Inhibition of p-glycoprotein activity by limonin and other secondary metabolites from citrus species in human colon and leukaemia cell lines

European Journal of Pharmacology 626 (2010) 139–145 Contents lists available at European Journal of Pharmacology Molecular and Cellular Pharmacology Inhibition of P-glycoprotein activity by limonin and other secondary metabolitesfrom Citrus species in human colon and leukaemia cell lines Mahmoud Zaki El-Readi Dalia Hamdan Nawal Farrag Assem El-Shazly Michael Wink a Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg, Germanyb Department of Pharmacognosy, Faculty of Pharmacy, Zagazig University, Zagazig 44519, Egypt P-glycoprotein (P-gp), a membrane transporter encoded by the MDR1 gene in human cells, mediates drug Received 22 July 2009 efflux from cells and plays a major role in causing multidrug resistance; which is one of the most accepted Received in revised form 2 September 2009 mechanisms for failure of chemotherapy in cancer treatment. In this study, we investigated the effects of Accepted 14 September 2009 nine naturally occurring compounds isolated from Citrus jambhiri Lush and Citrus pyriformis Hassk Available online 24 September 2009 (Rutaceae) for their potential to modulate the activity of P-gp in the multidrug-resistant human leukaemiacell line CEM/ADR5000. Limonin, deacetylnomilin, hesperidin, neohesperidin, stigmasterol and ß-sitosterol- O-glucoside inhibited the efflux of the P-gp substrate rhodamine 123 in a concentration-dependent manner.
Some of these compounds were more active than verapamil, which was used as a positive control. Treatment of drug-resistant Caco-2 cells with the most active C. jambhiri and C. pyriformis compounds increased their sensitivity to doxorubicin and completely reversed doxorubicin resistance, which agrees with a decreased P- gp activity. Limonin was the most potent P-glycoprotein inhibitor — when it was applied at a non-toxic concentration of 20 µM, it significantly enhanced doxorubicin cytotoxicity 2.98-fold (P < 0.001) and 2.2-fold(P < 0.001) in Caco2 and CEM/ADR5000 cells, respectively. These isolated Citrus compounds could beconsidered as good candidates for the development of novel P-gp/MDR1 reversal agents which may enhancethe accumulation and efficacy of chemotherapy agents.
2009 Elsevier B.V. All rights reserved.
Over-expression of multidrug resistance-proteins has been shown to cause cross-resistance to doxorubicin, Cancer is one of the most important diseases in the world, killing topotecan, mitoxantrone, methotrexate and many other chemother- about every fifth or sixth person in western countries. In 2008, it is apeutic agents (). A strategy to reverse multidrug estimated that there were over 12 million new cases of cancer resistance is to inhibit the activity of these transporters by co- diagnosed, 7 million deaths from cancer and 25 million persons alive administration of transport inhibitors together with the anticancer with cancer in the world (). Most cancer deaths arise agents. The inhibition of P-gp and other ABC transporters can increase because the tumours have metastasized and/or have become resistant the intracellular concentration of cytotoxic drugs.
to chemotherapy. When cancer patients are treated with a cytotoxic Uptake and/or efflux of isotopically-labeled drugs or rhodamine agent, the pharmacological goal is to deliver as much of an active drug 123 (Rho123) is used frequently for a functional P-gp assay in tumour as possible to the molecular target in the cancer cells and to cause cells (). Several classes of compounds that sufficient molecular damage that lead to cell death inhibit P-gp mediated efflux and enhance the accumulation and ). On the other hand, the occurrence of multiple drug resistance efficacy of anticancer compounds have been identified ( renders cells tolerant not only to the drug used in the actual ). Verapamil, phenothiazines, alkaloids like emetine and lobeline chemotherapy, but also to a broad spectrum of structurally and and other secondary functionally unrelated cytotoxic drugs as well.
metabolites, such as flavonoids like chrysin and biochanin A Drug-resistant cells often over-express P-gp; an ATP-binding ), coumarins () and terpenoids cassette (ABC) transporter (150–170 kDa), which pumps out lipo- have been reported as philic agents from cells that have entered them by free diffusion agents for overcoming multidrug resistance, and could be used aloneat very low concentrations or in combination to reverse multidrugresistance in vitro.
⁎ Corresponding author. Institut für Pharmazie und Molekulare Biotechnologie, Limonoids are typical secondary metabolites of Citrus species. The Universität Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany. Tel.: term limonoids was derived from the compound limonin +49 6221 54 4880; fax: +49 6221 54 4884.
E-mail address: (M. Wink).
This group of secondary metabolites exhibits a wide 0014-2999/$ – see front matter 2009 Elsevier B.V. All rights reserved.
doi: M.Z. El-Readi et al. / European Journal of Pharmacology 626 (2010) 139–145 range of biological properties including insecticidal, antimicrobial, polarity increased gradually by using dichloromethane followed by antimalarial, anticancer and a number of other pharmacological The pure isolated compounds were unequivocally identified using spectral data (mass spectrometry, such as EI/MS, In this study, we have investigated P-gp reversal activities of some FAB/MS and HRESI, 1H-NMR, 13C-NMR and 2D-NMR) and were secondary metabolites which were isolated from Citrus jambhiri and compared with authentic standards available at the Department of C. pyriformis by flow cytometry using Rho123 as a fluorescent P-gp Biology, IPMB, Heidelberg.
substrate in human leukaemia cells (CEM/ADR5000). We alsoinvestigated the cytotoxicity of these compounds in CEM/ADR5000 2.3. Cell culture (adriamycin resistant human leukaemia cell line, high expression ofP-gp), its parental cell line CCRF-CEM (adriamycin sensitive human Caco-2 cells were maintained in DMEM complete medium (L-glutamine, leukaemia cell line, no expression of P-gp) and Caco-2 (human colon 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, and adenocarcinoma cell line), which is used as a model for intestinal 100 µg/ml streptomycin), 1 mM sodium pyruvate and 1% non-essential epithelial cells with a relatively high expression of P-gp/MDR1. In amino acids. Human CCRF-CEM and CEM/ADR5000 leukaemia cells were addition, we investigated the ability of limonoids to increase maintained in RPMI complete medium. Cells were grown at 37 °C in a sensitivity of the resistant cells to doxorubicin and to reverse humidified atmosphere of 5% CO2. Drug-resistant CEM/ADR5000 cell lines were maintained in the absence of doxorubicin and resistance wasstabilized by 5 µg/ml doxorubicin treatment for 2 days, every 2 weeks, the 2. Materials and methods doxorubicin-resistant CEM/ADR5000 and Caco-2 cells over-expressed P-glycoprotein (P-gp) ). All experiments were performed with cells in the logarithmic growth phase.
Cell culture media, supplements and dimethyl sulphoxide (DMSO) 2.4. Rhodamine 123 efflux assay were purchased from Roth® (Karlsruhe, Germany). Other chemicalswere purchased from Sigma® (Taufkirchen, Germany) and Gibco® The activity of ABC transporters in cells can be measured by flow (Invitrogen; Karlsruhe, Germany).
cytometry using fluorescent dyes, such as Rho123, which is asubstrate for P-gp/MDR1 ( 2.2. Plant extraction and isolation Briefly, CEM/ADR5000 cells were incubated with 10 µg/ml ofRho123 in the dark on ice for 2 h. Afterwards the cells were washed The fruits of C. jambhiri Hassk and C. pyriformis Lush (Rutaceae) with fresh ice-cold medium to remove non-absorbed Rho123 and were collected in March 2004 from the Botanical Garden of the Faculty incubated with or without 32 µM of individual Citrus isolated of Agriculture in Mushtuher, Egypt. The plants were identified by compounds and verapamil as a positive control in culture medium Prof. B. Houlyel, Faculty of Agriculture, Benha University. The dried at 37 °C for 2 h. Cells were washed twice with ice-cold PBS and powdered peel of C. pyriformis (1.8 kg) and fresh peel of C. jambhiri analyzed using a FACSCalibur™ (Becton-Dickinson) equipped with a (5 kg) were exhaustively extracted with methanol to afford 508 g and 488-nm argon laser. The green fluorescence of Rho123 was measured 1750 g of total extracts after evaporation in vacuo at 40 °C, by a 530 nm band-pass filter (FL1) respectively. Both extracts were fractionated using different solvents Data were processed with the CellQuest™ software, only living starting with light petroleum (b.p. 60–80), dichloromethane and ethyl cells were taken into account. The assay was performed in the same acetate to afford 10 g, 15 g and 31 g for C. pyriformis and 20 g, 15 g and way with varying concentrations (32, 3.2, 0.32 and 0.032 µM) with 20 g for C. jambhiri crude extract, respectively.
the most active compounds to examine the dose-dependent effect of Light petroleum fraction obtained from C. jambhiri was chromato- these compounds.
graphed over silica gel column (2.5 × 150 cm, 200 g). The column wasgradiently eluted with benzene and the polarity was increased using 2.5. Cytotoxicity and cell proliferation assay chloroform and ethyl acetate. The collected fractions were monitoredby thin layer chromatography (TLC) using pre-coated silica gel GF254 Sensitivity of the cells to drugs was determined in triplicate using (Merck) and mixtures of chloroform/ethyl acetate (9:1, 8:2) for development, and the spots were visualized by spraying with 10% (MTT) cell viability assay ( H2SO4 followed by drying at 105 °C for 10 min. The column fractions Exponentially growing Caco-2 cells (2 × 104 cell/well) eluted with 20% chloroform in benzene were crystallized by were seeded in a 96-well plate Greiner Labortechnik® (Frickenhau- chloroform/methanol or chloroform/acetone to give 12 mg of white sen, Germany) and treated with various concentrations of com- amorphous powder represented as stigmasterol, a 100 mg yellowish pounds. Incubation was carried out at 37 °C for 24 h. Afterwards cells white needle-cottony shaped crystal which was identified as were incubated with 0.5 mg/ml MTT for 4 h. The formazan crystals nobiletin, 40 mg of dark yellow needle-shaped crystals of 5- were dissolved in 200 µl DMSO. The absorbance was detected at Hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone, and 25 mg of yellow 570 nm with a spectrophotometric plate reader (Tecan Safire II™ granules which was identified as 5-demethylnobiletin.
Hesperidin and neohesperidin (800 mg and 15 mg) were isolated CCRF-CEM and CEM/ADR500 cells were seeded at a density of from ethyl acetate fractions of C. jambhiri by using silica gel column 5 × 104 cells/well into 96-well plates and incubated with serial chromatography (150 × 4 cm, 300 g). The column was packed using dilutions of tested compounds. Cell viability of CCRF-CEM and CEM/ chloroform and then the polarity was increased gradually using ADR5000 was determined after 3 days using MTT.
methanol as a solvent system. Preparative thin layer chromatography(TLC) was used for further purification.
2.6. Measurement of doxorubicin cytotoxicity (reversal assay) Limonin (60 mg of white needle-shaped crystals), deacetylnomilin (40 mg of white small needle-shaped crystals) and β-sitosterol-O- Fully differentiated Caco-2 cells were seeded in a 96-well plate glucoside (75 mg of white amorphous powder) were isolated from C.
(2 × 104/well) and treated in their respective medium (100 µl) with pyriformis by using silica gel liquid column chromatography serial concentrations of doxorubicin with and without 20 µM of each (150 × 2.5 cm, 250 g) using petroleum ether as solvent with the tested compound, and incubated at 37 °C for 24 h. CEM/ADR5000 cells

M.Z. El-Readi et al. / European Journal of Pharmacology 626 (2010) 139–145 Fig. 1. Chemical structures of compounds isolated from Citrus species.
were seeded at a density of 5 × 104 cells/well into 96-well plates and Relative resistance for each tested compound was calculated using incubated with serial dilutions of doxorubicin with and without the following equation: 20 µM of limonin, and incubated for another 3 days. The metabolic Relative resistance = IC50 value obtained for the resistant cell line activity of each well was determined by the MTT assay ( (CEM/ADR5000)/IC50 value obtained for the sensitive parental cell line (CCRF -CEM).
2.7. Data analysis 3.1. Cytotoxicity and cell proliferation assay All experiments were repeated independently at least three times.
Data are presented as mean ± S.D. The statistical analysis between Six isolated Citrus compounds were tested for their growth inhibitory control and different treated groups (different compounds with activity in Caco-2, CEM/ADR5000 and CCRF-CEM leukaemia cells. The IC50 different concentrations) were calculated using one way "ANOVA" values are shown in . Neohesperidin (IC50=174.13±25.05 µM) followed by "Tukey's Multiple Comparison Test". P-values of less than and hesperidin (IC50 = 194.89 ± 43.87 µM) were the most active 0.05 were considered to represent statistical significance.
The IC50 was determined as the drug concentration resulting in a 50% reduction in cell numbers and IC50 values were calculated using a IC50 values and relative resistance of compounds isolated from Citrus in wild-type four parameter logistic curve (SigmaPlot® 11.0), and the graphs draw CCRF-CEM, multidrug-resistant CEM/ADR5000 and Caco-2 cells.
using GraphPad Prism® 5.0. Inhibition efficiency was calculated using the following equation: Inhibitory efficiency = (fluorescence intensity of citrus compound treated cells − fluorescence intensity of untreated control)/fluores- cence intensity of verapamil treated cells − fluorescence intensity of untreated control) %.
We introduced inhibition efficiency as indication of relative potency corrected by the potency of verapamil to normalize the effects of tested compounds.

M.Z. El-Readi et al. / European Journal of Pharmacology 626 (2010) 139–145 secondary metabolites in Caco-2 cells while ß-sitosterol-O-glucoside(IC50 = 133.81 ± 10.02 µM) and neohesperidin (IC50 = 168.71 ± 5.40 µM) were the most active compounds in CEM/ADR5000. Limonin minimally inhibited cell growth in all cells tested; IC50 were 519.77± 12.47 µM in Caco-2 cells, 284.77± 8.68 µM in CEM/ADR5000 and 159.44 ± 11.64 µM in CCRF-CEM cells. In general, the IC50 values weremuch lower in wild-type CCRF-CEM cells as compared to multidrug-resistant CEM/ADR500 cells.
3.2. Modulation of Rho123 efflux P-gp activity was determined by measuring the efflux of Rho123 in terms of fluorescence intensities using FACScan. P-gp over-expressingCEM/ADR5000 cells were used to determine P-gp inhibition activity of Fig. 2. Effect of 32 µM of isolated Citrus compounds on Rho123 retention in CEM/ the compounds isolated from Citrus (). The highest concen- ADR5000 cells. Rho123 fluorescence intensity was measured using FACScan. Data are tration (32 µM) for all the tested samples used in the efflux represented as means ± S.D. from three independent experiments. All tested com- experiments did not affect cell viability as it represents a non-toxic pounds show highly significantly different from the control (P < 0.001).
low concentration and short time incubation (2 h). Apparently,limonin, hesperidin, neohesperidin, deacetylnomilin, ß-sitosterol-O- glucoside and stigmasterol are P-gp substrates and inhibit Rho123 50 of doxorubicin in Caco-2 was decreased by a factor of 2.98, 2.43, 2.32 and 1.64 in combination with these compounds, respectively efflux efficiently as compared to verapamil and they have shown (indicating that the sensitivity of cells to doxorubicin is significant P-gp inhibitory activity compared with the control restored again. Reversal activity effect of the most active compound (P < 0.001) (). Rho123 retention of these compounds was limonin was confirmed in CEM/ADR5000 where IC increased 1.88, 1.64, 1.52, 1.49, 1.27 and 1.59-fold in comparison to 42.13 µM and 19.14 µM of doxorubicin, and doxorubicin with limonin, verapamil, respectively (P-gp inhibition of CEM/ADR5000 respectively. Cytotoxicity of doxorubicin was enhanced significantly cells by the tested compounds and verapamil was shown to be 2.2-fold in combination with 20 µM limonin (P < 0.001) ).
concentration-dependent (The fluorescence intensity ofRho123 was shifted rightward by these compounds in a concentra-tion-dependent manner and significant differences between each tested concentration for each compound (P < 0.001) were observed.
The histogram plots as determined by flow cytometry, suggest that Some Citrus secondary metabolites, which had been isolated from untreated cells do not retain Rho123 because P-gp can export Rho123 the aerial parts, have already been identified as potential modulators effectively. However, the fluorescence intensity signals shifted to the of P-gp with moderate activities. Previous studies were focused right field after treatment with Citrus secondary metabolites indicat- mainly on polymethoxylated flavones ing that cells now retain Rho123 ( ) and furanocoumarins ). In the presentstudy and as part of our ongoing search for natural P-gp inhibitors 3.3. Doxorubicin cytotoxicity (reversal assay) ) we examined the effects of threeother categories of natural compounds with different chemical Limonin, hesperidin, neohesperidin, deacetylnomilin, ß-sitosterol- structures and biological activities isolated from the peel of C. jambhiri O-glucoside and stigmasterol were selected as potential reversal and C. pyriformis. Bioactivity guided fractionation has led to the agents in doxorubicin insensitive Caco-2 cells. Co-incubation of isolation of 9 compounds (). Their activities on P-gp mediated doxorubicin with 20 µM of the six Citrus substances individually drug efflux were evaluated in human leukaemia cells (CEM/ADR5000) resulted in a significant increase in the cytotoxicity of doxorubicin.
Limonin, stigmasterol, ß-sitosterol-O-glucoside, and hesperidin Limonin and deacetylnomilin (limonoids), hesperidin, neohesper- showed highly significant reversal properties with P < 0.001 ().
idin (flavonoids), ß-sitosterol-O-glucoside and stigmasterol (sterols) Table 2Effect of 0.032–32 µM of individual isolated Citrus compounds and verapamil on Rho123 retention in CEM/ADR5000 cells.
Fluorescence intensity Mean ± S.D.
Data are means ± S.D. from three independent experiments.
Key: (c), (b) and (a) significantly different from the control (P < 0.05, P < 0.01 and P < 0.001) respectively.
Rho123 fluorescence intensity was measured using FACScan.
M.Z. El-Readi et al. / European Journal of Pharmacology 626 (2010) 139–145 Table 3Inhibitory efficiency of individual compounds isolated from Citrus on Rho123 efflux in CEM/ADR5000 cells related to 0.032–32 µM verapamil (positive control).
Inhibition efficiency Mean ± S.D.
Inhibitory efficiency calculated as percentage compared to verapamil (see ).
Data are means ± S.D. from three independent experiments.
Key: (c) and (a) significantly different from the control (P < 0.05 and P < 0.001), respectively.
Rho123 fluorescence intensity was measured using FACScan.
significantly reduced P-gp efflux in drug-resistant human leukaemia concentrations of limonin were achieved within 6 h and limonin could cells (CEM/ADR5000) at non-toxic concentrations (0.32–32 µM).
be detected even 24 h after consumption. The bioavailability and When tested in doxorubicin treated Caco-2 cells, these compounds persistence of limonin may help to explain why Citrus limonoids are substantially reverse doxorubicin resistance and restore doxorubicin potent long-acting anti-carcinogens preventing cancerous cells from cytotoxicity (P < 0.001) (). Limonin was the most active proliferating On the other hand, limonin can compound and significantly enhanced doxorubicin cytotoxicity in enhance glutathione-S-transferase activity and quinine reductase. It the CEM/ADR5000 cell line (P < 0.001) ).
could inhibit carcinogen-DNA adduct formation ).
A previous study had found potential chemopreventive agents in From our observations on the activities exerted by the tested orange juice that might account for the decreased colon tumour limonoids and other studies () it appears that certain incidence in patients regularly consuming orange juice rings in the limonoid skeleton may be critical for antineoplastic activity.
). In addition, other Citrus limonoids such as nomilin and Molecular changes in ring "A" can lead to a decrease or loss of anticancer obacunon could inhibit proliferation of many cancer cell lines activity (. This is the case for deacetylnomilin which shows low P- They enhance the cytotoxicity of vincristine, vinblastine gp inhibitor activity as compared to limonin with non-significant and taxol against L1210, KB-3-1 cells as well as in multidrug-resistant reversal activity (Changes in the ring "D" reveal no apparent loss KB-V1 cells. The limonoids have the advantage that they lack toxicity of biological activity. In addition, the furan ring and epoxide groups in in mammals even at higher dose so they could be used either in natural the limonoid skeleton are critical for the activity because they are able to fruit, citrus fortified with limonoids, or as purified forms of specific form covalent bonds with the active sites of proteins.
limonoids. In addition, they have the ability to induce specific Polyphenolic compounds interfere unselectively with proteins carcinogen-metabolizing enzymes ( through their phenolic hydroxyl groups that can dissociate into ) conducted absorption, metabolism and phenolate ions under physiological conditions. They form hydrogen bioavailability assays of Citrus limonoids using limonin glucoside on bonds with electronegative atoms of the peptide bonds of proteins or healthy volunteers. The results indicated that the highest plasma more stable ionic bonds with the positively charged side chains of Fig. 3. The histograms of flow cytometry shows that untreated cells (0.01% DMSO vehicle control CEM/ADR5000) do not retain Rho123 because P-gp can export Rho123 effectively.
For cells treated with different concentrations of (0.032, 0.32, 3.2 and 32 µM) limonin (A), hesperidin (B), neohesperidin (C) and stigmasterol (D), the fluorescence intensity signalsshifted to the right (pathochromic shift) indicating that cells now retain Rho123.

M.Z. El-Readi et al. / European Journal of Pharmacology 626 (2010) 139–145 Fig. 6. Dose response curve of doxorubicin effect on viability of Caco2 cell line with andwithout 20 µM of limonin using MTT assay. Doxorubicin cytotoxicity was enhanced Fig. 4. IC50 values of Caco2 cell line to doxorubicin with or without 20 µM reversal significantly 2.98-fold (P < 0.001), from IC50 3.04 µM (doxorubicin) to IC50 1.02 µM agents. Values expressed as mean ± S.D. Key: (⁎) and (⁎⁎⁎) significantly different from (doxorubicin with limonin).
the doxorubicin value (P < 0.05 and P < 0.001), respectively.
basic amino acids. These non-covalent interactions disturb the three- ing P-gp activity in comparison with verapamil (). This is in dimensional structure of proteins (conformation) and thus modulate agreement with previous studies which suggest that the number of their properties and activities. P-gp inhibition found for hesperidin methoxyl moieties is one of the determinants of P-gp-inhibitory and neohesperidin in this study may be influenced by such potency through enhancing uptake of [3H]vincristine into K562/ADM interactions ).
cells by nobiletin and other methoxyflavones ( Many glycosylated steroids, such as saponins, show anticancer, ). This could be explained by increasing the hepatoprotective, haemolytic, antiviral, secretolytic or other pharma- lipophilicity of molecules, which is one of the important factors to cological properties. The mechanism of cytotoxicity and anticancer determine the inhibitory potency on P-gp. In addition the position of activity is due to the ability of saponins to form a complex with methoxy moieties may be important in exerting the activities.
cholesterol in biomembrane, whereas the hydrophilic sugar chain The effects of hexamethoxyflavone, nobiletin and demethoxynobi- remains outside the biomembrane where it interacts with glycolipids letin on the uptake of Rho123 were weaker than that of verapamil at and glycoproteins. As a consequence, pores are formed in cell 32 µM (The reason for this discrepancy remains unclear, but it membrane bilayers, which make them leaky and which can induce is possible that hexamethoxyflavone, nobiletin and demethoxynobiletin apoptosis. In addition, the amphipathic nature of saponins can be used have additional effects that are independent of P-gp inhibition. This is in to enhance absorption of macromolecules and polar drugs through cell accordance with ) who investigated the binding site membranes. The balance between polar and non-polar functional of flavonoids on P-gp and demonstrated that flavonoids bind to vicinal groups are very important to exert their action. A hydrolysis of the ATP- and steroid-binding sites. have implied that glycoside will destroy the amphiphilic character leading to a decrease of pentamethoxyflavone is not transported by P-gp. Thus, methoxyflavone the toxicity. shows that sitosterol glucoside is more active than is considered to inhibit allosterically the function of P-gp.
the non-glucosylated stigmasterol ).
In conclusion, the present study demonstrates that some Citrus In the present study, we also assessed the effects of methoxy- compounds increase the uptake of Rho123 into multidrug resistance flavones on P-gp by measuring the potentiation of cellular Rho123 cells and exhibit multidrug-reversing effects and can be considered as accumulation at concentrations ranging between 0.032–32 µM. At good modulators of P-gp. Since they are present in a wide range of lower concentrations, some methoxyflavones were potent in revers- foods, beverages and supplements that show a low cytotoxicity, theycan be considered as promising lead compounds for the design of moreefficient multidrug resistance chemosensitizers or reversal agents.
Fig. 5. Reversal ratio as an indication of the effect of reversal agents on the cytotoxicityof doxorubicin in Caco-2 cells. 20 µM of limonin, stigmasterol, ß-sitosterol-O-glucoside, Fig. 7. Dose response curve of doxorubicin effect on viability of CEM/ADR5000 cell line hesperidin, neohesperidine, and deacetylnomilin with different concentrations of with and without 20 µM of limonin using MTT assay. Doxorubicin cytotoxicity was doxorubicin were added and these compounds significantly potentiated the cytotoxi- enhanced significantly 2.2-fold (P < 0.001), from IC50 42.13 µM (doxorubicin) to IC50 city of doxorubicin, 2.98, 2.43, 2.31, 1.63, 1.12 and 1.07-fold, respectively.
19.14 µM (doxorubicin with limonin).
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MATERIAL SAFETY DATA SHEET 1. IDENTIFICATION OF THE MATERIAL AND THE SUPPLIER Manufacturer's Product Code: 45025 250ml, 45001 1L, 45005 5L 45020 20L, 45200 200L, 45000 100L cube Dangerous Goods Class and Subsidiary Risk: None allocated Hazchem Code: None allocated Poisons Schedule Number: S5 Use: Diesel Fuel Additive Company: Fuel & Infrastructure Management Australasia Pty Ltd (ABN: 53 144 011 432)

Dementi till tidningen

Dementi till Helsingborgs Dagblad – begäran om omgående genmäle Anders Sultan kräver att tidningen går ut med en dementi samt att genmäle bereds. För det första så har Anders Sultan i Helsingborgs Dagblad utsatts för grovt förtal, då han i HD:s artikelserie utmålas som klandervärd och brottslig. För det andra så har Anders Sultan och Michael Zazzio inte fått avge sin sida av saken till tidningen. Anders Sultan nekades att få frågorna skriftligen inför intervju, samt så har journalisten ej ringt tillbaka till Michael Zazzio, talesman och styrelseordförande för Ion Silver då denne ville samtala kring den förestående artikeln. För det tredje så baseras HD:s tidningsartikel på uppgifter från en sluten facebooksida vilket strider mot de pressetiska reglerna. Anders Sultan har agerat som privatperson i ett slutet forum – och kan i detta forum enligt Europakonventionen för mänskliga rättigheter gällande yttrandefriheten – yttra precis vad han vill. Företaget Ion Silver är en egen juridisk person och kan i detta fall inte sammanblandas med privatpersonen Anders Sultan. För det fjärde så har Anders Sultan vetenskapliga belägg för sina påståenden. Till och med Anders Tegnell på Folkhälsomyndigheten sade för en och en halv vecka sedan, i ett telefonsamtal med Michael Zazzio: "Att det är känt att kolloidalt silver fungerar på de flesta virus" (lyssna på bifogad ljudfil: anders_tegnell.wav). Inte nog med att expertisen i Sverige vet detta. Det finns även ett hundratal MEDLINE-indexerade studier om kolloidalt silver och dess effekt på olika skadliga mikroorganismer såsom virus, bakterier, svamp samt encelliga organismer. Det finns även över 500 sidor användarberättelser som erfarenhetsmässigt även vittnar om positiva bieffekter i form av förbättrat hälsotillstånd när konsumenter har använt den silverhaltiga vattenreningsprodukten. Kolloidalt Silver, Ionosil säljs och marknadsförs som vattenreningsprodukt och får som sådan enligt kemikalieinspektionen ettiketteras som verksam mot mikroorganismer genom sitt innehåll av silver. För det femte så är kolloidalt silver ofarligt. Världshälsoorganisationen, WHO:s säkerhetsdata för rent silver, visar att man kan konsumera upp till 10 gram rent silver under en livstid om 70 år utan att överskrida myndighetens ‖nolleffektdos‖ – NOAEL, No Observed Adverse Effect Level – det vill säga den högsta dos som man kan exponeras för utan att riskera några som helst negativa hälsoeffekter. Det motsvarar ca 390 mikrogram per person och dag. Detta är enligt WHO en garanterat oskadlig dos för människan.