Effects of plant sterols on human multidrug transporters ABCB1 and ABCC1

The effects of dietary plant sterols on human drug efflux transporters P-glycoprotein (P-gp, ABCB1) and multidrug resistance protein 1 (MRP1, ABCC1) were investigated using P-gp-overexpressing human carcinoma KB-C2 cells and human MRP1 gene-transfected KB/MRP cells. The effects of natural phytosterols found in foods, herbs, and dietary supplements such as β-sitosterol, campesterol, stigmasterol, fucosterol, and z-guggulsterone were investigated. The accumulation of daunorubicin or rhodamine 123, fluorescent substrates of P-gp, increased in the presence of guggulsterone in KB-C2 cells. The efflux of rhodamine 123 from KB-C2 cells was inhibited by guggulsterone. Guggulsterone also increased the accumulation of calcein, a fluorescent substrate of MRP1, in KB/MRP cells. The ATPase activities of P-gp and MRP1 were stimulated by guggulsterone. These results suggest that guggulsterone, a natural dietary hypolipidemic agent have dual inhibitory effects on P-gp and MRP1 and the potencies to cause food-drug interactions.     

Energy-dependent accumulation of daunorubicin into subcellular compartments of human leukemia cells and cytoplasts.

Anthracycline accumulation was evaluated by flow cytometry or radiolabeled drug assays in cells and cytoplasts (enucleated cells) prepared from parental and multidrug-resistant human K562 leukemia cells. Treatment with energy inhibitors, such as dinitrophenol (DNP) or sodium azide/deoxyglucose, led to a marked decrease in daunorubicin accumulation in parental cells and cytoplasts. Another ionophore, monensin, also caused a significant decrease in daunorubicin accumulation; however, ATPase inhibitors ouabain, vanadate, and N-ethylamaleimide had little or no effect. The lysosomatropic agents chloroquine and methylamine caused a moderate decrease in anthracycline accumulation. Fluorescence microscopy showed that the DNP-sensitive daunorubicin uptake occurred in a nonnuclear subcellular compartment. Studies using increasing daunorubicin concentrations demonstrated fluorescence quenching that occurred in the nonnuclear, DNP-sensitive compartment. The effect of inhibitors on the accumulation of rhodamine 123 and acridine orange strongly implicated lysosomes as the principal compartment of this inhibitable daunorubicin accumulation. Cytoplasts from P-glycoprotein containing multidrug-resistant K562 cells demonstrated a verapamil-reversible, decreased daunorubicin accumulation that was observed in resistant whole cells. Verapamil pretreatment of cytoplasts from resistant cells revealed the subcellular DNP-sensitive uptake present in parental cytoplasts. These studies demonstrate that cytoplasts are an effective means to study drug transport in mammalian cells without nuclear drug binding. Parental K562 cells and cytoplasts exhibit an energy-dependent accumulation of daunorubicin into cytoplasmic organelles that is also present in resistant cells and cytoplasts when P-glycoprotein mediated efflux is inhibited.     

Understanding the molecular mechanisms of cancer prevention by dietary phytochemicals:From experimental models to clinical trials

Chemoprevention is one of the cancer prevention approaches wherein natural/synthetic agent(s) are prescribed with the aim to delay or disrupt multiple pathways and processes involved at multiple steps, i.e., initiation, promotion, and progression of cancer. Amongst environmental chemopreventive compounds, diet/beverage-derived components are under evaluation, because of their long history of exposure to humans, high tolerability, low toxicity, and reported biological activities. This compilation briefly covers and compares the available evidence on chemopreventive efficacy and probable mechanism of chemoprevention by selected dietary phytochemicals(capsaicin, curcumin, diallyl sulphide, genistein, green/black tea polyphenols, indoles, lycopene, phenethyl isocyanate, resveratrol, retinoids and tocopherols) in experimental systems and clinical trials. All the dietary phytochemicals covered in this review have demonstrated chemopreventive efficacy against spontaneous or carcinogen-induced experimental tumors and/or associated biomarkers and processes in rodents at several organ sites. The observed anti-initiating, anti-promoting and anti-progression activity of dietary phytochemicals in carcinogen-induced experimental models involve phytochemical-mediated redox changes, modulation of enzymes and signaling kinases resulting to effects on multiple genes and cell signaling pathways. Results from clinical trials using these compounds have not shown them to be chemopreventive. This may be due to our:(1) inability to reproduce the exposure conditions, i.e., levels, complexity, other host and lifestyle factors; and(2) lack of understanding about the mechanisms of action and agent-mediated toxicity in several organs and physiological processes in the host. Current research efforts in addressing the issues of exposure conditions, bioavailability, toxicity and the mode of action of dietary phytochemicals may help address the reason for observed mismatch that may ultimately lead to identification of new chemopreventive agents for protection against broad spectrum of exposures.     

6]-Gingerol inhibits metastasis of MDA-MB-231 human breast cancer cells

Gingerol (Zingiber officinale Roscoe, Zingiberaceae) is one of the most frequently and heavily consumed dietary condiments throughout the world. The oleoresin from rhizomes of ginger contains [6]-gingerol (1-[4′-hydroxy-3′-methoxyphenyl]-5-hydroxy-3-decanone) and its homologs which are pungent ingredients that have been found to possess many interesting pharmacological and physiological activities, such as anti-inflammatory, antihepatotoxic and cardiotonic effects. However, the effects of [6]-gingerol on metastatic processes in breast cancer cells are not currently well known. Therefore, in this study, we examined the effects of [6]-gingerol on adhesion, invasion, motility, activity and the amount of MMP-2 or -9 in the MDA-MB-231 human breast cancer cell line. We cultured MDA-MB-231 cells in the presence of various concentrations of [6]-gingerol (0, 2.5, 5 and 10 μM). [6]-Gingerol had no effect on cell adhesion up to 5 μM, but resulted in a 16% reduction at 10 μM. Treatment of MDA-MB-231 cells with increasing concentrations of [6]-gingerol led to a concentration-dependent decrease in cell migration and motility. The activities of MMP-2 or MMP-9 in MDA-MB-231 cells were decreased by treatment with [6]-gingerol and occurred in a dose-dependent manner. The amount of MMP-2 protein was decreased in a dose-dependent manner, although there was no change in the MMP-9 protein levels following treatment with [6]-gingerol. MMP-2 and MMP-9 mRNA expression were decreased by [6]-gingerol treatment. In conclusion, we have shown that [6]-gingerol inhibits cell adhesion, invasion, motility and activities of MMP-2 and MMP-9 in MDA-MB-231 human breast cancer cell lines.     

Microplate screening of the differential effects of test agents on Hoechst 33342, rhodamine 123, and rhodamine 6G accumulation in breast cancer cells that overexpress P-glycoprotein

A microplate screening method has been developed to evaluate the effects of test agents on the accumulation of the fluorescent P-glycoprotein (Pgp) substrates Hoechst 33342, rhodamine 123, and rhodamine 6G in multidrug-resistant (MDR) breast cancer cells that overexpress Pgp. All three substrates exhibit substantially higher accumulation in MCF7 non-MDR cells versus NCI/ADR-RES MDR cells, while incubation with 50 microM reserpine significantly reduces or eliminates these differences. Rhodamine 123 shows the lowest substrate accumulation efficiency in non-MDR cells relative to the substrate incubation level. The effects of several chemosensitizing agents and a series of paclitaxel analogs on the accumulation of each fluorescent substrate suggest that there are distinct differences in the substrate interaction profiles exhibited by these different agents. The described methods may be useful in Pgp-related research in the areas of cancer MDR, oral drug absorption, the blood-brain barrier, renal/hepatic transport processes, and drug-drug interactions.     

Diallyl sulfides in garlic activate both TRPA1 and TRPV1

We searched for novel agonists of TRP receptors especially for TRPA1 and TRPV1 in foods. We focused attention on garlic compounds, diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS). In TRPA1 or TRPV1 heterogeneously expressed CHO cells, all of those compounds increased [Ca²⁺]_i in concentration-dependent manner. The EC₅₀ values of DADS and DATS were similar to that of allyl isothiocyanate (AITC) and that of DAS was 170-fold larger than that of AITC. Maximum responses of these sulfides were equal to that of AITC. The EC₅₀ values of these compounds for TRPV1 were around 100 μM against that of capsaicin (CAP), 25.6 nM and maximum responses of garlic compounds were half to that of CAP. The Ca²⁺ responses were significantly suppressed by co-application of antagonist. We conclude that DAS, DADS, and DATS are agonist of both TRPA1 and TRPV1 but with high affinity for TRPA1.     

Stimulation of P-glycoprotein-mediated drug transport by prazosin and progesterone. Evidence for a third drug-binding site.

P-glycoprotein is a plasma membrane protein of mammalian cells that confers multidrug resistance by acting as a broad-specificity, ATP-dependent efflux transporter of diverse lipophilic neutral or cationic compounds. Previously, we identified two positively cooperative drug-binding sites of P-glycoprotein involved in transport [Shapiro, A. B. & Ling, V. (1997) Eur. J. Biochem. 250, 130-137]. The H site is selective for Hoechst 33342 and colchicine. The R site is selective for rhodamine 123 and anthracyclines. Substrate binding to one site stimulates transport by the other. In this paper, we show that prazosin and progesterone stimulate the transport of both Hoechst 33342 and rhodamine 123. Rhodamine 123 and prazosin (or progesterone) in combination stimulate Hoechst 33342 transport in an additive manner. In contrast, Hoechst 33342 and either prazosin or progesterone interfere with each other, so that the stimulatory effect of the combination on rhodamine 123 transport is less than that of each individually. Non-P-glycoprotein-specific effects of prazosin on membrane fluidity and permeability were excluded. These results indicate the existence of a third drug-binding site on P-glycoprotein with a positive allosteric effect on drug transport by the H and R sites. This allosteric site appears to be one of the sites of photoaffinity labeling of P-glycoprotein by [125I]iodoarylazidoprazosin [Safa, A. R., Agresti, M., Bryk, D. & Tamai, I. (1994) Biochemistry 33, 256-265] and is likely not to be capable of drug transport.     

Cytoplasmic orientation and two-domain structure of the multidrug transporter, P-glycoprotein, demonstrated with sequence-specific antibodies

The predicted cytoplasmic orientation and two-domain structure of the multidrug efflux pump P-glycoprotein were demonstrated with sequence-specific antibodies. We synthesized peptides corresponding to amino acid residues, Glu393-Lys408 (anti-P) and Leu1206-Thr1226 (anti-C) in P-glycoprotein from human mdr1 cDNA and used these peptides to produce polyclonal antibodies. From the primary structure of P-glycoprotein, and anti-C antibody is expected to recognize another position, Leu561-Thr581, in the duplicate structure of P-glycoprotein, but anti-P recognizes only one site. These antibodies bind to multidrug-resistant cells (KB-C2) with permeabilized plasma membrane but do not bind to nonpermeabilized KB-C2 cells or parental KB cells, supporting the predicted cytoplasmic orientation of these sequences. With immunoblotting of the membrane fractions from KB-C2 cells, a major 140-kDa polypeptide of the P-glycoprotein was detected with both anti-P and anti-C. Two minor polypeptides with molecular mass of 95 and 55 kDa were also detected. When membrane vesicles were digested mildly with trypsin, the amount of these two polypeptides increased. Anti-P detected only the 95-kDa polypeptide, and anti-C detected both 95- and 55-kDa polypeptides. Achromobacter lyticus protease I (lysyl endopeptidase) and Staphylococcus aureus V8 protease also produced two polypeptides with similar molecular weights. Absorption into lectin-agarose beads and labeling with [3H]glucosamine indicated that the 95-kDa polypeptide was glycosylated but that the 55-kDa polypeptide was not. These two polypeptides as well as P-glycoprotein were photoaffinity-labeled with a calcium channel blocker, [3H]azidopine, but most of the label was found in the 55-kDa polypeptide. The yield of labeled fragments from membrane vesicles photolabeled after digestion with trypsin was similar to that from membrane vesicles digested with trypsin after photolabeling. These data indicate 1) that the 95-kDa polypeptide is the fragment corresponding to the amino-terminal half of P-glycoprotein containing sugar chains; 2) that the 55-kDa polypeptide is the carboxyl-terminal half which was mainly labeled with [3H]azidopine; and 3) that P-glycoprotein has a relatively rigid structure with a small number of protease-sensitive sites and its global structure is not destroyed by tryptic cleavage.     

Analogs of a 4-aminothieno[2,3-d]pyrimidine lead (QB13) as modulators of P-glycoprotein substrate specificity

P-glycoprotein (P-gp) is an important factor in the development of multidrug resistance (MDR) in cancer cells. In literature reports, a thieno[2,3-d]pyrimidine (QB13) was described as P-gp modulator and opposed effects on the cell accumulation of distinct P-gp substrates were postulated. On the basis of this lead structure, a series of 2-alkylthio-4-aminothieno[2,3-d]pyrimidines was prepared and tested in a daunorubicin accumulation assay. Modulation of substrate specificity was shown for selected compounds in cytotoxicity (MTT) assays.     

N′-Isopropylnornicotine in Burley Tobacco (Nicotiana tabacum)

Allyl sulfides such as diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), typical flavor components of Allium vegetables, have been shown to inhibit benzo[a]pyrene (B[a]P)-induced carcinogenesis in animal models. As a possible mechanism of this inhibition, the effect of these volatile substances on cytochrome P450 (CYP)1 (CYP1A1, 1A2 and 1B1)-mediated bioactivation of B[a]P was investigated using a human hepatoma cell model (HepG2). DADS and DATS inhibited the B[a]P-induced ethoxyresorufin O-deethylase (EROD) activity, a marker enzyme for CYP1, by 30-90% and 70-95% at 100-1,000 μM concentration, respectively. The cell viability, an indicator of the capacity to inhibit B[a]P bioactivation, was increased by treatments of 100-1,000 μM DADS and 10-100 μM DATS. Immunoblot results indicated that the B[a]P inducible CYP1A2 protein was suppressed by 100-1,000 μM of DADS and 10-100 μM of DATS, but CYP1A1 and 1B1 were not detectable in any microsomes. Analysis of B[a]P metabolites revealed that the level of 7,8-diol formed was significantly reduced in the DADS and DATS treated microsomes as compared to the control. The level of 9,10-diol and 4,5-diol formed was also lowered by the allyl sulfide treatments. These results suggest that the protective mechanism of allyl sulfides on B[a]P-induced carcinogenesis is possibly related with the modulation of CYP1-mediated bioactivation of B[a]P.     

Modulation of cytochrome P4501-mediated bioactivation of benzo[a]pyrene by volatile allyl sulfides in human hepatoma cells.

Allyl sulfides such as diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), typical flavor components of Allium vegetables, have been shown to inhibit benzo[a]pyrene (B[a]P)-induced carcinogenesis in animal models. As a possible mechanism of this inhibition, the effect of these volatile substances on cytochrome P450 (CYP)1 (CYP1A1, 1A2 and 1B1)-mediated bioactivation of B[a]P was investigated using a human hepatoma cell model (HepG2). DADS and DATS inhibited the B[a]P-induced ethoxyresorufin O-deethylase (EROD) activity, a marker enzyme for CYP1, by 30-90% and 70-95% at 100-1,000 microM concentration, respectively. The cell viability, an indicator of the capacity to inhibit B[a]P bioactivation, was increased by treatments of 100-1,000 microM DADS and 10-100 microM DATS. Immunoblot results indicated that the B[a]P inducible CYP1A2 protein was suppressed by 100-1,000 microM of DADS and 10-100 microM of DATS, but CYP1A1 and 1B1 were not detectable in any microsomes. Analysis of B[a]P metabolites revealed that the level of 7,8-diol formed was significantly reduced in the DADS and DATS treated microsomes as compared to the control. The level of 9,10-diol and 4,5-diol formed was also lowered by the allyl sulfide treatments. These results suggest that the protective mechanism of allyl sulfides on B[a]P-induced carcinogenesis is possibly related with the modulation of CYP1-mediated bioactivation of B[a]P.     

Nutritional antioxidants and adaptive cell responses: an update

Many plant antioxidants, intaken through the daily diet or plant-derived dietary supplements, have been shown able to prevent free radical-related diseases by counteracting cell oxidative stress. However, it is now considered that the in vivo beneficial effects of these phytochemicals are unlikely to be explained just by their antioxidant capability. Several plant antioxidants exhibit hormetic properties, by acting as 'low-dose stressors' that may prepare cells to resist more severe stress. In fact, low doses of these phytochemicals activate cell signaling pathways (being the most prominent examples the modulation of the Nrf2/Keap1 pathway, the NF-κB pathway and the Sirtuin-FOXO pathway) but high doses are cytotoxic. Herein we review the adaptive responses induced by the most known plant hormetic antioxidants, which are sulforaphane, resveratrol, curcumin, flavonoids, green tea catechins and diallylsulphides, as well as the molecular mechanisms involved in such responses. Furthermore, this review outlines that the hormetic properties of these bioactive plant antioxidants might be successfully employed for realizing health-promoting dietary interventions especially in the field of neurodegenerative diseases and cancer.     

Antithrombotic and anticancer effects of garlic-derived sulfur compounds: a review

Garlic (Allium sativum L.) has a long history as being a food having a unique taste and odor along with some medicinal qualities. Modern scientific research has revealed that the wide variety of dietary and medicinal functions of garlic can be attributed to the sulfur compounds present in or generated from garlic. Although garlic produces more than 20 kinds of sulfide compounds from a few sulfur-containing amino acids, their functions are different from one another; e.g., allicin, methyl allyl trisulfide, and diallyl trisulfide have antibacterial, antithrombotic, and anticancer activities, respectively. The present paper reviews the physiological functions of garlic in the limited study fields of its antithrombotic and anticancer effects. Before describing these effects, however, we will discuss briefly some characteristics of garlic as a plant and some modes of absorption of orally-administered sulfur compounds from garlic.     

Signal transduction pathways leading to cell cycle arrest and apoptosis induction in cancer cells by Allium vegetable-derived organosulfur compounds: a review

Epidemiological studies continue to support the premise that dietary intake of Allium vegetables (e.g., garlic, onions and so forth) may lower the risk of various types of cancer. Anticarcinogenic effect of Allium vegetables is attributed to organosulfur compounds (OSCs) that are generated upon processing of these vegetables. Preclinical studies have provided convincing evidence to indicate that Allium vegetable-derived OSCs including diallyl sulfide, diallyl disulfide and diallyl trisulfide are highly effective in affording protection against cancer in laboratory animals induced by a variety of chemical carcinogens. Inhibition of carcinogen activation through modulation of cytochrome P450-dependent monooxygenases and/or acceleration of carcinogen detoxification via induction of phase II enzymes (glutathione transferases, quinone reductase, etc.) are believed to be responsible for protective effects of OSCs against chemically induced cancers. More recent studies have indicated that some naturally occurring OSC analogues can suppress proliferation of cancer cells in culture and inhibit growth of transplanted tumor xenografts in vivo by inducing apoptosis and/or by perturbing cell cycle progression. This review summarizes current knowledge on signal transduction pathways leading to perturbations in cell cycle progression and apoptosis induction by OSCs.     

6]-Gingerol prevents UVB-induced ROS production and COX-2 expression in vitro and in vivo

[6]-Gingerol, a naturally occurring plant phenol, is one of the major components of fresh ginger (Zingiber officinale Roscoe, Zingiberaceae) and has diverse pharmacologic effects. Here, we describe its novel anti-oxidant, anti-apoptotic, and anti-inflammatory activities in vitro and in vivo. In vitro, pre-treatment with [6]-gingerol reduced UVB-induced intracellular reactive oxygen species levels, activation of caspase-3, -8, -9, and Fas expression. It also reduced UVB-induced expression and transactivation of COX-2. Translocation of NF-kappaB from cytosol to nucleus in HaCaT cells was inhibited by [6]-gingerol via suppression of IkappaBalpha phosphorylation (ser-32). Examination by EMSAs and immunohistochemistry showed that topical application of [6]-gingerol (30 microM) prior to UVB irradiation (5 kJ/m(2)) of hairless mice, also inhibited the induction of COX-2 mRNA and protein, as well as NF-kappaB translocation. These results suggest that [6]-gingerol could be an effective therapeutic agent providing protection against UVB-induced skin disorders.     

Inhibitory effect of gingerol on the proliferation and invasion of hepatoma cells in culture

Effect of [6]-gingerol, a major pungent component in ginger, on the proliferation of a rat ascites hepatoma AH109A cells was investigated by measuring [³H]thymidine incorporation into acid-insoluble fraction of the cultured cells and that on the invasion by co-culturing the hepatoma cells with rat mesentery-derived mesothelial cells. [6]-Gingerol inhibited both the proliferation and invasion of hepatoma cells in a dose-dependent manner at concentrations of 6.25–200 μM (proliferation) and 50–200 μM (invasion). [6]-Gingerol accumulated cells in S phase and elongated doubling time of hepatoma cells, and increased the rate of apoptosis. Hepatoma cells previously cultured with hypoxanthine (HX) and xanthine oxidase (XO) or with hydrogen peroxide showed increased invasive activities. [6]-Gingerol suppressed the reactive oxygen species-potentiated invasive capacity by simultaneously treating AH109A cells with [6]-gingerol, HX and XO or with [6]-gingerol and hydrogen peroxide. Furthermore, [6]-gingerol reduced the intracellular peroxide levels in AH109A cells. These results suggest that the suppression of hepatoma cell proliferation by [6]-gingerol may be due to cell cycle arrest and apoptosis induction. They also suggest that the anti-oxidative property of [6]-gingerol may be involved in its anti-invasive activity of hepatoma cells.     

Hypolipidemic and antioxidant effects of curcumin and capsaicin in high-fat-fed rats

The beneficial hypolipidemic and antioxidant influences of the dietary spice compounds curcumin and capsaicin were evaluated. Curcumin, capsaicin, or their combination were included in the diet of high-(30%)-fat-fed rats for 8 weeks. Dietary high-fat-induced hypertriglyceridemia was countered by dietary curcumin, capsaicin, or their combination by 12%-20%. Curcumin, capsaicin, and their combination also produced a slight decrease in serum total cholesterol in these animals. Serum alpha-tocopherol content was increased by dietary curcumin, capsaicin, and their combination in high-fat-fed rats. Serum total thiol content in high-fat-fed animals and serum ascorbic acid in normal animals was elevated by the combination of curcumin and capsaicin. Hepatic glutathione was increased by curcumin, capsaicin, or their combination in normal animals. Hepatic glutathione and alpha-tocopherol were increased, whereas lipid peroxide level was reduced by dietary curcumin and combination of curcumin and capsaicin in high-fat-fed animals. Serum glutathione peroxidase and glutathione transferase in high-fat-fed rats were generally higher as a result of dietary curcumin, capsaicin, and the combination of curcumin and capsaicin. Hepatic glutathione reductase and glutathione peroxidase were significantly elevated by dietary spice principles in high-fat-fed animals. The additive effect of the 2 bioactive compounds was generally not evident with respect to hypolipidemic or antioxidant potential. However, the effectiveness of the combination was higher in a few instances.