Tomohiro Nabekura

Inhibition of P-glycoprotein function by tea catechins in KB-C2 cells

We studied the effects of tea catechins, (‐)‐epicatechin (EC), (‐)‐epigallocatechin (EGC), (‐)‐epicatechin gallate (ECG), and (‐)‐epigallocatechin gallate (EGCG) on the P‐glycoprotein (P‐gp) function in multidrug‐resistant P‐gp over‐expressing KB‐C2 cells. EC did not have any effects on cellular accumulation of P‐gp substrates, rhodamine‐123 and daunorubicin, but the other catechins increased the accumulation in the order of EGC

Inhibition of P-glycoprotein and multidrug resistance protein 1 by dietary phytochemicals.

PurposeFor the development of a safe and effective dual inhibitor of anticancer drug efflux transporters P-glycoprotein and multidrug resistance protein 1 (MRP1) to conquer multidrug resistance, we investigated the effects of dietary phytochemicals on the functions of P-glycoprotein and MRP1.MethodsThe effects of dietary phytochemicals on the functions of P-glycoprotein and MRP1 were investigated using P-glycoprotein-overexpressing human carcinoma KB-C2 cells and human MRP1 gene-transfected KB/MRP cells. The effects of natural compounds found in dietary supplements, herbs, and foods such as sesame, ginkgo, soybean, and licorice were evaluated.ResultsThe accumulation of daunorubicin, a fluorescent substrate of P-glycoprotein, increased in the presence of sesamin, ginkgolic acid, matairesinol, glycyrrhetinic acid, glabridin, and phyllodulcin in KB-C2 cells. Glycyrrhetinic acid and matairesinol also increased the accumulation of calcein, a fluorescent substrate of MRP1, in KB/MRP cells. KB-C2 and KB/MRP cells were sensitized to anticancer drugs by glycyrrhetinic acid, showing that glycyrrhetinic acid reverses multidrug resistance. The verapamil-stimulated P-glycoprotein ATPase activity was inhibited by glycyrrhetinic acid. Glycyrrhetinic acid stimulated the ATPase activity of MRP1.ConclusionThese results suggest that dietary phytochemicals, such as glycyrrhetinic acid found in licorice, have dual inhibitory effects on P-glycoprotein and MRP1 and might become useful to enhance the efficacy of cancer chemotherapy.

Inhibition of anticancer drug efflux transporter P-glycoprotein by rosemary phytochemicals

The effects of dietary antioxidative and chemopreventive rosemary phytochemicals on the function of the human drug efflux transporter P-glycoprotein (MDR1, ABCB1) and multidrug resistance protein 1 (MRP1, ABCC1) were investigated using P-glycoprotein-overexpressing human carcinoma KB-C2 cells and human MRP1 gene-transfected KB/MRP cells. The effects of natural phytochemicals found in rosemary such as carnosic acid, carnosol, rosmarinic acid, and ursolic acid were investigated. The accumulation of daunorubicin or rhodamine 123, fluorescent substrates of P-glycoprotein, in KB-C2 cells increased in the presence of carnosic acid, carnosol, and ursolic acid in a concentration-dependent manner. In contrast, carnosic acid, carnosol, rosmarinic acid, and ursolic acid had no effects on the accumulation of calcein, a fluorescent substrate of MRP1, in KB/MRP cells. The ATPase activities of P-glycoprotein were stimulated by carnosic acid, carnosol, and ursolic acid. KB-C2 cells were sensitized to vinblastine cytotoxicity by carnosic acid, showing that carnosic acid reverses multidrug resistance. These results suggest that rosemary phytochemicals, such as carnosic acid, have inhibitory effects on anticancer drug efflux transporter P-glycoprotein and may become useful to enhance the efficacy of cancer chemotherapy.

Nitric oxide participates in cataract development in selenite-treated rats

Purpose. The role of nitric oxide in the development of selenite-induced cataracts in rats was examined using nitric oxide synthase (NOS) inhibitors. Methods. Subcutaneous injection of sodium selenite was used to induce cataracts in rats, with or without pretreatment with NOS inhibitors. The anterior eye segment analysis system (EAS-1000, Nidek) was used to measure lens opacity. The glutathione content of the lenses was determined by an HPLC method and the Ca 2+ content by atomic absorption spectrometry. Nitrite, a stable metabolite of nitric oxide, was determined fluorometrically. NADPH-diaphorase activity staining and Western blot analysis were used to determine NOS levels. Results. Administration of the NOS inhibitor, N G -nitro-l-arginine methyl ester (l-NAME), inhibited lens opacification in selenite-treated rats. N G -nitro-d-arginine methyl ester, an inactive enantiomer of l-NAME, had no effect. Aminoguanidine, another NOS inhibitor, also inhibited the development of cataracts in a dose-dependent manner. On the other hand, l-arginine, a substrate of NOS, accelerated the development of cataracts. Although the opacification of the lenses was apparent approximately 3 days after selenite injection, the nitrite level was increased within one day. In addition, NOS was induced in the eye within one day of selenite injection. Conclusions. The present study demonstrated that NOS inhibitors prevented the development of cataracts in selenite-treated rats. The results also suggest that nitric oxide had an important role in the development of selenite-induced cataracts.

Delay of cataract development in hereditary cataract UPL rats by disulfiram and aminoguanidine

The UPL rat is a newly developed hereditary cataract model. We previously found that the administration of disulfiram, a dimer of diethyldithiocarbamate that possesses antioxidant activity, and aminoguanidine, which is known to inhibit inducible nitric oxide synthase, inhibits cataract development in selenite-induced cataract rats. In this study, we investigated the anti-cataract effects and mechanism of disulfiram and aminoguanidine on UPL rats. The opacities of UPL rat lenses, as documented by the anterior eye segment analysis system, EAS-1000 (Nidek, Aichi, Japan), increased from 39 days, and apparently mature cataracts were observed at 53 days. Accompanied with the increase in lens opacity, glutathione concentrations in UPL rat lenses decreased. The Na(+) to K(+) and water-insoluble to water-soluble protein ratios, as well as the Ca(2+) contents in UPL rat lenses increased with the development of cataracts. Oral administration of disulfiram and aminoguanidine delayed the lens opacification as well as the changes in glutathione, Na(+) to K(+) ratio, water-insoluble to soluble protein ratio, and Ca(2+) content in UPL rat lenses. The opacity and Ca(2+) content of UPL rat lenses were closely associated. The present study demonstrates that disulfiram and aminoguanidine have potency of the delay of cataract development in UPL rats, probably caused by inhibiting the rise in Ca(2+) levels.

Overcoming Multidrug Resistance in Human Cancer Cells by Natural Compounds

Multidrug resistance is a phenomenon whereby tumors become resistant to structurally unrelated anticancer drugs. P-glycoprotein belongs to the large ATP-binding cassette (ABC) transporter superfamily of membrane transport proteins. P-glycoprotein mediates resistance to various classes of anticancer drugs including vinblastine, daunorubicin, and paclitaxel, by actively extruding the drugs from the cells. The quest for inhibitors of anticancer drug efflux transporters has uncovered natural compounds, including (-)-epigallocatechin gallate, curcumin, capsaicin, and guggulsterone, as promising candidates. In this review, studies on the effects of natural compounds on P-glycoprotein and anticancer drug efflux transporters are summarized.

Effects of chemopreventive citrus phytochemicals on human P-glycoprotein and multidrug resistance protein 1.

The effects of dietary chemopreventive citrus phytochemicals on the drug efflux transporters P-glycoprotein (ABCB1) and multidrug resistance protein 1 (MRP1, ABCC1) were investigated using P-glycoprotein-overexpressing human carcinoma KB-C2 cells and human MRP1 gene-transfected KB/MRP cells. The effects of natural chemopreventive citrus phytochemicals, such as auraptene, nobiletin, citral, citronellal, limonene, limonin, and synephrine were examined. The accumulation of daunorubicin, a fluorescent substrate of P-glycoprotein, increased in the presence of auraptene and nobiletin in KB-C2 cells. Nobiletin also increased the accumulation of calcein, a fluorescent substrate of MRP1, in KB/MRP cells. The ATPase activity of P-glycoprotein was stimulated by auraptene and nobiletin. The ATPase activity of MRP1 was stimulated by nobiletin. These results suggest that chemopreventive citrus phytochemicals, such as nobiletin found in oranges, have inhibitory effects on P-glycoprotein and/or MRP1, and may cause food-drug interactions.

Bioavailability and anticataract effects of a topical ocular drug delivery system containing disulfiram and hydroxypropyl-beta-cyclodextrin on selenite-treated rats.

Purpose. To test the effect of aqueous eye drops containing a high concentration of disulfiram (DSF) in a cyclodextrin-based drug delivery system. This system increases both the drug solubility in aqueous eye drops and the permeability of drug into the rabbit eye, by the formation of a drug-cyclodextrin inclusion complex, and so enhances the ocular bioavailability and anti-cataract effect of DSF. Methods. The DSF and hydroxypropyl-β-cyclodextrin (HPβCD) inclusion (DSF/HPβCD) was studied using solubility methods, IR spectra and X-ray diffraction patterns. Suitable formulations for DSF eye drops were first identified by a trans-corneal penetration experiment in vitro. Finding a new p-bromophenacyl bromide (p-BPB) derivative reagent for diethyldithiocarbamic acid (DDC), which was a metabolite of DSF, allowed precise determination of the contents of DSF in aqueous humor. The ocular bioavailability was calculated by a transcorneal experiment of DSF in vivo. The lens opacity of a selenite-induced cataract in rat pups was monitored using a slit lamp with an anterior eye segment analysis system. Results. The formation of DSF/HPβCD inclusion and the addition of hydroxypropylmethylcellulose (HPMC), as a penetration enhancer, played very important roles in increasing the ocular bioavailability of DSF. DSF eye drops, with a formulation of 1.26% (w/v) DSF/HPβCD inclusion, 0.01% (w/v) HPMC, 0.005% (w/v) benzalkonium chloride and 0.9% (w/v) sodium chloride, inhibited the onset of selenite-induced cataracts effectively. Conclusions. The cyclodextrin-based drug delivery system enhances both the solubility of DSF in aqueous eye drops and permeability of the drug into the rabbit eye. DSF ocular bioavailability in rabbit aqueous humor exceeded those reported for the DSF ophthalmic preparation. DSF eye drops effectively prevent the development of selenite-induced cataracts.

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 beta-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.

Effects of natural nuclear factor-kappa B inhibitors on anticancer drug efflux transporter human P-glycoprotein.

Drug efflux transporter P-glycoprotein plays an important role in cancer chemotherapy. The nuclear factor-κB (NF-κB) transcription factors play critical roles in development and progression of cancer. In this study, the effects of natural compounds that can inhibit NF-κB activation on the function of P-glycoprotein were investigated using human MDR1 gene-transfected KB/MDR1 cells. The accumulation of daunorubicin or rhodamine 123, fluorescent substrates of P-glycoprotein, in KB/MDR1 cells increased in the presence of caffeic acid phenetyl ester (CAPE), licochalcone A, anacardic acid, celastrol, xanthohumol, magnolol, and honokiol in a concentration-dependent manner. In contrast, lupeol, zerumbone, thymoquinone, emodin, and anethol had no effects. The ATPase activities of P-glycoprotein were stimulated by CAPE, licochalcone A, anacardic acid, celastrol, xanthohumol, magnolol, and honokiol. Tumor necrosis factor (TNF)-α stimulated NF-κB activation was inhibited by CAPE, licochalcone A, anacardic acid, and xanthohumol. KB/MDR1 cells were sensitized to vinblastine cytotoxicity by CAPE, licochalcone A, anacardic acid, xanthohumol, magnolol, and honokiol, showing that these natural NF-κB inhibitors reverse multidrug resistance. These results suggest that natural compounds, such as CAPE, licochalcone A, and anacardic acid, have dual inhibitory effects on the anticancer drug efflux transporter P-glycoprotein and NF-κB activation, and may become useful to enhance the efficacy of cancer chemotherapy.