Effectiveness of Combinational Treatments for Alzheimer's Disease with Human Neural Stem Cells and Microglial Cells Over-Expressing Functional Genes.

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases. In AD patients, amyloid-ß (Aß) peptide-mediated degeneration of the cholinergic system utilizing acetylcholine (ACh) for memory acquisition is observed. Since AD therapy using acetylcholinesterase (AChE) inhibitors are only palliative for memory deficits without reversing disease progress, there is a need for effective therapies, and cell-based therapeutic approaches should fulfil this requirement. We established F3.ChAT human neural stem cells (NSCs) encoding the choline acetyltransferase (ChAT) gene, an ACh-synthesizing enzyme, HMO6.NEP human microglial cells encoding the neprilysin (NEP) gene, an Aß-degrading enzyme, and HMO6.SRA cells encoding the scavenger receptor A (SRA) gene, an Aß-uptaking receptor. For the efficacy evaluation of the cells, first, we established an appropriate animal model based on Aß accumulation and cognitive dysfunction. Among various AD models, intracerebroventricular (ICV) injection of ethylcholine mustard azirinium ion (AF64A) induced the most severe Aß accumulation and memory dysfunction. Established NSCs and HMO6 cells were transplanted ICV to mice showing memory loss induced by AF64A challenge, and brain Aß accumulation, ACh concentration and cognitive function were analyzed. All the transplanted F3.ChAT, HMO6.NEP and HMO6.SRA cells were found to survive up to 4 weeks in the mouse brain and expressed their functional genes. Combinational treatment with the NSCs (F3.ChAT) and microglial cells encoding each functional gene (HMO6.NEP or HMO6.SRA) synergistically restored the learning and memory function of AF64A-challenged mice by eliminating Aß deposits and recovering ACh level. The cells also attenuated inflammatory astrocytic (glial fibrillary acidic protein) response by reducing Aß accumulation. Taken together, it is expected that NSCs and microglial cells over-expressing ChAT, NEP or SRA genes could be strategies for replacement cell therapy of AD.

Intraocular Pressure-Lowering and Retina-Protective Effects of Exosome-Rich Conditioned Media from Human Amniotic Membrane Stem Cells in a Rat Model of Glaucoma.

Glaucoma is one of the most devastating eye diseases, since the disease can develop into blindness and no effective therapeutics are available. Although the exact mechanisms and causes of glaucoma are unknown, increased intraocular pressure (IOP) has been demonstrated to be an important risk factor. Exosomes are lipid nanoparticles secreted from functional cells, including stem cells, and have been found to contain diverse functional molecules that control body function, inhibit inflammation, protect and regenerate cells, and restore damaged tissues. In the present study, exosome-rich conditioned media (ERCMs) were attained via hypoxic culture (2% O2) of human amniotic membrane mesenchymal stem cells (AMMSCs) and amniotic membrane epithelial stem cells (AMESCs) containing 50 times more exosome particles than normoxic culture (20% O2) medium (NCM). The exosome particles in ERCM were confirmed to be 77 nm in mean size and contain much greater amounts of growth factors (GFs) and neurotrophic factors (NFs) than those in NCM. The glaucoma-therapeutic effects of ERCMs were assessed in retinal cells and a hypertonic (1.8 M) saline-induced high-IOP animal model. CM-DiI-labeled AMMSC exosomes were found to readily penetrate the normal and H2O2-damaged retinal ganglion cells (RGCs), and AMMSC-ERCM not only facilitated retinal pigment epithelial cell (RPEC) proliferation but also protected against H2O2- and hypoxia-induced RPEC insults. The IOP of rats challenged with 1.8 M saline increased twice the normal IOP (12-17 mmHg) in a week. However, intravitreal injection of AMMSC-ERCM or AMESC-ERCM (3.9-4.5 × 108 exosomes in 10 µL/eye) markedly recovered the IOP to normal level in 2 weeks, similar to the effect achieved with platelet-derived growth factor-AB (PDGF-AB, 1.5 µg), a reference material. In addition, AMMSC-ERCM, AMESC-ERCM, and PDGF-AB significantly reversed the shrinkage of retinal layers, preserved RGCs, and prevented neural injury in the glaucoma eyes. It was confirmed that stem cell ERCMs containing large numbers of functional molecules such as GFs and NFs improved glaucoma by protecting retinal cells against oxidative and hypoxic injuries in vitro and by recovering IOP and retinal degeneration in vivo. Therefore, it is suggested that stem cell ERCMs could be a promising candidate for the therapy of glaucoma.

The Neuroprotective Effects of Exosomes Derived from TSG101-Overexpressing Human Neural Stem Cells in a Stroke Model.

Although tissue-type plasminogen activator was approved by the FDA for early reperfusion of occluded vessels, there is a need for an effective neuroprotective drug for stroke patients. In this study, we established tumor susceptibility gene (TSG)101-overexpressing human neural stem cells (F3.TSG) and investigated whether they showed enhanced secretion of exosomes and whether treatment with exosomes during reperfusion alleviated ischemia-reperfusion-mediated brain damage. F3.TSG cells secreted higher amounts of exosomes than the parental F3 cells. In N2A cells subjected to oxygen-glucose deprivation (OGD), treatment with exosomes or coculture with F3.TSG cells significantly attenuated lactate dehydrogenase release, the mRNA expression of proinflammatory factors, and the protein expression of DNA-damage-related proteins. In a middle cerebral artery occlusion (MCAO) rat model, treatment with exosomes, F3 cells, or F3.TSG cells after 2 h of occlusion followed by reperfusion reduced the infarction volume and suppressed inflammatory cytokines, DNA-damage-related proteins, and glial fibrillary acidic protein, and upregulated several neurotrophic factors. Thus, TSG101-overexpressing neural stem cells showed enhanced exosome secretion; exosome treatment protected against MCAO-induced brain damage via anti-inflammatory activities, DNA damage pathway inhibition, and growth/trophic factor induction. Therefore, exosomes and F3.TSG cells can affect neuroprotection and functional recovery in acute stroke patients.

Effect of sorghum ethyl-acetate extract on benign prostatic hyperplasia induced by testosterone in Sprague-Dawley rats.

Benign prostatic hyperplasia (BPH) is commonly observed in men > 50 years worldwide. Phytotherapy is one of the many treatment options. Sorghum (Sorghum bicolor L.) contains various health-improving phytochemicals with antioxidant and inhibitory activities on cell proliferation, both in vitro and in vivo. To confirm the effects of Donganme sorghum ethyl-acetate extract (DSEE) on BPH, we induced BPH in Spragye-Dawley rats using exogenous testosterone. We measured prostate weight, examined prostrates histopathologically, and analyzed mRNAs associated with male hormones and proteins associated with cell proliferation in the prostate. DSEE inhibited weight gain of the prostate; decreased mRNA expressions of androgen receptor and 5α-reductase II; and improved histopathological symptoms, the protein-expressed ratio of Bax/Bcl-2, and the oxidative status of BPH induced by testosterone in SD rats. Therefore, DSEE may have potential as a preventive or therapeutic agent against BPH.

Toxicological evaluation and anti-inflammatory activity of a golden gelatinous sorghum bran extract.

Golden gelatinous sorghum (GGS) is rich in phytochemicals and anti-oxidants. We investigated the toxicity and anti-inflammatory properties of a GGS extract. We observed no toxic effects after a daily dose of up to 5000 mg/kg body weight of the GGS extract administered orally to rats for 14 d. The exposure of mice ears to 12-O-tetradecanoylphorbol-13-acetate (TPA) caused a marked increase in ear thickness, which was significantly inhibited by treating with the GGS extract; this inhibition of inflammatory response was clearly confirmed by a histological analysis. The TPA-induced mice ear edema model, indicated that treating with the GGS extract inhibited the expression levels of such inflammatory mediators as cyclooxygenase-2 and inducible nitric oxide synthase. The nitric oxide level in lipopolysaccharide (LPS)-induced Raw264.7 cells in vitro was lower in the GGS extract-treated group than in the LPS-only treated group. These results suggest that sorghum would be a safe, nontoxic product, and that the GGS extract possessed significant anti-inflammatory activity.

Repeated Intravenous Administration of Human Neural Stem Cells Producing Choline Acetyltransferase Exerts Anti-Aging Effects in Male F344 Rats.

Major features of aging might be progressive decreases in cognitive function and physical activity, in addition to withered appearance. Previously, we reported that the intracerebroventricular injection of human neural stem cells (NSCs named F3) encoded the choline acetyltransferase gene (F3.ChAT). The cells secreted acetylcholine and growth factors (GFs) and neurotrophic factors (NFs), thereby improving learning and memory function as well as the physical activity of aged animals. In this study, F344 rats (10 months old) were intravenously transplanted with F3 or F3.ChAT NSCs (1 × 106 cells) once a month to the 21st month of age. Their physical activity and cognitive function were investigated, and brain acetylcholine (ACh) and cholinergic and dopaminergic system markers were analyzed. Neuroprotective and neuroregenerative activities of stem cells were also confirmed by analyzing oxidative damages, neuronal skeletal protein, angiogenesis, brain and muscle weights, and proliferating host stem cells. Stem cells markedly improved both cognitive and physical functions, in parallel with the elevation in ACh levels in cerebrospinal fluid and muscles, in which F3.ChAT cells were more effective than F3 parental cells. Stem cell transplantation downregulated CCL11 and recovered GFs and NFs in the brain, leading to restoration of microtubule-associated protein 2 as well as functional markers of cholinergic and dopaminergic systems, along with neovascularization. Stem cells also restored muscular GFs and NFs, resulting in increased angiogenesis and muscle mass. In addition, stem cells enhanced antioxidative capacity, attenuating oxidative damage to the brain and muscles. The results indicate that NSCs encoding ChAT improve cognitive function and physical activity of aging animals by protecting and recovering functions of multiple organs, including cholinergic and dopaminergic systems, as well as muscles from oxidative injuries through secretion of ACh and GFs/NFs, increased antioxidant elements, and enhanced blood flow.

An Exosome-Rich Conditioned Medium from Human Amniotic Membrane Stem Cells Facilitates Wound Healing via Increased Reepithelization, Collagen Synthesis, and Angiogenesis.

Tissue regeneration is an essential requirement for wound healing and recovery of organs' function. It has been demonstrated that wound healing can be facilitated by activating paracrine signaling mediated by exosomes secreted from stem cells, since exosomes deliver many functional molecules including growth factors (GFs) and neurotrophic factors (NFs) effective for tissue regeneration. In this study, an exosome-rich conditioned medium (ERCM) was collected from human amniotic membrane stem cells (AMSCs) by cultivating the cells under a low oxygen tension (2% O2 and 5% CO2). The contents of GFs and NFs including keratinocyte growth factor, epidermal growth factor, fibroblast growth factor 1, transforming growth factor-ß, and vascular endothelial growth factor responsible for skin regeneration were much higher (10-30 folds) in the ERCM than in normal conditioned medium (NCM). In was found that CM-DiI-labeled exosomes readily entered keratinocytes and fibroblasts, and that ERCM not only facilitated the proliferation of keratinocytes in normal condition, but also protected against H2O2 cytotoxicity. In cell-migration assay, the scratch wound in keratinocyte culture dish was rapidly closed by treatment with ERCM. Such wound-healing effects of ERCM were confirmed in a rat whole skin-excision model: i.e., the wound closure was significantly accelerated, remaining minimal crusts, by topical application of ERCM solution (4 × 109 exosome particles/100 µL) at 4-day intervals. In the wounded skin, the deposition of collagens was enhanced by treatment with ERCM, which was supported by the increased production of collagen-1 and collagen-3. In addition, enhanced angiogenesis in ERCM-treated wounds was confirmed by increased von Willebrand factor (vWF)-positive endothelial cells. The results indicate that ERCM from AMSCs with high concentrations of GFs and NFs improves wound healing through tissue regeneration not only by facilitating keratinocyte proliferation for skin repair, but also activating fibroblasts for extracellular matrix production, in addition to the regulation of angiogenesis and scar tissue formation.

Antioxidative and Anti-Inflammatory Activities of Rosebud Extracts of Newly Crossbred Roses.

Oxidative stress and inflammation are basic pathogenic factors involved in tissue injury and pain, as well as acute and chronic diseases. Since long-term uses of synthetic steroids and non-steroidal anti-inflammatory drugs (NSAIDs) cause severe adverse effects, novel effective materials with minimal side effects are required. In this study, polyphenol content and antioxidative activity of rosebud extracts from 24 newly crossbred Korean roses were analyzed. Among them, Pretty Velvet rosebud extract (PVRE) was found to contain high polyphenols and to show in vitro antioxidative and anti-inflammatory activities. In RAW 264.7 cells stimulated with lipopolysaccharide (LPS), PVRE down-regulated mRNA expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and thereby decreased nitric oxide (NO) and prostaglandin E2 (PGE2) production. In a subcutaneous air-pouch inflammation model, treatment with PVRE decreased λ-carrageenan-induced tissue exudation, infiltration of inflammatory cells, and inflammatory cytokines such as tumor necrosis factor-α and interleukin-1ß concentrations, as achieved with dexamethasone (a representative steroid). Notably, PVRE also inhibited PGE2, similar to dexamethasone and indomethacin (a representative NSAID). The anti-inflammatory effects of PVRE were confirmed by microscopic findings, attenuating tissue erythema, edema, and inflammatory cell infiltration. These results indicate that PVRE exhibits dual (steroid- and NSAID-like) anti-inflammatory activities by blocking both the iNOS-NO and COX-2-PG pathways, and that PVRE could be a potential candidate as an anti-inflammatory material for diverse tissue injuries.

DK-MGAR101, an extract of adventitious roots of mountain ginseng, improves blood circulation by inhibiting endothelial cell injury, platelet aggregation, and thrombus formation.

Background: Since ginsenosides exert an anti-thrombotic activity, blood flow-improving effects of DK-MGAR101, an extract of mountain ginseng adventitious roots (MGAR) containing various ginsenosides, were investigated in comparison with an extract of Korean Red Ginseng (ERG). Methods: In Sprague-Dawley rats orally administered with DK-MGAR101 or ERG, oxidative carotid arterial thrombosis was induced with FeCl3 (35%), and their blood flow and occlusion time were measured. To elucidate underlying mechanisms, the cytoprotective activities on rat aortic endothelial cells (RAOECs) exposed to hydrogen peroxide (H2O2) were confirmed. In addition, the inhibitory activities of DK-MGAR101 and ERG on agonist-induced platelet aggregation, thromboxane B2 production, and ATP granule release from stimulated platelets as well as blood coagulation were analyzed. Results: DK-MGAR101 containing high concentrations of Rb1, Rg1, Rg3, Rg5, and Rk1 ginsenosides (55.07 mg/g) was more effective than ERG (ginsenosides 8.45 mg/g) in protecting RAOECs against H2O2 cytotoxicity. DK-MGAR101 was superior to ERG not only in suppressing platelet aggregation, thromboxane B2 production, and granule release, but also in delaying blood coagulation, FeCl3-induced arterial occlusion, and thrombus formation. Conclusions: The results indicate that DK-MGAR101 prevents blood vessel occlusion by suppressing platelet aggregation, thrombosis, and blood coagulation, in addition to endothelial cell injury.

Anti-proliferate and pro-apoptotic effects of 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyranone through inactivation of NF-kappaB in human colon cancer cells.

Many natural compounds have been shown to prevent cancer cell growth through the redox regulation of transcription factors. NF-kappaB, a redox transcription factor, has been implicated in the apoptotic cell death of several cancer cells. This study examined whether or nor 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyranone (DDMP) isolated from onions can modulate the activity of NF-kappaB, thereby induce the apoptotic cell death of colon cancer cells. Treatment with different DDMP concentrations (0.5-1.5 mg/mL) for various periods (0-48 h) inhibited the growth of colon cancer cells (SW620 and HCT116) followed by the induction of apoptosis in a dose dependent manner. It was also found that DDMP modulated tumor necrosis factor-alpha (TNF-alpha) and tetradeanoyl phorbol acetate (TPA)-induced NF-kappaB transcriptional and DNA binding activity. Moreover, DDMP suppressed the NF-kappaB target anti-apoptotic genes (Bcl-2), whereas it induced the expression of the apoptotic genes (Bax, cleaved caspase-3 and cleaved PARP). These results suggest that DDMP from onions inhibit colon cancer cell growth by inducing apoptotic cell death through the inhibition of NF-kappaB.

Estrogen receptor independent neurotoxic mechanism of bisphenol A, an environmental estrogen.

Bisphenol A (BPA), a ubiquitous environmental contaminant, has been shown to cause developmental toxicity and carcinogenic effects. BPA may have physiological activity through estrogen receptor (ER) -alpha and -beta, which are expressed in the central nervous system. We previously found that exposure of BPA to immature mice resulted in behavioral alternation, suggesting that overexposure of BPA could be neurotoxic. In this study, we further investigated the molecular neurotoxic mechanisms of BPA. BPA increased vulnerability (decrease of cell viability and differentiation, and increase of apoptotic cell death) of undifferentiated PC12 cells and cortical neuronal cells isolated from gestation 18 day rat embryos in a concentration-dependent manner (more than 50 microM). The ER antagonists, ICI 182,780, and tamoxifen, did not block these effects. The cell vulnerability against BPA was not significantly different in the PC12 cells overexpressing ER-alpha and ER-beta compared with PC12 cells expressing vector alone. In addition, there was no difference observed between BPA and 17-beta estradiol, a well-known agonist of ER receptor in the induction of neurotoxic responses. Further study of the mechanism showed that BPA significantly activated extracellular signal-regulated kinase (ERK) but inhibited anti-apoptotic nuclear factor kappa B (NF-kappaB) activation. In addition, ERK-specific inhibitor, PD 98,059, reversed BPA-induced cell death and restored NF-kappaB activity. This study demonstrated that exposure to BPA can cause neuronal cell death which may eventually be related with behavioral alternation in vivo. However, this neurotoxic effect may not be directly mediated through an ER receptor, as an ERK/NF-kappaB pathway may be more closely involved in BPA-induced neuronal toxicity.

Bee venom inhibits growth of human cervical tumors in mice.

We studied whether bee venom (BV) inhibits cervical tumor growth through enhancement of death receptor (DR) expressions and inactivation of nuclear factor kappa B (NF-κB) in mice. In vivo study showed that BV (1 mg/kg) inhibited tumor growth. Similar inhibitory effects of BV on cancer growth in primary human cervical cancer cells were also found. BV (1-5 µg/ml) also inhibited the growth of cancer cells, Ca Ski and C33Aby the induction of apoptotic cell death in a dose dependent manner. Agreed with cancer cell growth inhibition, expression of death receptors; FAS, DR3 and DR6, and DR downstream pro-apoptotic proteins including caspase-3 and Bax was concomitantly increased, but the NF-κB activity and the expression of Bcl-2 were inhibited by treatment with BV in tumor mice, human cancer cell and human tumor samples as well as cultured cancer cells. In addition, deletion of FAS, DR3 and DR6 by small interfering RNA significantly reversed BV-induced cell growth inhibitory effects as well as NF-κB inactivation. These results suggest that BV inhibits cervical tumor growth through enhancement of FAS, DR3 and DR6 expression via inhibition of NF-κB pathway.

Anti-arthritis effects of (E)-2,4-bis(p-hydroxyphenyl)-2-butenal are mediated by inhibition of the STAT3 pathway.

BACKGROUND AND PURPOSE: Products of Maillard reactions between aminoacids and reducing sugars are known to have anti-inflammatory properties. Here we have assessed the anti-arthritis effects of (E)-2,4-bis(p-hydroxyphenyl)-2-butenal and its possible mechanisms of action. EXPERIMENTAL APPROACH: We used cultures of LPS-activated macrophages (RAW264.7 cells) and human synoviocytes from patients with rheumatoid arthritis for in vitro assays and the collagen-induced arthritis model in mice. NO generation, iNOS and COX2 expression, and NF-κB/IKK and STAT3 activities were measured in vitro and in joint tissues of arthritic mice, along with clinical scores and histopathological assessments. Binding of (E)-2,4-bis(p-hydroxyphenyl)-2-butenal to STAT3 was evaluated by a pull-down assay and its binding site was predicted using molecular docking studies with Autodock VINA. KEY RESULTS: (E)-2,4-bis(p-hydroxyphenyl)-2-butenal (2.5-10 µg·mL(-1) ) inhibited LPS-inducedNO generation, iNOS and COX2 expression, and NF-κB/IKK and STAT3 activities in macrophage and human synoviocytes. This compound also suppressedcollagen-induced arthritic responses in mice by inhibiting expression of iNOS and COX2, and NF-κB/IKK and STAT3 activities; it also reduced bone destruction and fibrosis in joint tissues. A pull-down assay showed that (E)-2,4-bis(p-hydroxyphenyl)-2-butenal interfered with binding of ATP to STAT3. Docking studies suggested that (E)-2,4-bis(p-hydroxyphenyl)-2-butenal bound to the DNA-binding interface of STAT3 possibly inhibiting ATP binding to STAT3 in an allosteric manner. CONCLUSIONS AND IMPLICATIONS: (E)-2,4-bis(p-hydroxyphenyl)-2-butenal exerted anti-inflammatory and anti-arthritic effects through inhibition of the NF-κB/STAT3 pathway by direct binding to STAT3. This compound could be a useful agent for the treatment of arthritic disease.

Panax ginseng exerts antiproliferative effects on rat hepatocarcinogenesis.

It has been proposed that ginseng has chemopreventive effects against several types of cancer in animals and humans. However, the mechanisms underlying the chemopreventive activities of fresh ginseng against hepatocarcinogenesis have not yet been elucidated. Therefore, we hypothesized that these ginseng species may prevent hepatocarcinogenesis but that the chemopreventive mechanisms may differ by species. To determine the chemopreventive and therapeutic potential of 3 different types of fresh ginseng on hepatocarcinogenesis, Sprague-Dawley rats were injected with diethylnitrosamine and fed diets containing 2% Panax japonicus CA Meyer (JN), P. quinquefolius L (QQ), or P. ginseng CA Meyer (GS) for 10 weeks. Glutathione S-transferase P form (GST-P)-positive foci, a stable marker for rat hepatocarcinogenesis, were shown in all carcinogen-injected rats; but only the GS diet significantly reduced the area and number (62% and 68%, respectively; P < .05) of GST-P-positive foci compared with the diethylnitrosamine control group. In addition, the number of proliferating cell nuclear antigen-positive hepatocytes in the GST-P-positive area was significantly decreased in the GS group but not in the JN or QQ groups. Using cDNA microarray analyses to investigate the underlying molecular mechanisms, we observed that the p53 signaling pathway was altered by the GS diet and that the expression of Cyclin D1, Cyclin G1, Cdc2a, and Igf-1, which are involved in the p53 signaling pathway, was downregulated by the GS diet. Our data demonstrate, for the first time, that GS, but not JN or QQ, induces cell cycle arrest in hepatocarcinogenesis. This study suggests that fresh GS has potential chemopreventive effects and may prove to be a therapeutic agent against hepatocarcinogenesis.

Elm tree bark extract inhibits HepG2 hepatic cancer cell growth via pro-apoptotic activity.

Control of inflammation is widely accepted as an important strategy for cancer chemoprevention. Anti-inflammatory effects of bark extracts of elm tree (BEE) have been amply reported. Therefore, BEE may be a good candidate cancer chemopreventive agent. Considering the high incidence of hepatic cancer and limited therapeutic approaches for treating this disease, it is important to develop liver cancer-specific chemopreventive agents. To evaluate the chemopreventive potential of BEE, we investigated the growth inhibition effect of BEE on the HepG2 human hepatocellular carcinoma cell line. We performed a cell counting kit-8 assay to determine cell viability, and 4,6-diamino-2-phenylindole staining and flow cytometry to measure apoptotic cell death. Finally, the expression levels of pro- and anti-apoptotic proteins were measured. BEE inhibited the growth of HepG2 cells and induced apoptosis in a dose-dependent manner. Pro-apoptotic activity was promoted via the mitochondrial pathway of apoptosis, as demonstrated by the activation of pro-apoptotic proteins Bax, caspase-9, caspase-3, and poly (ADP-ribose) polymerase as well as the down-regulation of the anti-apoptotic protein Bcl-2. These results suggest that BEE may have potential use in hepatic cancer chemoprevention by suppressing cancer cell growth via pro-apoptotic activity.

Modifying effect of diallyl sulfide on colon carcinogenesis in C57BL/6J-ApcMin/⁺ mice.

Diallyl sulfide (DAS), a flavoring compound derived from garlic, is considered to have cancer chemopreventive potential in experimental animals and humans. This study was designated to examine possible chemopreventive effects of DAS on colon carcinogenesis using genetically engineered transgenic ApcMin/⁺ mice, a well-established animal model for familial adenomatous polyposis (FAP) and sporadic colorectal cancer. Male C57BL/6J-ApcMin/⁺ mice were divided into three groups. Animals of group 1 were placed on the basal diet (AIN-76A) as non-treated controls. Animals of groups 2 and 3 were given DAS- containing diets (in doses of 100 and 300 ppm, respectively). All mice were sacrificed at the end of week 10 of the experiment. Histopathological investigation revealed that the incidence of colonic polyps was decreased dose-dependently by 19% (13/16) in group 2 and by 32% (13/20) in group 3 compared to the 100% incidence (10/10) in group 1. The multiplicity of colonic polyps per mouse was also slightly decreased by DAS treatment (1.88 ± 0.35 in group 2 and 1.63 ± 0.36 in group 3) compared to 2.00 ± 0.39 in group 1. On the other hand, there were no significant differences in the numbers of total polyps per mouse in the small intestine between the groups. Taken together, we suggest that DAS may exert promising inhibitory effects on colon carcinogenesis in the transgenic ApcMin/⁺ mice.

Anti-inflammatory and arthritic effects of thiacremonone, a novel sulfur compound isolated from garlic via inhibition of NF-kappaB.

INTRODUCTION: Sulfur compounds isolated from garlic exert anti-inflammatory properties. We recently isolated thiacremonone, a novel sulfur compound from garlic. Here, we investigated the anti-inflammatory and arthritis properties of thiacremonone through inhibition of NF-kappaB since NF-kappaB is known to be a target molecule of sulfur compounds and an implicated transcription factor regulating inflammatory response genes. METHODS: The anti-inflammatory and arthritis effects of thiacremone in in vivo were investigated in 12-O-tetradecanoylphorbol-13-acetate-induced ear edema, carrageenan and mycobacterium butyricum-induced inflammatory and arthritis models. Lipopolysaccharide-induced nitric oxide (NO) production was determined by Griess method. The DNA binding activity of NF-kappaB was investigated by electrophoretic mobility shift assay. NF-kappaB and inducible nitric oxide synthetase (iNOS) transcriptional activity was determined by luciferase assay. Expression of iNOS and cyclooxygenase-2 (COX-2) was determined by western blot. RESULTS: The results showed that topical application of thiacremonone (1 or 2 microg/ear) suppressed the 12-O-tetradecanoylphorbol-13-acetate-induced (1 microg/ear) ear edema. Thiacremonone (1-10 mg/kg) administered directly into the plantar surface of hind paw also suppressed the carrageenan (1.5 mg/paw) and mycobacterium butyricum (2 mg/paw)-induced inflammatory and arthritic responses as well as expression of iNOS and COX-2, in addition to NF-kappaB DNA-binding activity. In further in vitro study, thiacremonone (2.5-10 microg/ml) inhibited lipopolysaccharide (LPS, 1 microg/ml)-induced nitric oxide (NO) production, and NF-kappaB transcriptional and DNA binding activity in a dose dependent manner. The inhibition of NO by thiacremonone was consistent with the inhibitory effect on LPS-induced inducible nitric oxide synthase (iNOS) and COX-2 expression, as well as iNOS transcriptional activity. Moreover, thiacremonone inhibited LPS-induced p50 and p65 nuclear translocation, resulting in an inhibition of the DNA binding activity of the NF-kappaB. These inhibitory effects on NF-kappaB activity and NO generation were suppressed by reducing agents dithiothreitol (DTT) and glutathione, and were abrogated in p50 (C62S)-mutant cells, suggesting that the sulfhydryl group of NF-kappaB molecules may be a target of thiacremonone. CONCLUSIONS: The present results suggested that thiacremonone exerted its anti-inflammatory and anti-arthritic properties through the inhibition of NF-kappaB activation via interaction with the sulfhydryl group of NF-kappaB molecules, and thus could be a useful agent for the treatment of inflammatory and arthritic diseases.

Effects of a preparation of combined glutathione-enriched yeast and rice embryo/soybean extracts on ethanol hangover.

The effects of a preparation of combined glutathione-enriched yeast (GEY) and rice embryo/soybean (RES) extracts (20:1), GEY/RES, on experimentally induced ethanol hangover were investigated in male Sprague-Dawley rats. To evaluate the preventive effects on hangover, rats were orally administered GEY/RES (50/2.5, 100/5, or 200/10 mg/kg) for 2 weeks. At 30 minutes after the final treatment, they were challenged with 3 mL/kg ethanol (15 mL of 20% in water/kg). The blood concentrations of alcohol and acetaldehyde were analyzed up to 7 hours postchallenge. Hepatic mRNA expression levels of alcohol-metabolizing enzymes, alcohol dehydrogenase (ADH), cytochrome P450 type 2E1 (CYP2E1), and aldehyde dehydrogenase (ALDH), were determined by real-time polymerase chain reaction. Additional rats were challenged with ethanol and, 60 minutes later, administered GEY/RES to evaluate alcohol clearance. Pretreatment with GEY/RES for 2 weeks reduced the blood concentrations of alcohol and acetaldehyde in a dose-dependent manner, lowering by 29.5% and 54.6% at the highest dose (200/10 mg/kg), respectively. The expressions of mRNAs for ADH and ALDH, the major alcohol-metabolizing enzymes, were markedly increased in the livers of rats administered GEY/RES for 2 weeks, whereas CYP2E1 mRNA was suppressed. Postchallenge treatment with GEY/RES enhanced the alcohol clearance rate by lowering blood concentrations of alcohol and acetaldehyde by 24% and 26.6%, respectively, for the highest dose group. GEY/RES remarkably eliminated 2,2-diphenyl-1-picrylhydrazyl hydrate radical and FeCl(3)-mediated lipid peroxidation in vitro and attenuated hepatic lipid accumulation following ethanol administration in vivo. Therefore, it is suggested that GEY/RES reduces the blood concentrations of alcohol and acetaldehyde not only by modulating alcohol-metabolizing enzymes, but also by exerting its antioxidant activity, and that GEY/RES could be a promising candidate for improvements of alcoholic hangover.

Inhibition of cell growth and induction of apoptosis via inactivation of NF-kappaB by a sulfurcompound isolated from garlic in human colon cancer cells.

Compounds such as S-allylmercaptocysteine, diallyl disulfide, and S-trityl-L-cysteine isolated from garlic have been known to be effective in chemoprevention. Nuclear transcription factor-kappaB (NF-kappaB) has been known to be an implicated factor in apoptotic cell death of several cancer cells. In this study, we investigated whether a sulfurcompound (named thiacremonone) isolated from garlic could modulate NF-kappaB activity and thereby induce apoptotic cell death of colon cancer cells. Treatment with different concentrations (30 - 150 microg/ml) of thiacremonone for various periods (0 - 48 h) inhibited colon cancer cell (SW620 and HCT116) growth followed by induction of apoptosis in a dose-dependent manner. We also found that thiacremonone modulated tumor necrosis factor-alpha (TNF-alpha) and tetradeanoyl phorbol acetate (TPA)-induced NF-kappaB transcriptional and DNA binding activity. Moreover, thiacremonone suppressed NF-kappaB target anti-apoptotic genes (Bcl-2, cIAP1/2, and XIAP) and inflammatory genes (iNOS and COX-2), whereas it induced apoptotic genes (Bax, cleaved caspse-3, and cleaved PARP) expression. These results suggest that a novel sulfurocompound from garlic inhibited colon cancer cell growth through induction of apoptotic cell death by modulating of NF-kappaB.