Flavonoids from the cultured cells of Glycyrrhiza echinata

Constituents of the cultured cells of Glycyrrhiza echinata have been investigated. Echinatin (4,4′-dihydroxy-2-methoxychalcone), a biosynthetically unique retrochalcone, and licodione (1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)-1,3-propanedione), a dibezoylmethane derivative, which is the possible precursor of echinatin, were obtained. The structures were determined by spectroscopic methods and syntheses. ¹H NMR of licodione revealed new features in chemical shifts of protons of diketonic and keto—enolic forms. 7,4′-Dihydroxyflavone, two of its prenyl derivatives and formononetin were also isolated. A discussion on retrochalcone biosynthesis is presented.     

Induction of isoflavonoid and retrochalcone branches of the flavonoid pathway in cultured Glycyrrhiza echinata cells treated with yeast extract.

Yeast extract-treated suspension cultures of a new cell line, AK-1, of Glycyrrhiza echinata were induced to produce an isoflavonoid phytoalexin (medicarpin) and metabolites of retrochalcone/flavone pathway (echinatin, licodione, and 7,4'-dihydroxyflavone). From these cells, putative full-length cDNAs encoding cytochrome P450s, (2S)-flavanone 2-hydroxylase and isoflavone 2'-hydroxylase, were cloned.     

Stimulation of chalcone synthase activity by yeast extract in cultured Glycyrrhiza echinata cells and 5-deoxyflavanone formation by isolated protoplasts

Chalcone synthase activity catalyzing the formation of naringenin (5-hydroxyflavanone) was detected in cell suspension cultures of Glycyrrhiza echinata. This activity rapidly increased by treatment of the cells with yeast extract, while non-treated cells showed a constant low activity. Isolated G. echinata protoplasts accumulated retrochalcone (echinatin) and its biosynthetic intermediate (licodione) during 24 h of culture. When the protoplasts were incubated with [¹⁴C(U)]phenylalanine, liquiritigenin (5-deoxyflavanone) was transiently labeled, indicating the induction of 6'-deoxychalcone synthase. The formation of liquiritigenin, in addition to naringenin, was observed when the crude extracts from the protoplasts were assaved for CHS activity.Abbreviations CHS chalcone synthase - YE yeast extract This paper is Part 52 in the series Studies on Plant Tissue Cultures. For Part 51, see Furuya T, Ushiyama M, Asada Y, Yoshikawa T, Orihara Y (1987) Phytochemistry: in press.     

Regulation of retrochalcone biosynthesis: Activity changes of O-methyltransferases in the yeast extract-induced Glycyrrhiza echinata cells

Three O-methyltransferases which catalyze S-adenosyl-L-methionine (SAM)-dependent O-methylation of licodione (LMT), flavone/flavonol (FMT), and caffeic acid (CMT) were separated from the callus culture of Glycyrrhiza echinata, and characteristic differences between their pH optima and Mg²⁺ requirement for activity were demonstrated. The activity of LMT, which is involved in retrochalcone (echinatin) biosynthesis, but not of FMT or CMT, was found to be stimulated when suspension-cultured G. echinata cells were treated with yeast extract (YE), which causes rapid production of echinatin in the cells. Cycloheximide suppressed both the YE-induced echinatin formation and LMT enhancement. The results indicate a selective induction of retrochalcone pathway in Glycyrrhiza cells in response to stress.Abbreviations SAM S-adenosyl-L-methionine - LMT, SAM licodione 2-O-methyltransferase - FMT, SAM flavone/flavonol O-methyltransferase - CMT, SAM caffeate 3-O-methyltransferase - OMT O-methyltransferase - CH cycloheximide - YE yeast extract This paper is Part 47 in the series Studies on Plant Tissue Cultures. For Part 46, see Ayabe S, Iida K, Furuya T (1986) Phytochemistry: in press     

Induction of Isoflavonoid and Retrochalcone Branches of the Flavonoid Pathway in Cultured Glycyrrhiza echinata Cells Treated with Yeast Extract

Yeast extract-treated suspension cultures of a new cell line, AK-1, of Glycyrrhiza echinata were induced to produce an isoflavonoid phytoalexin (medicarpin) and metabolites of retrochalcone/flavone pathway (echinatin, licodione, and 7,4′-dihydroxyflavone). From these cells, putative full-length cDNAs encoding cytochrome P450s,(2S)-flavanone 2-hydroxylase and isoflavone 2′-hydroxylase, were cloned.     

Induction of stress proteins by electromagnetic fields in cultured HL-60 cells.

HL-60 cells in culture were exposed for 2 h to a sinusoidal 0.1 or 1 mT (1 or 10 Gauss) magnetic field at 60 Hz and pulse labeled after exposure with radioactive isotopes by incubation by using either [(35)S]methionine, [(3)H]leucine, or [(33)P]phosphate. The radioactive labels were incorporated into cellular proteins through synthesis or phosphorylation. Proteins were extracted from electrostatically sorted nuclei, and the heat shock/stress proteins (sp) were analyzed for synthesis and phosphorylation by two-dimensional polyacrylamide gel electrophoresis. In the control cultures (no exposure to the magnetic field), sp 72c (cognate form) was faintly observed. A 0.1 mT exposure did not show sp metabolism to be different from that of the controls; however, after a 1 mT exposure of the HL-60 cells, sp 70i (inducible form) was synthesized ([(35)S]methionine incorporation). Sp 90 was not synthesized at either field level, but was phosphorylated ([(33)P]phosphate incorporation) in the 1 mT exposure. Sp 27 (isoforms a and b) was induced after a 1 mT exposure as reflected by labeling with [(3)H]leucine. These sps were not detected after a 0.1 mT exposure. After a 1 mT exposure and labeling with [(33)P], sp 27 isoforms b and c were phosphorylated whereas isoform 'a' was not observed. Sps 70i, 72c, and 90 were identified by commercial sp antibodies. Likewise, polypeptides a, b, and c were verified as sp 27 isoforms by Western blotting. Statistical evaluation of sp areas and densities, determined from fluorographs by Western-blot analysis, revealed a significant increase in sps 90 and 27a after a 1 mT magnetic field exposure. The 1 mT magnetic field interacts at the cellular level to induce a variety of sp species. Bioelectromagnetics 20:347-357, 1999. Published 1999 Wiley-Liss, Inc.     

NAD(P)H-dependent 6'-deoxychalcone synthase activity in Glycyrrhiza echinata cells induced by yeast extract

The crude extract prepared from Glycyrrhiza echinata cells treated with yeast extract catalyzed the formation of liquiritigenin (5-deoxyflavanone) and isoliquiritigenin (6'-deoxychalcone) in addition to naringenin (5-hydroxyflavanone) when incubated with 4-coumaroyl-CoA and malonyl-CoA in the presence of high concentrations (0.1 mM or higher) of NADPH. Incubation without NADPH, or with low concentrations (0.01 mM or lower), gave only naringenin as a reaction product. With NADH (1 mM), the major product was naringenin accompanied by a small quantity of liquiritigenin. The initial product of the assay with 1 mM NADPH was isoliquiritigenin, indicating a reaction catalyzed by 6'-deoxychalcone synthase (DOCS). Subsequent formation of liquiritigenin was attributed to the presence of chalcone isomerase in the crude extract. The results constitute the first demonstration in vitro of DOCS activity which, in G. echinata cells and other leguminous plants, is involved in the biosynthesis of retrochalcone and 5-deoxyisoflavonoid-derived phytoalexins.     

Chalcone isomerase isozymes with different substrate specificities towards 6'-hydroxy- and 6'-deoxychalcones in cultured cells of Glycyrrhiza echinata, a leguminous plant producing 5-deoxyflavonoids.

Three chalcone isomerase isozymes in Glycyrrhiza echinata (Fabaceae) were separated by chromatofocusing and partially purified to examine their substrate specificities. Two isozymes, one of which was elicitor-inducible, acted on both 6'-hydroxychalcone and 6'-deoxychalcone and presumably are involved in the legume-specific 5-deoxyflavonoid pathway, while another acted on only 6'-hydroxychalcone.     

Induction of gamma-glutamyltransferase in cultured brain cells

gamma-Glutamyltransferase is a membrane-bound enzyme widely distributed in animal tissues. This enzyme is involved in glutathione metabolism, but its exact biological function is still an open question. In rat brain cells in culture a depletion of L-cystine and L-glutamine or the addition of diethyl maleate to the culture medium leads to a decrease of intracellular glutathione concentration and to an increase of the specific activity of gamma-glutamyltransferase. The induction of the enzyme is inhibited by the addition of cycloheximide or actinomycin D to the culture medium.     

Up-regulation of soyasaponin biosynthesis by methyl jasmonate in cultured cells of Glycyrrhiza glabra

Exogenously applied methyl jasmonate (MeJA) stimulated soyasaponin biosynthesis in cultured cells of Glycyrrhiza glabra (common licorice). mRNA level and enzyme activity of beta-amyrin synthase (bAS), an oxidosqualene cyclase (OSC) situated at the branching point for oleanane-type triterpene saponin biosynthesis, were up-regulated by MeJA, whereas those of cycloartenol synthase, an OSC involved in sterol biosynthesis, were relatively constant. Two mRNAs of squalene synthase (SQS), an enzyme common to both triterpene and sterol biosyntheses, were also up-regulated by MeJA. In addition, enzyme activity of UDP-glucuronic acid: soyasapogenol B glucuronosyltransferase, an enzyme situated at a later step of soyasaponin biosynthesis, was also up-regulated by MeJA. Accumulations of bAS and two SQS mRNAs were not transient but lasted for 7 d after exposure to MeJA, resulting in the high-level accumulation (more than 2% of dry weight cells) of soyasaponins in cultured licorice cells. In contrast, bAS and SQS mRNAs were coordinately down-regulated by yeast extract, and mRNA accumulation of polyketide reductase, an enzyme involved in 5-deoxyflavonoid biosynthesis in cultured licorice cells, was induced transiently by yeast extract and MeJA, respectively.     

Induction of cold stability of microtubules in cultured tobacco cells

In suspension-cultured tobacco (Nicotiana tabacum) cells, we have often encountered cold-stable microtubules (MTs). The cold-stable MTs were found in the pelleted fraction of tobacco cell homogenates. These cold-stable MTs were shown to be accompanied by unidentified filamentous structures that extended along part of their length. However, during the early hours in culture such cold-stable MTs were never observed. They were detectable from 120 h after the beginning of subculture and then their numbers increased gradually. The number of cells with cold-stable MTs eventually accounted for more than 95% of the total population of cells at the stationary phase of culture. The rapid loss of cold stability of MTs occurred when such cells were transferred to fresh medium for subculture. However, if the fresh medium was supplemented with once-used medium, the cold stability of MTs was retained. The active agent in the medium appeared to be of low molecular weight and to be heat resistant. A similar activity was detected in a pectin hydrolyzate. When an inhibitor of protein kinase, either 6-dimethylaminopurine or staurosporin, was added to the cells at an early stage of culture, when cold-stable MTs were normally completely absent, most cells acquired cold-stable MTs. It appears that acquisition or loss of cold stability of MTs in tobacco cells is regulated by the action of a kinase/phosphatase or a phosphorylation/dephosphorylation system on some MT protein(s), such as a cold stabilizer of MTs, some unidentified MT-associated filamentous structure, or even tubulin itself.     

Induction of glycophorin gene expression in cultured murine erythroleukemia cells

In order to identify the mRNA for mouse glycophorin, mRNA was isolated from immature erythroid cells obtained from the spleens of anemic mice, translated in vitro in mRNA-dependent rabbit reticulocyte lysate, and then immunoprecipitated with a specific antiserum. Glycophorin mRNA was shown to be present only in erythroid cells. In immunofluorescent and in vitro translation studies, it was shown that glycophorin mRNA is absent in uninduced murine erythroleukemia (MEL) cells, but is induced in dimethylsulfoxide-treated differentiating cells.     

Cytotoxic and Antimigratory Activity of Retrochalcones from Glycyrrhiza echinata L. on Human Cancer Cells

Cytotoxic activity-guided fractionation studies on Glycyrrhiza echinata roots led to the isolation of eight compounds ( 1 – 8 ). Chemical structures of the isolates were identified by NMR and MS analysis. Among the tested molecules, retrochalcones namely echinatin ( 3 ) (IC₅₀=23.45–41.83 μM), licochalcone B ( 4 ) (IC₅₀=36.04–39.53 μM) and tetrahydroxylmethoxychalcone ( 5 ) (IC₅₀=7.09–80.81 μM) were the most active ones against PC3, MCF7 and HepG2 cells. Moreover, 5 exhibited selectivity on prostate cancer cells (SI: 5.19). Hoechst staining and Annexin V/PI binding assays as well as cell cycle analysis on the compounds 3 (23 μM) and 5 (5 and 7 μM) demonstrated that these retrochalcones induced apoptosis and significantly suppressed cell cycle in G₁ and G₂/M phases. Furthermore, 3 and 5 showed antimigratory effects on PC3 cells by wound healing assay. The results indicated that tested retrochalcones most particularly 5 could be potential anticancer drug candidates that prevent proliferation and migration of cancer cells.     

Effects of naphthalene metabolites on cultured cells from eye lens

Naphthalene is toxic to the eye and results in opacification of the lens. To investigate the metabolic events that may be occurring in the lens epithelial cells, a cell line of lens from a transgenic mouse was incubated with various metabolites of naphthalene. Naphthoquinone at 50 microM was toxic to most cells with a depletion of glutathione levels noted within 6 h of incubation. At 10 microM, naphthoquinone caused an increase in specific activity of the enzyme DT-diaphorase. This enzyme is thought to be a defense against quinones since semiquinone formation is thought to be lessened. Naphthalene-1,2-dihydrodiol at 50 microM also caused an increase in the specific activity of the DT-diaphorase, while at 10 microM no apparent change occurred in the cells. Although there was evidence of metabolic alterations in the cells with the metabolites of naphthalene, the protein profile by two-dimensional gel electrophoresis did not change and there was no indication of an increase in carbonyl formation in the soluble proteins of the cells. These experiments indicate that the metabolites of naphthalene can cause alteration in the metabolism of the lens cells but may not cause apparent changes in the major proteins within the lens epithelium.     

Metabolism of cytotoxic hydroxysterols in cultured cells. Chemical characterization of metabolites

The metabolism of labelled 7 alpha- and 7 beta-hydroxycholesterol was investigated in two lymphoma cell lines (YAC-1, RDM-4), in murine splenocytes and in HTC hepatoma cells. The structures of the metabolites in lymphoma cells were determined as 3 beta-esters of C14-C20 fatty acids by nuclear magnetic resonance and mass spectrometric studies. In hepatoma cells, more polar metabolites of 7 alpha- and 7 beta-hydroxycholesterol were detected whereas, in non-dividing lymphocyte cells, no metabolic transformation occurs. Therefore, metabolic transformation of the hydroxycholesterol is not required for the expression of their activity and the question of the physiological role of the metabolic products is raised.     

Induction of stilbene synthase by Botrytis cinerea in cultured grapevine cells

The interaction between Botrytis cinerea Pers. and grapevine (Vitis vinifera L.) was studied in a model system of reduced complexity. Cultured plant cells and fragments of fungal cell wall were used to simulate some of the processes taking place upon infection of grapevine with B. cinerea. A soluble glucan elicitor was prepared from the fungal cell wall by acid hydrolysis. Like the insoluble wall preparation, the soluble fragment derived from the cell wall acted upon plant cells in eliciting stilbene formation. In grapevine cells, the interaction with the fungus led to a dramatic shut-off general protein synthesis and to the selective formation of a small set of proteins involved in induced resistance. The proteins synthesized de novo with highest rates were stilbene synthase (StiSy) and l-phenylalanine ammonia-lyase (PAL). Stilbene synthase was purified to apparent homogeneity and its molecular properties were characterized. The enzyme is a homodimer with subunit M_r 43 000 and pl = 5.4. Although there were indications of the presence of isoenzymes, these were not distinguished by charge differences. In size, the grapevine StiSy shows microheterogeneity and differs from the appreciably larger enzyme prepared from peanut. Prior to induction by fungal attack, virtually no stilbenes are formed in the plant cell. Upon induction of the pathway leading to the stilbene resveratrol, StiSy activity determines the ratelimiting step in the metabolic sequence. The highly induced grapevine cells produce and secrete resveratrol and derivatives which are known to be fungistatic.Abbreviations PAL l-phenylalanine ammonia-lyase - SDS-PAGE sodium dodecyl sulfate-polyacrylamine gel electrophoresis - StiSy stilbene synthase (resveratrol forming) The authors thank Dr. Blaich, Bundesforschungsanstalt Geilweilerhof, Siebeldingen, F.R.G., for provision of callus culture. This paper is based on research supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.     

Induction of metallothionein synthesis in cultured cells derived from rabbit kidney

Metallothionein (MT) synthesis in rabbit kidney-derived RK-13 cells was studied. In response to Cd2+, RK-13 cells synthesized proteins closely similar in chromatographic and electrophoretic behaviors to the liver MTs induced in Cd2+-injected rabbit. These proteins were specifically immunoprecipitated by anti-mouse liver MT-II serum. The rate of RK-13 thionein (apoprotein of MT) synthesis rapidly increased after exposure to 1 microgram/ml of Cd2+, and reached the maximum in 7 h. The dose-response curve for the synthesis was biphasic; a sharp increase up to 0.5 microgram/ml and a slower increase at higher concentrations. RK-13 cells retained kidney-specific properties in terms of responsiveness of thionein synthesis to inducers; The MTs were inducible also by Zn2+ and probably by Hg2+, but not by dexamethasone. This system would therefore be a useful model in vitro for studying the regulation of MT synthesis in kidney cells.