In vitro assessment of intestinal permeability and hepatic metabolism of 4'-bromoflavone, a promising cancer chemopreventive agent.

1. The intestinal permeability and hepatic metabolism of the investigational cancer chemoprevention agent 4'-bromoflavone were investigated in vitro using human intestinal Caco-2 cell monolayers, human liver microsomes and human hepatocytes. Liquid chromatography-mass spectrometry and tandem mass spectrometry were used for quantitative analysis in support of the Caco-2 cell studies and for the characterization of metabolites of 4'-bromoflavone. 2. The Caco-2 cell model indicated that 4'-bromoflavone would be absorbed by the intestine at a moderate rate by means of direction-independent, passive diffusion. There was no indication of active transport or efflux. 3. Three monohydroxylated metabolites and one monohydroxylated, hydrated metabolite of 4'-bromoflavone were detected at relatively low levels in the human liver microsomal and hepatocyte incubations. The structures of these metabolites were confirmed by comparison with synthetic standards. Hydroxylation occurred on the A-ring of 4'-bromoflavone but not on the B-ring, probably due to deactivation of the B-ring by bromine. No phase II metabolites were detected following incubation of 4'-bromoflavone in these in vitro systems. 4. In conclusion, these studies predict that 4'-bromoflavone should show moderate oral bioavailability, and that it would probably be excreted as unchanged compound and monohydroxylated metabolites. The results might be helpful in the design of clinical trials and in the interpretation of pharmacokinetic studies of 4'-bromoflavone.

Deguelin inhibits the growth of colon cancer cells through the induction of apoptosis and cell cycle arrest.

As previously demonstrated, deguelin [(7aS, BaS)-13, 13a-dihydro-9,10-dimethoxy-3,3-dimethyl-3H-bis[1]benzo-pyrano[3,4-b:6',5'-e]pyran-7(7aH)-one mediates anti-proliferative properties in a variety of cell types. In the present study, deguelin was found to suppress the growth of HT-29 colon cancer cells with an IC(50) of 4.32 x 10(-8) M. The cells were arrested in the G1-S-phase of the cycle. Investigations of G1/S regulatory proteins by Western blot analyses showed an upregulation of p27, and decreased expression levels of cyclin E and CDK4. Furthermore, by 24 h, exposure to deguelin resulted in an increase in the hypophosphorylated form of Rb. Since hypophosphorylated pRb binds to and inactivates E2F1, additional studies were performed and downregulation of E2F1 was observed after 24 h of treatment with deguelin. These results are consistent with the observation that deguelin arrested cells in the G1-S- phase. In addition, based on ethidium bromide/acridine orange staining, detection of digoxigenin-labelled genomic 3'-OH DNA ends, and DNA laddering, it was found that deguelin exerts its growth inhibitory effects via the induction of apoptosis. Based on these data, the potential of deguelin to serve as a cancer chemotherapeutic agent for colon cancer may be suggested.

Pharmacokinetics of deguelin, a cancer chemopreventive agent in rats.

PURPOSE: To study the pharmacokinetics of deguelin, a naturally occurring potential cancer chemopreventive agent, in rats. METHODS: [3H]Deguelin was administered intravenously (i.v.) under anesthesia, and blood samples were collected over 24 h. [3H]Deguelin and metabolites were extracted from plasma with ethyl acetate, and quantified by HPLC. Data were analyzed with the WinNolin pharmacokinetic software package to determine pharmacokinetic parameters. A three-compartment first-order elimination model was used to fit the plasma concentration-time curve. In addition, deguelin concentrations in tissues after i.v. and intragastric (i.g.) administration were determined by HPLC, and excretion (feces and urine) was evaluated over a 5-day period after i.g. administration. RESULTS: Deguelin exhibited a mean residence time (MRT) of 6.98 h and terminal half-life (t1/2(gamma)) of 9.26 h. The area under the curve (AUC) and total clearance (Cl) were 57.3 ng.h/ml and 4.37 l/h per kg, respectively, with an apparent volume of distribution (V) and volume of distribution at steady-state (Vss) of 3.421 l/kg and 30.46 l/kg, respectively. Following i.v. administration, the relative levels of tissue distribution were as follows: heart > fat > mammary gland > colon > liver > kidney > brain > lung. Following i.g. administration, the relative levels of tissue distribution were as follows: perirenal fat > heart > mammary gland > colon > kidney > liver > lung > brain > skin. Within 5 days of i.g. administration, about 58.1% of the [3H]deguelin was eliminated via the feces and 14.4% via the urine. Approximately 1.7% of unchanged deguelin was found in the feces, and 0.4% in the urine. CONCLUSIONS: An initial pharmacokinetic investigation of deguelin showed that this rotenoid has a relatively long MRT and half-life in plasma in the rat. The compound distributed in the tissues and excreted as metabolites, mainly via the feces.

Biological effects of resveratrol.

Resveratrol (3,4',5-trihydroxy-trans-stilbene) is a common phytoalexin that is found in a few edible materials, such as grape skins, peanuts, and red wine. It has been speculated that dietary resveratrol may act as an antioxidant, promote nitric oxide production, inhibit platelet aggregation, and increase high-density lipoprotein cholesterol, and thereby serve as a cardioprotective agent. Based on epidemiological data, carcinogenesis and coronary heart disease are linked to dietary lifestyle and share a number of common pathways. Recently, it has been demonstrated that resveratrol can function as a cancer chemopreventive agent, and there has been a great deal of experimental effort directed toward defining this effect. Resveratrol has been reported to be estrogenic in transfected mammary cancer cells; however, there are conflicting results with respect to its actual estrogenic properties. In addition, resveratrol exhibits antiinflammatory, neuroprotective, and antiviral properties. In future work, some controversial in vitro biological effects need to be explored in animal models, and relevant physiological and pharmacological concentrations need to be used when assessing biological activities. This review focuses on various biological aspects of resveratrol and some issues that need to be addressed to gain a fuller appreciation of potential health benefits for human beings.

Cancer chemopreventive activity mediated by 4'-bromoflavone, a potent inducer of phase II detoxification enzymes.

Induction of phase II enzymes is an important mechanism of chemoprevention. In our search for novel cancer chemopreventive agents, 4'-bromoflavone (4'BF) was found to significantly induce quinone reductase (QR) activity in cultured murine hepatoma 1c1c7 cells (concentration to double activity: 10 nM) and effectively induce the alpha- and mu-isoforms of glutathione S-transferase in cultured H4IIE rat hepatoma cells with no observed toxicity. In short-term dietary studies, 4'BF was also shown to increase QR activity and glutathione levels in rat liver, mammary gland, colon, stomach, and lung in a dose-dependent manner. Induction mediated by 4'BF was bifunctional (induction of both phase I and phase II enzymes) and regulated at the transcriptional level, as revealed by transient transfection studies with plasmid constructs (pDTD-1097CAT, XRE-CAT, and ARE-CAT) and reverse transcription-PCR-based analysis of QR mRNA. In studies conducted with female Sprague Dawley rats, the effects of 4'BF on the relative induction levels of phase I and phase II enzyme activities were investigated in liver and mammary gland. Treatment with 4'BF and 7,12-dimethylbenz[a]anthracene (DMBA) or 4'BF alone did not significantly alter DMBA-induced cytochrome P4501A1 activity (phase I enzyme), but it significantly increased QR activity (phase II enzyme), compared with the DMBA treatment group. In addition, 4'BF was found to be a potent inhibitor of cytochrome P4501A1-mediated ethoxyresorufin-O-deethylase activity, with an IC50 of 0.86 microM. Furthermore, in studies conducted with cultured HepG2 or MCF-7 cells, 4'BF significantly reduced the covalent binding of metabolically activated benzo[a]pyrene to cellular DNA. On the basis of these results, a full-term cancer chemoprevention study was conducted with DMBA-treated female Sprague Dawley rats. Dietary administration of 4'BF (2000 and 4000 mg per kg of diet, from 1 week before to 1 week after DMBA) significantly inhibited the incidence and multiplicity of mammary tumors and greatly increased tumor latency. In summary, 4'BF can be viewed as a relatively simple, readily available, inexpensive compound that is a highly effective cancer chemopreventive agent. The full mechanism of action remains to be defined, but enhancement of detoxification pathways appears to be important.

Cancer chemopreventive activity mediated by deguelin, a naturally occurring rotenoid.

Deguelin, a natural product isolated from Mundulea sericea (Leguminosae), was shown previously to mediate strong inhibition of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity in cell culture and to reduce the formation of preneoplastic lesions when mouse mammary glands were exposed to 7,12-dimethylbenz(a)anthracene. As reported currently, deguelin was synthesized and evaluated for chemopreventive activity in the two-stage 7,12-dimethylbenz(a)anthracene/TPA skin carcinogenesis model with CD-1 mice and in the N-methylnitrosourea mammary carcinogenesis model with Sprague Dawley rats. In the mouse skin study, deguelin reduced tumor incidence from 60% in the control group to 10% in the group treated with a dose of 33 microg, and multiplicity was reduced from 4.2 in the control group to 0.1 in the treatment group. When the dose was increased 10-fold to 330 microg, no tumors were observed in the treatment group. These results correlated with the potential of deguelin to inhibit TPA-induced mouse epidermal ODC activity. When applied topically as a single dose in a time range of 2 h before to 2 h after TPA treatment, deguelin (384 microg) reduced ODC induction by TPA (6.17 microg) by more than 85%. Time course studies indicated that deguelin (33 microg) inhibited TPA (1.17 microg)-induced ODC activity by 70% without affecting the kinetics of induction over a period of 10 h. Complete inhibition of ODC induction was observed at a dose of 330 microg of deguelin. In the rat mammary tumorigenesis study, intragastric administration of 2 or 4 mg of deguelin/kg of body weight daily, 5 days/week, reduced tumor multiplicity from 6.8 tumors/rat in the control group to 5.1 or 3.2 tumors/animal, respectively. At the 4 mg of deguelin/kg of body weight dose level, the tumor latency period was significantly increased. Tumor incidence, however, was unaffected. These data indicate that deguelin exhibits cancer chemopreventive effects in skin and mammary tumorigenesis models and that additional studies are warranted to characterize the cancer chemopreventive or chemotherapeutic potential of this substance more fully.

Regulation of ornithine decarboxylase induction by deguelin, a natural product cancer chemopreventive agent.

Deguelin, a plant-derived rotenoid, mediates potent chemopreventive responses through transcriptional regulation of phorbol ester-induced ornithine decarboxylase (ODC) activity. To explore the mechanism of this effect, the activity of this compound was evaluated with a number of model systems. Using cultured mouse epidermal 308 cells, the steady-state levels of both 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ODC mRNA and c-fos were decreased by treatment with deguelin. ODC activity was also inhibited by bullatacin and various antimitotic agents (podophyllotoxin, vinblastine, and colchicine), but only deguelin and bullatacin were active as inhibitors of ODC levels in a TPA-independent c-Myc-mediated induction system using cultured BALB/c c-MycER cells. These results suggest that antimicrotubule effects, as mediated by rotenone, for example, are not responsible for inhibitory activity facilitated by deguelin. This was confirmed by use of an in vitro model of tubulin polymerization in which deguelin and a variety of other rotenoids were investigated and found to be inactive. As anticipated, however, NADH dehydrogenase was inhibited by these rotenoids. Moreover, inhibition of this enzyme correlated with a rapid depletion of ATP levels and potential to inhibit either TPA- or c-Myc-induced ODC activity. It therefore seems that deguelin-mediated interference with transient requirements for elevated energy can inhibit the induction of ODC activity and thereby yield a cancer chemopreventive response.