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Identification of Key Structural Characteristics of Schisandra chinensis Lignans Involved in P‑Glycoprotein Inhibition Jiří Slanina,†,▽ Gabriela Páchniková,‡,▽ Martina Č arnecká,† Ludmila Porubová Koubíková,‡,∥ Lenka Adámková,† Otakar Humpa,§ Karel Šmejkal,⊥ and Iva Slaninová*,‡ †

Department of Biochemistry, Faculty of Medicine, Masaryk University, Kamenice 5, Building A16, 625 00 Brno, Czech Republic Department of Biology, Faculty of Medicine, Masaryk University, Kamenice 5, Building A6, 625 00 Brno, Czech Republic § Central European Institute of Technology, Masaryk University, Kamenice 5, Building A4, 625 00 Brno, Czech Republic ⊥ Department of Natural Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Palackého 1-3, 612 42 Brno, Czech Republic ‡

S Supporting Information *

ABSTRACT: The aim of the present study was to determine the structural requirements for dibenzocyclooctadiene lignans essential for P-glycoprotein inhibition. Altogether 15 structurally related lignans isolated from Schisandra chinensis or prepared by modification of their backbone were investigated, including three pairs of enantiomers. P-Glycoprotein inhibition was quantified using a doxorubicin accumulation assay in human promyelotic leukemia HL60/MDR cells overexpressing P-glycoprotein. A preliminary quantitative structure−activity relationship analysis revealed three main structural features involved in P-glycoprotein inhibition: a 1,2,3-trimethoxy moiety, a 6-acyloxy group, and the absence of a 7-hydroxy group. The most effective inhibitors, (−)-gomisin N (1) and (+)-deoxyschizandrin [(+)-2], were selected for further evaluation of their effects. Both these lignans restored the cytotoxic effect of doxorubicin in HL60/MDR cells and when combined with a subtoxic concentration of this compound increased the proportion of G2/M cells significantly, which is a usual response to treatment with this anticancer drug.

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lignans, has been identified as inhibitors of P-gp and MRPs (multidrug resistance-related proteins).5−9 Such lignans are found almost exclusively in species of the two plant genera Schisandra and Kadsura.10 The main source of these lignans are the fruits of Schisandra chinensis (Turcz.) K. Koch (Schisandraceae), which are used widely in traditional Chinese medicine as a tonic and antitussive agent and in the treatment of lung, heart, and kidney disorders and used in health food products. Schisandra extracts are also clinically prescribed in mainland China for the treatment of hepatitis.11,12 The interaction of several dibenzocyclooctadiene lignans with P-gp was reported using competitive inhibition of photoaffinity labeling.13 Wan et al. observed reversion of Pgp-mediated multidrug resistance by gomisin A through noncompetitive inhibition of substrate−P-gp association.8 Schisandrin A (also known as deoxyschizandrin) showed the ability to reverse multidrug resistance in cancer cells by inhibition of P-gp expression.9 Our previous observations on lung cancer cells COR-L23/R overexpressing MRP1 protein confirmed the ability of dibenzocyclooctadiene lignans to overcome the resistance to doxorubicin.14

he ability of cancer cells to be cross-resistant to structurally and functionally unrelated anticancer drugs is known as multidrug resistance. Overexpression of the ATP binding cassette (ABC) transporters is responsible for most cases of cancer multidrug resistance (MDR) and chemotherapy failure.1,2 The most typical ABC transporter is P-glycoprotein (P-gp; 170 kDa), coded by the ABCB1 (MDR1) gene, which is responsible for the transportation out of cells of various xenobiotics including cytostatic compounds. A promising method for overcoming the resistance to anticancer drugs is the coadministration of compounds showing low toxicity and an ability to inhibit ABC transporters, i.e., the use of MDR inhibitors. In the last several decades, the search for effective and clinically applicable MDR inhibitors has occurred, and three generations of these agents were introduced into clinical trials. However, this has not led to any clear successes, especially because of the toxicity of these agents at doses necessary to block ABC transporters.3 As a result of unfavorable clinical outcome to previous MDR inhibitors, research has focused also on searching for naturally occurring MDR inhibitor candidates.3 Promising inhibitory activity of natural products on P-gp has been shown using plant phenols, especially curcumoids and flavonoids.4 Recently, another group of plant phenols, the dibenzocyclooctadiene © XXXX American Chemical Society and American Society of Pharmacognosy

Received: June 26, 2014

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dx.doi.org/10.1021/np500521v | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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activity) for both resistant HL60/MDR and parental HL60 cells were defined using a WST assay. The IC50 value of HL60/ MDR cells was approximately 200 times higher than that of parental HL60 cells (IC50 4.0 vs 0.02 μM). In order to confirm overexpression of MDR1 and to investigate expression of two other ABC transporters, MRP1 and MRP2 in both HL60/MDR and parental HL60 cells, RTPCR analysis was carried out. β-Actin was used as a control. The results showed that only the MDR1 gene is expressed in HL60/MDR cells. Neither MDR1 nor MRP1 and/or MRP2 genes were expressed in parental HL60 cells (Figure 2).

In contrast to verapamil, these lignans reveal antioxidative properties and show cardioprotective and hepatoprotective activities, which were demonstrated both on a cellular level and in mice.15−17 The cardioprotective effect of schisandrin B was shown using H9c2 cardiomyocytes.15 The experiments on mice demonstrated a protective effect of this compound against carbon tetrachloride-induced hepatotoxicity.16,17 The current knowledge of the structural aspects of P-gp inhibition by dibenzocyclooctadiene lignans is limited due to the small number of lignans that have been evaluated thus far. In the present study several such compounds were isolated from S. chinensis, and three pairs of enantiomers were prepared to better understand their structure−activity relationships of Pgp inhibition.

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RESULTS AND DISCUSSION Dibenzocyclooctadiene Lignans Investigated. The following dibenzocyclooctadiene lignans were isolated from the seeds of S. chinensis: gomisin N (1), (+)-deoxyschizandrin [(+)-2], tigloylgomisin P (3), (±)-γ-schizandrin [(±)-4], angeloylgomisin H (5), schisantherin C (6), gomisin G (7), gomisin J (8), wuweizisu C (9), gomisin A (10), and schizandrin (11), as described in the Experimental Section. Compounds (±)-4 and 8 were resolved into their enantiomers by chiral chromatography to give (+)-4, (−)-4, (+)-8, and (−)-8. The lignan (−)-2 was prepared by methylation of (−)-8. The structures of the lignans investigated are shown in Figure 1. Initial Biological Evaluation. The effect of dibenzocyclooctadiene lignans on doxorubicin accumulation and their ability to increase cytotoxic action of doxorubicin on resistant HL60/MDR cells were studied. IC50 values of doxorubicin (i.e., the concentration that induces a 50% inhibition of metabolic

Figure 2. RT-PCR analysis of ABC transporter expression: MDR1 (Pgp), MRP1, MRP2.

The presence of P-gp on the surface of HL60/MDR cells was demonstrated by flow cytometry analysis using a specific anti Pgp antibody (Figure 3).

Figure 3. P-gp on the surface of HL60/MDR cells. (A) Isotype control [PE/IgG1 (IOTest), Beckman Coulter]; (B) mouse monoclonal anti P-gp antibody conjugated with PE [anti-P-gp PE (IOTest) CD243, Beckman Coulter].

The inhibition of P-gp was studied by accumulation of the fluorescent substrate doxorubicin within resistant HL60/MDR cells. After 15 min of preincubation of the cells with the test lignans (25 μM), doxorubicin (10 μM) was added and the cells were incubated for another 60 min. The intensity of doxorubicin fluorescence within the cells was measured by flow cytometry. Previous studies have revealed that the lignan (+)-2 is a highly effective P-gp inhibitor.13,18 This finding was also supported by the present investigation, which indicated a high accumulation of doxorubicin within HL60/MDR cells treated with (+)-2. Therefore, (+)-2 was used to determine the most suitable concentration of the test lignans for the assessment of their P-gp inhibition potential. The doxorubicin accumulation was analyzed at various concentrations of (+)-2 as plotted in Figure 4. The doxorubicin accumulation was increased with rising concentrations of (+)-2 up to 25 μM almost linearly. Above this concentration, the doxorubicin accumulation increased at a slower pace. Accordingly, a 25 μM concentration of the test lignans was selected for the doxorubicin accumulation experiments.

Figure 1. Structures of compounds [tig = tigloyl (E-2-methyl-2butenoyl), bnz = benzoyl, ang = angeloyl (Z-2-methyl-2-butenoyl)]. B



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7-hydroxy group, the second best descriptor, significantly improved the correlation (R = 0.674, p = 0.036, Table 2). The negative impact of a hydroxy group at C-7 was evident from the partial correlation coefficient for this structural parameter in the one-parameter model (R = −0.324, Table 2). The threeparameter models were derived from the best two-parameter model. The use of two three-parameter models was found to improve the relationship determined. They included either the M-biaryl conformation (R = 0.807, p = 0.012) or 6-acyloxy group (R = 0.838, p = 0.006) as a third parameter. Since the latter model described the relationship more tightly, the optimal three-parameter model involved the following structural features: a 1,2,3-trimethoxy moiety, a 7-hydroxy group, and a 6-acyloxy group. The combination of four descriptors (1,2,3-trimethoxy, 7-hydroxy, 6-acyloxy, and M-biaryl) did not improve significantly the optimal three-parameter model. The correlation coefficient was only weakly improved (R = 0.853 vs 0.838), but the four-parameter model was less significant (p = 0.012 vs 0.006), and particularly, this model made two descriptors insignificant, the 7-hydroxy and 6-acyloxy groups, with p = 0.14 and 0.37, respectively. Therefore, the final model is represented by eq 1.

Figure 4. Relationship between concentration of (+)-2 and the ability to potentiate the accumulation of doxorubicin in HL60/MDR cells.

The accumulation of doxorubicin in combination with dibenzocyclooctadiene lignans related to the accumulation of doxorubicin alone is presented in Table 1. The accumulation was increased significantly on the effects of the lignans with a high degree of certainty according to the Dunnett test (p < 0.0001), but the effects of (±)-4 and 11 were less conclusive (p < 0.05). Under the conditions used, some lignans were more effective than verapamil, namely, 1 (p < 0.001), (−)-2 (p < 0.01), and (+)-2 (p < 0.05). Although several articles have reported the inhibition of P-gp transport activity by dibenzocyclooctadiene lignans,5−9 none of these investigations studied their quantitative structure−activity relationships (QSAR). To identify the structural characteristics of lignans essential for potent P-gp inhibition, the results were analyzed by a stepwise multiple regression. The data given in Table 1 were used for construction of a QSAR model. The structural characteristics of interest were the biaryl configuration and the 1,2,3-trimethoxy, 2,3-methylenedioxy, 3,12dihydroxy, 12,13,14-trimethoxy, 12,13-methylenedioxy, 6-acyloxy, and 7-hydroxy groups. First, a one-parameter model for each descriptor was calculated. The partial correlation coefficients and p-values for one-parameter models are given in Table 2. The best correlation was obtained with a 1,2,3trimethoxy moiety (R = 0.444, p = 0.112). Subsequently, twoparameter models were constructed on the base of the best one-parameter model. For two-parameter models, addition of a

% doxorubicin accumulation = 181 ( ±14) + 74 ( ±18) 1,2,3‐tri‐OCH3 − 92 (± 22) 7‐OH + 73 ( ±25) 6‐acyloxy

(1)

The relation between the P-gp inhibition measured (Table 1) and the inhibition calculated according to eq 1 is shown in Figure 5. The least-squares regression showed a very good correlation between the calculated and measured data (R = 0.838). The square of the correlation coefficient (R2 = 0.702) indicated that 70% of the variation may be explained by three significant variables. The five most effective dibenzocyclooctadiene lignans found have two of three important structural characteristics, i.e., the presence of a 1,2,3-trimethoxy moiety, a 6-acyloxy group, and/ or the absence of a 7-hydroxy group, but none of them met all

Table 1. Percentage of Doxorubicin (10 μM) Accumulation in HL60/MDR Cells after Pretreatment with Lignans (25 μM) and the Correlation Matrix for Multiple Regressiona compound verapamil 1 (−)-2 (+)-2 3 (+)-4 (−)-4 5 6 7 (−)-8 (+)-8 9 10 11

% accumulation

Mbiaryl

1,2,3trimethoxy

2,3methylenedioxy

3,12dihydroxy

12,13,14trimethoxy

12,13methylenedioxy

6acyloxy

7hydroxy

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0 0 1 0 1 0 1 0 0 0 1 0 1 1

1 1 1 1 1 0 1 1 0 0 0 0 1 1

0 0 0 0 0 1 0 0 1 0 0 1 0 0

0 0 0 0 0 0 0 0 0 1 1 0 0 0

0 1 1 0 0 1 0 0 1 0 0 0 0 1

1 0 0 1 1 0 0 1 0 0 0 1 1 0

0 0 0 1 0 0 0 1 1 0 0 0 0 0

0 0 0 1 0 0 1 1 1 0 0 0 1 1

239 283 277 264 234 232 152 202 201 199 191 156 187 162 125

30 33 24 28 14 38 24 12 19 27 27 14 21 13 17

Accumulation of racemic (±)-4 was 124 ± 10%. The values of accumulation (in %) are means ± SD of at least four independent experiments performed in triplicate.

a

C



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Table 2. Partial Correlation Coefficient (R) and p-Values for Three Consecutive Multiparameter Modelsa one-parameter model

two-parameter model

substituent

R

p

R

p

three-parameter model R

p

1,2,3-trimethoxy 7-hydroxy 6-acyloxy M-biaryl 2,3-methylenedioxy 3,12-dihydroxy 12,13-methylenedioxy 12,13,14-trimethoxy

0.444 −0.324 0.075 −0.267 −0.286 −0.272 0.219 −0.020

0.112 0.259 0.798 0.355 0.321 0.347 0.453 0.946

1st parameter 0.674 0.448 0.630 0.450 0.450 0.449 0.444

0.036 0.291 0.062 0.297 0.297 0.289 0.299

1st parameter 2nd parameter 0.838 0.807 0.690 0.690 0.678 0.674

0.006 0.012 0.080 0.080 0.092 0.096

a One-parameter model, two-parameter model, and three-parameter model describing the effect of lignans on doxorubicin accumulation in HL60/ MDR overexpressing P-glycoprotein. The three-parameter model: % accumulation = 181 (±14) + 74 (±18) 1,2,3-tri-OCH3 − 92 (±22) 7-OH + 73 (±25) 6-acyloxy.

(Table 2, R = −0.020). A methylenedioxy group attached to the B ring correlated better with increased accumulation of doxorubicin than the trimethoxy group, but the correlation was found to be statistically insignificant (Table 2, R = 0.219, p = 0.45). Four pairs of lignans [10 vs 11, (−)-4 vs 9, (+)-2 vs (+)-4, and 1 vs (−)-2], differing from each other only in the substitution on the aromatic B ring, were selected for direct comparison. Compounds with a methylenedioxy group attached to the B ring showed increased doxorubicin accumulation for three of these four pairs, with only (+)-2, having a trimethoxy group, being more effective than (+)-4. A paired Student’s t test was applied to the accumulation percentages of these four pairs of compounds, and no significant differences were found (p = 0.539). Interestingly, compound 5 differs from the substantially less active 11 (202% vs 125% doxorubicin accumulation) only in having a nonpolar, quite bulky angeloyl group attached to C-14 of the B ring. Lignan 5 was found to be the most active among those lignans containing only one of the three parameters enhancing doxorubicin accumulation. This demonstrates that substitution at C-14 may markedly enhance the effectiveness of P-gp inhibition. Lignan 10 was previously isolated from the fruits of S. chinensis by activity-guided fractionation as a compound that effectively blocked the function of P-gp.8 However, only moderate or weak P-gp inhibition by 10 was found in the present work, which is also consistent with some studies published previously.9,13 This QSAR work showed that the absence of a 7-hydroxy group is important for the potent activity of the lignans investigated. This effect is not surprising since the absence of the hydroxy group results in an increase of the hydrophobicity of inhibitors, which is essential for their uptake into the lipid bilayer. Compounds 3 and 5, containing a 7-hydroxy group, are relatively polar lignans, but they remain effective due to the presence of other functional groups that enhance activity. Despite the small number of lignans possessing a 6-acyloxy group among the set of molecules investigated, the QSAR model revealed the marked potentiation of activity by a 6acyloxy group in the final three-parameter model. The importance of acylation at C-6 was not obvious directly from the percentages of doxorubicin accumulation in a oneparameter QSAR model, because all three lignans carrying a 6-acyloxy group also have a 7-hydroxy group, which decreases the activity. The positive effect of esterification at C-6 shown in the present study corresponds to a previous observation, in

Figure 5. Correlation between observed versus calculated doxorubicin accumulation in HL60/MDR cells induced by the test compounds. The calculated data were obtained by the final three-parameter QSAR model. The squared correlation coefficient was 0.702.

three structural features necessary for increasing doxorubicin accumulation. Thus, the use of a QSAR method can predict that there are enhanced inhibitors that meet all structural requirements. The QSAR data indicated that a 1,2,3-trimethoxy moiety is a key component for effective and potent P-gp inhibition. The eight most active lignans found contain this functionality on the aromatic A ring (Table 1). Even a small change in substitution of this crucial structural component decreases activity. This may be seen from the superior activity of (−)-2, containing a 1,2,3trimethoxy substituent (doxorubicin accumulation 277%), in comparison to (−)-4 (accumulation 152%), with a 1-methoxy2,3-methylenedioxy moiety. Another example showing the importance of the 1,2,3-trimethoxy moiety is the clear difference in potentiation of doxorubicin accumulation between 1 (accumulation 283%) and 9 (accumulation 187%). The most active, 1, is again substituted with a 1,2,3-trimethoxy moiety, whereas 9 contains a 1-methoxy-2,3-methylenedioxy functionality. The lignan 8 differs from the two enantiomers of 2 in the absence of methyl groups at C-3 and C-11. The negative contribution of hydroxy groups at these two positions was evident from the comparisons of (−)-2 versus (−)-8 and (+)-2 versus (+)-8 (Table 1). This indicates that at least a methoxy group attached to C-3 is essential for the potent inhibition of Pgp. Despite the importance of a 1,2,3-trimethoxy moiety attached to the aromatic A ring, substitution of the B ring with a 12,13,14-trimethoxy moiety had no significant effect on accumulation according to the one-parameter QSAR model D



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Figure 6. Viability of the cells treated for 48 h by doxorubicin (DOXO) at concentrations of 0.02, 0.06, and 0.2 μM in the presence or absence of the lignans or verapamil (V) at a concentration of 25 μM. The data show means ± SD of at least three independent experiments. The viability of cells after treatment with doxorubicin alone was compared with viability after treatment with doxorubicin in combination with the test lignans or verapamil (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

The present results based on a one-parameter QSAR model showed that lignans possessing a P-biaryl configuration are more active than their counterparts with an M-biaryl configuration, but this difference was not significant statistically (Table 1). As shown above, the biaryl configuration was a component in a statistically significant three-parameter model, but the final three-parameter model, which included a 6-acyloxy group as a parameter instead of biaryl configuration, was more accurate. Due to the preparation of three pairs of enantiomers, the importance of absolute configuration was evaluated directly by using a t test for each pair of enantiomers. There is no doubt that (+)-4 increased the accumulation of doxorubicin substantially more effectively than (−)-4 (p < 0.0001), but conversely, (−)-8 exceeded the effect of (+)-8 (p < 0.01). The difference between the effects of (+)-2 and (−)-2 was only slightly (ca. 10%) in favor of (−)-2. The significant difference in activity between the enantiomers of 4 may be explained by the fact that only (+)-4, but not (−)-4, contains the activity enhancing the trimethoxy moiety attached to the aromatic A ring. Dibenzocyclooctadiene Lignans Overcome the Resistance to Doxorubicin in HL60/MDR Cells. The present results demonstrated clearly that lignans increased the accumulation of doxorubicin in resistant cells. To determine whether the lignans increase the sensitivity of HL60/MDR cells to doxorubicin, cytotoxicity tests were performed. The viability of cells treated for 48 h with three different concentrations of doxorubicin (0.02, 0.06, and 0.2 μM) was determined in the presence or absence of the lignan 1 or (+)-2 and verapamil (used as a positive control) at a concentration of 25 μM using the propidium iodide exclusion assay. The lignans or doxorubicin alone exhibited a toxicity up to 10% dead cells, while a combination of lignans with doxorubicin at all concentrations led to an increased proportion of dead cells on comparing to doxorubicin alone (Figure 6). The present results indicate that the test lignans restored the cytotoxic action of doxorubicin in resistant cells. The effects of three enantiomeric lignan pairs and the active lignan 1 (25 μM) in combination with three concentrations of doxorubicin (0.02, 0.2, and 0.6 μM) on cell proliferation were analyzed using the WST assay. Table 3 shows the viability of the cells treated 48 h with doxorubicin alone or doxorubicin in combination with lignans or verapamil related to an untreated control. Lignans 1, (+)-2, (−)-2, and (±)-4 significantly increased the toxicity of doxorubicin at a concentration of 0.2 μM, while all lignans with the exception of (−)-4 and (+)-8 were effective in combination with doxorubicin at a

which synthetic 6-acyl derivatives of lignans inhibited P-gp in Kb-V1 multidrug resistant human cervix carcinoma cells.19 The ester moiety of cyclooctadiene lignans may be attached to C-6 from either of the two stereochemically distinct sides of the cyclooctene ring. The isomeric lignans 3 and 6 differ only in the orientation at C-6 and C-7, and therefore they can be used for a direct comparison. Compound 3 was slightly more active than compound 6 (234% vs 201% doxorubicin accumulation). Effect of Absolute Configuration. The interaction between P-gp and chiral ligands is an interesting field of research. Only a few reports have been made on the comparison of the P-gp inhibitory activity of enantiomers. For example, enantiomers of verapamil, omeprazole, and propranolol displayed equal affinities to P-gp, suggesting that their recognition of P-gp is nonchiral.20−22 On the other hand, stereoselective P-gp inhibition by mefloquine was observed in rat cells but not in human cells. The (+)-stereoisomer of mefloquine was up to 8-fold more effective than its antipode in increasing cellular accumulation of [3H]vinblastine in GPNT (rat brain capillary endothelial) cells, while in Caco-2 (human colon carcinoma) cells, both enantiomers were equally effective.23 It was also found that mouse P-gp could distinguish between two enantiomeric cyclic peptides.24 Dibenzocyclooctadiene lignans are chiral compounds containing at least two asymmetric carbons at C-7 and C-8. Moreover, they exhibit axial chirality, which arises from restricted free rotation around the single bond connecting the two aromatic rings. These compounds have been isolated mostly from Schisandra species in the optically active form. However, some of these, such as (±)-4, have been isolated as a racemate.25 In a previous study, (+)-2 and (±)-4 were identified as the most potent inhibitors of multidrugresistance-associated protein (MRP1) among nine lignans evaluated. Since the common structural feature of both active lignans is a M-biaryl configuration and the structurally similar lignan, 1, possessing an opposite P-biaryl configuration was less active, it was suggested that the absolute configuration of lignans may play an important role in the modulation of ABC transporter activity.14 Using chiral HPLC, it was found that all lignans isolated in the present study are pure enantiomers except for racemic (±)-4 and 8, in which (−)-8 predominates. To investigate the effect of absolute configuration of lignans on P-gp inhibition more comprehensively, three pairs of enantiomers (2, 4, and 8) were prepared, as described in the Experimental Section and the Supporting Information. E



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increase of cells arrested in the G2/M phase (16%, Figure 7C) was observed. Neither (+)-2 (12.1%, Figure 7D) nor 1 (8.8%, Figure 7E) alone had any effect on the cell cycle. Both lignans in combination with a lower concentration of doxorubicin increased the proportion of G2/M cells significantly: (+)-2 with doxorubicin by 67.9% (Figure 7F) and 1 with doxorubicin by 36.9% (Figure 7G). In addition to the cell cycle profile, also the sub-G1 peak corresponding to apoptotic and/or necrotic cells was observed upon treatment with doxorubicin at a concentration of 0.2 μM and in cells treated with a combination of lignans and doxorubicin at a concentration 0.02 μM (Figure 7B, F, and G). Since the cell cycle arrest in the G2/M phase is usually a cellular response to doxorubicin treatment, it can be assumed that these lignans potentiate the effect of doxorubicin in doxorubicin-resistant HL60/MDR cells. In the present study, a preliminary structure−activity relationship of dibenzocyclooctadiene lignans as P-gp inhibitors has been determined. The three main structural characteristics involved in P-gp inhibition were identified: a 1,2,3-trimethoxy moiety, a 6-acyloxy group, and the absence of a 7-hydroxy group. The differences in activity between the enantiomers of 4 and the structural requirement for a trimethoxy moiety located only on the A ring, but not on the B ring, have provided evidence that the interaction between an active lignan of this type and P-gp is stereospecific (Figure 8). The potentiation of the doxorubicin effect on HL60/MDR cells was shown in cytotoxicity assays and cell cycle analysis. Since none of the lignans tested met all three structural requirements for potent P-gp inhibition, the present results may stimulate the rational design of more efficient molecules. Among the lignans investigated, 1 and (+)-2 were found to be the most promising P-gp modulators due to their strong increases of the doxorubicin accumulation and potentiation of the cytotoxicity shown for doxorubicin. These lead compounds might be improved further by substitution at carbon C-6 and by modification of the aromatic B ring.

Table 3. Percentage of Viability of HL60/MDR Cells on Treatment with Doxorubicin and/or Doxorubicin in Combination with Selected Lignansa doxorubicin (μM) compound

0

control (DMSO)

96 ± 2

1 (+)-2 (−)-2 (±)-4 (+)-4 (−)-4 (+)-8 (−)-8 verapamil

128 92 144 88 102 106 85 78 93

± ± ± ± ± ± ± ± ±

0.02

28 11 23 13 26 8 10 8 12

109 121 108 106 117 94 100 100 73 78

± ± ± ± ± ± ± ± ± ±

0.2 9 12 30 12 0 27 16 9 34 12

83 42 52 48 41 66 97 94 52 29

± ± ± ± ± ± ± ± ± ±

12 11* 4* 1* 13* 41 7 17 8 10**

0.6 91 39 43 39 36 38 81 73 30 4

± ± ± ± ± ± ± ± ± ±

14 13* 2** 2* 19* 14* 14 2 8* 5***

a

Data are expressed as percentages relative to untreated cells and are means ± SD of two independent experiments performed in triplicate. The statistical analysis compared samples of selected lignans (or verapamil) in combination with doxorubicin to doxorubicin only at the same concentration (*p < 0.05, **p < 0.01, ***p < 0.001).

concentration of 0.6 μM. Verapamil enhanced the effect of doxorubicin at concentrations of 0.2 and 0.6 μM. The above-mentioned results demonstrated the strong potentiation of doxorubicin cytotoxicity by most test lignans and confirmed the results obtained in accumulation studies, in which the lignans (+)-4 and (−)-8 were more potent than their enantiomers. A G2/M cell cycle arrest is a typical consequence of doxorubicin treatment.14,26 Therefore, the ability of 1 or (+)-2 (25 μM) was tested in combination with doxorubicin at a concentration of 0.02 μM to increase the proportion of resistant HL60/MDR cells in the G2/M phase. Doxorubicin at a concentration of 0.2 μM greatly increased the percentage of cells in the G2/M phase (52%, versus 11% in DMSO only treated cells, Figure 7A,B), while at a 10 times lower concentration of doxorubicin (0.02 μM), only a weak

Figure 7. Cell cycle analysis. Lignan 1 or (+)-2 (25 μM) in combination with doxorubicin at a concentration of 0.02 μM increased the proportion of G2/M significantly. The sub-G1 peak that could display apoptotic and/or necrotic cells is apparent (B, F, G: a). Control (A), doxorubicin 0.2 μM (B), doxorubicin 0.02 μM (C), (+)-2 (D), 1 (E), (+)-2 + doxorubicin 0.02 μM (F), 1 + doxorubicin 0.02 μM (G). F



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A stock solution of each lignan (1 mg/50 μL) was prepared in dimethyl sulfoxide (DMSO; Sigma-Aldrich Corp.) and stored at −20 °C. In biological experiments, the lignans were used in a concentration range of 0.025−75 μM. Cell Lines and Cultivation Conditions. Two cancer cell lines were used: HL60 (human promyelotic leukemia) obtained from the European Collection of Animal Culture (ECAC, Salisbury, U.K.) and its multidrug-resistant subline HL60/MDR derived from parental HL60 cells after treatment with doxorubicin, which was obtained from Prof. B. Sarkadi (Semmelweis University Medicine, National Blood Centre, Membrane Research Group, Hungarian Academy of Science, Budapest, Hungary). The cells were grown in RPMI 1640 supplemented with 2 mM glutamine, 10% fetal calf serum, 100 IU/ mL penicillin, and 100 μg/mL streptomycin (PAA Laboratories, Austria). Cells were incubated at 37 °C under 5% CO2 in a highhumidity atmosphere and subcultured three times a week. RNA Extraction and RT-PCR. RT-PCR was carried out to analyze the mRNA levels of MRP1, MRP2, and P-gp (MDR1) as described previously.14 Briefly, total RNA was extracted using the RNAeasy Mini kit (Quiagen) according to the manufacturer’s instructions. PCR was performed using gene-specific primers for MRP1 (forward: 5′GGTGCTTCCCACGGAGG-3′; reverse: 5′-TCAACCACAAAACTGCAGCC-3′), MRP2 (forward: 5′-GACATCAGAAATAGAGACC-3′; reverse 5′-CTACTCCATCAATGATAATCTGACC-3′), and MDR1 (forward: 5′-AGTGGTTCAGGTGGCTCT-3′; reverse: 5′-TTCTGTCTTGGGCTTGTG-3′). The β-actin gene was coamplified and served as a control (forward: 5′TTCCAGCCTTCCTTCCTGGG-3′; reverse: 5′-TTGCGCTCAGGAGGAGCAAT-3′). All primers were synthesized by Invitrogen. Expected product sizes were 182 bp for MRP1, 341 bp for MDR1, 322 bp for MRP2, and 250 bp for β-actin. PCR amplification was performed on a GeneAmp PCR System 9700 (Applied Biosystems). PCR products were run on 2% agarose gels containing 0.6 μg/mL of ethidium bromide and photographed under a TCX20 UVtransiluminator (Vilber Lourmat, France). Immunodetection of MDR1 Protein. The presence of MDR1 protein at the surface of HL60/MDR cells was studied using flow cytometry analysis of living cells stained with mouse monoclonal anti P-gp antibody conjugated with PE (IOTest) CD243 (P-gp; Beckman Coulter). As an isotype control mouse PE/IgG1 (IOTest, Beckman Coulter) was used. An excitation wavelength of 488 nm and an emission wavelength of 575 nm (FL2) were used. A minimum of 10 000 cells were counted for each sample. Drug Accumulation Assay. Since doxorubicin is a fluorescent substance, its content in cells can be measured directly by flow cytometry. For the drug accumulation assays HL60/MDR cells at a concentration of 2 × 106 per mL grown in 150 μL of medium in 96well plates were preincubated in medium containing either a test compound (25 μM) or verapamil (25 μM) for 15 min. Then doxorubicin (10 μM) was added, and the cells were incubated for an additional 60 min. The cells were collected and washed with ice-cold PBS. The intracellular doxorubicin content was determined using a Cytomics FC 500 flow cytometer (Beckman Coulter, Inc., CA, USA) at an excitation wavelength of 488 nm and an emission wavelength of 575 nm (FL2). A minimum of 10 000 cells were counted for each sample. Results were expressed as a percentage ratio of the mean fluorescence of doxorubicin in the presence of a modulator to the mean fluorescence of doxorubicin without a modulator. Determination of Antiproliferative Activity. Cell Viability Assay. The cell viability assay was based on the exclusion of propidium iodide (PI; Sigma-Aldrich, St. Louis, MO, USA) by the intact viable cells. The cells were plated in 12-well tissue culture test plates (Orange Scientific, Braine-I’Alleud, Belgium) at 7 × 104 cells/mL of medium and treated with test compounds at a concentration of 25 μM in combination with doxorubicin at concentrations of 0.02, 0.06, and 0.2 μM. After 48 h of incubation PI was added, and immediately after addition, the percentage of dead (PI-positive) cells was detected using a Cytomics FC 500 flow cytometry system (Beckman Coulter, Inc.) with channel FL3 (emission at 620 nm). A total number of 10 000

Figure 8. Summary of the structural features of dibenzocyclooctadiene lignans influencing the inhibition of P-gp in HL60/MDR cells. The plus-circles specify the positive contribution of structural elements to P-gp inhibition, and the minus-circles show the negative impact on Pgp inhibition.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO P-2000 polarimeter in a 1 dm tube filled with chloroform solution at the D line of sodium. CD spectra were recorded using a JASCO J-815 spectropolarimeter in methanol loaded into a cuvette with a path length of 10 mm. 1H and 13C NMR spectra were recorded on a Bruker AVANCE 300 MHz instrument or a Bruker AVANCE III 500 MHz spectrometer equipped with 5 mm Prodigy cryoprobe or a Bruker AVANCE III 700 MHz instrument equipped with 5 mm TXO cryoprobe. All samples were dissolved and measured in CDCl3 at 298 K. Chemical shifts are reported in ppm relative to tetramethylsilane. The compounds were identified on the basis of 1H, 13C, 13C APT, 1H−1H COSY, 1H−13C HSQC, and 1 H−13C HMBC experiments. High-resolution mass spectra were recorded on a MicrOTOF-QII (Bruker, Germany) mass spectrometer, operated in a positive electrospray ionization mode. Plant Material. The seeds of Schisandra chinensis were collected in the surroundings of Vladivostok (Russia) and were imported by Herbaton Ltd. (Klčov, Slovakia). The seeds were identified by Ing. Pavel Musil (head of the Centre of Medicinal Plant of Masaryk University), and a voucher specimen (No. 728-12) was deposited in the Department of Biochemistry, Faculty of Medicine, Masaryk University, Brno. The seeds were air-dried for a week at ambient temperature and then powdered with a mechanical grinder. Extraction and Isolation. The lignans 1, (+)-2, 3, (±)-4, 5, 8, 9, 10, and 11 were isolated from seeds of S. chinensis as described in the Supporting Information. The isolation of 6 and 7 is described in a previous paper.27 The lignans were identified by using 1H and 13C NMR, ESIMS, and CD experiments and by comparison of experimental data with those described in the literature.25,28−38 Characterization of compounds is given in the Supporting Information. The structure of 1 was also verified using X-ray crystallographic analysis.39 Preparation of (+)-4, (−)-4, (+)-8, (−)-8, and (−)-2. Racemic (±)-4 (18 mg) was separated into the corresponding enantiomers on a short semipreparative Chiralcel OD column (50 × 10 mm i.d., Daicel) with a mobile phase containing 0.125% (v/v) propane-2-ol in nhexane. A sample injection was 1−2 mg of (±)-4. As a result of semipreparative chiral chromatography, 7.8 mg of (+)-4 and 5.2 mg of (−)-4 were obtained. Compound 8 (20 mg) was resolved into enantiomers by repeating the separation on the Chiralcel OD column (50 × 10 mm i.d., sample injection 1 mg) using a mixture of nhexane−propane-2-ol (99:1) as a mobile phase. After evaporation of the solvent, 2.0 mg of (+)-8 and 15.7 mg of (−)-8 were obtained. The lignan (−)-2 (4.7 mg) was prepared by methylation of (−)-8 (6.7 mg) with methyl iodide (0.1 mL) and K2CO3 (9.5 mg) in acetone under reflux. G



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cells were analyzed for each sample. The viability of cells after treatment with doxorubicin alone was compared with the viability after treatment with doxorubicin in combination with the test compounds or verapamil (positive control). WST Assay. Antiproliferative activity was assessed using a WST assay on 96-well plates (Nunc A/S, Rockilde, Denmark). The assay is based on the reduction of WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, Na salt) by viable cells. The amount of the formazan dye generated by the activity of dehydrogenases within cells is therefore directly proportional to the number of living cells. All cells were seeded at a density of 5 × 104 cells per mL (100 μL/per well) and incubated at 37 °C in a medium containing doxorubicin, modulators (test compounds or verapamil), or doxorubicin with modulators for 48 h. After incubation 10 μL of working WST-1 solution [1 mM WST (Serva, Heidelberg, Germany)] and 0.2 mM MMPM [(1-methoxy-5-methylphenazinium methyl sulfate; Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA)] were added to 100 μL of fresh medium in each well, and cells were incubated for a further 4 h under normal cell culture conditions. At the end of the incubation periods the optical density was read at 450 nm using a DTX 880 multimode detector (Beckman Coulter, Inc.). Each concentration of each of the test compounds was examined in four replicate wells. The experiment was repeated at least three times. Cell Cycle Analysis. Approximately 5 × 105 cells/mL (HL60/ MDR) were treated with doxorubicin at a concentration of 0.2 μM and/or 0.02 μM either alone or in combination with 1 and/or (+)-2 at a concentration of 25 μM and cultivated under standard conditions for 48 h. Cells were harvested into ice-cold PBS and fixed with 70% ethanol for 30 min at room temperature. After washing in PBS, the cells were incubated with RNase A (17 μg/mL) at 37 °C for 30 min and then stained with PI (3 μg/mL) for 10 min (in darkness, at room temperature). The cell cycle profile was analyzed using a Cytomics FC 500 flow cytometer using the FL3 channel (emission at 620 nm), and 10 000− 20 000 events were acquired. Data from each sample were saved as separate flow cytometry standard (FCS) files using CXP analysis software (Beckman Coulter, Inc.) and were analyzed for cell cycle phases using Multicycle AV for Windows software (Phoenix Flow System, San Diego, CA, USA). DNA content analysis included the determination of the percentage of G1, G2/M, and S phases and subG1 fractions. Statistical Analysis. All statistical analysis was performed with Statistica version 10 (StatSoft software). The data for doxorubicin accumulation were from at least four independent experiments performed in triplicate. Statistical comparison of doxorubicin accumulation induced by the test compound versus the control group and the verapamil group was done using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Stepwise multiple regression analysis was used to find the relation between the percentage of P-gp inhibition and the descriptors in a QSAR model. The least-squares regression analysis was used to determine the correlation between the measured data and calculated percentage of doxorubicin accumulation. A paired Student’s t test was applied to compare the effects of enantiomers or pairs of test lignans differing only in one structural parameter. Cytotoxicity data were evaluated from at least three independent experiments performed in triplicate (PI-exclusion assay) or two independent experiments performed in quadruplicate (WST assay). The results were analyzed using Student’s t test.

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Article

AUTHOR INFORMATION

Corresponding Author

*Tel: +420549496985. Fax: 549491327. E-mail: ipokorna@ med.muni.cz. Present Address

Public Health Institute Ostrava, Partyzánské Náměsti ́ 7, 702 00 Ostrava, Czech Republic.

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Author Contributions ▽

J. Slanina and G. Páchniková contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Prof. B. Sarkadi (Semmelweis University Medicine, National Blood Centre, Membrane Ressearch Group, Hungarian Academy of Science, Budapest, Hungary) for providing HL60/MDR cells and Mrs. Z. Prokopová and I. ́ Komárková for laboratory assistance. O. Julinek, Institute of Chemical Technology, Prague, Czech Republic, is acknowledged for optical rotation measurements. This work was supported by Masaryk University Projects of Specific Research MUNI/A/0938/2013 and MUNI/A/0954/2013.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The isolation procedure for the lignans investigated and their characteristic details, including circular dichroism and NMR spectra, are available free of charge via the Internet at http:// pubs.acs.org. H



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