Aberrant GATA2 epigenetic dysregulation induces a GATA2/GATA6 switch in human gastric cancer.

Six GATA transcription factors play important roles in eukaryotic development. Among these, GATA2, an essential factor for the hematopoietic cell lineage, exhibits low expression in human gastric tissues, whereas GATA6, which is crucial for gastrointestinal development and differentiation, is frequently amplified and/or overexpressed in human gastric cancer. Interestingly, we found that GATA6 was overexpressed in human gastric cancer cells only when GATA2 expression was completely absent, thereby showing an inverse correlation between GATA2 and GATA6. In gastric cancer cells that express high GATA6 levels, a GATA2 CpG island is hypermethylated, repressing expression in these cells. In contrast, GATA6 expression is undetectable in GATA2-overexpressing gastric cancer cells, which lack GATA2 DNA methylation. Furthermore, PRC2 complex-mediated transcriptional silencing of GATA6 was observed in the GATA2-overexpressing cells. We also show that the GATA2 and PRC2 complexes are enriched within the GATA6 locus, and that the recruitment of the PRC2 complex is impaired by disrupting GATA2 expression, resulting in GATA6 upregulation. In addition, ectopic GATA2 expression significantly downregulates GATA6 expression, suggesting GATA2 directly represses GATA6. Furthermore, GATA6 downregulation showed antitumor activity by inducing growth arrest. Finally, we show that aberrant GATA2 methylation occurs early during the multistep process of gastric carcinogenesis regardless of Helicobacter pylori infection. Taken together, GATA2 dysregulation by epigenetic modification is associated with unfavorable phenotypes in human gastric cancer cells by allowing GATA6 expression.

Epigenetic regulation of RNA polymerase III transcription in early breast tumorigenesis.

RNA polymerase III (Pol III) transcribes medium-sized non-coding RNAs (collectively termed Pol III genes). Emerging diverse roles of Pol III genes suggest that individual Pol III genes are exquisitely regulated by transcription and epigenetic factors. Here we report global Pol III expression/methylation profiles and molecular mechanisms of Pol III regulation that have not been as extensively studied, using nc886 as a representative Pol III gene. In a human mammary epithelial cell system that recapitulates early breast tumorigenesis, the fraction of actively transcribed Pol III genes increases reaching a plateau during immortalization. Hyper-methylation of Pol III genes inhibits Pol III binding to DNA via inducing repressed chromatin and is a determinant for the Pol III repertoire. When Pol III genes are hypo-methylated, MYC amplifies their transcription, regardless of its recognition DNA motif. Thus, Pol III expression during tumorigenesis is delineated by methylation and magnified by MYC.

Disruption of CTCF/cohesin-mediated high-order chromatin structures by DNA methylation downregulates PTGS2 expression.

The CCCTC-binding factor (CTCF)/cohesin complex regulates gene transcription via high-order chromatin organization of the genome. De novo methylation of CpG islands in the promoter region is an epigenetic hallmark of gene silencing in cancer. Although the CTCF/cohesin complex preferentially targets hypomethylated DNA, it remains unclear whether the CTCF/cohesin-mediated high-order chromatin structure is affected by DNA methylation during tumorigenesis. We found that DNA methylation downregulates the expression of prostaglandin-endoperoxide synthase 2 (PTGS2), which is an inducible, rate-limiting enzyme for prostaglandin synthesis, by disrupting CTCF/cohesin-mediated chromatin looping. We show that the CTCF/cohesin complex is enriched near a CpG island associated with PTGS2 and that the PTGS2 locus forms chromatin loops through methylation-sensitive binding of the CTCF/cohesin complex. DNA methylation abolishes the association of the CTCF/cohesin complex with the PTGS2 CpG island. Disruption of chromatin looping by DNA methylation abrogates the enrichment of transcriptional components, such as positive elongation factor b, at the transcriptional start site of the PTGS2 locus. These alterations result in the downregulation of PTGS2. Our results provide evidence that CTCF/cohesin-mediated chromatin looping of the PTGS2 locus is dynamically influenced by the DNA methylation status.

Novel fusion transcripts in human gastric cancer revealed by transcriptome analysis.

Gene fusion is involved in the development of various types of malignancies. Recent advances in sequencing technology have facilitated identification of gene fusions and have stimulated the research of this field in cancer. In the present study, we performed next-generation transcriptome sequencing in order to discover novel gene fusions in gastric cancer. A total of 282 fusion transcript candidates were detected from 12 gastric cancer cell lines by bioinformatic filtering. Among the candidates, we have validated 19 fusion transcripts, which are 7 inter-chromosomal and 12 intra-chromosomal fusions. A novel DUS4L-BCAP29 fusion transcript was found in 2 out of 12 cell lines and 10 out of 13 gastric cancer tissues. Knockdown of DUS4L-BCAP29 transcript using siRNA inhibited cell proliferation. Soft agar assay further confirmed that this novel fusion transcript has tumorigenic potential. We also identified that microRNA-coding gene PVT1, which is amplified in double minute chromosomes in SNU-16 cells, is recurrently involved in gene fusion. PVT1 produced six different fusion transcripts involving four different genes as fusion partners. Our findings provide better insight into transcriptional and genetic alterations of gastric cancer: namely, the tumorigenic effects of transcriptional read-through and a candidate region for genetic instability.

Testican-1-mediated epithelial-mesenchymal transition signaling confers acquired resistance to lapatinib in HER2-positive gastric cancer.

Human epidermal growth factor receptor 2 (HER2)-directed treatment using trastuzumab has shown clinical benefit in HER2-positive gastric cancer. Clinical trials using lapatinib in HER2-positive gastric cancer are also currently underway. As with other molecularly targeted agents, the emergence of acquired resistance to HER2-directed treatment is an imminent therapeutic problem for HER2-positive gastric cancer. In order to investigate the mechanisms of acquired resistance to HER2-directed treatment in gastric cancer, we generated lapatinib-resistant gastric cancer cell lines (SNU216 LR) in vitro by chronic exposure of a HER2-positive gastric cancer cell line (SNU216) to lapatinib. The resultant SNU216 LR cells were also resistant to gefitinib, cetuximab, trastuzumab, afatinib and dacomitinib. Interestingly, SNU216 LR cells displayed an epithelial-mesenchymal transition (EMT) phenotype and maintained the activation of MET, HER3, Stat3, Akt and mitogen-activated protein kinase signaling in the presence of lapatinib. Using gene expression arrays, we identified the upregulation of a variety of EMT-related genes and extracellular matrix molecules, such as Testican-1, in SNU216 LR cells. We showed that the inhibition of Testican-1 by small interfering RNA decreased Testican-1-induced, MET-dependent, downstream signaling, and restored sensitivity to lapatinib in these cells. Furthermore, treatment with XAV939 selectively inhibited ß-catenin-mediated transcription and Testican-1-induced EMT signaling, leading to G1 arrest. Taken together, these data support the potential role of EMT in acquired resistance to HER2-directed treatment in HER2-positive gastric cancer, and provide insights into strategies for preventing and/or overcoming this resistance in patients.

Suppression of cyclooxygenase-2 expression of skin fibroblasts by wogonin, a plant flavone from Scutellaria radix.

Previously, wogonin (5,7-dihydroxy-8-methoxyflavone) was found to suppress proinflammatory enzyme expression including cyclooxygenase-2 (COX-2), contributing to in vivo anti-inflammatory activity against skin inflammation. However, the detailed effect on each skin cell type has not been understood. Therefore, present investigation was carried out to find the effect of wogonin on inflammation associated gene expression from skin fibroblasts in culture using reverse transcriptase-polymerase chain reaction. As a result, it was found that wogonin (10-100 microM) clearly down-regulated COX-2 expression from NIH/3T3 cells treated with 12-O-tetradecanoylphorbol 13-acetate, interleukin-1beta or tumor necrosis factor-alpha. But, the expression levels of COX-1, interleukin-1beta and fibronectin were not significantly affected. This finding was well correlated with significant reduction of prostaglandin E2 (PGE2) production by wogonin. As a comparison, NS-398 (selective cyclooxygenase 2 inhibitor) did not suppress COX-2 expression and other gene levels, while PGE2 production was potently reduced at 0.1-10 microM. All these results suggest that COX-2 down-regulation of skin fibroblasts may be, at least in part, one of anti-inflammatory mechanisms of wogonin against skin inflammation.

Protection against ultraviolet B- and C-induced DNA damage and skin carcinogenesis by the flowers of Prunus persica extract.

The ethanol extract of the flowers of Prunus persica (Ku-35) (50-200 microg/ml) was found to inhibit UVB- as well as UVC-induced DNA damage measured by the COMET assay in the skin fibroblast cell (NIH/3T3). In addition, Ku-35 inhibited UVB- or UVC-induced lipid peroxidation, especially against UVB-induced peroxidation at higher than 10 microg/ml. We also evaluated the protective effect of Ku-35 against UVB-induced non-melanoma skin cancer in mice. Ku-35 was applied topically before UVB exposure, and its effects on tumor incidence (% of mice with tumors) and tumor multiplicity (number of tumors per mouse) were evaluated. The application of Ku-35 clearly resulted in a delay of tumor development compared to the control. In tumor incidence, 100% mice in the control group and the low dose treatment of Ku-35 had tumors, whereas 94.1% of the mice had tumors after the high dose treatment of Ku-35 at the end of experiment (28 weeks). In tumor multiplicity, low and high treatments of Ku-35 resulted in 25.9 and 53.9% reduction at the end of the experiment (P<0.05, one-way analysis of variance (ANOVA)). The present data indicate that Ku-35 protects against photogenotoxicity in NIH/3T3 fibroblasts. The possible action mechanism of Ku-35 may be through its anti-oxidant activity without pro-oxidant effect. Ku-35 can also show a delay of tumor development against UVB-induced skin carcinogenesis. These results suggest that Ku-35 extract may be useful for protecting UV-induced DNA damage and carcinogenesis when topically applied.

The basis for IL-2-induced IL-2 receptor alpha chain gene regulation: importance of two widely separated IL-2 response elements.

The interleukin-2 receptor alpha (IL-2Ralpha) chain is an essential component of high-affinity IL-2 receptors. Accordingly, IL-2Ralpha expression helps to regulate T cell growth and other lymphoid functions. Lineage-restricted and activation-dependent IL-2Ralpha transcription is controlled by three upstream positive regulatory regions (PRRs). We now describe an additional IL-2 response element, PRRIV, within intron 1, in humans and mice. PRRIV activity requires GAS motifs that bind Stat5 proteins and additional upstream HMG-I(Y) binding sites. Moreover, IL-2 induces the binding of HMG-I(Y), Stat5a, and Stat5b in vivo to PRRIV and PRRIII, which also functions as an IL-2 response element. Thus, the IL-2 inducibility of the IL-2Ralpha gene is unexpectedly mediated by two widely separated regulatory Stat5-dependent elements, located both upstream and downstream of the transcription initiation sites.

Cyclooxygenase-2 inhibitory cerebrosides from phytolaccae radix.

A mixture of cerebrosides, called poke-weed cerebrosides, was purified from Phytolaccae Radix (Phytolaccaceae) and characterized as 1-O-beta-D-glucopyranosides of phytosphingosine type ceramides comprised of a common long chain base (2S,3S,4R,8Z)-2-amino-8-octadecene-1,3,4-triol and fatty acids. The fatty acyl chain of ceramide moieties was determined as (2R)-2-hydroxypentacosanoic acid, (2R)-2-hydroxylignoceric acid, (2R)-2-hydroxytricosanoic acid, (2R)-2-hydroxybehenic acid, (2R)-2-hydroxypalmitic acid, and palmitic acid. The pokeweed cerebroside inhibited the cyclooxygenase-2 dependent phase of prostaglandin D2 generation in bone marrow-derived mast cells in a concentration dependent manner with an IC50 of 6.2 microg/ml.

Effects of prenylated flavonoids and biflavonoids on lipopolysaccharide-induced nitric oxide production from the mouse macrophage cell line RAW 264.7.

Certain flavonoid derivatives possess anti-inflammatory activity in vitro and in vivo. Besides their antioxidative properties and effects on the arachidonic acid metabolism including cyclooxygenase/lipoxygenase inhibition, some flavones and flavonols were previously found to show inhibitory activity on nitric oxide production by inducible nitric oxide synthase (iNOS; NOS type 2) through suppression of iNOS induction. As part of our continuing investigations, the effects of unique and minor flavonoids (prenylated flavonoids and biflavonoids) on nitric oxide production from lipopolysaccharide-induced macrophage cell line (RAW 264.7) were evaluated in order to establish their inhibitory activity on NO production and correlate this action with their in vivo anti-inflammatory potential. Among the derivatives tested, prenylated compounds including morusin, kuwanon C, and sanggenon D and biflavonoids such as bilobetin and ginkgetin were found to inhibit NO production from lipopolysaccharide (LPS)-induced RAW 264.7 cells at > 10 microM. Inhibition of nitric oxide production was mediated by suppression of iNOS enzyme induction but not by direct inhibition of iNOS enzyme activity. An exception was echinoisoflavanone that inhibited iNOS enzyme activity (IC50 = 83 microM) and suppressed iNOS enzyme induction as well. While most prenylated derivatives showed cytotoxicity to RAW cells at 10-100 microM, all biflavonoids tested were not cytotoxic. Since nitric oxide (NO) produced by inducible NO synthase (iNOS) plays an important role in inflammatory disorders, inhibition of NO production by these flavonoids may contribute, at least in part, to their anti-inflammatory and immunoregulating potential in vivo.

Transcriptional activation of the human manganese superoxide dismutase gene mediated by tetradecanoylphorbol acetate.

Transcriptional activation of human manganese superoxide dismutase (MnSOD) mRNA induced by a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was examined to identify the responsive transcriptional regulator. The effect of various deletions and mutations within the 5'-flanking region of the human MnSOD gene promoter was evaluated using the luciferase reporter system in A549 human lung carcinoma cells. Deletion of a region between -1292 and -1202 nucleotides upstream of the transcription start site abolished TPA-responsive induction, whereas deletion of the putative binding sequence for NF-kappaB or AP-1 did not. The region between -1292 and -1202 contains a cAMP-responsive element-like sequence, TGACGTCT, which we identified as the manganese superoxide dismutase TPA-responsive element, MSTRE. Site-specific mutation of the MSTRE abolished the TPA-responsive induction, validating the critical role of this sequence. We detected specific MSTRE activity from nuclear extracts and demonstrated by antibody supershift assay that this activity is closely related to CREB-1/ATF-1. TPA treatment rapidly induced phosphorylation of the CREB-1/ATF-1-like factor via the protein kinase C pathway. These results led us to conclude that the human MnSOD gene having the promoter construct used in this study is induced by TPA via activation of a CREB-1/ATF-1-like factor and not via either NF-kappaB or AP-1. In addition, we found that this induction was blocked by inhibitors of flavoproteins and NADPH oxidases, indicating involvement of enhanced generation of superoxide radical anion as an upstream signal.

A novel cerebroside from lycii fructus preserves the hepatic glutathione redox system in primary cultures of rat hepatocytes.

We previously reported the isolation of a novel cerebroside (1-O-(beta-D-glucopyranosyl)-(2S,3R,4E,8Z)-2-N-palmityloc tadecasphinga-4,8-diene; LCC) from the fruits of Lycium chinense MILL. (Solanaceae) which protected primary cultured rat hepatocytes from the toxicity induced by carbon tetrachloride (CCl4). The present study was conducted to determine the mechanism(s) by which LCC might exert its hepatoprotective activity. To determine the effect of LCC on the glutathione (GSH) redox system, we measured the activities of enzymes involved in the system as well as the levels of hepatic mitochondrial GSH and malondialdehyde (MDA). The hepatotoxicant, CCl4, routinely decreased levels of total and reduced GSH. The levels of these compounds were significantly maintained at the levels of the control cultures following treatment with LCC. The decreased activities of glutathione reductase and glutathione peroxidase in CCl4-injured rat hepatocytes were significantly increased by the treatment of LCC. Furthermore, the elevated levels of MDA seen in CCl4-injured rat hepatocytes were reduced after treatment with LCC in a concentration dependent manner over a range of 1-10 microM. From these results, we postulate that LCC may preserve the hepatic mitochondrial level of GSH by scavenging reactive oxygen species produced during CCl4-induced toxicity and thereby reduce lipid peroxidation and cellular damage.

Nongenomic stimulation of nitric oxide release by estrogen is mediated by estrogen receptor alpha localized in caveolae.

Acute administration of 17beta-estradiol (E(2)) exerts antiatherosclerotic effects in healthy postmenopausal women. The vasoprotective action of E(2) may be partly accounted for by a rapid increase in nitric oxide (NO) levels in endothelial cells (ECs). However, the signaling mechanisms producing this rise are unknown. In an attempt to address the short-term effect of E(2) on endothelial NO production, confluent bovine aortic endothelial cells (BAECs) were incubated in the absence or presence of E(2), and NO production was measured. Significant increases in NO levels were detected after only 5 min of E(2) exposure without a change in the protein levels of endothelial NO synthase (eNOS). This short-term effect of estrogen was significantly blunted by various ligands which decrease intracellular Ca(2+) concentration. Furthermore, plasma membrane-impermeable BSA-conjugated E(2) (E(2)BSA) stimulated endothelial NO release, indicating that in the current system the site of action of E(2) is on the plasma membrane rather than the classical nuclear receptor. The partial antagonist tamoxifen did not block E(2)-induced NO production; however, a pure estrogen receptor alpha (ERalpha) antagonist ICI 182,780 completely inhibited E(2)-stimulated NO release. The binding of E(2) to the membrane was confirmed using FITC-labeled E(2)BSA (E(2)BSA-FITC). Western blot analysis showed that plasmalemmal caveolae possess ERalpha in addition to well-known caveolae-associated proteins eNOS and caveolin. This study demonstrates that the nongenomic and short-term effect of E(2) on endothelial NO release is Ca(2+)-dependent and occurs via ERalpha localized in plasmalemmal caveolae.

Effects of naturally occurring flavonoids on nitric oxide production in the macrophage cell line RAW 264.7 and their structure-activity relationships.

Flavonoids affect the inflammatory process of the mammalian system and possess anti-inflammatory as well as immunomodulatory activities in vitro and in vivo. Since nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is one of the inflammatory mediators, the effects of various naturally occurring flavonoids on NO production in LPS-activated RAW 264.7 cells were evaluated in vitro. Flavonoids such as apigenin, wogonin, luteolin, tectorigenin, and quercetin inhibited NO production, as measured by nitrite formation at 10-100 microM. The most active among 26 flavonoid derivatives tested were apigenin, wogonin, and luteolin, having IC50 values of 23, 17, and 27 microM, respectively, while AMT, a synthetic selective iNOS inhibitor, had an IC50 value of 0.09 microM. In contrast, flavanones, such as naringenin, and flavonoid glycosides, such as apiin, did not demonstrate significant inhibition up to 100 microM. These results clearly indicated that a C-2,3 double bond might be important, and that the potency of inhibition depended upon the substitution patterns of the flavonoid molecules. The inhibitory activity of flavonoids was not due to direct inhibition of iNOS enzyme activity because they did not reasonably inhibit iNOS activity, as measured by [3H]citrulline formation from [3H]arginine, up to 100 microM. In contrast, wogonin and luteolin concentration-dependently reduced iNOS enzyme expression, when measured by western blotting, at 10-100 microM. All these results clearly demonstrated that certain flavonoids inhibit NO production in lipopolysaccharide-activated RAW 264.7 cells, and their inhibitory activity might be due to reduction of iNOS enzyme expression.

Cell death and cytoskeletal alterations in cultured hepatic fat-storing cells induced by 6-hydroxydopamine.

We studied the effects of the neurotoxin, 6-hydroxydopamine (6-OHDA), on cultured fat-storing cells (FSCs) and hepatocytes. If either FSCs or hepatocytes were exposed to 6-OHDA for 4 hr, the neurotoxicant induced cell death in FSCs but not in hepatocytes. We decided to investigate why hepatocytes were refractile to injury from 6-OHDA while FSCs were labile. The activity of antioxidant enzymes within FSCs grown in vitro is remarkably lower than the activity in hepatocytes. Indeed, some specific antioxidant enzymes in FSCs were undetectable by our assays, but were easily detected in hepatocytes. Furthermore, the profile of antioxidant activity in FSCs was found to be almost identical to the profiles seen in cultured fibroblasts or myocytes. However, indirect immunolocalization of tyrosine hydroxylase in FSCs using anti-tyrosine hydroxylase antibodies was negative. Mazindol, a dopaminergic receptor antagonist, did not alleviate the toxicity of 6-OHDA suggesting that FSCs do not appear to possess a dopaminergic receptor. When the cell morphology of FSCs was examined by an indirect immunofluorescence technique, treatment of FSCs with 6-OHDA at a concentration of 200 microM for 2 hr modified the organization of alpha-smooth muscle actin into an irregular punctate pattern. Indeed, we found that the effects of 6-OHDA on cytoskeletal alterations and on the cell viability of FSCs were irreversible. These data suggest that : (1) 6-OHDA can cause irreparable injury to FSCs, but not hepatocytes; (2) hepatocytes are specially adapted to withstand an oxidative attack in contrast to FSCs. fibroblast and myocytes; (3) FSCs resemble other somatic cells in their low levels of antioxidant enzymes; and (4) this low profile of antioxidant activity may be responsible for the cell death and cytoskeletal alterations observed in FSCs in response to 6-OHDA.

Vitexicarpin, a flavonoid from the fruits of Vitex rotundifolia, inhibits mouse lymphocyte proliferation and growth of cell lines in vitro.

Certain flavonoids having a C-2,3-double bond were reported to show an inhibitory activity against T-lymphocyte proliferation, but not against B-lymphocyte proliferation in vitro. In the course of these studies, vitexicarpin (3',5-dihydroxy-3,4',6,7-tetramethoxyflavone) isolated from the fruits of Vitex rotundifolia was found to show potent inhibition against lymphocyte proliferation. Vitexicarpin inhibited T-lymphocyte proliferation as well as B-lymphocyte proliferation at > 0.1 microM. IC50's were approximately 0.7 microM both for T- and B-cell proliferation. The inhibitory activity of vitexicarpin was reversible. Vitexicarpin also inhibited the growth of certain cancer cell lines, EL-4 and P815.9 (IC50 = 0.25-0.3 microM). These results suggest that vitexicarpin may be a potential therapeutic agent involved in inflammatory/immunoregulatory disorders such as rheumatoid arthritis and lymphomas.

Zeaxanthin dipalmitate from Lycium chinense has hepatoprotective activity.

We previously reported the isolation of zeaxanthin and zeaxanthin dipalmitate using bioactivity-guided fractionation to discover hepatoprotective components of Lycium chinense against carbon tetrachloride induced hepatotoxicity. The present study was designed to uncover the effects of zeaxanthin dipalmitate on hepatic parenchymal and nonparenchymal cells in vitro. Uptake of [3H]thymidine by cultured rat Ito cells in response to zeaxanthin dipalmitate was measured. Collagen synthesis was assessed by the collagenase digestion method. The effects of zeaxanthin dipalmitate on the formation of nitric oxide (NO) and the release of tumor necrosis factor-alpha (TNF-alpha) from Kupffer cells and peritoneal macrophages were also assayed. Zeaxanthin dipalmitate showed a significant hepatoprotective activity against carbon tetrachloride toxicity. Cellular malondialdehyde (MDA) levels declined significantly with the treatment of the compound in a concentration dependent manner. Zeaxanthin dipalmitate significantly inhibited the uptake of [3H]thymidine by Ito cells. Zeaxanthin dipalmitate also reduced collagen synthesis in Ito cells by 65.1% (p < 0.05) as compared to untreated controls. The formation of NO in either Kupffer cells or in peritoneal macrophages was significantly decreased by zeaxanthin dipalmitate in a concentration dependent manner. The release of TNF-alpha was somewhat less affected by the compound. From these results, we conclude that zeaxanthin dipalmitate exerts a potent hepatoprotective activity by inhibiting Ito cell proliferation, collagen synthesis and by inhibiting certain biochemical functions of Kupffer cells.

Anticlastogenic effect of flavonoids against mutagen-induced micronuclei in mice.

14 flavonoids, including flavone and flavonol derivatives, were tested for their anticlastogenic effect against induction of micronuclei by benzo[a]pyrene in polychromatic erythrocytes of mice. When each flavonoid was administered orally, together with intraperitoneally administered benzo[a]pyrene, most flavonol derivatives showed an anticlastogenic effect. The data suggest that the 2,3-double bond and 3,5,7-hydroxyl groups in the flavonoid molecules may be essential to produce anticlastogenic effects against benzo[a]pyrene. Galangin, one of the active compounds, and (-)-epicatechin, a weak one, were administered to mice in order to compare their anticlastogenic effect against 3 different kinds of carcinogens: ethyl methanesulfonate, 7,12-dimethylbenz[a]anthracene, and adriamycin. Galangin showed a stronger anticlastogenic effect than (-)-epicatechin against ethyl methanesulfonate and 7,12-dimethylbenz[a]anthracene. However, there was no significant effect against adriamycin-induced micronuclei by both compounds. Our study indicates that most flavonoids are anticlastogenic agents. Their anticlastogenic effects are apparently independent of their own clastogenic activities. Furthermore, their anticlastogenic activities do not apply universally to all types of genotoxic chemicals.

Pharmacological activities of water extracts of Umbelliferae plants.

In order to evaluate the pharmacological activities of Chinese medicine, nine Umbelliferae plants were selected and their restoring activity against dexamethasone-induced disorders, liver protective activity, antimicrobial activity, anti-inflammatory activity and antimutagenic activity were tested and compared. Angelica dahurica. Angelica acutiloba and Ostericum koreanum showed various activities in these tests at the dose used in this study.

Synthesis and pharmacological evaluation of new topical anti-inflammatory steroids.

The purpose of this research was to develop new topical steroid derivatives showing reduced systemic effects. Pregna-16 alpha,17-carboxycyclic acetal derivatives have been recently synthesized by reacting triamcinolone with methyl acetylalkanoate in the presence of a catalytic amount of perchloric acid. In testing for the anti-inflammatory activity of the compounds, rat cotton-pellet granuloma inhibition bioassay and mouse croton-oil-induced ear oedema inhibition bioassay were employed. One of the synthesized compounds, (22R)-9 alpha-fluoro-11 beta,21-dihydroxy-3,20-dioxo-16 alpha, 17-(methyl, methoxycarbonylmethyl)methylenedioxy-1,4-pregnadiene (I), showed more or less the same activity as shown by prednisolone in the granuloma inhibition test. However, compound I showed higher activity in the ear oedema inhibition test when applied topically (ID50 = 0.002 mg), as compared to prednisolone (ID50 = 0.006 mg) and triamcinolone (ID50 = 0.026 mg). When compound I was applied to mice, and thymus involution was measured for judging systemic effects, it was found that compound I did not show any significant thymus involution up to 0.1 mg/mouse (systemic administration) and 0.5 mg/mouse (topical administration). Because of its significantly reduced systemic effects, this compound is a promising topical anti-inflammatory steroid.