Estrogen modulation properties of mangiferin and quercetin and the mangiferin metabolite norathyriol.

Mango fruit contain many bioactive compounds, some of which are transcription factor regulators. Estrogen receptor alpha (ERα) and beta (ERß) are two regulators of gene transcription that are important in a variety of physiological processes and also in diseases including breast cancer. We examined the ability of the mango constituents quercetin, mangiferin, and the aglycone form of mangiferin, norathyriol, to activate both isoforms of the estrogen receptor. Quercetin and norathyriol decreased the viability of MCF-7 breast cancer cells whereas mangiferin had no effect on MCF-7 cells. We also determined that quercetin and mangiferin selectively activated ERα whereas norathyriol activated both ERα and ERß. Despite quercetin, mangiferin and norathyriol having similar polyphenolic structural motifs, only norathyriol activated ERß, showing that bioactive agents in mangoes have very specific biological effects. Such specificity may be important given the often-opposing roles of ERα and ERß in breast cancer proliferation and other cellular processes.

High-throughput screening of potential inhibitors for the metabolism of the investigational anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid.

By screening potential inhibitors of drug metabolism using the in vitro models, potential drug-drug interactions in vivo may be predicted with the use of appropriate pharmacokinetic principles. This study aimed to develop a rapid screening system using human liver microsomes to efficiently identify the potential inhibitors of DMXAA metabolism. Initial IC50 was estimated by using a two-point method, and then Ki values were determined if required and compared with those initial IC50 values. More than 100 compounds including known substrates and inhibitors of human uridine diphosphate glucuronosyltransferases (UGTs) and cytochrome P450 (CYP), anti-cancer drugs and xanthenone analogues were screened for their inhibitory effect on DMXAA glucuronidation and 6-methylhydroxylation in human liver microsomes. Both metabolites of DMXAA, DMXAA acyl glucuronide (DMXAA-G) and 6-hydroxymethyl-5-methylxanthenone-4-acetic acid (6-OH-MXAA), formed in human liver microsomes were quantitated by validated HPLC methods. The results indicated that there was a significant relationship (r2 = 0.966, P < 0.001) between the two-point IC50 values and the apparent Ki values for 20 compounds showing significant inhibitory effects on DMXAA metabolism, suggesting the usefulness of the two-point determination for the initial screening of compounds. This study has been completed using a strategy for rapid HPLC analysis and thus provided early access to detailed information for potential inhibitors of DMXAA metabolism and allows for further DMXAA-drug interaction studies.

Effects of anticancer drugs on the metabolism of the anticancer drug 5,6-dimethylxanthenone-4-acetic (DMXAA) by human liver microsomes.

AIMS: To investigate the effects of various anticancer drugs on the major metabolic pathways (glucuronidation and 6-methylhydroxylation) of DMXAA in human liver microsomes. METHODS: The effects of various anticancer drugs at 100 and 500 microM on the formation of DMXAA acyl glucuronide (DMXAA-G) and 6-hydroxymethyl-5-methylxanthenone-4-acetic acid (6-OH-MXAA) in human liver microsomes were determined by high performance liquid chromatography (h.p.l.c.). For those anticancer drugs showing significant inhibition of DMXAA metabolism, the inhibition constants (Ki) were determined. The resulting in vitro data were extrapolated to predict in vivo changes in DMXAA pharmacokinetics. RESULTS: Vinblastine, vincristine and amsacrine at 500 microM significantly (P < 0.05) inhibited DMXAA glucuronidation (Ki = 319, 350 and 230 microM, respectively), but not 6-methylhydroxylation in human liver microsomes. Daunorubicin and N-[2-(dimethylamino)-ethyl]acridine-4-carboxamide (DACA) at 100 and 500 microM showed significant (P < 0.05) inhibition of DMXAA 6-methylhydroxylation (Ki = 131 and 0.59 microM, respectively), but not glucuronidation. Other drugs such as 5-fluoroucacil, paclitaxel, tirapazamine and methotrexate exhibited little or negligible inhibition of the metabolism of DMXAA. Pre-incubation of microsomes with the anticancer drugs (100 and 500 microM) did not enhance their inhibitory effects on DMXAA metabolism. Prediction of DMXAA-drug interactions in vivo based on these in vitro data indicated that all the anticancer drugs investigated except DACA appear unlikely to alter the pharmacokinetics of DMXAA, whereas DACA may increase the plasma AUC of DMXAA by 6%. CONCLUSIONS: These results indicate that alteration of the pharmacokinetics of DMXAA appears unlikely when used in combination with other common anticancer drugs. However, this does not rule out the possibility of pharmacokinetic interactions with other drugs used concurrently with this combination of anticancer drugs.

MRP2, a human conjugate export pump, is present and transports fluo 3 into apical vacuoles of Hep G2 cells.

The multidrug resistance protein 2 (MRP2, symbol ABCC2) transports anionic conjugates and certain amphiphilic anions across the apical membrane of polarized cells. Human hepatoma Hep G2 cells retain hepatic polarity and form apical vacuoles into which cholephilic substances are secreted. Immunofluorescence microscopy showed that human MRP2 was expressed in the apical vacuole membrane of polarized Hep G2 cells, whereas the isoform MRP3 was localized to the lateral membrane. Expression of both MRP2 and MRP3 was confirmed by immunoblotting and reverse transcription PCR. Fluo 3 secretion into the apical vacuoles was inhibited by cyclosporin A but not by selective inhibitors of multidrug resistance 1 P-glycoprotein. In addition, carboxyfluorescein, rhodamine 123, and the fluorescent bile salt derivatives ursodeoxycholyl-(Nepsilon-nitrobenzoxadiazolyl)-lysine and cholylglycylamido-fluorescein were secreted into the apical vacuoles; the latter two probably via the bile salt export pump. We conclude that MRP2 mediates fluo 3 secretion into the apical vacuoles of polarized Hep G2 cells. Thus the function of human MRP2 and the action of inhibitors can be analyzed by the secretion of fluorescent anions such as fluo 3.

Induction of calcium release from sarcoplasmic reticulum of skeletal muscle by xanthone and norathyriol.

1. Effects of xanthone and its derivative, 1,3,6,7-tetrahydroxyxanthone (norathyriol), on Ca2+ release and ryanodine binding were studied in isolated sarcoplasmic reticulum (SR) vesicles from rabbit skeletal muscle. 2. Both xanthone and norathyriol dose-dependently induced Ca2+ release from the actively loaded SR vesicles which was blocked by ruthenium red, a specific Ca2+ release inhibitor, and Mg2+. 3. Xanthone and norathyriol also dose-dependently increased apparent [3H]-ryanodine binding. Norathyriol, but not xanthone, produced a synergistic effect on binding activation when added concurrently with caffeine. 4. In the presence of Mg2+, which inhibits ryanodine binding, both caffeine and norathyriol, but not xanthone, could restore the binding to the level observed in the absence of Mg2+. 5. Xanthone activated the Ca(2+)-ATPase activity of isolated SR vesicles dose-dependently reaching 70% activation at 300 microM. 6. When tested in mouse diaphragm, norathyriol potentiated the muscle contraction followed by twitch depression and contracture in either a Ca(2+) -free bathing solution or one containing 2.5 mM Ca2+. These norathyriol-induced effects on muscle were inhibited by pretreatment with ruthenium red or ryanodine. 7. These data suggest that xanthone and norathyriol can induce Ca2+ release from the SR of skeletal muscle through a direct interaction with the Ca2+ release channel, also known as the ryanodine receptor.

Inhibition of adenylate cyclase in perfusion mouse palate by secalonic acid D.

Glucocorticoids (GC) induce cleft palate (CP) in the offspring of exposed pregnant mice. Glucocorticoids induce prostaglandin (PG) synthesis inhibition via reduced arachidonic acid (AA) release from membranes, and this results from inhibition of phospholipase A2. This metabolic event is associated with reduced palatal cAMP levels in the prefusion stages (d 13 of gestation). The mycotoxin secalonic acid D (SAD) induces CP in the offspring born to treated mothers, elevates maternal plasma corticosterone levels, and reduces prefusion palatal cAMP levels. In addition, an increase in cAMP was noted in the postfusion period (d 15 of gestation). Since exogenous AA given simultaneously to GC-exposed mothers may protect against GC-induced CP in the offspring, such a possibility was tested for SAD. Pregnant CD1 mice given a teratogenic dose of SAD (30 mg/kg, ip, on gestational d 11) were simultaneously treated with maximal tolerated doses of AA (200 mg/kg, sc, on gestational d 11, 12, and 13). At term, no significant reduction in SAD-induced CP was seen as a result of AA treatment. To evaluate if SAD-induced alterations in palatal cAMP are due to reduced palatal membrane-associated adenylate cyclase (AC) activity during pre- and postfusion periods, SAD-treated mothers were sacrificed at 12-h intervals between gestational d 13.5 and 15.5, palate shelves were collected from the fetuses, and AC activity (cAMP formed/mg protein/min) was assayed in the presence or absence of the enzyme stimulator, sodium fluoride (NaF). Although SAD did not alter unstimulated AC activity, it significantly reduced the NaF-induced stimulation of enzyme activity in the prefusion period. This inhibition could not be reversed by excess GTP in the incubation mixture. Since NaF stimulation of AC indicates post-receptor-site function involving GTP-binding and catalytic units, and since addition of GTP failed to correct SAD-induced alteration of NaF stimulation of the enzyme, it is suggested that SAD may inhibit the AC sensitivity to stimuli by its effect on the catalytic unit in a manner that does not affect enzymic basal activity.

Stimulation of macrophage tumouricidal activity by 5,6-dimethyl-xanthenone-4-acetic acid, a potent analogue of the antitumour agent flavone-8-acetic acid.

The new antitumour drug 5,6-dimethylxanthenone-4-acetic acid (5,6-MeXAA; NSC 640488) was 14-fold more potent than the investigational chemotherapeutic drug flavone-8-acetic acid (NSC 347512) in stimulating tumouricidal activity in cultures of resident murine peritoneal macrophages. The tumouricidal activity of thioglycollate-elicited and Bacillus Calmette-Guérin-primed macrophages was also significantly enhanced by 5,6-MeXAA. Stimulation of macrophage tumouricidal activity by 5,6-MeXAA was not affected by inhibitors of superoxide and nitric oxide production, but was reduced by cyclosporin A, an inhibitor of protein secretion. Inhibitors of neutral proteases had no effect. Cortisone, dexamethasone, indomethacin, dibutyryl cAMP, prostaglandin E2 and prostacyclin, but not prostaglandin F2 alpha, inhibited stimulation, suggesting the involvement of tumour necrosis factor-alpha (TNF). However, antibodies to TNF did not inhibit stimulation. The results suggest that 5,6-MeXAA acts on macrophages in a manner similar to that of endotoxin, utilizing a pathway which includes arachidonic acid metabolism and requiring cell-cell contact with target cells for a tumouricidal effect.

Chemical constituents of Gentianaceae XIX: CNS-depressant effects of swertiamarin.

CNS activity of swertiamarin, a secoiridoid glucoside from Swertia chirata, was evaluated. An apparent anomaly, associated with the unanticipated finding that the alcoholic extracts (excluding mangiferin) of S. chirata significantly reversed the mangiferin-induced CNS-stimulating effects in albino mice and rats, was resolved. The results indicate that swertiamarin and mangiferin antagonize each other in vivo and thereby reverse their CNS effects.