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Influence of combinations of digitonin with selected phenolics, terpenoids, and alkaloids on the expression and activity of P-glycoprotein in leukaemia and colon cancer cells.


Article history:

Received 3 December 2012

Received in revised form 2 June 2013

Accepted 26 July 2013





Multidrug resistance

Colon cells



P-glycoprotein (P-gp or MDR1) is an ATP-binding cassette (ABC) transporter. It is involved in the efflux of several anticancer drugs, which leads to chemotherapy failure and multidrug resistance (MDR) in cancer cells. Representative secondary metabolites (SM) including phenolics (EGCG and thymol), terpenoicls (menthol. aromadendrene,13-sitosterol-0-glucoside, and 13-carotene), and alkaloids (glaucine, harmine, and sanguinarine) were evaluated as potential P-gp inhibitors (transporter activity and expression level) in P-gp expressing Caco-2 and CEM/ADR5000 cancer cell lines. Selected SM increased the accumulation of the rhodamine 123 (Rhol 23) and calcein-AM (CAM) in a dose dependent manner in Caco-2 cells, indicating that they act as competitive inhibitors of P-gp. Non-toxic concentrations of [beta]-carotene (40 [micro]M) and sanguinarine (1 [micro]M) significantly inhibited Rho 123 and CAM efflux in CEM/ADR5000 cells by 222.42% and 259.25% and by 244.02% and 290.16%, respectively relative to verapamil (100%). Combination of the saponin digitonin (5 [micro]M), which also inhibits P-gp, with SM significantly enhanced the inhibition of P-gp activity. The results were correlated with the data obtained from a quantitative analysis of MDR1 expression. Both compounds significantly decreased mRNA levels of the MDR1 gene to 48% (p < 0.01) and 46% (p < 0.01) in Caco-2, and to 61% (p < 0.05) and 1% (p < 0.001) in CEM/ADR5000 cells, respectively as compared to the untreated control (100%). Combinations of digitonin with SM resulted in a significant down-regulation of MDR1. Our findings provide evidence that the selected SM interfere directly and/or indirectly with P-gp function. Combinations of different P-gp substrates, such as digitonin alone and together with the set of SM, can mediate MDR reversal in cancer cells.

[c] 2013 Elsevier GmbH. All rights reserved.


ATP-binding cassette (ABC) transporters are transmembrane proteins, which can transport a wide variety of compounds across cell membranes utilizing the energy from ATP hydrolysis. P-glycoprotein (P-gp or MDR1) is a member of the ABC transporter subfamily B and encoded by the ABCB 1 gene. It plays a crucial role in the development of multidrug resistance (MDR) by effluxing therapeutical agents out of cancer cells (Gottesman et al. 2002). An overexpression of P-gp has been frequently observed in drug resistant tumour cells and has been proposed as a cause for the failure of a broad range of anti-cancer drugs (Dean et al. 2001). Most of the commonly used chemotherapeutic agents are P-gp substrates which carry a positive charge (or are neutral) at physiological pH, are cyclic and hydrophobic molecules, such as anthracyclines (e.g. cloxorubicin), Vinca alkaloids (e.g. vinblastine), epipodophyllotoxins (e.g. etoposide), and taxanes (e.g. paclitaxel) (Sauna et al. 2007). Substances that completely, or partly, inhibit P-gp function and/or expression may prevent the undesirable efflux of anticancer agents from MDR cancer cells. Combining P-gp inhibitors with chemotherapeutical agents might be a strategy to counteract MDR in cancer cells (Wink et al. 2012).

There have been several attempts to overcome MDR by developing effective P-gp inhibitors. Calcium channel blockers (verapamil), calmodulin inhibitors (phenothiazines), nonpolar cyclic oligopeptides with immunosuppressant activity (cyclosporin A and the cyclosporin D analogue Valspodar PSC 833) have been identified as P-gp inhibitors (Tan et al. 2000). These inhibitors can successfully reverse MDR in vitro, but their efficacy in animal studies and clinical trials has been disappointing because of their close-limiting toxicity (Anuchapreeda et al. 2002).

A number of plant secondary metabolites (SM) have been identified, which inhibit P-gp, reverse MDR, and sensitize cancer cells to chemotherapy without or fewer undesired toxic effects (Wink et al. 2012). These findings suggest that plant SM provide a diversity of promising MDR reversal agents.

In this study, we investigated the potential inhibition of P-gp by representative SM from different chemical classes including phenolics (EGCG and thymol), terpenoids (menthol, aromaden-drene, [beta]-sitosterol-O-glucoside, and [bata]-carotene), and alkaloids (glaucine, harmine, and sanguinarine) (Fig. 1). Our previous results indicated that this set of SM, either alone or in two (Eid et al. 2012b) or three drug combinations, enhanced the sensitivity of MDR cancer cells to doxorubicin (Eid et al. 2012b,c). To understand the possible underlying mechanisms, we have analyzed in this study whether this set of SM interferes with P-gp in MDR cancer cells. The intracellular accumulation of rhodamine 123 (Rho123) and calcein-AM (CAM) (which are used frequently in functional assays of P-gp) in the presence or absence of SM was examined in colon cancer (Caco-2) and leukaemia (CEM/ADR5000) cells, which overexpress P-gp, and in CCRF-CEM cells from which CEM/ADR5000 had been selected. The possibility to down-regulate P-gp expression in these cancer cell lines was also investigated. Furthermore, we explored whether combinations of two P-gp substrates (such as the saponin digitonin plus a given SM) could inhibit or down-regulate P-gp in a synergistic fashion.

Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were applied on our data sets in order to reduce dimensionality and evaluate which variables were more effective for classifying the selected SM according to their degree of MDR inhibitory activity. These analyses showed that the variables; RR (the reversal ratio of doxorubicin cytotoxicity) (Eid et al. 2012b) and 1E (inhibitory activity of P-gp efflux; this study) in different resistant cells are able to distinguish moderate and strong MDR inhibitors.

Materials and methods Chemicals and reagents Digitonin ([greater than or equal to]98%), epigallocatechin gallate EGCG (99.5%), menthol [greater than or equal to]99%), thymol ([greater than or equal to]99.5%), aromadendrene ([greater than or equal to]97%), 13-carotene ([greater than or equal to]99%), glaucine ([greater than or equal to]98%), harmine ([greater than or equal to]95%), sanguinarine ([greater than or equal to]97%), Rhol 23, CAM, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MU; [greater than or equal to]98%) were purchased from Sigma--Aldrich GmbH, Germany. [bata]-Sitosterol-0-glucoside ([greater than or equal to]95%) was isolated from Citrus spec. and characterized by Dr. D. Ham-clan (IPMB, Heidelberg University). DMEM and RPMI1640 media, non-essential amino acid (NEAA), pyruvate, penicillin, streptomycin, foetal bovine serum (FBS), trypsin-EDTA, L-glutamine, and dimethyl sulfoxide (DMSO) were purchased from Gibco[R] lnvitro-gen; Germany.

Cell lines

Cell culture experiments were performed with Caco-2 (human colorectal adenocarcinoma) cells, obtained from DSMZ (German Collection of Microorganisms and Cell Cultures), CCRF-CEM and CEM/ADR5000 adriamycin resistant leukaemia cells, obtained from Prof. T. Efferth, (Department of Pharmaceutical Biology, University of Mainz, Germany). Caco-2 cells were cultivated in complete DMEM medium with Glutamax (Invitrogen/Gibco, Karlsruhe, Germany) supplemented with 10% FBS (BioChrom KG, Berlin, Germany), 100 U/ml penicillin, and 100 [micro]g/ml streptomycin, sodium pyruvate (1%), and 1% NEAA.CCRF-CEM and CEM/ADR5000 cells were maintained in RPM11640 supplemented with 10% FBS, 100 [micro]/ml penicillin, 100[micro]g/ml streptomycin, and 1% L-glutamine. Cells were kept at 37 [degrees]C, 5% C[O.sub.2], and 95% humidity. All experiments were performed with cells in the logarithmic growth phase.

Sample preparation

Pure SM were dissolved in dimethylsulfoxide (DMSO) at a concentration of 10 mM (stock solution) and stored at -80 [degrees]C until use. Each stock solution was serially diluted in the respective medium before use. The maximum final concentration of DMSO did not exceed 0.05% in the cell culture media.

Cytotoxicity assay

To determine the dose-dependent cytotoxicity, 2 x [10.sup.4] cells/well of Caco-2 cells were seeded in 96-well plates (Greiner Labortechnik) and incubated for 24h. The cytotoxicity of SM (digitonin, EGCG, menthol, thymol, aromadendrene, [bata]-sitosterol-0-glucoside, [bata]-carotene, glaucine, harmine, and sanguinarine) was determined using the MIT assay (Mosmann 1983). The cells were incubated with 100 ill of medium containing different concentrations of individual compounds. After 24 h, 0.5 mg/m1 of MIT was added. Cells were incubated for an additional 4h. The formazan crystals (produced by the cells) were dissolved in 100 [micro]l DMSO and shaken for 10 min. Then the absorbance was measured at 570 nm using a Tecan Safire 11 Reader (Tecan Crailsheim, Germany). For CCRF-CEM and CEM/ADR5000 (cells grown in suspension), 3 x [10.sup.4] cells/well were incubated with test compounds for 48 h and the MIT assay was carried out as outlined above.

Quenching test

In order to insure that SM did not bias the results of the Rhol 23 and CAM accumulation assay, these compounds were analyzed for possible quenching effects in a pilot experiment. Different concentrations of the SM were mixed with Rho123 and CAM solution and the fluorescence was measured and compared with Rho123 and CAM without test compounds.

Activity of P-gp in Caco-2 cells

The influence of selected SM on the accumulation of Rho123 and CAM was measured by fluorospectroscopy, as described previously (El-Readi et al. 2010). Briefly, Caco-2 cells (2 x 103 cells/well in 96-well plates) were incubated at 37 C until a confluent monolayer was formed after 4-6 days. Then cells were pre-incubated for 30 min at 37 [degrees]C with different concentrations of SM, with and without digitonin (5[micro]M). Verapamil, a known P-gp inhibitor, was used as a positive control. Cells were then incubated for 90 min with either Rho 123 (1[micro]/ml), or CAM (150 nM); afterwards they were washed twice with ice-cold PBS. Rhol 23 and CAM fluorescence was measured at excitation/emission wavelengths of 485/535 nm using a Tecan Safire II[TM] spectrofluorometer (Tecan Crailsheim, Germany).

Activity of P-gp in CCRF-CEM and CEM/ADR5000 cells

Suspended cultured CCRF-CEM and CEM/ADR5000 cells (1 x [10.sup.4] cells/m1) were treated with the selected SM in final concentrations of 40[micro]M, except harmine (25 [micro]M) and sanguinarine (1 [micro]M), with and without digitonin (5 [LM), and incubated for 90 min at 37 [degrees]C. Then the cells were washed twice with ice-cold PBS. Rho123 (10[micro]M final concentration) or CAM (2.5 [micro]M final concentration) was added, and the cells were incubated for 90min at 37 [degrees]C. Afterwards the cells were washed twice in ice cold PBS. Cells were re-suspended in PBS for the measurement. Verapamil was used as a positive control; and set to 100%. The fluorescence intensity of Rho123 and CAM was measured by flow cytometry using a Beckton-Dickinson FACScan instrument. The fluorescence intensity of treated cells was normalized by calculating the relative fluorescence intensity (inhibitory efficiency) as follows:

Inhibitory efficiency = [RFU.sub.SM] - [RFU.sub.untreated contril] / [RFU.sub.verapamil] - [RFU.sub.untreated contril] %

RFU is a relative fluorescence intensity unit. The resulting value was considered as relevant, if the percentage was >10%.

Real-time RT-PCR

Real time RT-PCR was carried out to quantify the mRNA level of the ABCB1 gene in Caco-2 and CEM/ADR5000 cells after treatment for 48 h with 40 p.M of selected SM (25 [micro]M harmine and 1 p.M sanguinarine) with and without 511M digitonin. In summary, the total cellular RNA was isolated from treated cells using the RNeasy[R] Midi kit (Qiagen; Hilden, Germany) according to the manufacturer's instructions. The purity of the RNA was checked photometrically at 0D2601280 and electrophoresis (1.5% agarose gel) was applied to evaluate the quality and purity of RNA. RNA was converted to cDNA by reverse transcriptase (Invi trogen/Gibco, Karlsruhe, Germany), employing random hexamer primers. The cDNA was quantified by LightCyclerTm technology (Roche Applied Science; Mannheim, Germany) using [bata]-microglobulin (132mg) as a housekeeping gene (Albermann et al. 2005). PCR amplification was carried out in a 20 [micro]l reaction volume containing 5 [micro]l 1:5 diluted cDNA, 1x Light-Cycler-FastStart DNA Master SYBR Green I (Roche Applied Science; Mannheim, Germany), and 0.511M of each primer. All samples were run in triplicate in a LightCycler capillary with an initial denaturation at 95 C for 10 s, annealing at 62 C for 5 s and elongation at 72 C for 9s. Expression levels were normalized relative to the transcription level of 132-microglobulin (132 mg). Data analysis was performed with LightCycler3 data analysis Software, Version 3.5.28 (Roche Applied Science; Mannheim, Germany) using a calibrator and standard curve. REST 2008, Relative Expression Software Tool Version 2.0.7 (Corbett Research Pty. Ltd.), was used for analysis of gene expression data from quantitative real-time PCR experiments.

Statistical analysis

All assays were carried out in triplicate and replicated 3 times. All data are expressed as mean [+ or -] standard deviation (SD). The IC50 values were calculated from the dose-response curves using a four parameter logistic fitting curve (SigmaPlot[R] 11.0). Graphs were drawn using GraphPad Prism[R] software (GraphPad Prism[R] 5.01, GraphPad Software, Inc., CA, USA). One-way analysis of variance and Bonferroni's post hoc test were used to analyze differences between the sets of data. A p-value <0.05 was considered significant.

Once the data of MDR inhibitory activity of SM and their combinations had been obtained, PCA and HCA analyses (Past software) were used to classify the SM into groups with similar activity behaviour and associate them with their chemical structure (Cherrington and Smart 1971; Di Ciaccio et al. 2012; Reigosa Roger and Nuria 2002). Similarities between selected SM (distance) were calculated according to squared Euclidean distances (Reigosa Roger and Nuria 2002).


Determination of the highest sub-lethal concentration of secondary metabolites In a first set of experiments we determined the IC50 values of each SM in Caco-2 and CEM/ADR5000 cells using the MTI cell viability assay (Eid et al. 2012c). Based on the cytotoxicity results in our experimental set, 40 [micro]M of each SM (except 25 pM harmine and 1 [micro]M sanguinarine) were selected as non-toxic concentrations for the P-gp assays. Additionally, a concentration of 5 [1M (<IC20) digitonin was non-toxic and already found to be optimal in combination protocols (Eid et al. 2012a,b).

Selected secondary metabolites are substrates for P-gp

After the incubation of Caco-2 cells with different concentrations (0.1-200 [micro]M) of EGCG, thymol, menthol, aromadendrene, 13-sitosterol-0-glucoside, 13-carotene, glaucine, harmine, and sanguinarine (0.01-20 p.M) and fluorescent substrates, the intracellular fluorescence intensity of Rhol 23 and CAM was significantly increased in a dose dependent manner, indicating that corresponding SM are P-gp substrates which prevent the export of the dyes from the cells (Fig. 2A and B; Table 1). The order of the inhibitory efficiency (average of both fluorescent probes) of 40 [micro]M of SM relative to verapamil (100%) was glaucine > sanguinarine > harmine >[bata]-carotene > EGCG >[bata]-sitosterol-O-glucoside > aromadendrene and > thymol > menthol (Table 1).

In CEM/ADR5000 cells, Rhol 23 and CAM retention was significantly increased after treatment with SM (40 [micro]M), harmine (25 [micro]M), and sanguinarine (1 [micro]M) as shown in the FACS histograms (Fig. 3A and B). The order of inhibitory activities was glaucine > sanguinarine > EGCG > [bata]-carotene > harmine> thymol >[beta]-sitosterol-0-glucoside > and menthol > aromadendrene (Table 1). However, these compounds do not show an effect on the parent sensitive leukaemia CCRF-CEM cells which does not overexpress P-gp (Fig. 4A).

A combination of digitonin with secondary metabolites can further enhance Rho 123 and CAM retention

Digitonin (alone and in combination with our SM test set) can increase doxorubicin toxicity in resistant cancer cells (Eid et al. 2012b). In the next set of experiments we tried to clarify if the increased toxicity of doxorubicin was due to the inhibition by P-gp efflux pump, which would lead to retention of doxorubicin. The accumulation of Rho123 and CAM was assessed in Caco-2 and CEM/ADR5000 cells after treatment with the set of SM in combination with 5 [micro]M digitonin. The intracellular accumulation of Rhol 23 and CAM was significantly enhanced in Caco-2 cells in a dose dependent manner (Fig. 2A and B). Fig. 2 shows that a synergistic interaction is clearly visible at higher concentrations of the combinations especially when the ratio of SM to digitonin was increased. However, in low concentrations of SM the interactions were rather additive or antagonistic. The synergistic effect of EGCG, thymol, 3-sitosterol-0-glucoside, and harmine, in combination with digitonin, represent the highest EF (between 1.45 and 1.98) and IE (between 220% and 469%) for both fluorescent probes (Table 1).

In CEM/ADR5000 cells thymol, menthol, and harmine in combination with digitonin, showed the highest EF (between 1.39 and 1.93) and IE (between 202% and 338%) for both fluorescent probes (Table 1; Fig. 3). Our results indicate that the selected SM inhibited the drug efflux via P-gp in CEM/ADR5000 cells to variable degrees; furthermore, combinations of digitonin with SM show prominent inhibitory effect on drug efflux. In contrast, these combinations did not reveal a significant effect in CCRF-CEM cells (Fig. 4B).

Quantification of mRNA level of ABCB1 gene by RT-PCR

In a last set of experiments we investigated the effect of SM, either alone or in combination with digitonin, on the expression of P-gp using RT-PCR. Fig. 5 shows that mRNA levels of the ABCB1 gene were significantly reduced in Caco-2 and CEM/ADR5000 cells after treatment with SM, especially with [beta]-carotene and EGCG in Caco-2, and sanguinarine and harmine in CEM/ADR5000 cells. Digitonin significantly potentiated the down-regulation, especially by sanguinarine, harmine, and [beta]-carotene, in both cell lines (Fig. 5).

Structure activity relationships

The main idea of PCA is to minimize the dimensionality of the data set to simplify the variance-covariance structure Uohnson and Wichern 1988). PCA produces few numbers of uncorrelated significant principal components (PCs) through the linear transformation of the set of variables of the original data. This transformation provides a geometrical rotation of the coordinate system from the original data. Statistically, these projections introduce a lot of information that can be visualized using microcomputers to achieve a more detailed study of the data structure (Cherrington and Smart 1971: Di Ciaccio et al. 2012). The direction of the first principal corn-ponent axis represents the maximum residual variance. The second maximum variance is given by the second principal component, which is orthogonal to the first one and so on.

In this study, before applying the PCA method, the MDR inhibitory activity of selected SM could be compared with each other in the same experiment. It is difficult to obtain a good classification of this compound set. The first two principal components recorded 98.49% of total variance in the data (PC1 = 89.82% and PC2 = 8.67%), as observed from PCA analysis. The first principal component was plotted against the second component to present the most informative score plot (Fig. 6A).This projection provides a reasonably accurate presentation of the higher order space because it conserves 98.49% of the total variance. The PCA scatter diagram for the first two principal components (PCI against PC2) shows that the selected SM are separated into two groups, A and B. Group A contains the moderately active SM, while group B contains the strongly active SM.

The results of HCA were similar to PCA, as shown in the dendrogram (Fig. 6B). Actually, the dendrogram provides information about the chemical behaviour and confirms the PCA data. Fig. 6B shows the selected SM on vertical lines and the horizontal lines represent the similarity values between two SM, single SM and SM-group, and between groups of SM. From Fig. 6B we can observe that the two groups, A and B, have distant similarity values. Groups A and B in Fig. GB represented the same SM in groups A and B in PCA analysis (Fig. 6A). Both PCA and FICA exactly classified the 9 selected SM or their combinations into two groups in the same manner. According to these classifications (PCA and FICA), the variables of reversal ratio and inhibitory efficiency of P-gp efflux pump can be used for a distinctive separation of investigated compounds into moderately and strongly active SM.

By the same way PCA and HCA were applied on SM combinations with digitonin (Fig. 7A and B). The first two principal components recorded 95.43% of the total data variance (PCI = 78.70% and PC2 = 16.73%) as observed in PCA analysis. PCA and HCA classified the activity of SM combinations into two groups with patterns similar to individual SM activity (Fig. 7A and B).


A number of plant secondary metabolites that can inhibit P-gp and re-sensitize resistant tumour cells in vitro have been described (El-Readi et al. 2010; Gyemant et at. 2006; Li et at. 2011; Limtrakul et al. 2007; Ma and Wink 2008; Moller et al. 2006; Wink 2007,2008: Wink et al. 2012). Some of these agents have been successfully employed in preclinical and in clinical studies (Anuchapreeda et at. 2002; Yu et al. 2007). However, some of these reversal agents did not work in vivo or some had apparent severe side effects (Dantzig et al. 2001). Therefore, new and better natural reversal agents or combinations of reversal agents are still needed. In the current study, we investigated whether combinations of two SM (differing in chemical structure and mode of action) can inhibit P-gp function and expression.

In this study, we used P-gp expressing Caco-2 and CEM/ADR5000 cell lines in comparison to CCRF-CEM cells which have no enhanced P-gp levels (Efferth et at. 2002; van de Waterbeemd 2004) to assess the effect of the representative SM (with and without digitonin) on P-gp activity and expression. As compared to the calcium antagonist verapamil, sanguinarine, glaucine, harmine, 13-carotene, and EGCG appear to be the most potent inhibitors of P-gp function (Figs. 2 and 3; Table 1). Since Rho123 and CAM are known to be good substrates for P-gp, and the time of cell exposure to selected SM in these experiments was short (2 h), we conclude that the selected SM mediate a retention of Rho123 and CAM by inhibiting P-gp.

Table 1
Influence of selected SM ([micro]M) with reversal activity on
intracellular rhodamine 123 and calcein accumulation in Caco-2
and CEM/ADR5000 cells. The data are represented as mean
[+ or -] SD of inhibitory efficiency (IE) related to the effect
of 20 [micro]M verapamil (100%). The ability of 5 [micro]M
digitonin to enhance the effect of combined SM is indicated by
the enhancement factor (EF).


                       Rhodamine                              Calcein

                     [IE.sub.SM]  [IE.sub.SM+Dig]    EF   [IE.sub.SM]


Thymol              111.09 [+ or  220.00 [+ or -]  1.98  121.29 [+ or
                        -] 10.29            22.12   (c)      -] 10.23

Menthol              91.45 [+ or  124.18 [+ or -]  1.36   78.39 [+ or
                         -] 9.30            11.32             -] 7.20


Aromadendrene       110.18 [+ or  137.82 [+ or -]  1.25  123.71 [+ or
                        -] 10.48            13.20            -] 10.92


[beta]-Sitosterol-  144.54 [+ or  240.00 [+ or -]  1.66  138.06 [+ or
O-glucoside             -] 14.23            24.91   (b)      -] 14.20


[beta]-Carotene     236.36 [+ or  345.45 [+ or -]  1.46  209.68 [+ or
                        -] 24.03            35.29   (c)      -] 17.33


EGCG                240.18 [+ or  469.09 [+ or -]  1.95  195.16 [+ or
                        -] 23.12             4523   (c)      -] 16.34


Glaucine            256.36 [+ or  330.91 [+ or -]  1.29  246.77 [+ or
                        -] 26.20            32.12            -] 23.93

Harmine             234.55 [+ or     340.00 [+ or  1.45  233.87 [+ or
                        -] 21.95          -]35.10   (b)      -] 21.59

Sanguinarine        238.18 [+ or  341.82 [+ or -]  1.44  241.93 [+ or
                        -] 22.73            31.00   (b)      -] 25.01



                    [IE.sub.SM+Dig]    EF   [IE.sub.SM]


Thymol              195.16 [+ or -]  1.61  192.48 [+ or
                              18.23   (a)      -] 11.67

Menthol             146.29 [+ or -]  1.87  198.11 [+ or
                              13.81   (c)       -] 5.74


Aromadendrene       190.32 [+ or -]  1.54  160.04 [+ or
                              18.27            -] 13.37


[beta]-Sitosterol-  232.26 [+ or -]  1.68  185.85 [+ or
O-glucoside                   19.21   (b)      -] 14.16


[beta]-Carotene     340.32 [+ or -]  1.62  222.42 [+ or
                              31.78   (c)      -] 16.05


EGCG                356.45 [+ or -]  1.83  231.61 [+ or
                              32.12   (c)      -] 12.33


Glaucine            330.65 [+ or -]  1.34  265.84 [+ or
                              30.20   (b)      -] 23.14

Harmine             396.77 [+ or -]  1.70  145.23 [+ or
                              37.19   (c)       -] 0.68

Sanguinarine        332.26 =t 28.18  1.37  244.02 [+ or
                                      (b)      -] 23.85


                     [IE.sub.SM+Dig]    EF   [IE.sub.SM]


Thymol               277.28 [+ or -]  1.44  166.93 [+ or
                               26.31   (a)      -] 12.08

Menthol              282.70 [+ or -]  1.43  158.80 [+ or
                               26.53   (a)       -] 4.91


Aromadendrene        193.81 [+ or -]  1.21  169.31 [+ or
                                8.14            -] 15.32


[beta]-Sitosterol-   219.22 [+ or -]  1.18  172.36 [+ or
O-glucoside                    18.48            -] 18.42


[beta]-Carotene      287.39 [+ or -]  1.29  259.23 [+ or
                                4.90   (a)       -] 4.12


EGCG                305.1 1 [+ or -]  1.32  256.89 [+ or
                               14.29   (a)      -] 23.13

Glaucine             395.08 [+ or -]  1.49  303.70 [+ or
                               25.90   (a)      -] 13.96

Harmine              202.39 [+ or -]  1.39  242.33 [+ or
                               19.77   (a)      -] 12.38

Sanguinarine         387.30 [+ or -]  1.59  290.16 [+ or
                               28.41   (a)       -] 8.66

                    [IE.sub.SM+Dig]    EF


Thymol              322.96 [+ or -]  1.93
                              10.03   (b)

Menthol             284.08 [+ or -]  1.79
                               5.03   (c)


Aromadendrene       300.15 [+ or -]  1.77
                              14.69   (b)


[beta]-Sitosterol-  353.67 [+ or -]  2.05
O-glucoside                   16.67   (b)


[beta]-Carotene     359.53 [+ or -]  1.39
                              15.13   (b)


EGCG                   361.83 [+ or  1.41
                             -]7.36   (a)


Glaucine               385.72 [+ or  1.27
                             -]8.12   (a)

Harmine             338.02 [+ or -]  1.39
                              19.77   (b)

Sanguinarine        391.80 [+ or -]  1.35
                              12.82   (b)

[IE.sub.SM] inhibitory efficiency of secondary metabolites.

[IE.sub.SM+Dig] inhibitory efficiency of secondary metabolites
in combination with digitonin.

EF enhancement factor.

(a) p < 0.05.

(b) p < 0.01.

(c) p < 0.001.

Moreover, the resistant cell lines were further sensitized by exposure to digitonin-SM combinations (Figs. 2 and 3). These data suggest that two drug combinations are even more effective than single P-gp inhibitors. These results are in agreement with our previous studies, in which we found that the same SM and their combination with digitonin (Eid et al. 2012c) also increased the cytotoxicity of doxorubicin in Caco-2 and CEM/ADR5000 cells (Eid et al. 2012b). In conclusion, all our data indicate that selected SM alone, and in combination with digitonin, can increase intracellular drug levels by modulating P-gp transport activity. However, the mechanism by which selected SM caused P-gp inhibition is not deducible from the present data.

Generally, P-gp modulators act as competitive inhibitors by binding to the membrane protein or by indirect mechanisms related to the expression of the P-gp gene and/or phosphorylation of the transport protein (Wink 2008, 2012). We assume that lipophilic terpenoids (thymol, menthol, aromadendrene, [beta]-sitosterol-O-glucoside, and [beta]-carotene), steroids (including digitonin), and alkaloids (glaucine, harmine, and sanguinarine) probably act as competitive inhibitors of P-gp in cancer cells (Wink 2008; Wink et al. 2012). However, EGCG, a polar polyphenol, can effectively interact directly with P-gp by forming hydrogen and ionic bonds with amino acid side chains of the protein, thus interfering with the 3D structure of P-gp (conformation) and inhibiting its activity (Wink 2008; Wink and Van Wyk 2008). Furthermore, it has been reported that EGCG can modulate P-gp activity by binding to NBD2, thereby preventing the binding of ATP and subsequently the energy-dependent efflux of the drug (Qian et al. 2005).

The synergistic enhancement of transport inhibition by thymol, menthol, aromadendrene, and EGCG in combinations with digitonin (Table 1) is apparently related to their amphiphilic character. Digitonin forms a complex with its lipophilic terpenoid moiety with cholesterol in the biomembrane; additionally it binds to glycoproteins and glycolipids of the cell membrane with its sugar side chain. This leads to a severe tension of the biomembrane and influences membrane permeability. By this mechanism, saponin might enhance the uptake of polar drugs, such as EGCG, in digitonin combinations (Wink 2008, 2012). In addition to P-gp, Caco-2 cells express the metabolizing enzyme CYP3A and nuclear receptors such as pregnane X receptor (PXR), which regulate the expression of ABCB1 and CYP3. P-gp and CYP3A interact co-ordinately at the intestinal barrier and limit drug absorption. Therefore, the modulation (inhibition or induction) of P-gp by natural products is expected to modulate CYP3A (El-Reacli et al. 2013). In the present study, the substrates (Rhol 23 and calcein) that were used to evaluate P-gp function are substrates for P-gp but not CYP3A or PXR. Thus, our data would not indicate that the tested compounds are CYP3A or PXR modulators. However, their potential to regulate CYP3A or PXR is very likely, which may present an additional mechanism for the synergistic interactions that however requires further studies.

The potential modulation of P-gp expression in resistant cancer cells by the selected SM had not been previously studied. This is the first report which shows that some SM can down-regulate the expression of P-gp in human cells (Fig. 5). Decreasing both expression and transport activity of P-gp provides a more attractive therapeutic target than the direct inhibition of P-gp efflux activity alone (Jin et al. 2000). Again the combination of digitonin with selected SM was more effective than the SM alone in down-regulating P-gp in both cell lines (Fig. 5A and B).

Furthermore, PCA and HCA analyses of MDR inhibitory activity of SM and their combinations (Figs. 6 and 7) shows that the inhibitors can be classified into two main groups: GA for moderate and GB for strong interactions. In both experimental sets, group A includes the terpenoids (mono-, di-, and triterpenes) either alone (Fig. 6) or in combination with digitonin (Fig. 7).

All compounds with a terpenoid skeleton (Fig. 1) belong to the group A, except [beta]-carotene. Regarding the behaviour of terpenoids, all of them had moderate levels of activity, observed just at the highest concentrations. In fact, all of these compounds target the cell membrane and interact with cholesterol and phospholipicls. Digitonin also acts on the same target as these compounds, thus potentiating their effects in combinations. [beta]-Carotene was one of the most active compounds in the experimental set (Figs. 5 and 6). As in our previous study, carotenoids showed strong MDR reversal activity when combined with cytotoxic agents (Eid et al. 2012a). The same effect can be seen in P-gp functional assays, since carotenoids are more effective than verapamil (Eid et al. 2012a).

Group B represent SM with strong activity: it includes alkaloids (sanguinarine, harmine, and glaucine), a polyphenol (EGCG), as well as a tetraterpene ([beta]-carotene). The alkaloids are lipophilic and positively charged molecules. These characteristic chemical properties qualify them as strong P-gp inhibitors. EGCG is a polyphenol which can form hydrogen and ionic bonds with P-gp. The combinations of digitonin with alkaloids were more potent than saponin-terpenoid combinations (Fig. 6B).

In conclusion, our results indicate that selected SM can both directly inhibit the P-gp efflux function and modulate P-gp expression. Additionally, this study provides evidence that digitonin, in combination with SM, leads to a stronger inhibition of ABC transporters as when applied alone. There are possibilities to prove in vitro whether with drug combinations synergistic or additive effects can be expected (Berenbaum 1989: Wagner and Ulrich-Merzenich 2009). The reversal ratio and inhibitory efficiency of P-gp efflux pump are responsible variables for the classification of SM into moderate (A) and strong (B) active groups (PCA analysis). Experiments in animal models are needed to confirm that the combination strategy also works under in vivo conditions because all in vivo effects of drug combinations depend on their pharmacokinetic and pharmacodynamics behaviour.

0944-7113/$--see front matter 0 2013 Elsevier GmbH. All rights reserved.

Safaa Yehia Eid (a), *, Mahmoud Zaki El-Readi (a), (b), Essam Eldin Mohamed Nour Eldin (c), Sameer Hassan Fatani (c), Michael Wink (a),*

(a) Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, lm Neuenheimer Feld 364, 69120 Heidelberg, Germany

(b) Department of Biochemistry, Faculty of Pharmacy, Al-Azhar University, 71524 Assiut, Egypt

(c) Department of Biochemistry, Faculty of Medicine, 6707 Umm Al-Qura University, Saudi Arabia

* Corresponding authors. Tel.: +49 6221 54 4880: fax: +49 6221 544884. E-mail addresses:, (S.Y. Eid), (M. Wink).


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Author:Eid, Safaa Yehia; El-Readi, Mahmoud Zaki; Eldinc, Essam Eldin Mohamed Nour; Fatanic, Sameer Hassan;
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EUGE
Date:Dec 15, 2013
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