Inhibition of Myb-dependent gene expression by the sesquiterpene lactone mexicanin-I.

The c-myb proto-oncogene encodes a transcription factor that is highly expressed in the progenitor cells of the hematopoietic system, where it regulates the expression of genes important for lineage determination, cell proliferation and differentiation. There is strong evidence that deregulation of c-myb expression is involved in the development of human tumors, particularly of certain types of leukemia, and breast and colon cancer. The c-Myb protein is therefore an interesting therapeutic target. Here, we have investigated the potential of natural sesquiterpene lactones (STLs), a class of compounds that are active constituents of a variety of medicinal plants, to suppress Myb-dependent gene expression. We have developed a test system that allows screening of compounds for their ability to interfere with the activation of Myb target genes. Using this assay system, we have identified the STL mexicanin-I as the first cell-permeable, low-molecular-weight inhibitor of Myb-induced gene expression.

Hypoglycaemic constituents of Stachytarpheta cayennensis leaf.

The aqueous infusion (tea) of Stachytarpheta cayennensis leaves is used ethnomedically in Peru, Nigeria and other tropical countries for the management of diabetes. Oral administration (p. o.) of aqueous (125 mg/kg) and methanolic (2000 mg/kg) extracts of the leaves to alloxan-diabetic rats showed significant blood glucose reductions by 43 and 53%, respectively, at the end of a 4 hour period similar to the strong effect of glibenclamide (5 mg/kg, P. O.). The methanolic extract was successively partitioned into ethyl acetate, butanol and water fractions, and the same test showed that the butanol fraction (2000 mg/kg) had the highest (50%) hypoglycaemic activity at 4 hours after oral administration. It was also the most active fraction when tested in vitro [insulin release from an insulin secreting cell line (INS-1)] and was also active in normal rats and rats made hyperglycaemic by a glucose load. Its activity was comparable to that of glibenclamide (positive control) in these models. This active butanol fraction was subjected to chromatographic subfractionation; some subfractions reduced hyperglycaemia in alloxan-diabetic rats to 60 and 78% and induced insulin release from the INS-1 cells; other subfractions, however, gave hyperglycaemic activities IN VIVO and inhibition of insulin release from the INS-1 cells. Three major compounds of the butanol fraction were isolated and characterised as 6beta-hydroxyipolamide, ipolamide and isoverbascoside; they increased insulin secretion from INS-1 cells to 125, 128 and 127%, respectively, whereas glibenclamide increased insulin secretion to 157%. The results justify the ethnomedical use of the plant in the management of diabetes and suggest that the butanol fraction and some of its isolated constituents mediate their actions primarily by stimulating insulin release directly.

Inhibitory effects of helenalin and related compounds on 5-lipoxygenase and leukotriene C(4) synthase in human blood cells.

The sesquiterpene lactone helenalin, which can be isolated from several plant species of the Asteraceae family, is a potent anti-inflammatory and antineoplastic agent. In agreement, alcohol extracts of these plants are used for local external treatment of inflammatory conditions. Since leukotrienes are important mediators in inflammatory processes, the inhibitory effects of helenalin and some derivatives on leukotriene (LT) biosynthesis were studied. Treatment of human platelets with helenalin provoked irreversible inhibition of LTC(4) synthase in a concentration- and time-dependent manner with an IC(50) of 12 microM after a 60 min preincubation. 11alpha,13-Dihydrohelenalin acetate was less potent. Interestingly, individual donors could be divided into two distinct groups with respect to the efficacy of helenalin to suppress platelet LTC(4) synthase. In human granulocytes, helenalin inhibited both the 5-lipoxygenase (IC(50) 9 microM after 60 min preincubation) and LTC(4) synthase in a concentration- and time-dependent fashion. In contrast, the drug was without effect on LTA(4) hydrolase. The GSH-containing adducts (2beta-(S-glutathionyl)-2,3-dihydrohelenalin and 2beta-(S-glutathionyl)-2,3,11alpha,13-tetra hydrohelenalin acetate) did not significantly inhibit LTC(4) synthase. The present results indicate a mechanism for the anti-inflammatory effect of helenalin and related compounds.

The influence of glutathione and cysteine levels on the cytotoxicity of helenanolide type sesquiterpene lactones against KB cells.

The biological activities of sesquiterpene lactones have been attributed to their reactivity with the cysteine residues of functional proteins forming covalent bonds via Michael type addition. In the present study we investigated the influence of different L-cysteine (cys) and glutathione (GSH) concentrations on the cytotoxicity of the sesquiterpene lactones (STLs) helenalin, 11alpha,13-dihydrohelenalin acetate and chamissonolide against KB cells. Due to the significantly higher reactivity of the alpha-methylene-gamma-lactone (ML) towards cys as compared with the cyclopentenone (CP) site at physiological pH, addition of 20, 50 and 100 molar equivalents of cys decreased the cytotoxicity of helenalin and chamissonolide, whereas the cytotoxicity of 11alpha,13-dihydrohelenalin acetate remained unaffected. In contrast, the influence of GSH addition on the cytotoxicity of 11alpha,13-dihydrohelenalin acetate depends on the concentration of GSH added. Concentration-effect curves obtained for chamissonolide and GSH resembled the decline in cytotoxicity after cys addition. Helenalin showed a biphasic shape of the concentration-effect curve for the 100:1 GSH/helenalin ratio resembling at higher doses the chamissonolide and in lower doses the 11alpha,13-dihydrohelenalin acetate curve at 50-fold excess. These results can be explained by the different reactivity and equilibrium conditions for thiol addition of the two reactive centers of bifunctional STLs in cellular test systems and verified a clear correlation between the different reactivity of their electrophilic centers and the observed biological effects in in-vitro cell systems.

Cysteine 38 in p65/NF-kappaB plays a crucial role in DNA binding inhibition by sesquiterpene lactones.

Sesquiterpene lactones (SLs) have potent anti-inflammatory properties. We have shown previously that they exert this effect in part by inhibiting activation of the transcription factor NF-kappaB, a central regulator of the immune response. We have proposed a molecular mechanism for this inhibition based on computer molecular modeling data. In this model, SLs directly alkylate the p65 subunit of NF-kappaB, thereby inhibiting DNA binding. Nevertheless, an experimental evidence for the proposed mechanism was lacking. Moreover, based on experiments using the SL parthenolide, an alternative mode of action has been proposed by other authors in which SLs inhibit IkappaB-alpha degradation. Here we report the construction of p65/NF-kappaB point mutants that lack the cysteine residues alkylated by SLs in our model. In contrast to wild type p65, DNA-binding of the Cys(38) --> Ser and Cys(38,120) --> Ser mutants is no longer inhibited by SLs. In addition, we provide evidence that parthenolide uses a similar mechanism to other SLs in inhibiting NF-kappaB. Contrary to previous reports, we show that parthenolide, like other SLs, inhibits NF-kappaB most probably by alkylating p65 at Cys(38). Although a slight inhibition of IkappaB degradation was detected for all SLs, the amount of remaining IkappaB was too low to explain the observed NF-kappaB inhibition.

Glutathione adducts of helenalin and 11 alpha,13-dihydrohelenalin acetate inhibit glutathione S-transferase from horse liver.

The 2-mono- and 2,13-bis-glutathionyl adducts of helenalin and the 2-monoglutathionyl adduct of 11 alpha,13-dihydrohelenalin acetate were previously shown to be formed by spontaneous Michael addition at physiological pH. In living cells, glutathione (GSH) conjugation of many types of electrophilic agents is catalysed by a family of GSH S-transferase enzymes (GST). The capability of a glutathione S-transferase from horse liver to catalyze the reaction of helenalin and other helenanolides with GSH was investigated. The enzyme did not accelerate GSH conjugation of helenalin, 11 alpha,13-dihydrohelenalin, or 2-deacetyl-6-deoxychamissonolide. The GSH-adducts, formed by spontaneous reaction, were found to be inhibitors of this enzyme. Free helenalin, a potent inhibitor of many enzymes containing free sulfhydryl groups, did not show any inhibitory activity on GST. It was thus demonstrated that GSH-adducts of sesquiterpene lactones possess their own specific biological activity. Two further enzymes using GSH as substrate, glutathione reductase and glyoxalase I, were not influenced by free helenalin or its GSH-adducts.

Functional and energetic consequences of chronic myocardial creatine depletion by beta-guanidinopropionate in perfused hearts and in intact rats.

Oral feeding with the creatine analogue beta-guanidinopropionate (beta-GP) reduces myocardial phosphocreatine and creatine concentrations by about 80%in vitro, this is accompanied by reduced contractile performance. We hypothesized, thus, that beta-GP feeding leads to hemodynamic changes in vivo characteristic of heart failure. beta-GP was fed to Wistar rats for up to 8 weeks. In isolated hearts, function was measured isovolumically, myocardial energetics were followed with (31)P-NMR spectroscopy. In vivo hemodynamics were measured with Millar-Tip-catheters and an electromagnetic flow probe. Beta-GP feeding did not alter heart weight. In vitro, diastolic pressure-volume curves indicated structural left ventricular dilatation, and a 36% reduction of left ventricular developed pressure was found; phosphocreatine was reduced by approximately 80%, ATP unchanged and creatine kinase reaction velocity ((31)P-MR saturation transfer) decreased by approximately 90%. The total creatine pool (high-pressure liquid chromatography) was reduced by up to approximately 70%. In contrast to in vitro findings, in vivo cardiac hemodynamics (including left ventricular developed pressure, d P/d t(max), cardiac output and peripheral vascular resistance) at rest and during acute volume loading showed no alterations after beta-GP feeding. The only functional impairment observed in vivo was a 14% reduction of maximum left ventricular developed pressure during brief aortic occlusion. In the intact rat, cardiac and/or humoral compensatory mechanisms are sufficient to maintain normal hemodynamics in spite of a 90% reduction of creatine kinase reaction velocity. However, chronic beta-GP feeding leads to structural left ventricular dilatation.

Helenanolide type sesquiterpene lactones. Part 5: the role of glutathione addition under physiological conditions.

Sesquiterpene lactones (STLs) are known to exert most of their numerous biological activities through inhibition of enzymes and other functional proteins by forming covalent bonds with free cysteine residues in these macromolecules. The question arises how these drugs can alkylate such vital target structures instead of being quickly deactivated by reaction with the cysteine group of glutathione (GSH) which is present in high concentrations in all cells. We have measured in this study the pH dependent kinetics of GSH addition to the cyclopentenone and alpha-methylene-gamma-lactone group of helenanolide type sesquiterpene lactones using UV-spectrophotometry. The reaction with GSH at physiological pH proceeds very quickly but is reversible so that a fraction of STL molecules will always be available for reaction with protein targets. In agreement with these chemical data, helenalin-mono- and -bis-glutathionyl adducts were demonstrated to inhibit the nuclear transcription factor NF-kappaB at concentrations similar to the free sesquiterpene lactone.

Inhibition of transcription factor NF-kappaB by sesquiterpene lactones: a proposed molecular mechanism of action.

Many sesquiterpene lactones (SLs) possess considerable anti-inflammatory activity. They inhibit the transcription factor NF-kappaB by selectively alkylating its p65 subunit probably by reacting with cysteine residues. Here we assayed 28 sesquiterpene lactones for their ability to inhibit NF-kappaB. The majority of the potent NF-kappaB inhibitors possess two reactive centers in form of an alpha-methylene-gamma-lactone group and an alpha,beta- or alpha,beta,gamma,delta-unsaturated carbonyl group. Based on computer molecular modelling we propose a molecular mechanism of action, which is able to explain the p65 selectivity of the SLs and the observed correlation of high activity with alkylant bifunctionality. A single bifunctional SL molecule can alkylate the cysteine residue (Cys 38) in the DNA binding loop 1 (L1) and a further cysteine (Cys 120) in the nearby E' region. This cross link alters the position of tyrosine 36 and additional amino acids in such a way that their specific interactions with the DNA become impossible. We also created a model for monofunctional SLs.

The anti-inflammatory sesquiterpene lactone helenalin inhibits the transcription factor NF-kappaB by directly targeting p65.

The sesquiterpene lactone helenalin is a potent anti-inflammatory drug whose molecular mechanism of action remains unclear despite numerous investigations. We have previously shown that helenalin and other sesquiterpene lactones selectively inhibit activation of the transcription factor NF-kappaB, a central mediator of the human immune response. These drugs must target a central step in NF-kappaB pathway, since they inhibit NF-kappaB induction by four different stimuli. It has previously been reported that sesquiterpene lactones exert their effect by inhibiting degradation of IkappaB, the inhibitory subunit of NF-kappaB. These data contradicted our report that IkappaB is not detectable in helenalin-treated, ocadaic acid-stimulated cells. Here we use confocal laser scanning microscopy to demonstrate the presence of IkappaB-released, nuclear NF-kappaB in helenalin-treated, tumor necrosis factor-alpha stimulated cells. These data show that neither IkappaB degradation nor NF-kappaB nuclear translocation are inhibited by helenalin. Rather, we provide evidence that helenalin selectively alkylates the p65 subunit of NF-kappaB. This sesquiterpene lactone is the first anti-inflammatory agent shown to exert its effect by directly modifying NF-kappaB.

Agonist-specific modulation of glucocorticoid receptor-mediated transcription by immunosuppressants.

Although the immunosuppressive drugs FK506, rapamycin and cyclosporin A have been reported to potentiate transcriptional activation mediated by a non-saturating concentration of the glucocorticoid receptor agonist dexamethasone, the precise mechanism(s) underlying these responses remains unclear. The murine L-929-derived LMCAT cell line stably transfected with the mouse mammary tumor virus promoter-chloramphenicol acetyl transferase reporter gene construct was utilized in the present study to further investigate the mechanism(s) underlying this dexamethasone potentiation as well as the possible agonist specificity of this potentiation. The present data demonstrate that pretreatment (2 h) of LMCAT cells with 10 microM FK506, rapamycin or cyclosporin A results in the potentiation of reporter gene transcription mediated not only by dexamethasone (approximately 12-fold), but also by hydrocortisone (approximately 6-fold) and triamcinolone acetonide (approximately 2.5-fold). In sharp contrast, the data show for the first time that pretreatment with any one of these immunosuppressive drugs suppresses (approximately 2-8-fold) the transcriptional responses mediated by corticosterone, deoxycorticosterone, and cortexolone. Pretreatment of intact LMCAT cells with FK506 increases the subsequent whole cell specific binding of [3H]dexamethasone, but does not increase specific cytoplasmic binding when the tritiated agonist is added directly to cytosolic extracts prepared from the pretreated cells. These data suggest that the FK506-mediated potentiation of the transcriptional responses induced by some agonists, like dexamethasone, may be related to the ability of this immunosuppressant to inhibit the membrane-associated multidrug resistance (MDR) P-glycoprotein, which actively extrudes some steroids from cells. Identical pretreatment with FK506 has no detectable effect on the subsequent whole cell specific binding of [3H]corticosterone, a steroid which is not effectively extruded by the MDR pump. Two additional MDR pump inhibitors, verapamil and quinidine, potentiate (30-fold) the dexamethasone-mediated transcriptional response as expected, but have no detectable effects on a corticosterone-mediated transcriptional response. Unlike immunosuppressive drugs, these ion channel blockers do not bind to receptor-associated immunophilins (FK506-binding proteins or cyclophilins). Collectively, these results suggest that immunosuppressants potentiate a dexamethasone-mediated transcriptional response in LMCAT cells by inhibiting efflux of this steroid. In contrast, these drugs appear to suppress a corticosterone-mediated transcriptional response by a different mechanism, perhaps one involving their binding to glucocorticoid receptor-associated immunophilins.

Helenalin, an anti-inflammatory sesquiterpene lactone from Arnica, selectively inhibits transcription factor NF-kappaB.

Alcoholic extracts prepared form Arnicae flos, the collective name for flowerheads from Arnica montana and A. chamissonis ssp. foliosa, are used therapeutically as anti-inflammatory remedies. The active ingredients mediating the pharmacological effect are mainly sesquiterpene lactones, such as helenalin, 11alpha,13-dihydrohelenalin, chamissonolid and their ester derivatives. While these compounds affect various cellular processes, current data do not fully explain how sesquiterpene lactones exert their anti-inflammatory effect. We show here that helenalin, and, to a much lesser degree, 11alpha,13-dihydrohelenalin and chamissonolid, inhibit activation of transcription factor NF-kappaB. This difference in efficacy, which correlates with the compounds' anti-inflammatory potency in vivo, may be explained by differences in structure and conformation. NF-kappaB, which resides in an inactive, cytoplasmic complex in unstimulated cells, is activated by phosphorylation and degradation of its inhibitory subunit, IkappaB. Helenalin inhibits NF-kappaB activation in response to four different stimuli in T-cells, B-cells and epithelial cells and abrogates kappaB-driven gene expression. This inhibition is selective, as the activity of four other transcription factors, Oct-1, TBP, Sp1 and STAT 5 was not affected. We show that inhibition is not due to a direct modification of the active NF-kappaB heterodimer. Rather, helenalin modifies the NF-kappaB/IkappaB complex, preventing the release of IkappaB. These data suggest a molecular mechanism for the anti-inflammatory effect of sesquiterpene lactones, which differs from that of other nonsteroidal anti-inflammatory drugs (NSAIDs), indomethacin and acetyl salicylic acid.

Trans-retinoic acid and glucocorticoids synergistically induce transcription from the mouse mammary tumor virus promoter in human embryonic kidney cells.

Human embryonic kidney (K293) cells transfected with a mouse mammary tumor virus (MMTV) promoter-luciferase reporter construct (pHH-Luc) were utilized to investigate the potential effects of trans-retinoic acid (tRA), either by itself or in combination with glucocorticoid (GC) hormones, on a well-characterized, GC-sensitive transcriptional response. tRA or the synthetic GC hormone dexamethasone induced transcription from the MMTV promoter in a dose-dependent manner, with 1 micromol tRA and 1 micromol dexamethasone alone causing a four- to six-fold and a 40-fold induction of basal transcription, respectively. Simultaneous treatment with 1 micromol dexamethasone and 1 micromol tRA resulted in a synergistic transcriptional response that was 120-fold higher than basal level and 2.5 times the predicted response, based on a simple additive effect of both agonists. tRA does not appear to mediate this synergistic transcriptional response by enhancing GC receptor (GR) binding capacity, affinity, or nuclear translocation. tRA was unable to potentiate GC-induced transcriptional activity from a minimal GC response element (GRE), and GC were unable to potentiate tRA-induced transcriptional activity from a minimal retinoic acid response element (RARE). These data rule out direct protein-protein interactions between GC and retinoid receptors as a mechanism for the observed synergism. tRA also synergized with aldosterone-induced, mineralocorticoid receptor (MR)-mediated, transcriptional activation of the MMTV promoter, resulting in a response that was 1.7 times the predicted additive response. The MMTV GRE located between -187 and -165 was required for GC-induced and synergistic activation of the MMTV promoter, whereas sequences located within -151 to +5 were sufficient for tRA-induced transcription from the MMTV promoter. Mutation of a consensus RARE half-site (CCAAGT) identified at position -65 to -60 within the MMTV-LTR did not affect either tRA-induced transcriptional activation or synergism with GC. We propose that the tRA-induced transcriptional response from the MMTV promoter, as well as synergism with GC, may be mediated by the activation or induction of a factor(s) that either directly binds to the MMTV promoter or indirectly stabilizes binding of another transcription factor to these sequences.

Differential effects of agonist and antagonists on autoregulation of glucocorticoid receptors in a rat colonic adenocarcinoma cell line.

The relative abilities of a potent glucocorticoid receptor (GR) agonist (RU 28362), a weak GR agonist (aldosterone) and a potent GR antagonist (RU 38486) to promote in vivo activation/transformation and subsequent down-regulation of GR mRNA and protein levels were compared using the PROb rat colonic adenocarcinoma cell line. In vivo activation, which is followed immediately by nuclear translocation, by these ligands (1 microM) was evaluated in terms of their abilities to deplete cytoplasmic GR protein levels after a 30 min incubation period. Western blotting experiments with the anti-GR monoclonal antibody BuGR2 demonstrated that a brief incubation with RU 28362 resulted in nearly complete depletion of cytoplasmic GR, whereas incubation with aldosterone resulted in a 50% depletion of the cytoplasmic GR protein. Incubation with RU 38486 was even more effective than aldosterone in promoting this key step in the GR pathway. Prolonged treatment (18 h) with RU 28362 resulted in significant down-regulation of GR mRNA and total cellular GR protein levels. Similar incubation with aldosterone resulted in a transient decrease in the GR mRNA level and also down-regulated the total GR protein level. Although prolonged incubation with RU 38486 did not result in detectable down-regulation of the GR mRNA level, this antagonist very effectively down-regulated total cellular GR protein levels. Taken collectively, these data demonstrate that agonists capable of promoting in vivo activation (and subsequent nuclear translocation) of GR are also effective at down-regulating GR at both the mRNA and protein levels. Although the antagonist RU 38486 is also capable of down-regulating GR protein levels by shortening the half-life of the receptor, it appears to be incapable of altering the rate of transcription of the GR gene. Glucocorticoid target tissue sensitivity may thus be decreased via two independent mechanisms: agonist-induced repression of GR gene transcription; and/or ligand-induced degradation of total cellular GR protein levels.

Helenanolide-type sesquiterpene lactones--III. Rates and stereochemistry in the reaction of helenalin and related helenanolides with sulfhydryl containing biomolecules.

The reactivity of the two potential Michael addition sites of the helenanolide-type sesquiterpene lactone helenalin towards the physiological thiols glutathione (GSH) and cysteine (cys) in aqueous solution was investigated by 1H NMR spectroscopic experiments. In the presence of one molar equivalent of GSH, the reaction was shown to occur with high regio- and stereoselectivity at the beta-position of C-2 in the cyclopentenone ring. Addition to the exocyclic methylene group at the lactone ring was found to occur in the presence, of GSH in molar ratios over 1:1, but proceeded at a rate 10 times smaller than at C-2 leading to the 2 beta,13(11 beta)-bis-glutathionyl adduct. In contrast, addition of free cys highly favoured the exocyclic methylene group. Addition of GSH to the cyclopentenone of 11 alpha, 13-dihydrohelenalin (plenolin) showed the same characteristics as observed with helenalin while 2 alpha-acetoxy-2,3-dihydro-4 beta H-helenalin (chamissonolide) did not form an adduct when incubated with an equimolar amount of GSH. Explanations for the observed differences in reactivity of the two potential reaction sites based on MO computations are given and implications for the biological activity of this type of sesquiterpene lactones are discussed.

Structure-cytotoxicity relationships of some helenanolide-type sesquiterpene lactones.

This study deals with the cytotoxicity of helenanolide-type (10 alpha-methylpseudoguaianolide) sesquiterpene lactones. We determined the influence of substitution patterns on the toxicity of 21 helenanolides to a cloned Ehrlich ascites tumor cell line, EN2. Within a series of helenalin esters, the acetate (2) and isobutyrate (3) were more toxic than helenalin itself (1). Esters with larger acyl groups (tiglate 4 and isovalerate 5) exhibited a decreased toxicity compared with the parent alcohol (1). Similar relationships were observed between the 6,8-diastereomer of helenalin, mexicanin I (6) and its acetate (7) and isovalerate (8). In contrast, cytotoxicity within a series of 11 alpha, 13-dihydrohelenalin esters (9-12) was shown to be directly related to the size and lipophilicity of the ester side chain, dihydrohelenalin (9) being the least toxic compound in this group. Investigation of several 2,3-dihydrohelenalin derivatives (13-21) with 2 alpha-hydroxy-4-oxo- and 2 alpha,4 alpha-dihydroxy- or -O-acyl-substituted cyclopentane rings (arnifolins and chamissonolides, respectively), for which no pharmacological data have been reported so far, revealed further interesting influences of the substitution pattern on cytotoxicity. The results may be interpreted in terms of lipophilicity and steric effects on the accessibility of the reactive sites considered responsible for biological activity.

Potential mechanisms underlying autoregulation of glucocorticoid receptor mRNA levels in the DHD/K12/PROb rat colonic adenocarcinoma cell line.

The DHD/K12/PROb rat colonic epithelial cell line, which was originally derived from a chemically induced adenocarcinoma, expresses functional glucocorticoid receptors (GR) and has been reported to be growth inhibited by glucocorticoid agonists. In the present study the potential mechanisms underlying corticosteroid-mediated autoregulation of GR mRNA levels in this colonic cell line were investigated. The GR mRNA levels in the various treatment groups were quantitated via the ribonuclease protection assay using a specific 32P-cRNA probe. Time-course experiments demonstrated that in contrast to several other cell lines that are also growth inhibited by glucocorticoids, treatment of confluent monolayers of PROb cells with the pure GR agonist RU 28362 (1 microM) elicits a rapid and significant (65%) down-regulation of GR mRNA levels that is sustained for at least 36 h. This down-regulation, which is also elicited to a lesser extent by weaker GR agonists including corticosterone and aldosterone, is blocked by the GR antagonist RU 38486. The protein synthesis inhibitor cycloheximide was utilized to demonstrate that the initial phase (6 h) of agonist-mediated down-regulation occurs independently of ongoing protein synthesis, although new protein synthesis, perhaps of the GR protein itself, is required to maintain this down-regulation. Although agonist-mediated down-regulation in these cells probably occurs primarily at the level of GR gene transcription, inhibition of ongoing RNA synthesis with actinomycin D or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) demonstrated that during the initial phase (1 h) of this down-regulation, but not following maximal (18 h) down-regulation, RU 28362 treatment also significantly reduces the stability of the GR mRNA.

Decreased helenalin-induced cytotoxicity by flavonoids from Arnica as studied in a human lung carcinoma cell line.

The effect of the flavones apigenin, luteolin, hispidulin and eupafolin, and of the flavonols kaempferol, quercetin, 6-methoxykaempferol and patuletin from Arnica spp. on the cytotoxicity of the sesquiterpene lactone helenalin was studied in the human lung carcinoma cell line GLC4 using the microculture tetrazolium (MTT) assay. The tumour cells were exposed to the test compounds for 2h. Helenalin concentrations around its control IC(50) value, 0.5 µM, were combined with flavonoid concentrations ranging from 0.01 to 20 µM. At non-toxic concentrations, up to 10µM, all flavonoids except kaempferol significantly reduced the helenalin-induced cytotoxicity. Hispidulin and patuletin displayed their modulating effect on helenalin-induced cytotoxicity in the broadest concentration range. The strongest effect was found with 5 and 10µM hispidulin, 0.05 µM quercetin, and 1 µM patuletin, increasing the IC(50) value of helenalin with circa 40%. No dose-dependency was found in the concentration range tested.

Cytotoxicity of flavonoids and sesquiterpene lactones from Arnica species against the GLC4 and the COLO 320 cell lines.

The cytotoxicity of 21 flavonoids and 5 sesquiterpene lactones, as present in Arnica species, was studied in GLC4, a human small cell lung carcinoma cell line, and in COLO 320, a human colorectal cancer cell line, using the microculture tetrazolium (MTT) assay. Following continuous incubation, most flavonoids showed moderate to low cytotoxicity, as compared with the reference compound cisplatin (IC50 = 1.1 microM against GLC4 and 2.9 microM against COLO 320). Their IC50 values varied from 17 to > 200 microM. The most toxic compound was the flavone jaceosidin. Of the sesquiterpene lactones tested, helenalin, possessing both the reactive alpha-methylene-gamma-lactone moiety and a reactive alpha,beta-unsubstituted cyclopentenone ring, displayed the strongest cytotoxicity. For 2 h exposure, its IC50 value was 0.44 microM against GLC4 and 1.0 microM against COLO 320. COLO 320 was more sensitive than GLC4 for many flavonoids (especially for flavones), but more resistant to the cytotoxic effect of the sesquiterpene lactones bearing an exocylic methylene group fused to the lactone function.