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As part of an ongoing search for plant-derived compounds that inhibit the activation of NF-kappaB, the methanol extract of the aerial parts of Isodon excisus was found to have significant inhibitory effects on the activation of NF-kappaB in murine macrophage RAW264.7 cells. Bioactivity-guided isolation of the extract yielded five new diterpenoids, excisusin A-E (1-5), along with seven known compounds, inflexarabdonin I (6), inflexarabdonin G (7), inflexin (8), inflexanin A (9), inflexanin B (10), inflexinol (11), and inflexarabdonin A (12). The structures were determined by analysis of the spectroscopic data including 2D NMR. All of the isolates were evaluated for their inhibitory effects on LPS-induced NF-kappaB activation and nitric oxide production in RAW264.7 cells.

Nine terpenylated coumarins, namely 7-[(E)-3',7'-dimethyl-6'-oxo-2',7'-octadienyl]oxy-coumarin (1), schinilenol (2), schinindiol (3), collinin (4), 7-[(E)-7'-hydroxy-3',7'-dimethy-locta-2',5'-dienyloxy]-coumarin (5), 8-methoxyanisocoumarin (6), 7-(6'R-hydroxy-3',7'-dimethyl-2'E,7'-octadienyloxy)coumarin (7), (E)-4-methyl-6-(coumarin-7'-yloxy)hex-4-enal (8), and aurapten (9), along with a 4-quinolone alkaloid (10) and integrifoliodiol (11), were isolated from the leaves of Zanthoxylum schinifolium. Of the isolates, compounds 4 and 7 potentially inhibited NO production in lipopolysaccharide (LPS)-stimulated macrophage RAW264.7 cells, with IC50 values of 5.9 ± 0.8 and 18.2 ± 1.8 μM, respectively. Furthermore, compounds 4 and 7 dose-dependently reduced the LPS-induced iNOS expression. Moreover, pre-incubation of cells with 4 and 7 significantly suppressed LPS-induced COX-2 protein expression. In addition, compounds 4, 7, 8, and 10 showed strong α-glucosidase inhibitory effects, with IC50 values of 92.1 ± 0.7, 90.6 ± 0.9, 78.2 ± 0.2, and 82.4 ± 0.8 μM, respectively. Compounds 1, 5, and 11 displayed moderate effects with IC50 values of 161.6 ± 0.3, 164.4 ± 1.1, and 155.4 ± 0.9 μM, while acarbose, a positive control, possessed an IC50 value of 121.5 ± 1.0 μM. This is the first investigation on the α-glucosidase inhibitory effect of components from Zanthoxylum schinifolium. Further studies should be made on active compounds.

In this study, we evaluated the anti-inflammatory activity of the soluble ethyl acetate fraction and chemical components of the stem bark of Passiflora foetida (Passifloraceae). Ten flavonoids (1-10) were isolated by various chromatographic techniques, and their structures were determined based on spectroscopic analyses by using nuclear magnetic resonance (NMR). Luteolin (2) and chrysoeriol (3) showed the most potent inhibition of nitric oxide (NO) production in macrophage cell line, RAW264.7, with half maximal inhibitor concentration (IC50) values of 1.2 and 3.1 μM, respectively. These compounds suppressed lipopolysaccharide (LPS)-induced inducible NO synthase (iNOS) expression at the transcription level. Our research indicates that the stem bark of P. foetida has significant anti-inflammatory properties, suggesting that its flavonoids may have anti-inflammatory benefits.

Many natural products have been shown to have an inhibitory effect on nitric oxide (NO), and are used as chemotherapy agents for inflammation disease. The current study was designed to evaluate the anti-inflammatory activity of chemical components from the leaves of Ampelopsis cantoniensis. Sixteen compounds (1-16) were isolated and identified. Phloretin (5) and 5,7,3',5'-tetrahydroxyflavanone (16) inhibited nitric oxide (NO) production with IC50 values of 5.2, and 18.5 μM, respectively. The inhibitory effect of compounds 5 and 16 were accompanied by dose-dependent decreases in LPS-induced nitric oxide synthase (iNOS) in RAW 264.7 cells, respectively. This study investigated the significant anti-inflammatory properties of isolated compounds from the leaves of A. cantoniensis for the first time. The findings demonstrate that A. cantoniensis could be used beneficially in the treatment of inflammation disease.

Celastrol, a quinone methide triterpenoid, was isolated as an inhibitor of NF-kappaB from Celastrus orbiculatus. This compound dose-dependently inhibited a variety of stimuli-induced NF-kappa B-regulated gene expression and the DNA-binding of NF-kappa B in different cell lines without affecting DNA-binding activity of AP-1. Preincubation of celastrol completely blocked the LPS-, TNF-alpha-, or PMA-induced degradation and phosphorylation of I kappa B alpha. Importantly, celastrol inhibited IKK activity and the constitutively active IKK beta activity in a dose-dependent manner without either affecting the NF-kappa B activation induced by RelA over-expression or directly suppressing the DNA-binding of activated NF-kappa B. However, mutation of cysteine 179 in the activation loop of IKK beta abolished sensitivity towards to celastrol, suggesting that celastrol suppressed the NF-kappa B activation by targeting cysteine 179 in the IKK. To verify that celastrol is a NF-kappa B inhibitor, we investigated its effect on some NF-kappa B target genes expressions. Celastrol prevented not only LPS-induced mRNA expression of iNOS and TNF-alpha, but also TNF-alpha-induced Bfl-1/A1 expression, a prosurvival Bcl-2 homologue. Consistent with these results, celastrol significantly suppressed the production of NO and TNF-alpha in LPS-stimulated RAW264.7 cells, and increased the cytotoxicity of TNF-alpha in HT-1080 cells. We also demonstrated that celastrol showed anti-inflammatory and anti-tumor activities in animal models. Taken together, this study extends our understanding on the molecular mechanisms underlying the anti-inflammatory and anti-cancer activities of celastrol and celastrol-containing medicinal plant, which would be a valuable candidate for the intervention of NF-kappa B-dependent pathological conditions.

Migration is an essential feature of metastatic cancer cells. To understand how motility is regulated in hepatocellular carcinoma, we analyzed gene expression profiles of mouse model cell lines we established from transgenic mice carrying SV40 large T antigen. A non-motile HC9 cell line was isolated from mouse liver tumors, and two additional cell lines, HCM1 and HCM4, were derived from HC9 cells. We found that both HCM1 and HCM4 cells were substantially more migratory than HC9, and that HCM1 generated tumor nodules in nude mice. In contrast to HCM4 cells that exhibited mesenchymal cell-type gene expression similar to HC9 cells, HCM1 cells appeared to have undergone a mesenchymal-amoeboidal transition. Thus, HCM1 and HCM4 cells have distinct migration and gene expression patterns, and together with HC9 cells, they can serve as model cell lines for understanding how migration is acquired and controlled in hepatocellular carcinoma.

Agonists of peroxisome proliferator-activated receptor gamma (PPAR gamma) are of interest as a treatment for diabetes, which prompted the identification of a new class of non-TZD PPAR gamma agonist. Moreover, compound 14c has displayed the most active agonistic activity with an EC50 value of 50 nM, in addition to exhibiting a new binding mode in the X-ray cocrystal structure.

NK cells are a key component of innate immune systems, and their activity is regulated by cytokines and hormones. Adiponectin, which is secreted from white adipose tissues, plays important roles in various diseases, including hypertension, cardiovascular diseases, inflammatory disorders, and cancer. In this study the effect of adiponectin on NK cell activity was investigated. Adiponectin was found to suppress the IL-2-enhanced cytotoxic activity of NK cells without affecting basal NK cell cytotoxicity and to inhibit IL-2-induced NF-κB activation via activation of the AMP-activated protein kinase, indicating that it suppresses IL-2-enhanced NK cell cytotoxicity through the AMP-activated protein kinase-mediated inhibition of NF-κB activation. IFN-γ enhances NK cell cytotoxicity by causing an increase in the levels of expression of TRAIL and Fas ligand. The production of IFN-γ, one of the NF-κB target genes in NK cells, was also found to be suppressed by adiponectin, accompanied by the subsequent down-regulation of IFN-γ-inducible TRAIL and Fas ligand expression. These results clearly demonstrate that adiponectin is a potent negative regulator of IL-2-induced NK cell activation and thus may act as an in vivo regulator of anti-inflammatory functions.

A new sorbicillinoid polyketide, dihydrotrichodimerol (2), along with known trichodimerol (1), and rezishanones C (3) and D (4) were isolated by bioassay-guided fractionation from an unidentified fungal strain. Dihydrotrichodimerol (2) specifically activated peroxisome proliferator-activated receptor gamma with an ED50 value of 80 ng/ml as measured by a transactivation assay using a chimeric hPPAR/GAL4 system without affecting peroxisome proliferator-activated receptors alpha and sigma. On the other hand, compounds 1 and 2 suppressed the production of tumor necrosis factor-alpha and nitric oxide in LPS-stimulated RAW264.7 cells to a similar extent.

Nuclear factor-kappaB (NF-kappaB) and the signaling pathways that regulate its activity have become a focal point for intense drug discovery and development efforts. NF-kappaB regulates the transcription of a large number of genes, particularly those involved in immune, inflammatory, and antiapoptotic responses. In our search for NF-kappaB inhibitors from natural resources, we identified cardamomin, 2',4'-dihydroxy-6'-methoxychalcone, as an inhibitor of NF-kappaB activation from Alpinia conchigera Griff (Zingiberaceae). In present study, we demonstrated the effect of cardamomin on NF-kappaB activation in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and LPS-induced mortality. This compound significantly inhibited the induced expression of NF-kappaB reporter gene by LPS or tumor necrosis factor (TNF)-alpha in a dose-dependent manner. LPS-induced production of TNF-alpha and NO as well as expression of inducible nitric-oxide synthase and cyclooxygenase-2 was significantly suppressed by the treatment of cardamomin in RAW264.7 cells. Also, cardamomin inhibited not only LPS-induced degradation and phosphorylation of inhibitor kappaBalpha (IkappaBalpha) but also activation of inhibitor kappaB (IkappaB) kinases and nuclear translocation of NF-kappaB. Further analyses revealed that cardamomin did not directly inhibit IkappaB kinases, but it significantly suppressed LPS-induced activation of Akt. Moreover, cardamomin suppressed transcriptional activity and phosphorylation of Ser536 of RelA/p65 subunit of NF-kappaB. However, this compound did not inhibit LPS-induced activation of extracellular signal-regulated kinase and stress-activated protein kinase/c-Jun NH(2)-terminal kinase, but significantly impaired activation of p38 mitogen-activated protein kinase. We also demonstrated that pretreatment of cardamomin rescued C57BL/6 mice from LPS-induced mortality in conjunction with decreased serum level of TNF-alpha. Together, cardamomin could be valuable candidate for the intervention of NF-kappaB-dependent pathological condition such as inflammation.

Background Previous studies have shown that TIMP-2 overexpression is a useful therapeutic tool for inhibiting tumor growth and invasion in animals. However, it has not been reported whether genetic manipulation for TIMP-2 overexpression can induce an inhibitory effect on spontaneous metastasis from the primary tumor site to other organs such as lungs or lymph nodes in an animal model. Methods The present studies describe the effects of retrovirus-mediated TIMP-2 gene transfer into human breast cancer cell lines on the in vitro invasion of the tumor cells or the in vivo growth in nude mouse. Here we also used retroviral-mediated TIMP-2 overexpression by intratumoral injection for suppression of metastasis in human breast carcinoma established in the mammary fat pad of nude mice. Results As expected, overexpression of TIMP-2 inhibited matrix metalloprotenase (MMP) activity and invasion of the tumor cells. Also, the growth rate of tumors grafted with the breast cancer cells transduced with the retrovirus vector encoding TIMP-2 cDNA was significantly slower than the growth rate of tumors grafted with the breast cancer cells transduced with a control retrovirus vector. Furthermore, single intratumoral injection of the TIMP-2 retrovirus-producing cells into human breast tumor tissue established in mammary fat pads of nude mice showed a dramatic decrease in size and number of lung metastatic tumors. Conclusions Retrovirus-mediated TIMP-2 gene transfer into human breast cancer cells is able to down-regulate invasion and show that tumor-derived angiogenesis is reduced. In this model, retroviral-mediated transduction of TIMP-2 cDNA into a limited population of human tumor cells inhibits tumor growth and prevents distant pulmonary metastasis. These results indicate that it may not be necessary to deliver and express these genes in every single tumor cell as long as the level of expression in a limited number of transduced cells is sufficient to prevent the excessive breakdown of the extracellular matrix. Copyright © 2004 John Wiley & Sons, Ltd.

Three guaianolide sesquiterpene lactones, 3alpha,4alpha-epoxyrupicolins C-E, together with six known sesquiterpenes, artemisolide, 3-methoxytanapartholide, deacetyllaurenobiolide, moxartenolide as well as arteminolides B and D were isolated by bioassay-guided fractionation from the methanol extract of the aerial parts of Artemisia sylvatica using the NF-kappaB mediated reporter gene assay. All isolated compounds displayed inhibitory activity on the LPS-induced NF-kappaB activation, NO production, and TNF-alpha production with IC50 values of 0.49-7.17, 1.46-6.16, and 3.19-27.76 microM, respectively, in RAW264.7 cells. It was also established that arteminolide B suppressed the expression of NF-kappaB target genes such as iNOS and COX-2. This is the first report of NF-kappaB inhibitory activities of these compounds and supports the pharmacological use of Artemisia sylvatica, which has been employed as an herbal medicine for the treatment of inflammation.

Sauchinone, a known lignan, was isolated from the root of Saururus chinensis as an active principle responsible for inhibiting the production of NO in LPS-stimulated RAW264.7 cells by activity-guided fractionation. Sauchinone dose-dependently inhibited not only the production of NO, but also the expression of iNOS mRNA and protein in LPS-stimulated RAW 264.7 cells. Furthermore, sauchinone prevented LPS-induced NF-kappaB activation, which is known to play a critical role in iNOS expression, assessed by a reporter assay under the control of NF-kappaB. However, an electrophoretic mobility shift assay (EMSA) demonstrated that sauchinone did not suppress the DNA-binding activity of NF-kappaB or the degradation of IkappaB-alpha induced by LPS. Further analysis revealed that transactivation activity of RelA subunit of NF-kappaB was dose-dependently suppressed in the presence of sauchinone. Taken together, our results suggested that sauchinone could inhibit production of NO in LPS-stimulated RAW264.7 cells through the suppression of NF-kappaB by inhibiting transactivation activity of RelA subunit.

The sesquineolignans, saucerneol D and saucerneol E were isolated from the roots of Saururus chinensis together with four known lignans, manassantin A, manassantin B, (-)-saucerneol methyl ether, and (+)-saucernetin. Structure elucidation was based on the analysis of spectroscopic data and anti-inflammatory activity was studied using HeLa cells transfected with NF-kappaB reporter construct. All compounds except for (+)-saucernetin inhibited NF-kappaB dependent reporter gene expression with IC50 values of 2.5-16.9 microM.

Four ent-kaurane diterpenoids including two known, ent-7alpha,14beta-dihydroxykaur-16-en-15-one (1) and ent-18-acetoxy-7alpha-hydroxykaur-16-en-5-one (2), and two new, ent-1beta-acetoxy-7alpha,14beta-dihydroxykaur-16-en-15-one (3) and ent-18-acetoxy-7alpha,14beta-dihydroxykaur-16-en-15-one (4), were isolated from the leaves of Croton tonkinensis in a search for inhibitors of NF-kappaB activation and nitric oxide production. These ent-kauranoids inhibited LPS-induced NF-kappaB activation in murine macrophage RAW264.7 cells at IC50 values between 0.07 and 0.42 microM. Consistently, the ent-kauranoids markedly reduced LPS-induced NO production in a comparable concentration-dependent manner.

The human CD99 protein is a 32-kDa type I transmembrane glycoprotein, while CD98 is a disulfide-linked 125-kDa heterodimeric type II transmembrane glycoprotein. It has been previously shown that CD99 and CD98 oppositely regulate β1 integrin signaling, though the mechanisms by which this regulation occurs are not known. Our results revealed that antibody-mediated crosslinking of CD98 induced FAK phosphorylation at Y397 and facilitated the formation of the protein kinase Cα (PKCα)-syntenin-focal adhesion kinase (FAK), focal adhesions (FAs), and IPP-Akt1-syntenin complex, which mediates β1 integrin signaling. In contrast, crosslinking of CD99 disrupted the formation of the PKCα-syntenin-FAK complex as well as FA via FAK dephosphorylation. The CD99-induced dephosphorylation of FAK was apparently mediated by the recruitment of Src homology region 2 domain-containing phosphatase-2 (SHP2) to the plasma membrane and subsequent activation of its phosphatase activity. Further consequences of the activation of SHP2 included the disruption of FAK-talin and talin-β1 integrin interactions and attenuation in the formation of the IPP-Akt1-syntenin complex at the plasma membrane, which resulted in reduced cell-ECM adhesion. This report uncovers the molecular mechanisms underlying the inverse regulation of β1 integrin signaling by CD99 and CD98 and may provide a novel therapeutic approach to treat inflammation and cancer.

One new β-carboline alkaloid 7-methoxy-(9H-β-carbolin-1-il)-(E)-1-propenoic acid (1) together with 9-methoxycanthin-6-one (2) and 9-hydroxycanthin-6-one (3) were isolated from the hairy-root cultures of Eurycoma longifolia. The effects of these compounds on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 cells were investigated. Compound 1 strongly inhibited the production of NO while 2 and 3 having weak or inactive effect. Consistently, compound 1 decreased the expression of cyclooxygenase-2 and inducible nitric oxide synthase.

Two human breast cancer cell lines of differing invasive and metastatic potential, MDA-MB-435 and MCF7, were examined using subtractive suppression hybridization in a search for any genes associated with metastasis. Of the 17 cDNAs identified as being differentially expressed genes, it was determined that syntenin was overexpressed in metastatic MDA-MB-435 cells. Expression analysis showed that the expression level of syntenin was well correlated with invasive and metastatic potential in various human breast and gastric cancer cell lines. Moreover, gastric tumor tissues exhibited a much higher syntenin mRNA expression than their normal counterparts. Syntenin-transfected MCF7 cells migrated more actively, and showed an increased invasion rate relative to vector-transfectants or parental MCF7 in vitro, without evidencing any effect on the adhesion to fibronectin, type I collagen and laminin. Similarly, the forced expression of syntenin to human gastric cancer cell line Az521 increased its migratory and invasive potential in vitro. Syntenin-expressing MCF7 cells were associated with the appearance of numerous cell surface extensions and with pseudopodia formation on collagen I, suggesting that syntenin may be involved in the signaling cascade to actin-reorganization. Mutation study suggested that PDZ2 domain of syntenin could be an essential role in its stimulatory effect on the cell migration. This is the first demonstration that syntenin, a PDZ motif-containing protein, can be overexpressed during the metastatic progression of human breast and gastric cancer cells and that it can function as a metastasis-inducing gene.

isolation, structural elucidation and antiinflammatory activity of a new megastigmane glycoside, galloyl linarionoside A (I), along with 13 known compounds

Two new compounds, erythroformide (I) and 3β-acetoxynorcassamide (II), are isolated together with four known compounds and their effects on endothelial tube formation on matrigel are investigated.

3-Deoxysappanchalcone (3-DSC), isolated from Caesalpinia sappan (Leguminosae), is a chalcone that exerts a variety of pharmacological activities. In the present study, we demonstrated that 3-DSC exerts anti-inflammatory activity in murine macrophages by inducing heme oxygenase-1 (HO-1) expression at the translational level. Treatment of RAW264.7 cells with 3-DSC induced HO-1 protein expression in a dose- and time-dependent manner without affecting HO-1 mRNA expression. Mitogen-activated protein kinase inhibitors or actinomycin D, a transcriptional inhibitor, did not block 3-DSC-mediated HO-1 induction. However, 3-DSC-mediated HO-1 induction was completely blocked by treatment with cycloheximide, a translational inhibitor, or rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR). Strikingly, 3-DSC increased the phosphorylation level of mTOR downstream target molecules such as eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and S6 kinase 1 (S6K1), as well as AKT in a dose- and time-dependent manner, suggesting that the 3-DSC induces HO-1 expression by activating the AKT/mTOR pathway. Consistent with the notion that HO-1 has anti-inflammatory properties, 3-DSC inhibited the production of nitric oxide (NO) and interleukin (IL)-6 in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Inhibition of HO-1 activity by treatment with tin protoporphyrin IX, a specific HO-1 inhibitor, abrogated the inhibitory effects of 3-DSC on the production of NO and IL-6 in LPS-stimulated RAW264.7 cells. Taken together, 3-DSC may be an effective HO-1 inducer at the translational level that has anti-inflammatory effects, and a valuable compound for modulating inflammatory conditions.

In the search for new naturally occurring angiogenic inhibitor, we found that culture broths from two unidentified fungal strains exerted potent inhibitory activities on capillary-like tube formation of human umbilical vein endothelial cells (HUVEC) in vitro. Two active compounds were isolated by bioassay-guided separation and their structures were identified as gliotoxin (1) and its derivative methylthiogliotoxin (2) by spectroscopic analyses. These compounds significantly inhibited the migration of HUVEC assessed by in vitro wounding migration assay and exhibited at least 10 times more potent inhibition of proliferation of HUVECs as compared with that of cancer cell lines such as HeLa, MCF-7, and KB 3-1 cells. Especially, gliotoxin having disulfide group exerted more potent activities than methylthiogliotoxin, suggesting that gliotoxin could be a useful compound for further study as an anti-angiogenic agent.

A methanolic extract of the whole plant of Isodon japonicus (Labiatae) showed potent inhibition on the LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in RAW264.7 cells. Four known kaurane diterpenes were isolated by activity-guided fractionation and their structures were identified as kamebanin (1), kamebacetal A (2), kamebakaurin (3), excisanin A (4). All compounds also inhibited the LPS-induced NF-kappaB activation as assessed by NF-kappaB reporter assay and electrophoretic mobility shift assay (EMSA). Compounds 2-4 showed comparable inhibitory effects on the LPS-induced production of NO and PGE2, and activation of NF-kappaB without affecting cell viability. These results suggest that kaurane diterpenes could exert their inhibitory effects on the production of NO and PGE2 through the suppression of NF-kappaB activation, and be partially responsible for the anti-inflammatory activities of the genus Isodon.

The biological activity of avermectin B components is superior to that of avermectin A components, which are derived from avermectin B by avermectin B 5-O-methyltransferase. Gene disruption, targeting avermectin B 5-O-methyltransferase gene in Streptomyces avermitilis, was carried out to obtain a strain of avermectin B producer. Phenotype analysis of the mutant with the disrupted O-methyltransferase gene showed that only avermectin B components were produced with a significant increase in production

Three new guaiane type sesquiterpenes were isolated from the methanolic extract of the fruits of Torilis japonica together with a known compound, torilin (1). Their structures were established as 11-acetoxy-8-isobutyryl-4-guaien-3-one (2), 11-acetoxy-8-methacrylyl-4-guaien-3-one (3), and 11-acetoxy-8-propionyl-4-guaien-3-one (4) by spectroscopic methods. These compounds inhibited lipopolysaccharide (LPS)-induced nitric oxide production in murine macrophages RAW 264.7 cells.

Avermectin O-methyltransferase gene was cloned from a cosmid clone covering the first module of polyketide synthase gene cluster of avermectin biosynthesis. Streptomyces lividans transformed with a DNA fragment containing avermectin O-methyltransferase gene efficiently convert milbemycin D to milbemycin G, indicating that biosynthetic genes of milbemycin and avermectin might complement each other. © Rapid Science Ltd. 1998

Microcell-mediated transfer of chromosome 6 into human melanoma cell lines C8161 and MelJuSo suppresses metastasis by at least 95% without affecting tumorigenicity. Subtractive hybridization and differential display were used to identify the molecule(s) responsible for suppressing metastasis in neo6/melanoma (neo6/C8161 and neo6/MelJuSo) hybrids. Seven cDNA clones exhibiting quantitatively or qualitatively higher expression in neo6/melanoma hybrids were obtained. These genes fell into 2 categories: 1) transcription-related genes (AP-2A, HMG-I(Y) and a novel isoform of nucleophosmin B23), which have previously been shown to regulate metastasis-associated genes; and 2) novel genes. One of the novel genes, designated KiSS-1, significantly suppressed metastasis of the human malignant melanoma cell lines MelJuSo and a highly metastatic subclone of C8161, C8161cl.9, following transfection and constitutive expression. Our results illustrate the power of subtractive hybridization and differential display to identify functional metastasis-controlling genes in human melanoma.Int. J. Cancer 71: 1035-1044, 1997. © 1997 Wiley-Liss Inc.

Metastasis is suppressed more than 95% following microcell-mediated transfer of a single copy of neomycin-tagged human chromosome 6 (neo6) into the human melanoma cell lines C8161 and MelJuSo. Concomitant with metastasis suppression is upregulation of NME1 (Nm23-H1) mRNA and protein expression. The purposes of this study were to determine whether NME1 expression was responsible for metastasis suppression in neo6/melanoma hybrids, and whether genes on chromosome 6 regulate NME1. Using neo6/C8161 cells, transfection of CAT reporter constructs linked to the NME1 promoter failed to consistently induce CAT. Therefore, it does not appear that genes on chromosome 6 directly control transcription of NME1. Transfection and overexpression of NME1 in MelJuSo, under the control of the CMV promoter, resulted in 40-80% inhibition of lung metastasis following i.v. inoculation of 2´10 cells. Only one transfectant of C8161 subclone 9 (C8161cl.9) cells was suppressed for metastasis. Control transfections with pCMVneo or pSV2neo did not suppress metastasis in either cell line. Taken together, these data suggest that NME1 can reduce metastatic potential of some human melanoma cells; but, this inhibitory activity appears to be independent of the metastasis suppression following introduction of chromosome 6 into C8161 and MelJuSo human melanoma cell lines.

Four new lanostane triterpenes, butyl lucidenate P (I), butyl lucidenate D2 (II), butyl lucidenate E2 (III), and butyl lucidenate Q (IV), are isolated along with eleven known compounds from the fruiting bodies of Ganoderma lucidum.

Arginase II has recently reported as a novel therapeutic target for the treatment of cardiovascular diseases such as atherosclerosis. In the course of screening plants used in natural medicines as arginase II inhibitory activity, a methanol extract of Scutellaria indica showed significant inhibitory effect. Further fractionation and repeated column chromatography led to the isolation of a new flavan-type (1), and seven known compounds (2–8). The chemical structures of isolated compounds were elucidated based on extensive 1D and 2D NMR spectroscopic data. The isolates 1–8 were investigated in vitro for their arginase II inhibitory activity using enzyme solution prepared from kidney of anesthetized C57BL/6 mice. Compounds 3 and 5 significantly inhibited arginase II activity with IC50 values of 25.1 and 11.6 μM, respectively, whereas the other compounds were apparently inactive.

Background Microcell-mediated transfer of chromosome 6 into human C8161 and MelJuSo melanoma cell suppresses their ability to metastasize by at least 95% without affecting their tumorigenicity. This observation demonstrates that the ability to metastasize is a phenotype distinct from tumor formation and suggests that tumorigenic cells acquire metastatic capability only after accumulating additional genetic defects. These results also imply that mutations of genes on chromosome 6 are among those late genetic changes responsible for metastatic potential. They further suggest that a melanoma metastasis-suppressor gene(s) is encoded on chromosome 6 or is regulated by genes on chromosome 6. Purpose Our objective was to identify the gene(s) responsible for the suppression of metastasis in chromosome 6/melanoma cell hybrids. Methods A modified subtractive hybridization technique was used to compare the expression of messenger RNAs (mRNAs), via an analysis of complementary DNAs (cDNAs), in metastatic cells (C8161 or MelJuSo) and nonmetastatic hybrid clones (neo6/C8161 or neo6/MelJuSo). Results A novel cDNA, designated KiSS-1, was isolated from malignant melanoma cells that had been suppressed for metastatic potential by the introduction of human chromosome 6. Northern blot analyses comparing mRNAs from a panel of human melanoma cells revealed that KiSS-1 mRNA expression occurred only in nonmetastatic melanoma cells. Expression of this mRNA in normal heart, brain, liver, lung, and skeletal muscle was undetectable by northern blot analysis. Weak expression was found in the kidney and pancreas, but the highest expression was observed in the placenta. The KiSS-1 cDNA encodes a predominantly hydrophilic, 164 amino acid protein with a polyproline-rich domain indicative of an SH3 ligand (binds to the homology 3 domain of the oncoprotein Src) and a putative protein kinase C-alpha phosphorylation site. Transfection of a full-length KiSS-1 cDNA into C8161 melanoma cells suppressed metastasis in an expression-dependent manner. Conclusions These data strongly suggest that KiSS-1 expression may suppress the metastatic potential of malignant melanoma cells. Implications KiSS-1 may be a useful marker for distinguishing metastatic melanomas from nonmetastatic melanomas.

Progression of human melanoma toward increasingly malignant behavior is associated with several nonrandom chromosomal aberrations, most commonly involving chromosomes 1, 6, 7, 9, and 10. We previously showed that introduction of human chromosome 6 into the highly metastatic human malignant melanoma cell line C8161 completely suppressed metastasis without altering tumorigenicity (Welch DR, Chen P, Miele ME, et al., Oncogene 9:255–262, 1994). Alterations of chromosome 1 are the most frequent chromosome abnormality observed in melanomas, and they frequently arise late in tumor progression. The purpose of the study presented here was to compare the effects of chromosomes 1 and 6 on malignant melanoma metastasis. By using microcell-mediated chromosome transfer, single copies of neo-tagged human chromosomes 1 or 6 were introduced into the human melanoma cell line MelJuSo. The presence of the added chromosome was verified by G banding of karyotypes, fluorescence in situ hybridization, and screening for polymorphic markers on each chromosome. The incidence and number of metastases per lung after intravenous or intradermal injection of parental MelJuSo cells was significantly (P < 0.01) greater than those of hybrids containing either chromosome 1 or chromosome 6, although chromosome 1 was a less potent inhibitor of metastasis than chromosome 6. Cultures established from primary tumors and metastases remained neomycin resistant, suggesting that portions of the added chromosomes were retained. These results strengthen the evidence for the presence of a melanoma metastasis suppressor gene on chromosome 6. neo6/MelJuSo hybrids expressed 2.4- to 3.4-fold more of the melanoma differentiation-associated gene mda-6 (previously shown to be identical to WAF1/CIP1/Sdi1/CAP20) than parental metastatic cells. mda-6/WAF1 is among the candidate genes on chromosome 6. These results also demonstrate, for the first time, the existence of metastasis suppressor genes on human chromosome 1, although these genes appear to be less potent than the one encoded on chromosome 6. © 1996 Wiley-Liss, Inc.

A new homomonoterpene, l,3,3-trimethyl-7-oxabicyclo[3.1.1]hexa-9-en-10-oic acid, named madhusic acid A (1), together with ten known compounds (2-11) were isolated from the methanolic extract of the dried leaves of Madhuca pasquieri (Dubard) H. J. Lam. The structure of the new compound was elucidated on the basis of ID, 2D NMR (COSY, HMQC, and HMBC) and mass spectral analyses. We examined the effects of the isolated compounds against LPS-induced NO production in macrophage RAW264.7 cells and compound 2 showed effective activity with an IC50 value of 14.5 μM.

Four new phenolic compounds, caesalpiniaphenols A-D (1-4), together with eight known compounds were isolated from Caesalpinia sappan heartwood. The chemical structures were established mainly by NMR, MS, ECD, and Mosher's method. Compounds 4, 5, and 7 showed weak inhibitory activity against the LPS-induced NO production in macrophage RAW264.7 cells with IC(50) values of 12.2, 3.5, and 5.7 μM, respectively.

Elevated plasma cholesterol is a hallmark of numerous cardiovascular diseases that are closely linked to endothelial dysfunction indicating decreased nitric oxide (NO) production in the endothelium. It has been previously demonstrated that piceatannol-3'-O-β-D-glucopyranoside (PG) inhibits arginase activity and reciprocally regulates NO production. Here, we aimed to ascertain whether PG ameliorates vascular function in wild-type (WT) and atherogenic model mice [apolipoprotein E-null mice (ApoE-/-)] and to investigate the possible underlying mechanism. Preincubation of aortic vessels from WT mice fed a normal diet (ND) with PG attenuated vasoconstriction response to U46619 and phenylephrine (PE), while the vasorelaxant response to acetylcholine (Ach) was markedly enhanced in an endothelium-dependent manner. However, the endothelium-independent NO donor, sodium nitroprusside (SNP), did not change vessel reactivity. In thoracic aorta from ApoE-/- mice, a high-cholesterol diet (HCD) induced an increase in arginase activity, a decrease in NO release and an increase in reactive oxygen species generation that was reversed by treatment with PG. The effect of PG was associated with enhanced stability of the eNOS dimer and was not dependent on the expression levels of arginase II and eNOS proteins, although eNOS expression was increased in ApoE-/- mice fed an HCD. Furthermore, PG treatment attenuated the PE-dependent contractile response, and significantly improved the Ach-dependent vasorelaxation response in aortic rings from ApoE-/- mice fed an HCD. On the other hand, PG incubation neither altered the contractile response to a high K+ solution nor the relaxation response to SNP. When analyzing the L-arginine content using high-performance liquid chromatography, PG incubation increased the intracellular L-arginine concentration. PG administration in the drinking water significantly reduced fatty streak formation in ApoE-/- mice fed an HCD. These data indicate that PG improves the pathophysiology of cholesterol-mediated endothelial dysfunction. Therefore, we conclude that the development of PG as a novel effective therapy for preventing atherosclerotic diseases is warranted.

Four new lanostane triterpenes, butyl lucidenate P ( 1 ), butyl lucidenate D 2 ( 2 ), butyl lucidenate E 2 ( 3 ) and butyl lucidenate Q ( 4 ) along with 11 known compounds ( 5 – 15 ) were isolated from the fruiting bodies of Ganoderma lucidum . Their chemical structures were established mainly by 1D and 2D NMR techniques and mass spectrometry. Their anti-inflammatory activity was evaluated against LPS-induced NO production in macrophage RAW 264.7 cells. Compounds 1 , 3 , 4 , 9 , 10 and 15 showed inhibitory potency with IC 50 values of 7.4, 6.4, 4.3, 9.4, 9.2 and 4.5 μM, respectively. Compounds 1 , 3 and 15 dose-dependently reduced the LPS-induced iNOS expressions. Preincubation of cell with 1 , 3 and 15 significantly suppressed LPS-induced expression of COX-2 protein.

The abnormal proliferation and migration of vascular smooth muscle cell (VSMC) contributes importantly to the pathogenesis of atherosclerosis and restenosis. Here, we investigated the effects of eupatolide (EuTL), a sesquiterpene lactone isolated from the medicinal plant Inula britannica, on platelet-derived growth factor (PDGF)-induced proliferation and migration of primary rat aortic smooth muscle cells (RASMCs), as well as its underlying mechanisms. EuTL remarkably inhibited PDGF-induced proliferation and migration of RASMCs. Treatment of RASMCs with EuTL induced both protein and mRNA expression of heme oxygenase-1 (HO-1). SB203580 (a p38 inhibitor), SP600125 (a JNK inhibitor), U0126 (a MEK inhibitor) and LY294002 (a PI3K inhibitor) did not suppress EuTL-induced HO-1 expression; however, N-acetylcysteine (NAC, an antioxidant) blocked EuTL-induced HO-1 expression. Moreover, treatment of RASMCs with EuTL increased reactive oxygen species (ROS) accumulation and nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2); however, this translocation was also inhibited by NAC. NAC or inhibition of HO-1 significantly attenuated the inhibitory effects of EuTL on PDGF-induced proliferation and migration of RASMCs. Taken together, these findings suggest that EuTL could suppress PDGF-induced proliferation and migration of VSMCs through HO-1 induction via ROS-Nrf2 pathway and may be a potential HO-1 inducer for preventing or treating vascular diseases.

Platelets play a critical role in pathogenesis of cardiovascular disorders and strokes. The inhibition of platelet function is beneficial for the treatment and prevention of these diseases. In this study, we investigated the anti-platelet activity of erythro-(7S,8R)-7-acetoxy-3,4,3',5'-tetramethoxy-8-O-4'-neolignan (EATN), a neolignan isolated from Myristica fragrans, using human platelets. EATN preferentially inhibited thrombin- and platelet-activating factor (PAF)-induced platelet aggregation without affecting platelet damage in a concentration-dependent manner with IC50 values of 3.2 ± 0.4 and 3.4 ± 0.3 μM, respectively. However, much higher concentrations of EATN were required to inhibit platelet aggregation induced by arachidonic acid. EATN also inhibited thrombin-induced serotonin and ATP release, and thromboxane B2 formation in human platelets. Moreover, EATN caused an increase in cyclic AMP (cAMP) levels and attenuated intracellular Ca(2+) mobilization in thrombin-activated human platelets. Therefore, we conclude that the inhibitory mechanism of EATN on platelet aggregation may increase cAMP levels and subsequently inhibit intracellular Ca(2+) mobilization by interfering with a common signaling pathway rather than by directly inhibiting the binding of thrombin or PAF to their receptors. This is the first report of the anti-platelet activity of EATN isolated from M. fragrans.

Malabaricone C (Mal-C), isolated from nutmeg, is known to exert a variety of pharmacological activities. However, the effect of Mal-C on vascular smooth muscle cells (VSMCs) is unknown. This study examined the effect of Mal-C on proliferation and migration of primary rat aortic smooth muscle cells (RASMCs) as well as its underlying mechanisms. Treatment of RASMCs with Mal-C induced both protein and mRNA expression of heme oxygenase-1 (HO-1) in a dose- and time-dependent manner. Mal-C-mediated HO-1 induction was inhibited by treatment with actinomycin D or by cycloheximide. SB203580 (a p38 inhibitor), SP600125 (a JNK inhibitor), U0126 (a MEK inhibitor), and N-acetylcysteine (NAC, an antioxidant) did not suppress Mal-C-induced HO-1 expression. In contrast, LY294002 (a PI3K inhibitor) blocked Mal-C-induced HO-1 expression. Moreover, RASMCs treated with Mal-C exhibited activation of AKT in a dose- and time-dependent manner. Treatment of RASMCs with Mal-C increased nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2), which is a key regulator of HO-1 expression, and this translocation was also inhibited by LY294002. Consistent with the notion that HO-1 has protective effects against VSMCs, Mal-C remarkably inhibited platelet-derived growth factor (PDGF)-induced proliferation and migration of RASMCs. However, inhibition of HO-1 significantly attenuated the inhibitory effects of Mal-C on PDGF-induced proliferation and migration of RASMCs. Taken together, these findings suggest that Mal-C could suppress PDGF-induced proliferation and migration of RASMCs through Nrf2 activation and subsequent HO-1 induction via the PI3K/AKT pathway, and may be a potential HO-1 inducer for preventing or treating vascular diseases.

2-Deoxy-D-glucose (2-DG), a synthetic glucose analog that acts as a glycolytic inhibitor, is currently under clinical evaluation for targeting tumor cells. Tephrosin (TSN), a plant rotenoid, is known as an anticancer agent. In this study, we describe that the addition of TSN to 2-DG enhanced the cytotoxic activity of 2-DG against various types of cancer cells by accelerating ATP depletion and blunting autophagy. TSN increased the sensitivity of cancer cells to the cytotoxic effect of 2-DG. The combination of TSN and 2-DG induced acceleration of intracellular ATP depletion and the drastic activation of AMP-activated protein kinase (AMPK), which resulted in the inactivation of the mammalian target of rapamycin (mTOR) pathway. Of particular interest, TSN suppressed 2-DG-induced autophagy, a cell survival process in response to nutrient deprivation. We also showed that TSN inhibited 2-DG-induced activation of elongation factor-2 kinase (eEF-2K), which has been known to regulate 2-DG-induced autophagy. Inhibition of eEF-2K by RNA interference blunted 2-DG-induced autophagy and increased the sensitivity of cancer cells to the cytotoxic effect of 2-DG. The addition of TSN to 2-DG, however, did not enhance the cytotoxic activity of 2-DG by knockdown of eEF-2K, suggesting that inhibition of eEF-2K by tephrsoin could be a critical role in the potentiating effect of TSN on the cytotoxicity of 2-DG. Furthermore, we showed that the blunted autophagy and enhanced cytotoxicity of 2-DG was accompanied by the augmentation of apoptosis. These results show that TSN may be valuable for augmenting the therapeutic efficacy of 2-DG.

Inhibition of cell-cycle progression is a target for the treatment of cancer. 3-Oxoolean-12-en-27-oic acid (3-OOLA) has shown significant anticancer activity towards diverse cancer cells, but has not been investigated for non-small cell lung carcinoma (NSCLC) cells. In this study, we investigated the antiproliferative effect of 3-OOLA in NSCLC cell lines and its underlying mechanism.The MTT assay, bromodeoxyuridine (BrdU) incorporation assay, and flow cytometry were used for cell proliferation studies, and annexin V staining for apoptotic effects. Western blot analysis was used to evaluate expression of cell-cycle regulatory proteins, such as cyclins and cyclin-dependent kinases (CDKs).3-OOLA caused G0/G1 phase cell-cycle arrest without inducing apoptosis in NSCLC cells, and Western blot analyses demonstrated down-regulation of cyclin D1, cyclin E and phosphorylated Rb.3-OOLA inhibits cell proliferation of NSCLC cells by inducing cell-cycle arrest at G0/G1 through down-regulation of cyclin D1 and cyclin E.

A new alkaloid, methyl 2'-(7,8-dihydrosanguinarine-8-yl)acetate (1), together with six known alkaloids, stylopine (2), protopine (3), norchelidonine (4), chelidonine (5), berberine (6), and 8-hydroxydihydrosanguinarine (7), were isolated from Chelidonium majus. Their chemical structures were primarily established using 1D and 2D NMR techniques and mass spectrometry. The anti-inflammatory activity of the isolates was examined for their inhibitory effects on LPS-induced NO production in macrophage RAW264.7 cells. Among them, compounds 5 and 7 showed strong inhibitory activities toward the LPS-induced NO production in macrophage RAW264.7 cells with IC(50) values of 7.3 and 4.5 μM, respectively. In addition, compounds 5 and 7 inhibited the inductions of COX-2 and iNOS mRNA in dose-dependent manners, indicating that these compounds attenuated the syntheses of these transcripts at the transcriptional level.

KW-2149, a new mitomycin C (MMC) derivative, inhibited the growth of murine P388 leukemia in vitro at 20-fold lower concentrations than those of MMC. KW-2149 was also effective in inhibiting the growth of MMC-resistant P388 (P388/MMC) cells. To elucidate these characteristics of KW-2149, its uptake and efflux were compared with those of MMC in MMC-sensitive and -resistant P388 cells. Both MMC and KW-2149 accumulated rapidly in P388 cells after incubation at the concentration of 0.47 and 0.024 microM, respectively, which were the IC50 values at 1-h exposure. Although this concentration of KW-2149 was 20 times lower than that of MMC, its intracellular concentration was little more than that of MMC, suggesting that KW-2149 accumulated in the cells quite efficiently. The accumulated KW-2149 in the cells after 1-h treatment remained for as long as 24 h after the incubation of the cells in drug-free medium, suggesting that most of the intracellular KW-2149 or MMC was bound to cellular components. The ratios of resistance of P388/MMC cells to MMC and KW-2149 were 34 and 8.8, respectively, at 1-h exposure, suggesting that P388/MMC cells were partially resistant to KW-2149 in vitro. P388/MMC cells also showed partial resistance to cisplatin, Adriamycin, m-AMSA, and etoposide. The accumulation of MMC in P388/MMC cells was lower than that in P388 cells, although the size of the former cells was almost equal to that of the latter. As a result, the amount of DNA-bound MMC was lower in P388/MMC cells than in P388 cells, suggesting its involvement in the mechanisms of MMC resistance in P388/MMC cells.

To investigate the resistant mechanisms against MMC in human tumor cells, we isolated an MMC-resistant variant (HT-29/MMC) of HT-29 human colon carcinoma cells. HT-29/MMC cells showed 5-fold resistance to MMC as compared with the parental cell line but did not show cross-resistance to Adriamycin, vincristine, ACNU, bleomycin, or cisplatin. Treatment of the cells with dicoumarol, an inhibitor of DT-diaphorase, reduced the cytotoxicity of MMC in DT-diaphorase proficient HT-29 cells but not in HT-29/MMC cells. HT-29/MMC cells were 5 times more sensitive than HT-29 cells to menadione, which is detoxified by DT-diaphorase, DT-diaphorase was deficient in HT-29/MMC cells as determined by the enzyme activity and immunoblot analysis of the cytoplasmic proteins. Levels of cytochrome P-450 reductase and glutathione S-transferase, however, were comparable in both cell lines. The amount of [3H]-MMC found covalently bound to chromosomal DNA in HT-29/MMC cells was one-fourth that detected in HT-29 cells. Treatment with dicoumarol reduced the DNA-bound MMC in HT-29 cells but not in HT-29/MMC cells. These results indicate that the deficiency in DT-diaphorase, an activating enzyme of MMC, is one of the mechanisms of resistance in HT-29/MMC cells.

The NF-κB transcription factors control many physiological processes, including inflammation, immunity, and apoptosis. Its activity contributes to the development of various cell malignancies. NF-κB-inducing kinase (NIK) plays a pivotal role in NF-κB activation. However, the molecular mechanism to stabilize and activate NIK remains elusive, although it is known that cIAP1/2 (cellular inhibitor of apoptosis 1 and 2) ubiquitinate NIK for degradation. Here, we report a novel NF-κB-related zinc finger protein 91 (ZFP91) that stabilizes and activates NIK in a ubiquitination-dependent manner. We show that ZFP91 interacts with and promotes the Lys(63)-linked ubiquitination of NIK and subsequent processing of p100 to p52. The results of in vitro biochemical assays indicate that ZFP91 functions as an E3 ligase directly to NIK. Remarkably, the ubiquitination of NIK coincides with its Thr(559) phosphorylation. Furthermore, knockdown of ZFP91 expression by RNA interference inhibits the CD40 ligation-induced activation of NIK and p100 processing as well as the expression of noncanonical NF-κB target genes. These data clearly indicate that ZFP91 is an important regulator of the noncanonical NF-κB pathway.