana, has a single chiral carbon in its structure and exists naturally as a racemic mixture. Our previous study showed (S)-erypoegin K selectively exhibits potent anti-proliferative and apoptosis-inducing activity against human leukemia HL-60 cells. To identify the target molecule of (S)-erypoegin K, we employed the human cancer cell panel analysis (termed JFCR39) coupled with a drug sensitivity database of pharmacologically well-characterized drugs for comparison using the COMPARE algorithm. (S)-erypoegin K exhibited a similar profile to that of etoposide, suggesting the molecular target for erypoegin K may be topoisomerase II (Topo II). Subsequent experiments using purified human Topo IIα established that the (S)-isomer selectively stabilizes the cleavage complex composed of double-stranded plasmid DNA and the enzyme. Moreover, (S)-erypoegin K inhibited decatenation of kinetoplast DNA. Molecular docking studies clearly indicated specific binding of the (S)-isomer to the active site of Topo IIα involving hydrogen bonds that help stabilize the cleavage complex. (S)-erypoegin K displayed potent cytotoxic activity against two human gastric cancer cells GCIY and MKN-1 with IC50 values of 0.270 and 0.327 µM, respectively, and induced enzyme activities of caspase 3 and 9. Cell cycle analysis showed marked cell cycle arrest at G2 phase in both cell lines. (S)-erypoegin K also displayed significant antitumor activity toward GCIY xenografted mice. The present study suggests (S)-erypoegin K acts as a Topo II inhibitor to block the G2/M transition of cancer cells.

EPHB6 controls catecholamine biosynthesis by up-regulating tyrosine hydroxylase transcription in adrenal gland chromaffin cells.

EPHB6 is a member of the erythropoietin-producing hepatocellular kinase (EPH) family and a receptor tyrosine kinase with a dead kinase domain. It is involved in blood pressure regulation and adrenal gland catecholamine (CAT) secretion, but several facets of EPHB6-mediated CAT regulation are unclear. In this study, using biochemical, quantitative RT-PCR, immunoblotting, and gene microarray assays, we found that EPHB6 up-regulates CAT biosynthesis in adrenal gland chromaffin cells (AGCCs). We observed that epinephrine content is reduced in the AGCCs from male Ephb6-KO mice, caused by decreased expression of tyrosine hydroxylase, the rate-limiting enzyme in CAT biosynthesis. We demonstrate that the signaling pathway from EPHB6 to tyrosine hydroxylase expression in AGCCs involves Rac family small GTPase 1 (RAC1), MAP kinase kinase 7 (MKK7), c-Jun N-terminal kinase (JNK), proto-oncogene c-Jun, activator protein 1 (AP1), and early growth response 1 (EGR1). On the other hand, signaling via extracellular signal-regulated kinase (ERK1/2), p38 mitogen-activated protein kinase, and ELK1, ETS transcription factor (ELK1) was not affected by EPHB6 deletion. We further report that EPHB6's effect on AGCCs was via reverse signaling through ephrin B1 and that EPHB6 acted in concert with the nongenomic effect of testosterone to control CAT biosynthesis. Our findings elucidate the mechanisms by which EPHB6 modulates CAT biosynthesis and identify potential therapeutic targets for diseases, such as hypertension, caused by dysfunctional CAT biosynthesis.

Induction of enantio-selective apoptosis in human leukemia HL-60 cells by (S)-erypoegin K, an isoflavone isolated from Erythrina poeppigiana.

Erypoegin K, an isoflavone isolated from the stem bark of Erythrina poeppigiana, has potent apoptosis-inducing effect on human leukemia HL-60 cells. Erypoegin K has a chiral carbon at the C-2'' position of its furan ring and naturally occurs as a racemic mixture of (S)- and (R)-isomers. In the present study, we semi-synthesized (RS)-erypoegin K from genistein and separated the optical isomers by HPLC using a chiral column to characterize its apoptosis-inducing activity. Apoptotic cell death was assessed by analyzing caspase-3 and caspase-9 activation, nuclear fragmentation, and genomic DNA ladder formation. (S)-erypoegin K showed exclusive anti-proliferative and apoptosis-inducing activity, with an IC50 value of 90 nM, about 50% lower than that of its racemic mixture (175 nM). By contrast, no apoptosis-inducing activity was shown by the (R)-isomer. In addition, methylglyoxal accumulation in the culture medium was observed only in cells treated with (S)-erypoegin K. These results demonstrated that (S)-erypoegin K is a unique bioactive component that has potent apoptosis-inducing activity on HL-60 cells.

Peripheral blood cell-free DNA is an alternative tumor DNA source reflecting disease status in myelodysplastic syndromes.

Genetic alterations in myelodysplastic syndromes (MDS) are critical for pathogenesis. We previously showed that peripheral blood cell-free DNA (PBcfDNA) may be more sensitive for genetic/epigenetic analyses than whole bone marrow (BM) cells and mononuclear cells in peripheral blood (PB). Here we analyzed the detailed features of PBcfDNA and its utility in genetic analyses in MDS. The plasma-PBcfDNA concentration in MDS and related diseases (N = 33) was significantly higher than that in healthy donors (N = 14; P = 0.041) and in International Prognostic Scoring System higher-risk groups than that in lower-risk groups (P = 0.034). The concentration of plasma-/serum-PBcfDNA was significantly correlated with the serum lactate dehydrogenase level (both P < 0.0001) and the blast cell count in PB (P = 0.034 and 0.025, respectively). One nanogram of PBcfDNA was sufficient for one assay of Sanger sequencing using optimized primer sets to amplify approximately 160-bp PCR products. PBcfDNA (approximately 50 ng) can also be utilized for targeted sequencing. Almost all mutations detected in BM-DNA were also detected using corresponding PBcfDNA. Analyses using serially harvested PBcfDNA from an RAEB-2 patient showed that the somatic mutations and a single nucleotide polymorphism that were detected before allogeneic transplantation were undetectable after transplantation, indicating that PBcfDNA likely comes from MDS clones that reflect the disease status. PBcfDNA may be a safer and easier alternative to obtain tumor DNA in MDS.

New whole-body multimodality imaging of gastric cancer peritoneal metastasis combining fluorescence imaging with ICG-labeled antibody and MRI in mice.

BACKGROUND: Peritoneal metastasis is the most frequent pattern of recurrence after curative surgery for gastric cancer. However, such a recurrence is difficult to detect by conventional computed tomography (CT) and magnetic resonance imaging (MRI) at an early stage. To improve the sensitivity and specificity of diagnostic imaging for peritoneal metastasis, we developed a new type of multimodality imaging combining fluorescence imaging with near-infrared fluorophore (NIR)-labeled antibodies and MRI. METHODS: Dual optical imaging of peritoneal metastasis was carried out using luciferase-tagged gastric cancer cell lines and XenoLight CF750 or indocyanine green (ICG)-labeled anti-human epidermal growth factor receptor (EGFR) or CEA antibody as a probe in mice with Ivis in vivo imaging system. RESULTS: This whole-body fluorescent imaging system sensitively detected metastatic foci <1 mm in diameter in the peritoneal cavity noninvasively. Fluorescence imaging proved to be specific because the fluorescence signal was abolished by blocking with excess unlabeled antibody. Although this fluorescence imaging had higher sensitivity for detection of small-sized peritoneal metastases than MRI, it proved difficult to accurately determine organ distribution of the metastasis. We thus developed a multimodality imaging system by the fusion of the three-dimensional fluorescence image with the MRI image and demonstrated its improved diagnostic accuracy over either method alone. CONCLUSION: The present results suggest that multimodality imaging consisting of fluorescence imaging with NIR-labeled EGFR or CEA antibody and MRI allows sensitive, specific, and anatomically accurate detection of peritoneal metastasis noninvasively at an early stage.

[Studies on the Isolation and Molecular Mechanisms of Bioactive Phytochemicals].

Studies on the isolation and molecular mechanisms of phytochemicals with anti-tumor or anti-inflammatory properties are important to developing new drugs for cancer and neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. In the course of a study to screen bioactive isoflavones from Erythrina poeppigiana (Leguminosae), we isolated an isoflavone with potent apoptosis-inducing activity against human leukemia HL-60 cells. It was designated erypoegin K. The studies demonstrated an enantiomer, (S)-erypoegin K, displayed selective cytotoxic activity, was a novel inhibitor of topoisomerase II, and possessed anti-tumor activity both in vitro and in vivo. We identified other apoptosis-inducing isoflavones with the ability to inhibit glyoxalase I. Dimeric acridone alkaloids, carbazole alkaloids, and coumarin and quinoline derivatives-all obtained mainly from plants in the family Rutaceae-induced apoptosis of HL-60 cells via the production of reactive oxygen species and mitochondrial dysfunction. We also identified terpenoid coumarins, carbazole quinones, rotenoid derivatives, and quinolone alkaloids with anti-inflammatory activities. These compounds reduced nitric oxide (NO) production from RAW264.7 macrophage cells stimulated with lipopolysaccharides and interferon-γ. Some of the compounds displayed neuroprotective activity by reducing NO production. This review primarily describes our recent studies on erypoegin K, and other compounds with apoptosis-inducing and anti-inflammatory activities.

Chemical inducer of regucalcin attenuates lipopolysaccharide-induced inflammatory responses in pancreatic MIN6 ß-cells and RAW264.7 macrophages.

We previously isolated derrisfolin A, a novel rotenoid derivative, from the stems of Derris trifoliata Lour. (Leguminosae). Here, we report that derrisfolin A induces the expression of endogenous regucalcin (RGN) protein in both pancreatic MIN6 ß-cells and RAW264.7 macrophages. Induction of RGN expression by derrisfolin A or retrovirus-mediated gene transfer in MIN6 cells and RAW264.7 macrophages significantly decreased lipopolysaccharide (LPS)-induced mRNA expression of Nos2, Il1b, and Tnf via nuclear factor-κB activation; reduced LPS-induced apoptosis in MIN6 cells, accompanied by decreased production of nitric oxide, interleukin-1ß, and tumor necrosis factor-α; and attenuated generation of LPS-induced reactive oxygen species, malondialdehyde, and 3-nitrotyrosine in MIN6 cells. Additionally, in co-cultures of MIN6 cells with RAW264.7 macrophages in the presence of LPS, induction of RGN expression by derrisfolin A or retrovirus-mediated gene transfer in RAW264.7 macrophages attenuated apoptosis and oxidative/nitrosative stress in MIN6 cells. These results suggest that the induction of RGN expression in MIN6 cells was effective in suppressing LPS-induced inflammatory cytotoxicity and that in co-culture conditions, the induction of RGN expression in RAW264.7 macrophages blocked LPS-induced paracrine effects of RAW264.7 macrophages on inflammatory cytotoxicity in MIN6 cells. Our findings suggest that derrisfolin A, a chemical inducer of RGN, might be useful for developing a new drug against macrophage-associated ß-cell inflammation in type 2 diabetes.

Establishment and characterization of a noradrenergic adrenal chromaffin cell line, tsAM5NE, immortalized with the temperature-sensitive SV40 T-antigen.

We established a clonal adrenal medullary cell line, named tsAM5NE, from transgenic mice harbouring the temperature-sensitive Simian virus 40 large T-antigen gene, under the control of the tyrosine hydroxylase promoter. tsAM5NE cells conditionally grew at a permissive temperature of 33°C and exhibited the noradrenergic chromaffin cell phenotype. To understand the characteristics of tsAM5NE cells, we first examined the responsiveness of the cells to ligands of the GDNF (glial cell line-derived neurotrophic factor) family. tsAM5NE cells proliferated at the permissive temperature of 33°C in response to either GDNF or neurturin, but not artemin or persephin. At the non-permissive temperature of 39°C, GDNF or neurturin caused tsAM5NE cells to differentiate into neuron-like cells; however, the differentiated cells died in a time-dependent manner. Interestingly, LIF (leukaemia inhibitory factor) did not affect the GDNF-mediated cell proliferation at 33°C, but promoted the survival and differentiation of GDNF-treated cells at 39°C. In the presence of GDNF plus LIF, the morphological change induced by the temperature shift was associated with up-regulated expression of neuronal markers, indicating that the cells had indeed undergone neuronal differentiation. Thus, we demonstrated that tsAM5NE cells had the capacity to terminally differentiate into neuron-like cells in response to GDNF plus LIF when the oncogene was inactivated by the temperature shift. Thus, this cell line provides a useful model system for studying the mechanisms regulating neuronal differentiation.

Regucalcin enhances adipocyte differentiation and attenuates inflammation in 3T3-L1 cells.

Dysregulation of adipocyte differentiation and dysfunction play key roles in the pathogenesis of obesity and associated disorders such as diabetes and metabolic syndrome, and as such, a better understanding of the molecular mechanism of adipogenesis may help to elucidate the pathological condition of obesity and its associated disorders. Regucalcin (RGN) plays multiple regulatory roles in intracellular Ca2+ signaling pathways in mammalian cells. Here, we report that overexpression of RGN enhances lipid accumulation in 3T3-L1 adipocyte cells after adipogenic stimulation, accompanied by upregulation of adipocyte differentiation marker proteins. In contrast, genetic disruption of RGN inhibited adipogenic stimulation-induced differentiation of 3T3-L1 cells. Furthermore, RGN overexpression in differentiated 3T3-L1 adipocytes blocked inflammatory crosstalk between 3T3-L1 adipocytes and RAW264.7 macrophages in a transwell coculture system. Knockdown of RGN expression in cocultured 3T3-L1 adipocytes enhanced their susceptibility to RAW264.7 macrophage-mediated inflammation. These results suggest that RGN is required for 3T3-L1 adipocyte differentiation and that it exerts anti-inflammatory activity against 3T3-L1 adipocyte inflammation after coculture with RAW264.7 macrophages. Thus, RGN may be a novel regulator of adipocyte differentiation and act as a suppressor of inflammation in macrophage-infiltrated adipocyte tissue.

Cytotoxic activity of dimeric acridone alkaloids derived from Citrus plants towards human leukaemia HL-60 cells.

OBJECTIVES: Acridone alkaloids from Citrus and their derivatives show various kinds of biological activity. However, the anticancer activities of dimeric acridone alkaloids with unique structures and the molecular mechanism of these effects are poorly understood. METHODS: We investigated the cytotoxicity effects of dimeric acridone alkaloids isolated from Marsh grapefruit on human myeloid leukaemia HL-60 cells. KEY FINDINGS: Of the six dimeric acridone alkaloids tested, citbismine-E, the most potent, dose- and time-dependently decreased HL-60 cell viability by inducing apoptosis. The treatment of HL-60 cells with citbismine-E yielded a significant increase in levels of intracellular reactive oxygen species (ROS). Citbismine-E lowered the mitochondrial membrane potential and increased the activities of caspase-9 and -3. In addition, citbismine-E-induced apoptosis, decrease in mitochondrial membrane potential and caspase activation were significantly alleviated by pretreatment of the cells with antioxidant N-acetylcysteine (NAC). Citbismine-E induced intrinsic caspase-dependent apoptosis through ROS-mediated c-Jun N-terminal kinase activation. Citbismine-E-induced production of oxidative stress biomarkers, malondialdehyde and 8-hydroxy-2'-deoxyguanosine was also attenuated by pretreatment with NAC. CONCLUSIONS: Citbismine-E is a powerful cytotoxic agent against HL-60 cells that acts by inducing mitochondrial dysfunction-mediated apoptosis through ROS-dependent JNK activation. Citbismine-E also induced oxidative stress damage via ROS-mediated lipid peroxidation and DNA damage in HL-60 cells.

Structural analysis of the inhibitory effects of polyphenols, (+)-hopeaphenol and (-)-isohopeaphenol, on human SIRT1.

Human sirtuin 1 (hSIRT1) is a NAD+ -dependent deacetylase that regulates several cellular processes. Unlike resveratrol, natural polymeric phenolic compounds isolated from Vitaceae are mostly hSIRT1 inhibitors. The resveratrol tetramer, (+)-hopeaphenol ((+)-HP), and its geometric isomer, (-)-isohopeaphenol ((-)-iHP), were tested for inhibitory effects on purified hSIRT1 using a fluorescent derivative of peptide substrate p53-AMC (Fluor de Lys) and a cofactor NAD+ . The Lineweaver-Burk plots indicated that both (+)-HP and (-)-iHP were competitive inhibitors against NAD+ . Computer-assisted modeling of the binding of these molecules with hSIRT1 protein provided the most feasible conformation of the enzyme-inhibitor complex. © 2018 BioFactors, 45(2):253-258, 2019.

Dynamics of Circulating Tumor Cells Early After Targeting Therapy to Human EGFR-mutated Lung Cancers and HER2 Gene-amplified Gastric Cancers in Mice.

BACKGROUND/AIM: Dynamics of circulating tumor cells (CTCs) after molecular targeting therapy remain unclear. MATERIALS AND METHODS: We examined changes in CTC numbers and morphology early after targeting therapy in EGFR-mutated PC-9 human lung cancer and HER2-gene amplified GLM-1 gastric cancer mouse CTC models using a cytology-based semi-automated CTC detection platform. RESULTS: Erlotinib and T-DM1 inhibited cell growth mainly by induction of apoptosis in vitro. The number of CTCs detected 5-10 days after targeting therapy in mice was significantly increased compared to CTC numbers before therapy. The increased CTCs after therapy consisted of apoptotic CTCs and viable CTCs. This heterogeneous population of CTCs reflects well the cell population of the primary tumor disrupted by therapy. CONCLUSION: CTCs can be mobilized from the primary tumor due to tissue disruption in acute response to targeting therapy, suggesting potential usefulness of CTC monitoring as a predictor of therapeutic response in the clinical settings.

Tegafur and 5-fluorouracil levels in tears and changes in tear volume in long-term users of the oral anticancer drug S-1.

Eye problems are an adverse reaction sometimes found in chemotherapy. Although not life-threatening, they can reduce patients' quality of life. The highest incidence of eye problems is reported for the combination anticancer drug S-1 (tegafur-gimeracil-oteracil), and methods to prevent or treat the eye problems caused by this drug are presently lacking. To determine early detection methods and treatment for adverse ocular reactions, we measured changes in tear volume and levels of tegafur (FT) and 5-fluorouracil (5-FU), an active metabolite of FT, in the tears of patients with long-term use of S-1. A total of 11 patients receiving S-1 monotherapy as adjuvant chemotherapy after gastric cancer surgery were included. Tear volume and FT and 5-FU levels in tears were measured by liquid chromatography with tandem mass spectrometry during a maximum of 8 treatment cycles (48 weeks). For analysis, patients were divided into two groups: "watering eyes" (n=6, complaints of watering eyes at least once during the treatment period) and "no watering eyes" (n=5, no complaints of watering eyes). Both groups exhibited increased FT and 5-FU levels in tears upon initiation of S-1 treatment, and levels rapidly decreased upon discontinuation. Our findings suggest a relationship between FT level in tears and tear volume in patients with long-term S-1 use. The symptom of watering eyes may thus be linked to FT level in tears.

Neuronal differentiation elicited by glial cell line-derived neurotrophic factor and ciliary neurotrophic factor in adrenal chromaffin cell line tsAM5D immortalized with temperature-sensitive SV40 T-antigen.

To understand the characteristics of tsAM5D cells immortalized with the temperature-sensitive simian virus 40 large T-antigen, we first examined the responsiveness of the cells to ligands of the glial cell line-derived neurotrophic factor (GDNF) family. tsAM5D cells proliferated at the permissive temperature of 33 degrees C in response to either GDNF or neurturin, but not persephin or artemin. At the nonpermissive temperature of 39 degrees C, GDNF or neurturin caused tsAM5D cells to differentiate into neuron-like cells; however, the differentiated cells died in a time-dependent manner. Interestingly, ciliary neurotrophic factor (CNTF) did not affect the GDNF-mediated cell proliferation at 33 degrees C but promoted the survival and differentiation of GDNF-treated cells at 39 degrees C. In the presence of GDNF plus CNTF, the morphological change induced by the temperature shift was associated with up-regulated expression of various neuronal marker genes, indicating that the cells had undergone neuronal differentiation. In addition, tsAM5D cells caused to differentiate by GDNF plus CNTF at 39 degrees C became dependent solely on nerve growth factor (NGF) for their survival and neurite outgrowth. Moreover, upon treatment with GDNF plus CNTF, the dopaminergic phenotype was suppressed by the temperature shift. Thus, we demonstrated that tsAM5D cells had the capacity to differentiate terminally into neuron-like cells in response to GDNF plus CNTF when the oncogene was inactivated by the temperature shift. This cell line provides a useful model system for studying the role of a variety of signaling molecules for GDNF/CNTF-induced neuronal differentiation.

Autocrine TGF-beta signaling is required for the GDNF/CNTF-induced neuronal differentiation of adrenal chromaffin tsAM5D cells expressing temperature-sensitive SV40 T-antigen.

We recently established adrenal medullary cell line tsAM5D, which was immortalized by use of a temperature-sensitive mutant of the oncogene simian virus 40 large T-antigen. In the present study, when co-treated with glial cell line-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF), tsAM5D cells proliferated at the permissive temperature (33 degrees C) for the T-antigen expression and differentiated into neuron-like cells at the nonpermissive temperature (39 degrees C). Interestingly, in GDNF/CNTF-treated cultures, the addition of pan-specific transforming growth factor (TGF)-beta-neutralizing antibody did not affect the cell proliferation at 33 degrees C, but significantly reduced the survival of neuronally differentiated cells at 39 degrees C. Using real-time RT-PCR for analysis of GDNF/CNTF-treated cells, we found that the expression of mRNAs for TGF-beta1, TGF-beta2, and TGF-beta3 was up-regulated by the temperature shift. These results suggest that autocrine TGF-beta signaling is necessary for the survival of GDNF/CNTF-differentiated tsAM5D cells upon the temperature shift.

Induction of apoptosis in human leukaemia HL-60 cells by furanone-coumarins from Murraya siamensis.

To identify potential anti-tumour agents, we screened five furanone-coumarins isolated from Murraya siamensis Craib (Rutaceae) for their ability to inhibit the growth of human leukaemia HL-60 cells. Among the furanone-coumarins tested, murrayacoumarin B (compound 2) showed significant cytotoxicity against HL-60 cells. Fluorescence microscopy with Hoechst 33342 staining revealed that the percentage of apoptotic cells with fragmented nuclei and condensed chromatin increased in a time-dependent manner after treatment with murrayacoumarin B. Interestingly, this furanone-coumarin induced the loss of the mitochondrial membrane potential. In addition, treatment with murrayacoumarin B stimulated the activities of caspase-9 and caspase-3, and caspase-9 and caspase-3 inhibitors suppressed the apoptosis induced by murrayacoumarin B. These results suggest that murrayacoumarin B induced apoptosis in HL-60 cells through activation of the caspase9/caspase-3 pathway triggered by mitochondrial dysfunction.

Changes in tear volume and ocular symptoms of patients receiving oral anticancer drug S-1.

BACKGROUND: Most eye disorders are not fatal but may deteriorate the quality of life of a patient. The eye disorder that is most frequently reported in the cancer chemotherapy is associated with the combination of tegafur/gimeracil/potassium oxonate (S-1). However, preventive methods or treatment methods for the eye disorder have not yet been established. This study aimed to determine changes in tear volume and subjective ocular symptoms during the treatment period in patients receiving S-1 monotherapy for early detection of adverse effects in the eye and establishment of its treatment methods. METHODS: This study included eleven patients receiving S-1 monotherapy as a postoperative adjuvant chemotherapy for gastric cancer. Six subjective ocular symptoms including watering eyes were evaluated and changes in tear volume measured by the Schirmer's test in patients receiving S-1 during the treatment period. In the present study, the patients were divided into "no watering eyes" (patients not experienced watering eyes) group and "watering eyes" (patients experienced watering eyes even once) group. RESULTS: Six out of eleven patients developed watering eyes after receiving S-1 monotherapy. Among these, the earliest onset occurred on the 2nd week after oral administration. Watering eyes and eye discharge were highly related in patients having a trouble in daily life due to the decreased QOL. Changes in tear volume in the "watering eyes" group significantly increased compared to the "no watering eyes" group during the treatment period, especially when the patients had no subjective symptom of the increased tear volume. CONCLUSIONS: It is essential to prevent eye disorders including watering eyes as an adverse effect of S-1 administration. The present study recommends that the tear volume should be periodically measured using Schirmer's test, and the patient should be interviewed regarding the subjective ocular symptoms for the early detection of watering eyes caused by S-1 administration. If the tear volume can not be measured periodically, medical staffs should pay attention to the patient with eye discharge.

Regucalcin confers resistance to amyloid-ß toxicity in neuronally differentiated PC12 cells.

Amyloid-ß (Aß), a primary component of amyloid plaques, has been widely associated with the pathogenesis of Alzheimer's disease. The Ca2+-binding protein regucalcin (RGN) plays multiple roles in maintaining cell functions by regulating intracellular calcium homeostasis, various signaling pathways, and gene expression systems. Here, we investigated the functional role of RGN against Aß-induced cytotoxicity in neuronally differentiated PC12 cells. Overexpression of RGN reduced Aß-induced apoptosis by reducing mitochondrial dysfunction and caspase activation. It also attenuated Aß-induced reactive oxygen species production and oxidative damage and decreased Aß-induced nitric oxide (NO) overproduction, upregulation of inducible NO synthase by nuclear factor-κB, and nitrosative damage. Interestingly, the genetic disruption of RGN increased the susceptibility of neuronally differentiated PC12 cells to Aß toxicity. Thus, RGN possesses antioxidant activity against Aß-induced oxidative and nitrosative stress and may play protective roles against Aß-induced neurotoxicity in Alzheimer's disease.

Potent apoptosis-inducing activity of erypoegin K, an isoflavone isolated from Erythrina poeppigiana, against human leukemia HL-60 cells.

Erypoegin K is an isoflavone isolated from the stem bark of Erythrina poeppigiana. It contains a furan group at the A-ring of the core isoflavone structure and can inhibit the activity of glyoxalase I, an enzyme that catalyzes the detoxification of methylglyoxal (MG), a by-product of glycolysis. In the present study, we found that erypoegin K has a potent cytotoxic effect on human leukemia HL-60 cells. Its cytotoxic effect was much stronger than that of a known glyoxalase I inhibitor S-p-bromobenzylglutathione cyclopentyl diester. Conversely, erypoegin K demonstrated weak cytotoxicity toward normal human peripheral lymphocytes. The treatment of HL-60 cells with erypoegin K significantly induced caspase-3 activity, whereas the pretreatment of the cells with caspase-3 inhibitor suppressed erypoegin K-induced cell death. Furthermore, nuclear condensation and apoptotic genome DNA fragmentation were observed in erypoegin K-treated HL-60 cells. These results indicated that the observed cell death was mediated by apoptosis. In addition, the toxic compound MG was highly accumulated in the culture medium of erypoegin K-treated HL-60 cells, suggesting that cell apoptosis was triggered by extracellular MG. The present study showed that erypoegin K has a potent apoptosis-inducing effect on cancerous cell lines, such as HL-60.