Cancer chemopreventive properties of orally bioavailable flavonoids--methylated versus unmethylated flavones.

Poor oral bioavailability has been a major limitation for the successful use of dietary flavonoids as cancer chemopreventive agents. In this study, we examined fully methylated flavones as promising improved agents. In the human oral SCC-9 cancer cells, 5,7-dimethoxyflavone and 5,7,4'-trimethoxyflavone were both 10 times more potent inhibitors of cell proliferation (IC(50) values 5-8 microM) than the corresponding unmethylated analogs chrysin and apigenin. Flow cytometry indicated that both methylated flavones arrested the SCC-9 cells in the G1 phase with a concomitant decrease in the S phase, dramatically different from the unmethylated analogs, which promoted G2/M phase arrest. Both methylated compounds inhibited the proliferation of two other cancer cell lines with very little effect on two immortalized normal cell lines. Examination of additional flavone structures indicated that methylated flavones in general have antiproliferative properties. Finally, we demonstrated that 5,7-dimethoxyflavone, in contrast to its unmethylated analog chrysin, was well absorbed and had high oral bioavailability as well as tissue accumulation in vivo in the rat. Thus, fully methylated flavones appear to have great potential as cancer chemopreventive/chemotherapeutic agents, in particular in oral cancer.

Novel methoxylated flavone inhibitors of cytochrome P450 1B1 in SCC-9 human oral cancer cells.

Dietary polyphenols, including flavonoids, have been implied to have cancer preventive properties. Suggested mechanisms include inhibition of carcinogen-activating cytochrome P450 (CYP) transcription and activities. These studies have focused mainly on CYP1A1. However, CYP1B1 has recently been shown to be of particular importance in smoking-induced oral and oesophageal cancer. Previous observations in our laboratory demonstrated that methoxylated flavonoids may be effective inhibitors of CYP1A1 transcription and activity as well as being orally bioavailable. In this study, an initial screening of 19 methoxylated flavones, using the ethoxyresorufin de-ethylation assay in human oral squamous cell carcinoma SCC-9 cells pretreated with 1 microM benzo[a]pyrene, identified six strongly inhibitory compounds for further studies. The effect of these flavones on CYP1B1 mRNA expression was measured with quantitative branched DNA methodology. Four of the compounds--3',4'-dimethoxyflavone and 5,7,4'-trimethoxyflavone and, in particular, 7,3'-dimethoxyflavone and 7,4'-dimethoxyflavone--were potent inhibitors of CYP1B1 mRNA expression. Two of the more common unmethylated polyphenols--curcumin and quercetin--were also potent inhibitors. Whereas most unmethylated polyphenols, such as curcumin and quercetin, have very poor bioavailability, the high metabolic stability of the methoxylated flavones studied here suggests that these CYP1B1 inhibitors may also be effective in-vivo.

Improving metabolic stability of cancer chemoprotective polyphenols.

Dietary flavonoids and other polyphenols have the potential to be developed as effective food supplements as well as drugs for the prevention, as well as treatment of, cancer and other disease conditions. However, their very poor oral bioavailability, mainly due to extensive conjugation by glucuronidation and sulfation, is a severe limiting factor. First, this review shows the use of a simple, commercially available model system, the human hepatic S9 fraction, by which metabolic stability can be assessed effectively and accurately. Second, permethylation of the polyphenols effectively blocks the metabolic conjugation reactions, thereby dramatically increasing both metabolic stability and intestinal absorption, while maintaining or even increasing the biologic activities. Thus, permethylated polyphenols may have a future as chemoprotective agents.

Covalent binding of the flavonoid quercetin to human serum albumin.

Quercetin is an abundant flavonoid in the human diet with numerous biological activities, which may contribute to the prevention of human disease but also may be potentially harmful. Quercetin is oxidized in cells to products capable of covalently binding to cellular proteins, a process that may be important for its biological activities. In the present study, using radiolabeled drug and quantifying the products after electrophoretic separation, proteins to which oxidized quercetin is binding irreversibly were identified. The binding of quercetin to human serum albumin (HSA) in human blood and the effect of stimulation of neutrophilic myeloperoxidase on this binding were also measured. The in vitro binding of quercetin to eight proteins in the presence of catalytic amounts of horseradish peroxidase and hydrogen peroxide was highly selective for HSA. For all proteins the binding was dramatically decreased by reduced L-glutathione. In the blood samples, the release of neutrophilic myeloperoxidase by phorbol ester caused a 3-fold increase in the binding of quercetin to HSA. This study shows that quercetin in the presence of peroxidase/hydrogen peroxide covalently links to proteins with a particularly high affinity for HSA and that this also may occur in vivo after exposure to quercetin. This provides further insights into the complex behavior of this major dietary flavonoid.

Flavonoid glycosides inhibit oral cancer cell proliferation--role of cellular uptake and hydrolysis to the aglycones.

Epidemiologic evidence supports the view that dietary flavonoids exert protective effects in oral diseases, including cancer. However, the dietary forms of flavonoids, the flavonoid glycosides, are thought to be inactive, thus they must first be hydrolysed to their active aglycones. This may occur in the saliva in the oral cavity. We have examined if the flavonoid glycosides directly could affect cell proliferation, using the human oral squamous carcinoma SCC-9 cells. The cellular uptake and hydrolysis of the glycosides were assessed also. The four flavonoid glycosides tested each behaved differently. Genistin, the 7-glucoside of genistein, showed clear and consistent inhibition of cell proliferation, which appeared to be the result of rapid cellular uptake of the glucoside and hydrolysis to genistein. Spiraeoside, the 4'-glucoside of quercetin, showed a similar inhibition of cell proliferation, which also appeared to be associated with its hydrolysis to quercetin. Diosmin, the 7-rutinoside of diosmetin, surprisingly, was more potent and effective than diosmetin. In contrast, quercitrin, the 3-rhamnoside of quercetin, showed no effect and only minimal cellular uptake and no hydrolysis. In summary, dietary flavonoid glycosides may exert cellular effects in the oral cavity, but this varies greatly with the nature of the glycoside.

Evidence of covalent binding of the dietary flavonoid quercetin to DNA and protein in human intestinal and hepatic cells.

Quercetin-rich foods have the potential to prevent human disease. However, knowledge of its biological fate and mechanism of action is limited. This study extends previous observations of the oxidation of quercetin by peroxidases to quinone/quinone methide intermediates and, for the first time, demonstrates covalent binding of [14C]quercetin to macromolecules. This was first demonstrated using horseradish peroxidase and hydrogen peroxide with human liver microsomal protein to trap the intermediates. To extend this observation to the cellular level, human intestinal Caco-2 cells and hepatic Hep G2 cells were incubated for up to 2hr with [14C]quercetin, and cellular DNA and protein were isolated. The cellular uptake of [14C]quercetin was rapid, and the covalent binding of [14C]quercetin to DNA and protein was determined by liquid scintillation spectrometry after extensive purification. Both cell types demonstrated DNA binding with a maximum level of 5-15pmol/mg DNA. The level of covalent binding to protein was considerably higher in both cell types, 75-125pmol/mg protein. To determine potential specificity in the protein binding, Hep G2 cells were treated with [14C]quercetin, and the cell lysate was subjected to SDS-PAGE followed by staining and autoradiography. Several distinct radiolabeled protein bands did not correspond to the major Coomassie blue stained cellular proteins. We propose that this specific binding may mediate part of the antiproliferative and other cellular actions of quercetin.

Resveratrol transport and metabolism by human intestinal Caco-2 cells.

Resveratrol is a dietary constituent suggested to have protective effects against cancer as well as cardiovascular disease. The purpose of the study was to learn whether this agent could be absorbed in man and enter the systemic circulation. This was examined by measuring transport and metabolism of resveratrol (5-40 microM) by the human intestinal epithelial cell line Caco-2 cultured in Transwells. Transport across the Caco-2 monolayer occurred in a direction-independent manner with P(app) values of approximately 7 x 10(-6) cm s(-1), much higher than for the paracellular transport marker mannitol (approximately 0.4 x 10(-6) cm s(-1)), suggesting efficient absorption in-vivo. At the highest resveratrol concentration, the absorption increased, possibly due to saturation of metabolism. In sharp contrast to previous findings in the rat, the metabolism of resveratrol in Caco-2 cells involved mainly sulfation and, to a minor extent, glucuronidation. At low resveratrol concentrations, most of the sulfate conjugate was exported to the apical side, presumably by MRP2, which is well expressed in these cells. At high concentrations, there was a shift towards the basolateral side, possibly involving MRP3, which was recently shown also to be expressed in Caco-2 cells. These results indicate that absorption of resveratrol in-vivo may be high but with limited bioavailability due to efficient sulfate conjugation. Extensive accumulation of resveratrol in the Caco-2 cells, demonstrated in additional experiments, suggests enterocytes as a major target site for this cancer preventive agent.

Bioavailable flavonoids: cytochrome P450-mediated metabolism of methoxyflavones.

Methoxylated flavones were recently shown to be promising cancer chemopreventive agents. Their high metabolic stability compared with the hydroxylated analogs was shown in our laboratory using the human hepatic S9 fraction with cofactors for glucuronidation, sulfation, and oxidation. In the present study, the resistance of methoxylated flavones toward oxidative metabolism was investigated with human liver microsomes and recombinant cytochrome P450 (P450) isoforms. Among 15 methoxylated flavones investigated, the two partially methylated compounds, tectochrysin and kaempferide, were among the most susceptible to microsomal oxidation (Cl(int) 283 and 82 ml/min/kg). Of the fully methylated compounds, 5,7-dimethoxyflavone and 5-methoxyflavone were the most stable (Cl(int) 13 and 18 ml/min/kg, respectively), whereas 4'-methoxyflavone, 3'-methoxyflavone, 5,4'-dimethoxyflavone, and 7,3'-dimethoxyflavone were the least stable (Cl(int) 161, 140, 119, and 92 ml/min/kg, respectively), emphasizing the importance of the positions of the methoxy substituents in the flavone ring system. Among the five P450 isoforms tested, CYP1A1 showed the highest rate of metabolism of fully methylated compounds, followed by CYP1A2 and CYP3A4. CYP2C9 and CYP2D6 gave minimal disappearance of the parent compound. Finally, in incubations with hepatic S9 fraction with cofactors for oxidation and both conjugation reactions, partially methylated flavones, as expected, were much less metabolically stable than fully methylated flavones, confirming that oxidative demethylation is the rate-limiting metabolic reaction for fully methylated flavones only. In summary, the rate of oxidative metabolism of methoxylated flavones, mainly involving CYP1A1 and CYP1A2, varied widely, even between compounds with very similar structures.

Benzo[A]pyrene-induced oral carcinogenesis and chemoprevention: studies in bioengineered human tissue.

Oral cancer, originating from smoking-induced lesions of the basal cells in the complex stratified oral epithelium, is difficult to treat. Early detection of premalignant lesions, e.g., leukoplakia, has suggested the possibility of chemopreventive measures, such as topical application of antimutagenic/antiproliferative dietary or pharmaceutical agents. As an extension of a study in human oral epithelial cell monolayers, we determined the carcinogen, i.e., benzo[a]pyrene (BaP), transport, bioactivation, and DNA binding in a bioengineered human gingival epithelial tissue construct and the chemopreventive effects of dietary polyphenols. Short-term experiments showed that both types of compounds can traverse this tissue as well as be effectively taken up by the tissue. The model cigarette smoke carcinogen BaP very slowly, but to a great extent, accumulated in the tissue with maximal uptake at 24 h. Such exposure clearly resulted in DNA binding of BaP by the tissue. This DNA binding was associated with BaP-induced CYP1B1 as well as CYP1A1 expression, as evidenced by mRNA measurements. Cotreatment of the oral tissue with dietary polyphenols, including resveratrol and quercetin, and BaP, resulted in significant inhibition of the BaP-DNA binding. Using fluorescence microscopy as well as simultaneous autoradiography, we also demonstrated that quercetin indeed penetrates the entire stratified tissue layer, but that quercetin was also oxidized within the cells. Thus, this bioengineered oral tissue construct opens up improved ways of understanding and preventing/treating smoking-induced oral cancer.

5,7-Dimethoxyflavone downregulates CYP1A1 expression and benzo[a]pyrene-induced DNA binding in Hep G2 cells.

The objective of this study was to examine the ability of dietary polyphenols to inhibit cytochrome P450 (CYP) 1A1 expression and activity and benzo[a]pyrene (BaP) DNA binding, with the main emphasis on prevention of chemical-induced hepatic carcinogenesis. For this purpose we used Hep G2 cells, a good model of the normal human hepatocyte for CYP1A1 cell signaling. First, when these cells were exposed to a low concentration (1 microM) of BaP, DNA binding occurred, which dramatically increased after 6 h of treatment. BaP also dramatically induced CYP1A1 activity, protein expression and mRNA levels, the likely reason for the marked increase in DNA binding. Second, we screened 25 polyphenols with highly varying chemical structures for maximum ability to inhibit CYP1A1 activity in the Hep G2 cells. Highly varying responses were obtained, ranging from a 10-fold induction by some polyphenols to almost complete inhibition, in particular by 5,7-dimethoxyflavone (DMF), a flavonoid found in some tropical plants. Third, we examined the ability of DMF to inhibit DNA binding of BaP and the mechanisms involved. DMF (2-20 microM) inhibited BaP-induced DNA binding. DMF also inhibited BaP-induced CYP1A1 activity, CYP1A1 protein expression and mRNA levels. Moreover, DMF directly inhibited the catalytic activity of recombinant CYP1A1 with an IC50 of 0.8 microM. In conclusion, DMF was a highly potent inhibitor of BaP-induced DNA binding and CYP1A1 protein expression and activity in the Hep G2 cells. These properties may make DMF an effective chemoprotectant in chemical-induced liver cancer.

Flavonoid glucosides are hydrolyzed and thus activated in the oral cavity in humans.

Increasing epidemiological evidence supports the view that dietary flavonoids have protective roles in oral diseases, including cancer. However, the dietary forms of flavonoids, the flavonoid glycosides, must first be hydrolyzed to the aglycones, which is thought to occur mainly in the intestine. In the present study we tested whether this hydrolytic activity occurs in the oral cavity. Saliva was collected from human subjects, incubated with flavonoid glycosides, and analyzed for aglycone formation by HPLC. When quercetin 4'-glucoside or genistein 7-glucoside was incubated with human saliva, hydrolysis to quercetin and genistein, respectively, was detected within minutes. Studies of additional flavonoid glycosides demonstrated that glucose conjugates were rapidly hydrolyzed, but not conjugates with other sugars, i.e., rutin, quercitrin, and naringin. In a limited study of 17 subjects, the interindividual variability in the hydrolysis of genistein 7-glucoside was >20-fold. This supports the contention that salivary hydrolysis of certain flavonoid glucosides may be important in some individuals but not in others. Support for a bacterial contribution to this hydrolysis was obtained from the inhibitory effect of antibacterials in vivo and in vitro and from experiments with subcultured oral bacterial colonies. However, cytosol isolated from oral epithelial cells was also capable of effective hydrolysis. Dietary flavonoid glucosides may thus be hydrolyzed in the oral cavity by both bacteria and shedded epithelial cells to deliver the biologically active aglycones at the surface of the epithelial cells. The aglycones quercetin and genistein both potently inhibited proliferation of oral cancer cells. The large interindividual variability in this hydrolytic activity may be a factor that should be taken into consideration in future studies.

The beta-D-glucoside and sodium-dependent glucose transporter 1 (SGLT1)-inhibitor phloridzin is transported by both SGLT1 and multidrug resistance-associated proteins 1/2.

Phloridzin, a glucoside of the flavonoid-like polyphenol phloretin, has long been known to be a specific nontransportable inhibitor of the sodium-dependent glucose transporter SGLT1. The objective of this study was to determine whether efflux by multidrug resistance-associated protein (MRP) transporters might have masked the absorption by SGLT1 in previous studies. Various cells used as transport models were incubated with phloridzin (50 microM) in the absence and presence of 50 microM 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid (MK-571), a highly selective MRP1/MRP2 inhibitor, and the cellular uptake of phloridzin was measured by high performance liquid chromatography. The uptake of phloridzin by SGLT1-transfected Chinese hamster ovary (CHO) (G6D3) cells was 1.7-fold higher than that by parent CHO cells (p < 0.01). In the presence of MK-571, the uptake of phloridzin by CHO cells increased 3.7-fold (p < 0.001). MK-571 caused an 8.0-fold increase in the uptake of phloridzin by G6D3 cells (p < 0.0001). Thus, in the absence of MRP1 efflux, transport of phloridzin by SGLT1 was clearly demonstrated. Similar results were obtained for the glycosides of the flavonoids quercetin, genistein, and diosmetin. A significantly lower accumulation of phloridzin in MRP2-transfected Madin-Darby canine kidney (MDCK) cells compared with parent MDCK cells demonstrated that phloridzin was a substrate also for MRP2 (p < 0.05). This conclusion was further strengthened when MK-571 increased the uptake by MRP2-MDCK cells as much as 3.6-fold (p < 0.01). These results demonstrate that phloridzin, in contrast to previous notions, is transported by SGLT1. In addition, they demonstrate that this and other flavonoid glycosides unexpectedly are efficiently effluxed by both MRP1 and MRP2.

Induction of human UDP-glucuronosyltransferase UGT1A1 by flavonoids-structural requirements.

Recent studies in our laboratory in the human hepatic and intestinal cell lines Hep G2 and Caco-2 have demonstrated induction of UGT1A1 by the flavonoid chrysin (5,7-dihydroxyflavone) using catalytic activity assays and Western and Northern blotting. In the present study, we examined which features of the flavonoid structures were associated with induction of UGT1A1 and whether common drug-metabolizing enzyme inducers also produce this induction. We also determined whether flavonoid treatment affected sulfate conjugation and CYP1A1 activity. We used intact Hep G2 cells for these studies, with chrysin as the model substrate. Both glucuronidation and sulfation were measured. Hep G2 cells were pretreated for 3 days with 25 microM concentrations of 22 flavonoids (n = 4-12). Only four flavonoids demonstrated induction of glucuronidation similar to that of chrysin (i.e., 3-5-fold in the intact cells). These were acacetin, apigenin, luteolin, and diosmetin, all of which, like chrysin, are 5,7-dihydroxyflavones with varying substituents in the B-ring. 5-Hydroxy-7-methoxyflavone and 5-methyl-7-hydroxyflavone produced a modest 1.5 to 2-fold induction, whereas all other flavonoids examined were without effect. None of the flavonoids caused more than a modest change in sulfation activity (60-140% of control). In contrast, all tested 5,7-dihydroxyflavones and -flavonols induced CYP1A1 activity (ethoxyresorufin deethylation). Of seven common drug-metabolizing enzyme inducers only 3-methylcholanthrene and oltipraz showed modest induction of chrysin glucuronidation but not 2,3,7,8-tetrachlorodibenzo-p-dioxin or phenobarbital. Together, these results strongly suggest that the flavonoid induction of UGT1A1 is through a novel nonaryl hydrocarbon receptor-mediated mechanism.

High absorption but very low bioavailability of oral resveratrol in humans.

The dietary polyphenol resveratrol has been shown to have chemopreventive activity against cardiovascular disease and a variety of cancers in model systems, but it is not clear whether the drug reaches the proposed sites of action in vivo after oral ingestion, especially in humans. In this study, we examined the absorption, bioavailability, and metabolism of 14C-resveratrol after oral and i.v. doses in six human volunteers. The absorption of a dietary relevant 25-mg oral dose was at least 70%, with peak plasma levels of resveratrol and metabolites of 491 +/- 90 ng/ml (about 2 microM) and a plasma half-life of 9.2 +/- 0.6 h. However, only trace amounts of unchanged resveratrol (<5 ng/ml) could be detected in plasma. Most of the oral dose was recovered in urine, and liquid chromatography/mass spectrometry analysis identified three metabolic pathways, i.e., sulfate and glucuronic acid conjugation of the phenolic groups and, interestingly, hydrogenation of the aliphatic double bond, the latter likely produced by the intestinal microflora. Extremely rapid sulfate conjugation by the intestine/liver appears to be the rate-limiting step in resveratrol's bioavailability. Although the systemic bioavailability of resveratrol is very low, accumulation of resveratrol in epithelial cells along the aerodigestive tract and potentially active resveratrol metabolites may still produce cancer-preventive and other effects.