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Natural lignans from Arctium lappa modulate P-glycoprotein efflux function in multidrug resistant cancer cells.

ABSTRACT

Arctium lappa is a well-known traditional medicinal plant in China (TCM) and Europe that has been used for thousands of years to treat arthritis, baldness or cancer. The plant produces lignans as secondary metabolites which have a wide range of bioactivities. Yet, their ability to reverse multidrug resistance (MDR) in cancer cells has not been explored. In this study, we isolated six lignans from A. lappa seeds, namely arctigenin, matairesinol, arctiin, (iso)lappaol A, lappaol C, and lappaol F. The MDR reversal potential of the isolated lignans and the underlying mechanism of action were studied using two MDR cancer cell lines, CaCo2 and CEM/ADR 5000 which overexpress P-gp and other ABC transporters. In two-drug combinations of lignans with the cytotoxic doxorubicin, all lignans exhibited synergistic effects in CaCo2 cells and matairesinol, arctiin, lappaol C and lappaol F display synergistic activity in CEM/ADR 5000 cells. Additionally, in threedrug combinations of lignans with the saponin digitonin and doxorubicin MDR reversal activity was even stronger enhanced. The lignans can increase the retention of the P-gp substrate rhodamine 123 in CEM/ADR 5000 cells, indicating that lignans can inhibit the activity of P-gp. Our study provides a first insight into the potential chemosensitizing activity of a series of natural lignans, which might be candidates for developing novel adjuvant anticancer agents.

Keywords:

Arctium lappa

Lignans

Multidrug resistance

P-glycoprotein

Cancer cells

Introduction

Cancer cells are capable to develop resistance to a single drug, or a class of anti-cancer drugs. After having obtained resistance to a single drug, cells often also show cross-resistance to many other structurally and functionally unrelated drugs. This phenomenon is called multidrug resistance (MDR), which might explain why therapy that even combine multiple agents with different targets fail to improve therapeutic efficacy (Gottesman et al. 1994). The best known mechanism for MDR is the overexpression of ATP-binding cassette (ABC) transporter proteins in the cell membrane in cancer cells which can mediate the efflux of cytotoxic drugs and thus lower intracellular drug concentrations (Ambudkar et al. 1999). A major member of the ABC transporter family is P-glycoprotein (P-gp), which is involved in the MDR of cancer cells to several anticancer drugs. Thus, targeting P-gp is one approach to overcome and reverse MDR.

Medicinal plants produce a high diversity of secondary metabolites (SM) which include effective and new drugs for cancer treatment. Several attempts have been made to identify natural products with a variety of chemical structures which can inhibit ABC transporters (Wink et al. 2012).

Arctium lappa, commonly known as burdock, is an important medicinal plant in China (TCM) and Europe which has been used for thousands of years (Van Wyk and Wink 2004). A. lappa is a rich source of bioactive lignans. Recently, it was discovered that the natural lignans in A. lappa have promising anticancer potential. They can induce apoptosis in cancer cells and suppress tumor growth via decreasing tumor tolerance to glucose starvation (Marian et al. 2003). However, to our knowledge, no research on the potential of lignans from A. lappa to reverse MDR in cancer cells to chemotherapeutic drugs like doxorubicin has been published. As we had isolated a series of lignans from A. lappa seeds, we were interested to combine non-toxic lignans from A. lappa with clinically used chemotherapeutic agent to test their potential to reverse MDR of cancer cells.

CaCo2 grow as an adherent cell monolayer, which is a well-established in vitro model of the intestinal epithelium that highly expresses P-gp on its apical surface (Hunter et al. 1993). This cell line has been widely used to study a number of substrates and inhibitors of P-gp. CEM/ADR 5000 is a doxorubicin-resistant human T-lymphoblastic leukemia cell line derived from its parental doxorubicin-sensitive cell line, CCRF-CEM. CEM/ADR 5000 also overexpresses P-gp (Efferth et al. 2002). In the present study, we investigated the multidrug-resistant reversal potency of six lignans from A lappa seeds, namely arctigenin, matairesinol, arctiin, (iso)lappaol A, lappaol C and lappaol F using CEM/ADR 5000 and CaCo2 cell lines.

Materials and methods

Plant material

Seeds of Arctium lappa were collected from the Neuenheimer Feld, Heidelberg, Germany in July 2011. A voucher specimen of the plant was deposited in the Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany. The voucher number is P8243.

Extraction and isolation of lignans from Arctium lappa

The extraction and isolation of lignans from A. lappa extract were performed as described in our article: natural lignans from Arctium lappa as novel antiaging agents in Caenorhabditis elegans (submitted to Phytochemistry). Their structures have been identified as arctigenin, matairesinol, arctiin, a mixture of two isomers containing lappaol A and isolappaol A (we call it (iso)lappaol A), lappaol C and lappaol F (Fig. 1) by analysis of [sup.1]H-NMR, [sup.13]C-NMR, El-MS and ESI-MS and comparison with literature data (Han et al. 1994; Liu et al. 2003; Umehara et al. 1993). The isolated lignans were then dissolved in DMSO to prepare stock solutions of 10 mM, 20 mM and 100 mM for further tests.

Cell culture

CaCo2 and human T-cell lymphoma CEM/ADR 5000 were used in this study. Culture conditions were identical to those described previously (El-Readi et al. 2010).

Cytotoxicity assay

3-(4,5-DimethylthiazoI-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was carried out as established in our laboratory (El-Readi et al. 2010).

Two-drug combinations of doxorubicin with lignans

Three different concentrations ([IC.sub.10], [IC.sub.20], [IC.sub.30]) of lignans were combined with the cytotoxic doxorubicin (Sigma- Aldrich, GmbH, Germany) to measure whether lignans can increase the sensitivity of MDR cancer cells to doxorubicin. Briefly, CaCo2 and CEM/ADR 5000 cells were seeded in 96 well-plates and incubated with serial dilutions of doxorubicin as well as each lignans of different concentrations as I mentioned above. MTT assay was carried out to test the cytotoxicity of doxorubicin alone or in combination against CaCo2 and CEM/ADR 5000 cells.

Three-drug combinations of doxorubicin with lignans plus digitonin

In addition to two-drug combination assays, three-drug combinations of doxorubicin with lignans of different concentration ([IC.sub.10], [IC.sub.20], [IC.sub.30]) plus the saponin digitonin (Sigma- Aldrich, GmbH, Germany) were also performed. Briefly, CaCo2 and CEM/ADR 5000 cells were seeded in 96 well-plates and incubated with serial dilutions of doxorubicin and each lignan with different concentrations plus digitonin in a non-toxic concentration ([IC.sub.10]) as mentioned above. The corresponding cytotoxicity was determined with the MTT assay.

Analysis of combination effects

For the analysis of synergism or antagonism of drug combinations the combination index (CI) method was employed. The ranges of CI and the symbols were described by Chou (2006) in which CI <1, =1, and >1 indicate synergism, additive effect, and antagonism, respectively. The formula to calculate CI value is identical as in Eid et al. (2012b).

Rhodamine 123 accumulation assay

The function of ABC transporters in MDR cancer cells can be tested by flow cytometry using a fluorescent dye, rhodamine 123 (SigmaAldrich, GmbH, Germany), which is a substrate of P-gp (Anuchapreeda et al. 2002). Assays were performed following an established protocol (El-Readi et al. 2010). The standard P-gp inhibitor verapamil (20 [micro]M) (Sigma- Aldrich, GmbH, Germany) was used as a positive control. 1% DMSO was used as a solvent control. Three different concentrations of 20, 50 and 100 [micro]M of lignans were tested in this assay.

Statistical analysis

Data analysis was performed with Graphpad Prism 5.0 (Graphpad Software, San Diego, CA, USA). Data are presented as mean [+ or -] SD. Statistical comparisons between controls and different treatments were performed using a two-tailed unpaired Student's t-test. All experiments were performed at least three times. Differences between the data were considered significant when p < 0.05. The [IC.sub.50] value was determined as the amount of the substances needed to reduce cell viability by 50% and the [IC.sub.50] was calculated using Graphpad Prism 5.0.

Results

Cytotoxicity of lignans

The cytotoxicity of the six isolated lignans determined against the multidrug-resistant cell lines CEM/ADR 5000 and CaCo2 cells using the MTT assay. As shown in Table 1, the [IC.sub.50] values of all lignans indicate that they are hardly cytotoxic against CaCo2 cells whereas arctigenin and matairesinol showed moderate cytotoxicity against CEM/ADR 5000 cells.

Lignans increased the sensitivity of MDR cancer cells to doxorubicin combinations of two drugs

Based on the results of cytotoxicity assay, three non-toxic concentrations ([IC.sub.10], [IC.sub.20], [IC.sub.30]) of lignans were combined with doxorubicin. The IC50 values of doxorubicin alone or in two-drug combination in CaCo2 and CEM/ADR 5000 cell lines are documented in Tables 2 and 3, respectively. Additionally, the reversal ratio and the combination index (CI) were calculated for both cell lines (Tables 2 and 3; Fig. 2).

The nature of drug combinations was evaluated by CI analysis (see Materials and methods). Overall, CI values < 1, represent synergism, 1 = additive effects and >1 = antagonism. As shown in Tables 2 and 3, all lignans significantly decrease the IC50 values of doxorubicin against CaCo2 cells and CEM/ADR 5000 cells when combined with lignans. The respective CI values are lower than 1, indicating that the interactions are synergistic in CaCo2 cells. Among the lignans, the activity of arctiin and lappaol F was concentration-dependent. In contrast to the results in CaCo2 cells, matairesinol ([IC.sub.10]), arctiin, lappaol C and lappaol F ([IC.sub.10]. [IC.sub.20]. [IC.sub.30]) displayed synergistic effects in CEM/ADR 5000 cells. The synergistic effect of arctiin and lappaol C are concentration-dependent.

Digitonin enhanced Che synergistic interaction of lignans and doxorubicin in three-drug combinations

Considering the medium polarity of the lignans used, we designed a three-drug combination scheme in which the saponin digitonin was added to lignans--doxorubicin combination as a third partner. Digitonin has been shown to increase the membrane permeability of the cells and enhance the uptake of polar cytotoxic secondary metabolites (Fiskum et al. 1980). Three-drug combinations were synergistic in previous experiments (Eid et al. 2012b). We added digitonin to increase the intracellular concentration of lignans. The [IC.sub.10] dose of digitonin (5 [micro]M for CaCo2 cells, 1.5 [micro]M for CEM/ADR 5000 cells) was used in three-drug combinations. Compared to two-drug combinations, digitonin significantly increased the synergism of lignans in both MDR cell lines with a stronger effect in CEM/ADR 5000 cell line (Tables 2 and 3). As shown in Table 3, after the addition of digitonin, arctigenin, matairesinol and (iso)lappaol A showed synergistic activity together with doxorubicin compared with two-drug combination in which they only exhibited additive or slightly antagonistic effects in CEM/ADR 5000 cells. Similar results were observed for arctiin, lappaol C and lappaol F after addition of digitonin. They exhibited synergistic or strong synergistic effects in the three-drug combinations, whereas they showed a moderate synergism in the absence of digitonin.

Lignans increased the accumulation of rhodamine 123

Since the CEM/ADR 5000 is a multidrug resistant cell line that overexpresses P-gp, it was selected to test the effects of lignans on P-gp activity (Majumder et al. 2006). In the assay, we used rhodamine 123 as a fluorescent P-gp substrate that is known to be effluxed by P-gp (Anuchapreeda et al. 2002). The activity of P-gp was determined cytometrically via measuring the retention of rhodamine 123 in the cells. We also included the calcium channel blocker verapamil as a positive control, a commonly used P-gp inhibitor that has been shown to reverse drug resistance (Muller et al. 1994; Summers et al. 2004).

As illustrated in Fig. 3, all lignans shifted the fluorescence intensity of rhodamine 123 rightwards in a concentration-dependent manner compared to the control suggesting that lignans increased the retention of rhodamine 123 in the cells. The accumulation of rhodamine 123 which was measured via the fluorescent intensity in cells is documented in Fig. 4. All lignans, except lappaol C, significantly increased the retention of rhodamine 123 in the cells in a concentration-dependent manner compared to the control. These results demonstrated that lignans efficiently inhibited the rhodamine 123 efflux from cells, suggesting that most of the lignans can inhibit P-gp activities.

Discussion

In the present study, we found that the major lignans isolated from Arctium lappa seeds possess promising MDR reversal activities in CEM/ADR 5000 and CaCo2 cell lines, which overexpress P-gp and other ABC transporters. To our knowledge, this is the first study applying a series of natural lignans isolated from A. lappa in a combination with the chemotherapeutic drug doxorubicin to enhance the efficiency of active chemotherapeutics and even to reverse multidrug resistance in MDR cancer cells. Previous studies have demonstrated that lignans could promote the chemosensitivity of MDR cancer cells to commonly used chemotherapeutic drugs. For instance, Nabekura et al. reported that matairesinol increased the accumulation of daunorubicin and calcein (fluorescent substrates of P-gp and MRP1, separately) (Nabekura et al. 2008). Furthermore, matairesinol treatment could sensitize prostate cancer cells to tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL)--induced apoptosis (Peuhu et al. 2010). A recent study reported that the combination of arcdin with cisplatin could enhance the chemosensitivity of H460 cells (cisplatin-treated non-small-cell lung cancer cells) to cisplatin (Wang et al. 2014). Arctigenin improved the sensitivity of HepG2, Hela and K562 cancer cells to cisplatin through inhibition of signal transducers and activators of the transcription 3 (STAT3) signaling pathway (Yao et al. 2011).

Importantly, we found that all lignans enhanced the efficacy of doxorubicin and decreased the dose of doxorubicin needed to reach cytotoxicity in CaCo2 cells (Table 2). The CI value analysis indicated that all lignans exhibit synergism in CaCo2 cells (Fig. 2). Among these lignans, arctigenin, matairesinol, and lappaol F exhibited the strongest synergistic activities. However, arctigenin and (iso)lappaol A failed to exhibit synergistic effect in CEM/ADR 5000 cells (Table 3, Fig. 2).

To further explore the underlying mechanism of action of the lignans, we focused on P-gp in CEM/ADR 5000 cells, because overexpression of P-gp leads to multidrug resistance of cancer cells. We demonstrated that all lignans, except lappaol C, significantly retained rhodamine 123 in CEM/ADR 5000 cells as compared to the control (Figs. 3 and 4). The result indicates that isolated lignans inhibited the function of P-gp. Considering the synergistic effect for lappaol C in CaCo2 and CEM/ADR 5000 cells, we assume that lappaol C works through regulation of other ABC transporters instead of P-gp alone. Secondary metabolites can interfere with a large set of molecular targets in cells such as proteins, DNA, RNA and the cell membrane through covalent or non-covalent modifications (Wink 2008, 2011). Based on the structure of the lignans (Fig. 1) which possess several phenolic hydroxyl groups which can dissociate under physiological conditions (Wink 2008, 2011) (arctiin has normal hydroxyl groups in its glucose chain), it is likely that these lignans interact with P-gp by forming ionic and hydrogen bonds with amino residues of the protein and thus inhibit its functions. Our result is consistent with previous observations, highlighting the chemosensitizing activity of natural lignans.

Cell membranes are impermeable for polar or charged molecules. Saponins, such as digitonin, can disturb the membrane stability and enhance its permeability. In agreement with this, digitonin apparently increased the MDR reversal activity of lignans in three-drug combinations probably by facilitating the entry of polar lignans into the cells. It should be recalled that digitonin can enhance the apoptosis-inducing effect of doxorubicin in MDR cancer cells because digitonin itself can induce apoptosis and activate related signaling pathways (Eid et al. 2012a). Digitonin treatment leads to activation of caspases and loss of mitochondrial function in cancer cells (Ishisaka et al. 1998; Ricci et al. 2003).

In conclusion, our results suggest that the MDR reversal activity of lignans is mediated, at least in part, by a modulation of P-gp activity. Since these natural lignans isolated from A. lappa such as arctigenin, matairesinol and arctiin also occur in our diet like soybeans, they have no or very low toxic side effects. Some natural lignans have been tested in animals. For example, the administration of arctigenin strongly suppressed the PANC-1 tumor growth in nude mice (Esumi et al. 2006). Lappaol F treatment significantly inhibited the growth of Hela cell xenograft tumor in nude mice (Sun et al. 2014). These lignans might be excellent candidates as reversal agents and deserve more detailed exploration in the future to develop novel anticancer drugs or adjuvants. Further in vivo studies using these natural lignans in combination with chemotherapeutic agents for treatment of cancer are needed to confirm their efficacy.

ARTICLE INFO

Article history:

Received 10 November 2014

Accepted 5 December 2014

Acknowledgement

S.S. thanks the Chinese Scholar Council (CSC) for a PhD fellowship.

References

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http://doi.org/10.1016/j.phymed.2014.12.009

Shan Su, Xinlai Cheng, Michael Wink *

Institute of Pharmacy and Molecular Biotechnology. Heidelberg University, Im Neuenheimer Feld 364, D-69120, Heidelberg, Germany

* Corresponding author. Tel.: +49 (0) 6221 544881; fax: +49 (0) 6221 544884.

E-mail address: wink@uni-heidelberg.de, Michael.Wink@urz.uni-heidelberg.de (M. Wink).

Table 1
[IC.sub.50 ]values ([micro]M) of lignans and doxorubicin (a positive
control) against multidrug-resistant CEM/ADR 5000 and CaCo2 cells.
The [IC.sub.50] values were calculated by Graphpad Prism 5.0. Data
are represented as mean [+ or -] SD. Data were obtained from three
independent experiments.

                 CaCo2                     CEM/ADR 5000

Arctigenin        696.90 [+ or -] 76.66       1.46 [+ or -] 0.20
Matairesinol      562.30 [+ or -] 52.19       6.34 [+ or -] 0.79
(lso)lappaol A    284.20 [+ or -] 38.44     181.40 [+ or -] 2.42
Arctiin          >5000                      350.00 [+ or -] 14.98
Lappaol C        1862.10 [+ or -] 158.44   2041.74 [+ or -] 245.51
Lappaol F        >5000                      302.00 [+ or -] 30.22
Doxorubicin         4.06 [+ or -] 0.28       40.13 [+ or -] 2.60
Digitonin          25.80 [+ or -] 2.57        12.5 [+ or -] 0.96

Table 2
Cytotoxicity of doxorubicin (Dox.) against CaCo2 cells, either alone
or in two-drug or three-drug combinations. Data were obtained from
three independent experiments and represented as mean [+ or -] SD.
CI < 0.1 means very strong synergism (+++++), 0.1-0.3 strong
synergism (++++), 0.3-0.7 synergism (+++), 0.7-0.85 moderate
synergism (++), 0.85-0.9 slight synergism (+), 0.9-1.10 nearly
additivity ([+ or -]), 1.10-1.20 slight antagonism (-), 1.20-1.45
moderate antagonism (--), 1.45-3.3 antagonism (----), 3.3-10 strong
antagonism (----), and >10 very strong antagonism (-----) (Chou
2006). NR = not relevant.

                             Two-drug combinations with lignans

                             [IC.sub.50]          Reversal ratio
                             [micro]M of Dox.

Doxorubicin alone            4.06 [+ or -] 0.28   1.00

Arctigenin
80 [micro]M ([IC.sub.10])    1.46 [+ or -] 0.15   2.79
120 [micro]M ([IC.sub.20])   1.40 [+ or -] 0.41   2.90
160 [micro]M ([IC.sub.30])   1.23 [+ or -] 0.43   3.29

Matairesinol
80 [micro]M ([IC.sub.10])    1.39 [+ or -] 0.29   2.92
120 [micro]M ([IC.sub.20])   1.19 [+ or -] 0.23   3.41
160[micro]M ([IC.sub.30])    1.19 [+ or -] 0.20   3.42

Arctiin
80 [micro]M ([IC.sub.10])    2.80 [+ or -] 0.30   1.45
160 [micro]M ([IC.sub.20])   2.61 [+ or -] 0.75   1.55
320 [micro]M ([IC.sub.30])   2.10 [+ or -] 0.06   1.93

(Iso)lappaol A
80 [micro]M ([IC.sub.10])    1.75 [+ or -] 0.08   2.32
160 [micro]M ([IC.sub.20])   1.40 [+ or -] 0.36   2.91
200 [micro]M ([IC.sub.30])   1.01 [+ or -] 0.31   4.01

Lappaol C
320 [micro]M ([IC.sub.10])   2.05 [+ or -] 0.59   1.98
480 [micro]M ([IC.sub.20])   1.60 [+ or -] 0.54   2.54
650 [micro]M ([IC.sub.30])   1.36 [+ or -] 0.27   2.99

Lappaol F
40 [micro]M ([IC.sub.10])    2.50 [+ or -] 0.38   1.63
80 [micro]M ([IC.sub.20])    2.24 [+ or -] 0.47   1.82
320 [micro]M ([IC.sub.30])   1.97 [+ or -] 0.42   2.06

                             Two-drug combinations with lignans

                             CI     Interpretation

Doxorubicin alone            NR     NR

Arctigenin
80 [micro]M ([IC.sub.10])    0.47   +++
120 [micro]M ([IC.sub.20])   0.52   +++
160 [micro]M ([IC.sub.30])   0.53   +++

Matairesinol
80 [micro]M ([IC.sub.10])    0.48   +++
120 [micro]M ([IC.sub.20])   0.51   +++
160[micro]M ([IC.sub.30])    0.58   +++

Arctiin
80 [micro]M ([IC.sub.10])    0.70   ++
160 [micro]M ([IC.sub.20])   0.67   +++
320 [micro]M ([IC.sub.30])   0.57   +++

(Iso)lappaol A
80 [micro]M ([IC.sub.10])    0.71   ++
160 [micro]M ([IC.sub.20])   0.91   [+ or -]
200 [micro]M ([IC.sub.30])   0.95   [+ or -]

Lappaol C
320 [micro]M ([IC.sub.10])   0.68   +++
480 [micro]M ([IC.sub.20])   0.65   +++
650 [micro]M ([IC.sub.30])   0.68   +++

Lappaol F
40 [micro]M ([IC.sub.10])    0.62   +++
80 [micro]M ([IC.sub.20])    0.56   +++
320 [micro]M ([IC.sub.30])   0.54   +++

                             Three-drug combinations with
                             lignans plus digitonin

                             [IC.sub.50]          Reversal ratio
                             [micro]M of Dox.

Doxorubicin alone            4.06 [+ or -] 0.28    1.00

Arctigenin
80 [micro]M ([IC.sub.10])    0.58 [+ or -] 0.01    7.01
120 [micro]M ([IC.sub.20])   0.39 [+ or -] 0.03   10.47
160 [micro]M ([IC.sub.30])   0.32 [+ or -] 0.01   12.85

Matairesinol
80 [micro]M ([IC.sub.10])    0.66 [+ or -] 0.03    6.20
120 [micro]M ([IC.sub.20])   0.45 [+ or -] 0.10    9.00
160 [micro]M ([IC.sub.30])   0.38 [+ or -] 0.07   10.74

Arctiin
80 [micro]M ([IC.sub.10])    0.86 [+ or -] 0.01    4.71
160 [micro]M ([IC.sub.20])   0.72 [+ or -] 0.02    5.64
320 [micro]M ([IC.sub.30])   0.64 [+ or -] 0.01    6.36

(Iso)lappaol A
80 [micro]M ([IC.sub.10])    0.91 [+ or -] 0.16    4.48
160 [micro]M ([IC.sub.20])   0.49 [+ or -] 0.03    8.2
200 [micro]M ([IC.sub.30])   0.37 [+ or -] 0.03   11.08

Lappaol C
320 [micro]M ([IC.sub.10])   1.24 [+ or -] 0.14    3.29
480 [micro]M ([IC.sub.20])   0.97 [+ or -] 0.12    4.17
650 [micro]M ([IC.sub.30])   0.75 [+ or -] 0.03    5.38

Lappaol F
40 [micro]M ([IC.sub.10])    1.66 [+ or -] 0.02    2.44
80 [micro]M ([IC.sub.20])    1.59 [+ or -] 0.14    2.55
320 [micro]M ([IC.sub.30])   1.49 [+ or -] 0.09    2.72

                             Three-drug combinations with
                             lignans plus digitonin

                             CI     Interpretation

Doxorubicin alone            NR     NR

Arctigenin
80 [micro]M ([IC.sub.10])    0.45   +++
120 [micro]M ([IC.sub.20])   0.46   +++
160 [micro]M ([IC.sub.30])   0.50   +++

Matairesinol
80 [micro]M ([IC.sub.10])    0.50   +++
120 [micro]M ([IC.sub.20])   0.52   +++
160 [micro]M ([IC.sub.30])   0.57   +++

Arctiin
80 [micro]M ([IC.sub.10])    0.42   +++
160 [micro]M ([IC.sub.20])   0.40   +++
320 [micro]M ([IC.sub.30])   0.40   +++

(Iso)lappaol A
80 [micro]M ([IC.sub.10])    0.70   ++
160 [micro]M ([IC.sub.20])   0.88   +
200 [micro]M ([IC.sub.30])   0.99   +

Lappaol C
320 [micro]M ([IC.sub.10])   0.67   +++
480 [micro]M ([IC.sub.20])   0.69   ++
650 [micro]M ([IC.sub.30])   0.73   ++

Lappaol F
40 [micro]M ([IC.sub.10])    0.61   +++
80 [micro]M ([IC.sub.20])    0.60   +++
320 [micro]M ([IC.sub.30])   0.62   +++

Table 3
The cytotoxicity of doxorubicin against CEM-ADR 5000 cells either
alone, in two-drug or in three-drug combinations. Data were obtained
from three independent experiments and represented as mean [+ or -]
SD. For the interpretation of drug combinations see Table 2.

                             Two-drug combination with lignans

                             [IC.sub.50] [micro]M of Dox.

Doxorubicin alone            40.13 [+ or -] 2.60
Arctigenin
0.2 [micro]M ([IC.sub.10])   31.69 [+ or -] 3.59
0.4 [micro]M ([IC.sub.20])   27.59 [+ or -] 5.04
0.8 [micro]M ([IC.sub.30])   21.40 [+ or -] 2.92

Matairesinol
1 [micro]M ([IC.sub.10])     27.56 [+ or -] 4.50
2 [micro]M ([IC.sub.20])     25.74 [+ or -] 4.74
3 [micro]M ([IC.sub.30])     22.90 [+ or -] 3.49

Arctiin
1 [micro]M ([IC.sub.10])     29.53 [+ or -] 4.57
4 [micro]M ([IC.sub.20])     24.52 [+ or -] 3.04
8 [micro]M ([IC.sub.30])     20.47 [+ or -] 3.27

(Iso)lappaol A
40 [micro]M ([IC.sub.10])    28.20 [+ or -] 4.33
100 [micro]M ([IC.sub.20])   21.02 [+ or -] 3.32
140 [micro]M ([IC.sub.30])   15.35 [+ or -] 3.21

Lappaol C
100 [micro]M ([IC.sub.10])   23.45 [+ or -] 5.86
200 [micro]M ([IC.sub.20])   19.53 [+ or -] 4.14
300 [micro]M ([IC.sub.30])   17.43 [+ or -] 2.71

Lappaol F
1 [micro]M ([IC.sub.10])     35.51 [+ or -] 2.43
20 [micro]M ([IC.sub.20])    30.97 [+ or -] 3.26
40 [micro]M ([IC.sub.30])    29.52 [+ or -] 1.34

                             Two-drug combination with lignans

                             Reversal ratio   CI     Interpretation

Doxorubicin alone            1.00             NR     NR
Arctigenin
0.2 [micro]M ([IC.sub.10])   1.27             0.92   [+ or -]
0.4 [micro]M ([IC.sub.20])   1.46             0.96   [+ or -]
0.8 [micro]M ([IC.sub.30])   1.87             1.08   [+ or -]

Matairesinol
1 [micro]M ([IC.sub.10])     1.46             0.84   ++
2 [micro]M ([IC.sub.20])     1.56             0.9    [+ or -]
3 [micro]M ([IC.sub.30])     1.75             1.04   [+ or -]

Arctiin
1 [micro]M ([IC.sub.10])     1.36             0.74   ++
4 [micro]M ([IC.sub.20])     1.64             0.62   +++
8 [micro]M ([IC.sub.30])     1.97             0.53   +++

(Iso)lappaol A
40 [micro]M ([IC.sub.10])    1.42             0.92   [+ or -]
100 [micro]M ([IC.sub.20])   1.91             1.07   [+ or -]
140 [micro]M ([IC.sub.30])   2.62             1.15   -

Lappaol C
100 [micro]M ([IC.sub.10])   1.71             0.63   +++
200 [micro]M ([IC.sub.20])   2.06             0.58   +++
300 [micro]M ([IC.sub.30])   2.30             0.58   +++

Lappaol F
1 [micro]M ([IC.sub.10])     1.13             0.89   +
20 [micro]M ([IC.sub.20])    1.30             0.84   ++
40 [micro]M ([IC.sub.30])    1.36             0.87   +

                             Three-drug combination with lignans
                             plus digitonin

                             [IC.sub.50] [micro]M of Dox.

Doxorubicin alone            40.13 [+ or -] 2.60
Arctigenin
0.2 [micro]M ([IC.sub.10])   22.94 [+ or -] 3.64
0.4 [micro]M ([IC.sub.20])   20.43 [+ or -] 5.31
0.8 [micro]M ([IC.sub.30])    7.15 [+ or -] 0.64

Matairesinol
1 [micro]M ([IC.sub.10])     19.90 [+ or -] 3.29
2 [micro]M ([IC.sub.20])     14.87 [+ or -] 2.83
3 [micro]M ([IC.sub.30])      8.16 [+ or -] 1.26

Arctiin
1 [micro]M ([IC.sub.10])      8.64 [+ or -] 2.31
4 [micro]M ([IC.sub.20])      6.72 [+ or -] 0.35
8 [micro]M ([IC.sub.30])      2.33 [+ or -] 0.26

(Iso)lappaol A
40 [micro]M ([IC.sub.10])     9.70 [+ or -]3.16
100 [micro]M ([IC.sub.20])    4.43 [+ or -] 0.84
140 [micro]M ([IC.sub.30])    3.66 [+ or -] 0.78

Lappaol C
100 [micro]M ([IC.sub.10])   12.31 [+ or -] 2.67
200 [micro]M ([IC.sub.20])    9.13 [+ or -] 2.83
300 [micro]M ([IC.sub.30])    4.36 [+ or -] 1.61

Lappaol F
1 [micro]M ([IC.sub.10])     21.43 [+ or -] 3.52
20 [micro]M ([IC.sub.20])    11.07 [+ or -] 2.83
40 [micro]M ([IC.sub.30])     5.20 [+ or -] 2.11

                             Three-drug combination with lignans
                             plus digitonin

                             Reversal ratio   CI     Interpretation

Doxorubicin alone             1.00            NR     NR
Arctigenin
0.2 [micro]M ([IC.sub.10])    1.75            0.83   ++
0.4 [micro]M ([IC.sub.20])    1.96            0.90   [+ or -]
0.8 [micro]M ([IC.sub.30])    5.65            0.84   ++

Matairesinol
1 [micro]M ([IC.sub.10])      1.46            0.77   ++
2 [micro]M ([IC.sub.20])      2.71            0.80   ++
3 [micro]M ([IC.sub.30])      4.95            0.78   ++

Arctiin
1 [micro]M ([IC.sub.10])      4.66            0.34   +++
4 [micro]M ([IC.sub.20])      5.97            0.29   ++++
8 [micro]M ([IC.sub.30])     17.2             0.20   ++++

(Iso)lappaol A
40 [micro]M ([IC.sub.10])     4.13            0.58   +++
100 [micro]M ([IC.sub.20])    9.11            0.78   ++
140 [micro]M ([IC.sub.30])   11.14            0.98   [+ or -]

Lappaol C
100 [micro]M ([IC.sub.10])    3.26            0.48   +++
200 [micro]M ([IC.sub.20])    4.40            0.44   +++
300 [micro]M ([IC.sub.30])    9.20            0.38   +++

Lappaol F
1 [micro]M ([IC.sub.10])      1.88            0.66   +++
20 [micro]M ([IC.sub.20])     3.65            0.45   +++
40 [micro]M ([IC.sub.30])     7.71            0.38   +++
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Author:Su, Shan; Cheng, Xinlai; Wink, Michael
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Feb 15, 2015
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