Potential of decursin to inhibit the human cytochrome P450 2J2 isoform.

FCT 7936

No. of Pages 6, Model 5G

14 May 2014 Food and Chemical Toxicology xxx (2014) xxx–xxx 1

Contents lists available at ScienceDirect

Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox 4 5

Potential of decursin to inhibit the human cytochrome P450 2J2 isoform

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Boram Lee a,1, Zhexue Wu a,1, Sang Hyun Sung b, Taeho Lee a, Kyung-Sik Song a, Min Young Lee a,⇑, Kwang-Hyeon Liu a,⇑ a b

College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 702-701, Republic of Korea College of Pharmacy and Research Institute of Pharmaceutical Science, Seoul National University, Seoul 151-742, Republic of Korea

a r t i c l e

i n f o

Article history: Received 25 November 2013 Accepted 11 April 2014 Available online xxxx Keywords: CYP2J2 Cytotoxicity Decursin Drug interactions Human liver microsomes

a b s t r a c t CYP2J2 enzyme is highly expressed in human tumors and carcinoma cell lines, and epoxyeicosatrienoic acids, CYP2J2-mediated metabolites, have been implicated in the pathologic development of human cancers. To identify a CYP2J2 inhibitor, 50 natural products obtained from plants were screened using astemizole as a CYP2J2 probe substrate in human liver microsomes. Of these, decursin noncompetitively inhibited CYP2J2-mediated astemizole O-demethylation and terfenadine hydroxylation activities with Ki values of 8.34 and 15.8 lM, respectively. It also showed cytotoxic effects against human hepatoma HepG2 cells in a dose-dependent manner while it did not show cytotoxicity against mouse hepatocytes. The present data suggest that decursin is a potential candidate for further evaluation for its CYP2J2 targeting anti-cancer activities. Studies are currently underway to test decursin as a potential therapeutic agent for cancer. Ó 2014 Elsevier Ltd. All rights reserved.

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1. Introduction

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CYP2J2 is the only member of the human 2J subfamily, and, unlike other cytochrome P450 (P450) isozymes, it is predominantly expressed in extrahepatic tissues including the heart, skeletal muscle, placenta, small intestine, kidney, lung, pancreas, bladder, and brain (Enayetallah et al., 2004; Zeldin et al., 1997, 1996). CYP2J2 plays a major role in the metabolism of some xenobiotics, such as albendazole (Wu et al., 2013b), astemizole (Matsumoto et al., 2003, 2002; Matsumoto and Yamazoe, 2001), terfenadine (Lafite et al., 2007; Rodrigues et al., 1995), and ebastine (Liu et al., 2006). It is also responsible for the formation of epoxyeicosatrienoic acids (EETs) from arachidonic acid, an endogenous metabolite (Wu et al., 1996). CYP2J2 is also highly expressed in tumor tissues and promotes tumor growth and proliferation (Chen et al., 2011; Jiang et al., 2005b, 2009). Several studies have implicated CYP2J2 and its EET metabolites in the pathological development of human cancers for both solid tumors and hematological malignancies (Chen et al., 2009a, 2011; Freedman et al., 2007; Jiang et al., 2005b, 2007b). These findings suggest that inhibition of CYP2J2-mediated EETs biosynthesis may represent a new approach for the treatment of

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⇑ Corresponding authors. Tel.: +82 53 950 8567; fax: +82 53 950 8557. 1

E-mail addresses: [email protected] (M.Y. Lee), [email protected] (K.-H. Liu). These authors contributed equally to this work.

human cancers. Several CYP2J2 inhibitors such as 17-octadecynoic acid (Jiang et al., 2007a), terfenadine derivatives (Chen et al., 2009a, 2011) and let-7b (Chen et al., 2012) showed significant antitumor effects in vitro and in vivo by reducing EET biosynthesis. These inhibitors have been demonstrated to reduce proliferation, migration, invasion and adhesion, to promote apoptosis of cancer cells (Chen et al., 2009a, 2011; Jiang et al., 2007a, 2005b; Nithipatikom et al., 2010), to inhibit tumor growth and metastasis, and to prolong survival in tumor-bearing mice (Chen et al., 2009a, 2011). However, little data are available on the inhibitors of CYP2J2 enzyme to date (Lafite et al., 2006; Lee et al., 2012; Liu, 2011; Ren et al., 2013; Wu et al., 2013a). To identify a CYP2J2 inhibitor, 50 natural products obtained from medicinal plants were screened using astemizole as a CYP2J2 probe substrate in human liver microsomes. We further evaluated cancer cell-specific cytotoxicity for the compounds that inhibited CYP2J2 to confirm the potential anti-cancer effects.

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2. Materials and methods

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2.1. Materials

79

Astemizole, O-desmethyl astemizole, terfenadine, terfenadine alcohol and hydroxyebastine were purchased from Toronto Research Chemicals (North York, Canada). Glucose-6-phosphate (G6P), glucose-6-phosphate dehydrogenase (G6PDH), and b-nicotinamide adenine dinucleotide phosphate (NADP+) were obtained from Sigma–Aldrich (St. Louis, MO). Fifty natural compounds (Table 1) were a gift from the Institute for Korea Traditional Medical Industry (Daegu, Korea).

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http://dx.doi.org/10.1016/j.fct.2014.04.020 0278-6915/Ó 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Lee, B., et al. Potential of decursin to inhibit the human cytochrome P450 2J2 isoform. Food Chem. Toxicol. (2014), http:// dx.doi.org/10.1016/j.fct.2014.04.020

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Pooled human liver microsomes (HLMs, H161, 5 mg/ml) and human cDNAexpressed CYP2J2 were purchased from BD Biosciences (San Jose, CA). Eight-week-old male ICR mice were purchased from Daehan Bio Link Co. (Incheon, Korea). All animal management procedures were conducted in accordance with the standard operation protocols established by Kyungpook National University. Cell Counter Kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). Solvents were high performance liquid chromatography (HPLC) grade, and the other reagents and chemicals were of analytical grade (P98%).

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2.2. CYP2J2 inhibitor screening

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All incubations were performed in triplicate, and data are presented as average values. The inhibitory potential of 50 chemicals on CYP2J2-mediated astemizole O-demethylation activity was determined using pooled HLMs in the absence and presence of test compounds. In brief, the incubation mixtures (final volume, 100 ll) containing pooled HLMs (0.25 mg/ml), astemizole (1 lM), and test compound (5 lg/ml) were preincubated for 5 min at 37 °C. The reaction was initiated by addition of NADPH-generating system (1.3 mM NADP+, 3.3 mM G6P, 3.3 mM MgCl2, and 500 unit/ml G6PDH) after a 5-min pre-incubation. To determine the inhibitory potentials (Ki values) of decursin for CYP2J2-mediated astemizole O-demethylation in HLMs, decursin (0–100 lM) was added to reaction mixtures containing different concentrations of astemizole (0.2, 1, and 2 lM) or terfenadine (0.1, 0.2, and 0.5 lM). In addition, to determine the IC50 values of decursin for CYP2J2-catalyzed astemizole O-demethylation and terfenadine hydroxylation in HLMs or human cDNA-expressed CYP2J2, decursin (0–50 lM) was added to reaction mixtures containing concentrations of astemizole 1 lM or terfenadine 1 lM. After pre-incubation at 37 °C, the reactions were maintained for 20 min in a thermoshaker. The reactions were terminated by the addition of 50 ll of cold methanol containing 100 nM mebendazole (internal standard, IS) into the mixtures. After mixing and centrifuging at 13,000g for 5 min at 4 °C, aliquots of the supernatants (1 ll) were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described previously (Yoon and Liu, 2011). The reaction rates were linear with incubation time and microsomal protein amount under these conditions.

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2.3. LC–MS/MS analysis

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The concentration of O-desmethylastemizole was measured by LC–MS/MS as described previously (Yoon and Liu, 2011), using a Thermo TSQ Vantage Triplestage quadruple mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with an electrospray ionization interface to generate protonated ions [M+H]+. The compounds were separated on a reversed-phase column (Luna C18, 2 mm i.d. 50 mm, 5-lm particle size; Phenomenex, Torrance, CA, USA) with an isocratic mobile phase consisting of acetonitrile and water [30/70 (v/v)] containing 0.1% (v/v) formic acid. The mobile phase was eluted using a Thermo Accela HPLC system (Thermo Fisher Scientific) at a flow rate of 0.2 ml/min. The mass spectrometer was operated in positive ionization mode and calibrated using the manufacturer’s calibration mixture. The operating conditions were as follows: capillary temperature, 350 °C; vaporizer temperature, 300 °C; ionization voltage, 4000 V; and collision gas (argon) pressure, 1.5 mTorr. The collision energy was set to 35 and 17 eV for O-desmethyl astemizole and mebendazole (IS), respectively. Quantitation was performed by selected reaction monitoring (SRM) of the precursor ion [M+H]+ and the related product ion for O-desmethylastemizole, using the internal standard (1 lM mebendazole) to establish peak area ratios. Ions were detected by monitoring the transitions of m/z 445 ? 204 for O-desmethylastemizole, m/z 488 ? 452 for terfenadine alcohol, m/z 486 ? 167 for hydroxyebastine and m/z 296 ? 264 for IS. The analytical data were processed by Xcalibur (version 2.1) software. The lower limit of quantitation for the analyte was 1.0 nM. The interassay precision for the analyte was less than 15%.

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2.4. Isolation of mouse hepatocytes

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Primary mouse hepatocytes were isolated from mouse liver by the two-step EDTA and collagenase perfusion method (Li et al., 2010). After the mouse was anesthetized, the liver was perfused with Krebs-Henseleit buffer without Ca2+ and SO42 (115 mM NaCl, 25 mM NaHCO3, 5.9 mM KCl, 1.18 mM MgCl2, 1.23 mM NaH2PO4, 6 mM glucose, 0.1 mM EDTA) through the hepatic portal vein to rinse out the blood (flow: at 7–9 ml/min for 5 min). Next, the liver was perfused with Krebs-Henseleit buffer without Ca2+ and SO24 containing 0.02% (w/v) collagenase and 0.1 mM CaCl2

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Table 1 Compounds investigated in the CYP2J2 inhibition assay using human liver microsomes. No

Compound

Origins

Purity

% Inhibition

No

Compound

Origins

Purity

% Inhibition

1 2

6-Gingerol Acankoreoside A

>98% >99%

27% 17%

27 28

Forsythoside A Fraxinellon

Forsythia koreana Nakai Dictamnus dasycarpus Turcz.

>98% >98%

39% 25%

3

Acankoreoside E

>99%

18%

29

Gentiopicrin

Gentiana lutea L.

>98%

–

4 5

Acetoside Aconitine Arbutin Arecoline Atropine Aurantioobtusin Azulene

Zingiber officinale Roscoe Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. Plantago asiatica L. Aconitum carmichaeli Debeaux Arctostaphylos Uva-ursi L. Sprengel Areca catechu L. Atropabelladonna Linne Senna tora (L.) Roxb.

>97.3% >98% >98% >99% >99% >99%

16% 22% 14% 23% 46% 10%

30 31 32 33 34 35

Geraniin Ginkgolide A Ginkgolide B Ginkgolide C Huzhangoside A Huzhangoside C

Euphorbia pekinensis Rupr. Ginkgo biloba L. Ginkgo biloba L. Ginkgo biloba L. Anemone rivularis Buch – Ham. Anemone rivularis Buch – Ham.

>95% >98% >98% >98% >99% >95%

19% 1% – 1% – 6%

Lactarius deterrimus

>99%

29%

36

Imperatorin

>98%

28%

a

>95% >98%

21% 17%

>95%

50%

>98%

13%

>90% >98%

– –

>98% >98%

13% –

>98%

–

>100% >97%

– –

7 8 9 10 11 12

Bacitracin A Berberine

Bacillus licheniformis Berberis heterophylla

>97% >98%

– 34%

37 38

Isoalantolactone Loganin

13

Betulin

>97%

8%

39

Machilin A

14

Betulinic acid

>99%

13%

40

Magnolin

15 16

Bicuculline Bilobalide

Betula platyphylla var. japonica(Miq.) Hara Zizyphus jujuba var. inermis (Bunge) Rehder Corydalis chaerophylla Ginkgo biloba L.

>97% >98%

–

41 42

Nodakenin NotoginsenosideR1

17 18

Brucine Byakangelicin

Strychnos nux–vomica Angelicae Dahuricae Radix

>98% >85.2%

– –

43 44

Obacunone PlatycodinD

19

Byakangelicol

Angelicae Dahuricae Radix

>99.2%

–

45

PraeruptorinA

20 21

Camphene Chiisanoside

>95% >93%

– 4%

46 47

Prosapogenin CP6 Rengyol

22

Dammaradienyl acetate Decursin Decursinol Ecdysterone Forsythin

Salvia officinalis Eleutherococcus senticosus (Rupr. & Maxim.)Maxim. Helenii Radix

Angelica dahurica (Fischer) Bentham et Hooker F. Inula helenium L. Cornus officinalis Siebold et Zucc. Machilus thunbergii Sieb. et Zucc. Machilus thunbergii Sieb. et Zucc. Angelica gigas N. Panax notoginseng (Burk) F. H. Chen Dictamnus dasycarpus Turcz. Platycodon grandiflorum (Jacq) Nakai Angelica decursiva (Miq.) Franch. et Savat. Anemone rivularis Buch.-Ham Forsythia koreana Nakai

>95%

17%

48

Rengyolone

Forsythia koreana Nakai

>96%

–

Angelica gigas N. Angelica gigas N. Silene repens Patrin Forsythia koreana Nakai

>99% >99% >99% >98%

66% 27% 11% 8%

49 50 51

Sennoside A Sennoside B Hydroxyebastine

Senna alexandrina Mill. Senna alexandrina Mill.

>98% >98%

– 1% 50%

23 24 25 26


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B. Lee et al. / Food and Chemical Toxicology xxx (2014) xxx–xxx until the liver appeared to be softened. The liver was then removed and gently minced, and the obtained cells were dispersed in medium (DMEM; Life Technologies, NY) containing 10% (v/v) fetal bovine serum (FBS), 1% (v/v) penicillin/streptomycin 100 solution (Life Technologies, NY). The solution containing mixed cells and debris was filtered through a 100-lm filter. Subsequently, the filtrate was centrifuged at 50 g for 3 min at 4 °C; the cells were washed with DMEM three times and then seeded in collagen-coated plates.

156

2.5. Cell culture

157 158 159 160 161

The mouse hepatocytes and HepG2 cells were maintained in DMEM high glucose (4.5 g/L) supplemented with 10% (v/v) FBS, 1% (v/v) penicillin/streptomycin 100 solution, 1 lg/ml insulin, and 10 12 M dexamethasone for 12 h at 37 °C in a humidified atmosphere (5% CO2). 24 h prior to the experiments, the cells were incubated with fresh DMEM without FBS.

162

2.6. Cell viability assay

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Cell viability was detected using CCK-8 kit. HepG2 cells and mouse hepatocytes were cultured in 96-well plates. The cells were treated with different concentrations of decursin as indicated (0–50 lM). The CCK-8 solution was added to each well at a 1:10 dilution followed by further 3-h incubation at 37 °C. Absorbance was measured at 450 nm with a microplate reader (Bio Tek Instruments Inc., VT). All values are expressed as the mean (±SE) and were converted from absolute counts to a percentage of the control.

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2.7. Western blot analysis

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The cell homogenates (30 lg of protein) were separated using 10% (w/v) SDS– polyacrylamide gel electrophoresis and then transferred to PVDF (polyvinylidene fluoride) transfer membranes. The blots were then washed in Tris-buffered solution containing Tween-20 [TBST, 10 mM Tris–HCl (pH 7.6), 150 mM NaCl, 0.05% (v/v) Tween-20], blocked with 5% (w/v) skim milk powder in TBST for 1 h, and incubated for 12 h with the appropriate primary antibody at the dilution recommended by the supplier (1:1000). The membranes were then washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibody (1:5000) for 12 h. The bands were visualized using an enhanced chemiluminescence detection system (Thermo Scientific, San Jose, CA) in accordance with the manufacturer’s protocols.

181

2.8. Data analysis

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The CYP2J2-mediated activities in the presence of inhibitors were expressed as a percentage of the corresponding control values. The IC50 values (concentration of the inhibitor causing 50% inhibition of the original enzyme activity) were determined from the following equation using the WinNonlin software (Pharsight, Mountain View, CA): percentage of control activity = 100 [1 (I/(I + IC50))], where I is the concentration of inhibitor, and IC50 is the inflection point on the curve (Kim et al., 2006). The apparent kinetic parameters for the inhibitory potential (Ki) were first estimated by graphical methods, such as Lineweaver–Burk, Dixon and secondary reciprocal plots, and were finally determined by non-linear least squares regression analysis, based on the best enzyme inhibition model (Segel, 1975) using the WinNonlin software. In our experiments, the inhibition data were consistently best fitted by the competitive or noncompetitive inhibition model, via the Akaike information criterion and Schwartz criteria. The models tested included pure and partial competitive inhibition, non-competitive inhibition, mixed-type inhibition, and uncompetitive inhibition (Liu et al., 2004).

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3. Results and discussion

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CYP2J2 is an enzyme that is mainly found in extrahepatic tissue, with predominant expression in the cardiovascular system and cardiomyocytes (Node et al., 1999; Wu et al., 1997) as well as in human cancer tissues and cell lines (Jiang et al., 2009, 2007b). This enzyme is responsible for the formation of epoxyeicosatrienoic acid (EET) from arachidonic acid (Zeldin, 2001). Recently, Jiang et al. (2007a,b) reported that CYP2J2-derived EET may play important roles in promoting invasion and metastasis of human cancers through down-regulation of metastatic suppressor genes and upregulation of metastatic enhancing genes. Therefore, CYP2J2 might be a potential target for therapy of human cancers. However, little data are presently available on inhibitors of CYP2J2 enzyme (Lafite et al., 2006; Wu et al., 2013a; Yoon and Liu, 2011). In this study, we screened 50 natural products obtained from plants for their inhibitory potential on CYP2J2 enzyme. The effects of natural products on the catalytic activities of human CYP2J2

140 120

% of Control Activity

149 150 151 152 153 154 155

100 80 60 40 20 0

199 200 201 202 203 204 205 206 207 208 209 210 211 212 213

0

5

10

15

20

25

30

35

40

45

50

Compound number Fig. 1. Inhibitory effects of 50 compounds (5 lg/ml) on CYP 2J2-mediated astemizole O-demethylation activity [decursin (#23), hydroxyebastine (#51)].

Table 2 Inhibitory effect (IC50) of decursin and hydroxyebastine on CYP2J2-catalyzed astemizole O-demethylation and terfenadine hydroxylation activity in human liver microsomes (HLMs). Compound Decursin Hydroxyebastine a

Astemizole a

6.95 lM 10.9 lMa

Terfenadine 17.1 lMa 2.03 lMa

Averages of triplicate determinations (n = 3).

Fig. 2. Chemical structure of decursin.

were investigated in human liver microsomes (HLMs). Decursin (Fig. 1) inhibited CYP2J2-mediated astemizole O-demethylation activity at a 5 lg/ml concentration (>60%), whereas the other compounds showed negligible or weak inhibitory effects. In addition, the inhibitory effect of decursin on CYP2J2-mediated astemizole O-demethylation activity was not changed after preincubation (20 min) with microsomes in the presence of NADPH generating system, suggesting that decursin is not a time dependent inhibitor (Fig. S1) (Kim et al., 2006). We performed further experiment for the estimation of IC50 value of decursin because decursin showed stronger inhibitory potential on CYP2J2 activity than hydroxyebastine (50% inhibition), known-CYP2J2 inhibitor. Decursin moderately inhibited CYP2J2-catalyzed astemizole O-demethylation and terfenadine hydroxylation activity in a concentration-dependent manner, with an IC50 values of 6.95 and 17.1 lM, respectively (Table 2), which are comparable to the IC50 values of thelephoric acid [IC50 = 3.23 lM, (Wu et al., 2013a)] and haloperidol [IC50 = 14.5 lM, (Liu, 2011)]. Decursin also inhibited CYP2J2-catalyzed astemizole O-demethylation and terfenadine hydroxylation activity with an IC50 value of 10.18 and 1.8 lM, respectively, in recombinant CYP2J2 isoform. We also estimated the inhibitory


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A. Astemizole O-demethylation

B. Terfenadine hydroxylation

Ki 8.34 µM 1.5

1/V(pmol/min/mg protein)

Ki 15.8 µM

none Decursin 2 µM Decursin 5µM Decursin 20 µM Decursin 50 µM

1.2

none Decursin 10 µM Decursin 20 µM Decursin 50 µM Decursin 100 µM

0.9

0.6

0.9

0.6 0.3 0.3

0.0

0.0 0

1

2

3

4

5

0

2

5

10

1/[S] Fig. 3. Representative Lineweaver–Burk plots for inhibition of CYP2J2-catalyzed astemizole O-demethylation (A) and terfenadine hydroxylation (B) by decursin in pooled human liver microsomes (HLMs). An increasing concentration of astemizole (0.2, 1, and 2 lM) (A) or terfenadine (0.1, 0.2, and 0.5 lM) (B) was incubated with HLMs (0.25 mg/ ml, Gentest H161) and an NADPH-generating system at 37 °C for 20 min in the presence or absence of decursin. The inhibition data were fit to a noncompetitive inhibition model. The Data are shown as mean ± S.D. (n = 3).

Cell viability (%)

(A) HepG2

(B) Hepatocyte

120

120

90

90

**

60

** **

30

0

60

0

1

5

10

50

30

0

0

1

5

10

50

Decursin (µM) Fig. 4. Effect of decursin on cell viability in HepG2 cells and primary cultured mouse hepatocytes. HepG2 cells (A) and mouse hepatocytes (B) were incubated with decursin at the indicated concentrations for 24 h. Cell viability was measured by CCK-8 assay. The values are expressed as the mean ± SE of triplicate experiments (n = 3).

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potential of hydroxyebastine, well-known CYP2J2 inhibitor (Yoon and Liu, 2011), and compared it with that of decursin. Hydroxyebastine inhibited CYP2J2-catalyzed astemizole O-demethylation and terfenadine hydroxylation activity in a concentration-dependent manner, with an IC50 values of 10.9 and 2.03 lM, respectively (Table 2). Hydroxyebastine (IC50 = 2.03 lM) more strongly inhibited the CYP2J2-mediated terfenadine alcohol formation than decursin (IC50 = 17.1 lM), whereas decursin (IC50 = 6.95 lM) more strongly inhibited the CYP2J2-catalyzed O-desmethylastemizole formation than hydroxyebastine (IC50 = 10.9 lM) (Table 2). As decursin inhibited CYP2J2 enzyme activity, we sought to clarify the mechanism of inhibition. The Lineweaver–Burk plots and secondary reciprocal plots indicated that decursin inhibited CYP2J2 enzyme activity with an apparent Ki value of 8.34 and 15.8 lM on CYP2J2-catalyzed astemizole O-demethylation and terfenadine hydroxylation, respectively (Fig. 3). Its inhibitory potential, however, was less potent than that of danazole [Ki = 0.02 lM, (Lee et al., 2012)], telmisartan [Ki = 0.19 lM, (Ren et al., 2013)] and flunarizine [Ki = 0.13 lM, (Ren et al., 2013)], a strong CYP2J2 inhibitor. The Lineweaver–Burk plot intersected above the x-axis (Segel, 1975; Seo et al., 2008; Waldrop, 2009), indicating that decursin inhibited the reaction in

Fig. 5. Effect of decursin on CYP2J2 expression in HepG2 cells and primary cultured mouse hepatocytes. Primary cultured mouse hepatocytes and HepG2 cells were homogenized and the levels of CYP2J2 were determined through western blot analysis (A). HepG2 cells were treated with different concentration of decursin for 24 h and the levels of CYP2J2 were determined (B). The figure shown is a representative of three experiments.

a noncompetitive manner over the substrate range from 0.2 to 2 lM astemizole (Fig. 3A) and 0.1 to 0.5 lM terfenadine (Fig. 3B). To estimate the relevance of CYP2J2 inhibition by decursin, the expected relative inhibitory potential was compared in mice with a known plasma concentration of decursin (Li et al., 2012). For this purpose, the potency of inhibition was calculated relative to the concentration ([I]/Ki), a measure of the potency of an inhibitor (Boobis, 1995; Liu et al., 2004). Maximum plasma concentration


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for decursin was 1.64 lM when decursin was administered via oral gavage (single dose, 240 mg/kg) (Li et al., 2012). Decursin showed a low [I]/Ki value with a value of 0.19, indicative of a low possibility of drug interactions with CYP2J2 substrate drugs such as albendazole (Wu et al., 2013b), astemizole (Matsumoto et al., 2003) and ebastine (Liu et al., 2006). However, recent studies have pointed out that decursin (25 mg/kg) significantly increased the systemic exposure of theophylline, a CYP1A2 substrate drug, in rats (Chae et al., 2012), even though decursin has a weak inhibitory effect on CYP1A2 activity (Ki = 67.6 lM) (Abd El-Aty et al., 2008). Therefore, in vivo interaction studies of decursin must be further evaluated to rule out the possible inhibitory potential of decursin Q2 with CYP2J2 substrates (see Fig. 2). Several studies have reported that CYP2J2 enzyme is highly expressed in human tumors and carcinoma cell lines (Chen et al., 2011; Jiang et al., 2005a), and selective inhibitors of CYP2J2 had significant antitumor effects in vitro and in vivo (Chen et al., 2011, 2009b). We evaluated the cytotoxic effect of decursin, which showed an inhibitory effect against CYP2J2 enzyme. To analyze the cancer-specific cytotoxic effect of decursin, HepG2 cells and mouse hepatocytes were treated with dimethyl sulfoxide (DMSO) or various concentrations (1, 5, 10 or 50 lM) of decursin for 24 h, and cell viability was subsequently analyzed by CCK-8 assay. As shown in Fig. 4, decursin showed cytotoxicity against HepG2 cells in a dose-dependent manner with an IC50 value of 5.48 lM, while it did not show significant cytotoxicity against mouse hepatocytes at the indicated concentration. To address the differing cytotoxicity of decursin, we analyzed CYP2J2 expression by western blotting. HepG2 cells showed high CYP2J2 expresseion while mouse hepatocytes did not (Fig. 5A). In addition, the CYP2J2 expression levels were not affected by the decursin treatment in different concentration (Fig. 5B). Therefore, we concluded that although decursin directly block the activity of already expressed CYP2J2, decursin does not affect to its expression. Indeed, previous study suggested that very strong and selective CYP2J2 expression was detected in a variety of human cancer cell lines and undetectable in adjacent normal tissues and nontumoric human cell lines (Jiang et al., 2005b). Additionally, it has been known that forced overexpression of CYP2J2 markedly accelerated proliferation and protection of carcinoma cells (Jiang et al., 2005b). Therefore, we concluded that decursin plays its anticancer effects thorough inhibition of CYP2J2 activity without influences in the expression of CYP2J2 in HepG2 cells. Previous studies suggested that decursin induced apoptosis in various types of cancer cells including prostate cancer (Choi et al., 2011), bladder and colon cancer (Kim et al., 2010), and breast cancer (Jiang et al., 2007a) through the regulation of the activity of their intracellular targets such as ERK1/2 (Kim et al., 2010), estrogen receptor (Jiang et al., 2007a), cyclooxygenase-2-dependent survivin (Ahn et al., 2010), Pin 1 (Kim et al., 2013) and so on. Therefore, our results imply that the differing cytotoxicities of decursin may be due to different expression levels of CYP2J2. In other words, CYP2J2 may be a tumor-specific target for cancer therapy. In conclusion, we identified decursin as a CYP2J2 inhibitor through screening of natural products. Decursin inhibited CYP2J2-mediated astemizole O-demethylation and terfenadine hydroxylation activity with Ki value of 8.34 and 15.8 lM in a noncompetitive inhibition mode. Decursin also showed cytotoxic effects against human hepatoma cells in a dose-dependent manner, whereas it did not show significant cytotoxicity against mouse hepatocytes. Finally, given that CYP2J2 may represent a potential target for therapy of human cancers (Chen et al., 2011; Jiang et al., 2005a), studies are currently underway to test decursin as a potential therapeutic agent for cancer.

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Conflict of Interest The authors declare that there are no conflicts of interest.

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Transparency Document

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The Transparency document associated with this article can be found in the online version.

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Acknowledgements

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This work was supported by a grant from the Korea Health Technology R&D Project, Ministry of Health & Welfare (A111345) and the Cooperative Research Program for Agriculture Science and Technology (project PJ00948604), Rural Development Administration, Republic of Korea.

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Appendix A. Supplementary material

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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fct.2014.04.020.

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References

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Abd El-Aty, A.M., Shah, S.S., Kim, B.M., Choi, J.H., Cho, H.J., Hee, Y., Chang, B.J., Shin, H.C., Lee, K.B., Shimoda, M., Shim, J.H., 2008. In vitro inhibitory potential of decursin and decursinol angelate on the catalytic activity of cytochrome P-450 1A1/2, 2D15, and 3A12 isoforms in canine hepatic microsomes. Arch. Pharm. Res. 31, 1425–1435. Ahn, Q., Jeong, S.J., Lee, H.J., Kwon, H.Y., Han, I., Kim, H.S., Lee, H.J., Lee, E.O., Ahn, K.S., Jung, M.H., Zhu, S., Chen, C.Y., Kim, S.H., 2010. Inhibition of cyclooxygenase-2dependent survivin mediates decursin-induced apoptosis in human KBM-5 myeloid leukemia cells. Cancer Lett. 298, 212–221. Boobis, A.R., 1995. Prediction of inhibitory drug–drug interactions by studies in vitro, in: I.G.M.P.a.G.N. Gracchia (Ed.). Advances in Drug Metabolism in Man, Luxembourg; European Commission, pp. 513–539. Chae, J.W., Baek, I.H., Kwon, K.I., 2012. Effect of decursin on the pharmacokinetics of theophylline and its metabolites in rats. J. Ethnopharmacol. 144, 248–254. Chen, C., Li, G., Liao, W., Wu, J., Liu, L., Ma, D., Zhou, J., Elbekai, R.H., Edin, M.L., Zeldin, D.C., Wang, D.W., 2009a. Selective inhibitors of CYP2J2 related to terfenadine exhibit strong activity against human cancers in vitro and in vivo. J. Pharmacol. Exp. Ther. 329, 908–918. Chen, C., Wei, X., Rao, X., Wu, J., Yang, S., Chen, F., Ma, D., Zhou, J., Dackor, R.T., Zeldin, D.C., Wang, D.W., 2011. Cytochrome P450 2J2 is highly expressed in hematologic malignant diseases and promotes tumor cell growth. J. Pharmacol. Exp. Ther. 336, 344–355. Chen, F., Chen, C., Yang, S., Gong, W., Wang, Y., Cianflone, K., Tang, J., Wang, D.W., 2012. Let-7b inhibits human cancer phenotype by targeting cytochrome P450 epoxygenase 2J2. PLoS One 7, e39197. Chen, Y., Agarwal, S., Shaik, N.M., Chen, C., Yang, Z., Elmquist, W.F., 2009b. Pglycoprotein and breast cancer resistance protein influence brain distribution of dasatinib. J. Pharmacol. Exp. Ther. 330, 956–963. Choi, S.R., Lee, J.H., Kim, J.Y., Park, K.W., Jeong, I.Y., Shim, K.H., Lee, M.K., Seo, K.I., 2011. Decursin from Angelicagigas Nakai induces apoptosis in RC-58T/h/SA#4 primary human prostate cancer cells via a mitochondria-related caspase pathway. Food Chem. Toxicol. 49, 2517–2523. Enayetallah, A.E., French, R.A., Thibodeau, M.S., Grant, D.F., 2004. Distribution of soluble epoxide hydrolase and of cytochrome P450 2C8, 2C9, and 2J2 in human tissues. J. Histochem. Cytochem. 52, 447–454. Freedman, R.S., Wang, E., Voiculescu, S., Patenia, R., Bassett Jr., R.L., Deavers, M., Marincola, F.M., Yang, P., Newman, R.A., 2007. Comparative analysis of peritoneum and tumor eicosanoids and pathways in advanced ovarian cancer. Clin. Cancer Res. 13, 5736–5744. Jiang, C., Guo, J., Wang, Z., Xiao, B., Lee, H.J., Lee, E.O., Kim, S.H., Lu, J., 2007a. Decursin and decursinol angelate inhibit estrogen-stimulated and estrogenindependent growth and survival of breast cancer cells. Breast Cancer Res. 9, R77. Jiang, J., Greulich, H., Janne, P.A., Sellers, W.R., Meyerson, M., Griffin, J.D., 2005a. Epidermal growth factor-independent transformation of Ba/F3 cells with cancer-derived epidermal growth factor receptor mutants induces gefitinibsensitive cell cycle progression. Cancer Res. 65, 8968–8974. Jiang, J.G., Chen, C.L., Card, J.W., Yang, S., Chen, J.X., Fu, X.N., Ning, Y.G., Xiao, X., Zeldin, D.C., Wang, D.W., 2005b. Cytochrome P450 2J2 promotes the neoplastic phenotype of carcinoma cells and is up-regulated in human tumors. Cancer Res. 65, 4707–4715.

347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397


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Jiang, J.G., Fu, X.N., Chen, C.L., Wang, D.W., 2009. Expression of cytochrome P450 arachidonic acid epoxygenase 2J2 in human tumor tissues and cell lines. Ai Zheng 28, 93–96. Jiang, J.G., Ning, Y.G., Chen, C., Ma, D., Liu, Z.J., Yang, S., Zhou, J., Xiao, X., Zhang, X.A., Edin, M.L., Card, J.W., Wang, J., Zeldin, D.C., Wang, D.W., 2007b. Cytochrome p450 epoxygenase promotes human cancer metastasis. Cancer Res. 67, 6665–6674. Kim, H., Yoon, Y.J., Shon, J.H., Cha, I.J., Shin, J.G., Liu, K.H., 2006. Inhibitory effects of fruit juices on CYP3A activity. Drug Metab. Dispos. 34, 521–523. Kim, J.H., Jung, J.H., Kim, S.H., Jeong, S.J., 2013. Decursin exerts anti-cancer activity in MDA-MB-231 breast cancer cells via inhibition of the pin1 activity and enhancement of the Pin1/p53 Association. Phytother Res. Kim, W.J., Lee, S.J., Choi, Y.D., Moon, S.K., 2010. Decursin inhibits growth of human bladder and colon cancer cells via apoptosis, G1-phase cell cycle arrest and extracellular signal-regulated kinase activation. Int. J. Mol. Med. 25, 635–641. Lafite, P., Dijols, S., Buisson, D., Macherey, A.C., Zeldin, D.C., Dansette, P.M., Mansuy, D., 2006. Design and synthesis of selective, high-affinity inhibitors of human cytochrome P450 2J2. Bioorg. Med. Chem. Lett. 16, 2777–2780. Lafite, P., Dijols, S., Zeldin, D.C., Dansette, P.M., Mansuy, D., 2007. Selective, competitive and mechanism-based inhibitors of human cytochrome P450 2J2. Arch. Biochem. Biophys. 464, 155–168. Lee, C.A., Jones 3rd, J.P., Katayama, J., Kaspera, R., Jiang, Y., Freiwald, S., Smith, E., Walker, G.S., Totah, R.A., 2012. Identifying a selective substrate and inhibitor pair for the evaluation of CYP2J2 activity. Drug Metab. Dispos. 40, 943–951. Li, L., Zhang, J., Shaik, A.A., Zhang, Y., Wang, L., Xing, C., Kim, S.H., Lu, J., 2012. Quantitative determination of decursin, decursinol angelate, and decursinol in mouse plasma and tumor tissue using liquid–liquid extraction and HPLC. Planta Med. 78, 252–259. Li, W.C., Ralphs, K.L., Tosh, D., 2010. Isolation and culture of adult mouse hepatocytes. Methods Mol. Biol. 633, 185–196. Liu, K.H., 2011. Screening of potential anticancer compounds from marketed drugs: aripiprazole, haloperidol, miconazole, and terfenadine inhibit cytochrome P450 2J2. J. Life Sci. 21, 1558–1564. Liu, K.H., Kim, M.G., Lee, D.J., Yoon, Y.J., Kim, M.J., Shon, J.H., Choi, C.S., Choi, Y.K., Desta, Z., Shin, J.G., 2006. Characterization of ebastine, hydroxyebastine, and carebastine metabolism by human liver microsomes and expressed cytochrome P450 enzymes: major roles for CYP2J2 and CYP3A. Drug Metab. Dispos. 34, 1793–1797. Liu, K.H., Lee, Y.M., Shon, J.H., Kim, M.J., Lee, S.S., Yoon, Y.R., Cha, I.J., Shin, J.G., 2004. Potential of pranlukast and zafirlukast in the inhibition of human liver cytochrome P450 enzymes. Xenobiotica 34, 429–438. Matsumoto, S., Hirama, T., Kim, H.J., Nagata, K., Yamazoe, Y., 2003. In vitro inhibition of human small intestinal and liver microsomal astemizole O-demethylation: different contribution of CYP2J2 in the small intestine and liver. Xenobiotica 33, 615–623. Matsumoto, S., Hirama, T., Matsubara, T., Nagata, K., Yamazoe, Y., 2002. Involvement of CYP2J2 on the intestinal first-pass metabolism of antihistamine drug, astemizole. Drug Metab. Dispos. 30, 1240–1245. Matsumoto, S., Yamazoe, Y., 2001. Involvement of multiple human cytochromes P450 in the liver microsomal metabolism of astemizole and a comparison with terfenadine. Br. J. Clin. Pharmacol. 51, 133–142.

Nithipatikom, K., Brody, D.M., Tang, A.T., Manthati, V.L., Falck, J.R., Williams, C.L., Campbell, W.B., 2010. Inhibition of carcinoma cell motility by epoxyeicosatrienoic acid (EET) antagonists. Cancer Sci. 101, 2629–2636. Node, K., Huo, Y., Ruan, X., Yang, B., Spiecker, M., Ley, K., Zeldin, D.C., Liao, J.K., 1999. Anti-inflammatory properties of cytochrome P450 epoxygenase-derived eicosanoids. Science 285, 1276–1279. Ren, S., Zeng, J., Mei, Y., Zhang, J.Z., Yan, S.F., Fei, J., Chen, L., 2013. Discovery and characterization of novel, potent, and selective cytochrome P450 2J2 inhibitors. Drug Metab. Dispos. 41, 60–71. Rodrigues, A.D., Mulford, D.J., Lee, R.D., Surber, B.W., Kukulka, M.J., Ferrero, J.L., Thomas, S.B., Shet, M.S., Estabrook, R.W., 1995. In vitro metabolism of terfenadine by a purified recombinant fusion protein containing cytochrome P4503A4 and NADPH-P450 reductase. Comparison to human liver microsomes and precision-cut liver tissue slices. Drug Metab. Dispos. 23, 765–775. Segel, I.H., 1975. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. Wiley Classics Library, New York. Seo, K.A., Kim, H., Ku, H.Y., Ahn, H.J., Park, S.J., Bae, S.K., Shin, J.G., Liu, K.H., 2008. The monoterpenoids citral and geraniol are moderate inhibitors of CYP2B6 hydroxylase activity. Chem. Biol. Interact. 174, 141–146. Waldrop, G.L., 2009. A qualitative approach to enzyme inhibition. Biochem. Mol. Biol. Educ. 37, 11–15. Wu, S., Chen, W., Murphy, E., Gabel, S., Tomer, K.B., Foley, J., Steenbergen, C., Falck, J.R., Moomaw, C.R., Zeldin, D.C., 1997. Molecular cloning, expression, and functional significance of a cytochrome P450 highly expressed in rat heart myocytes. J. Biol. Chem. 272, 12551–12559. Wu, S., Moomaw, C.R., Tomer, K.B., Falck, J.R., Zeldin, D.C., 1996. Molecular cloning and expression of CYP2J2, a human cytochrome P450 arachidonic acid epoxygenase highly expressed in heart. J. Biol. Chem. 271, 3460–3468. Wu, Z., Lee, B.R., Song, K.S., Liu, K.H., 2013a. Inhibitory potential of thelephoric acid on CYP2J2 activities in humna liver microsomes. J. Life Sci. 23, 1126–1132. Wu, Z., Lee, D., Joo, J., Shin, J.H., Kang, W., Oh, S., Lee do, Y., Lee, S.J., Yea, S.S., Lee, H.S., Lee, T., Liu, K.H., 2013b. CYP2J2 and CYP2C19 Are the major enzymes responsible for metabolism of albendazole and fenbendazole in human liver microsomes and recombinant P450 assay systems. Antimicrob. Agents Chemother. 57, 5448–5456. Yoon, Y.J., Liu, K.H., 2011. Potential of hydroxyebastine and terfenadine alcohol to inhibit the human cytochrome P450 2J2 isoform. J. Korean Soc. Appl. Biol. Chem. 54, 659–666. Zeldin, D.C., 2001. Epoxygenase pathways of arachidonic acid metabolism. J. Biol. Chem. 276, 36059–36062. Zeldin, D.C., Foley, J., Goldsworthy, S.M., Cook, M.E., Boyle, J.E., Ma, J., Moomaw, C.R., Tomer, K.B., Steenbergen, C., Wu, S., 1997. CYP2J subfamily cytochrome P450s in the gastrointestinal tract: expression, localization, and potential functional significance. Mol. Pharmacol. 51, 931–943. Zeldin, D.C., Foley, J., Ma, J., Boyle, J.E., Pascual, J.M., Moomaw, C.R., Tomer, K.B., Steenbergen, C., Wu, S., 1996. CYP2J subfamily P450s in the lung: expression, localization, and potential functional significance. Mol. Pharmacol. 50, 1111– 1117.

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