Supplemental material to this article can be found at:

http://jpet.aspetjournals.org/content/suppl/2009/03/16/jpet.109.152017.DC1.html
0022-3565/09/3293-908–918
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 329, No. 3
U.S. Government work not protected by U.S. copyright 152017/3473809
JPET 329:908–918, 2009 Printed in U.S.A.

Selective Inhibitors of CYP2J2 Related to Terfenadine Exhibit

Strong Activity against Human Cancers in Vitro and in Vivo□S

Chen Chen, Guiling Li, Wanmin Liao, Jun Wu, Liu Liu, Ding Ma, Jianfeng Zhou,
Reem H. Elbekai, Matthew L. Edin, Darryl C. Zeldin, and Dao Wen Wang
Department Internal Medicine and the Institute of Hypertension, Tongji Hospital, Tongji Medical College, Huazhong University
of Science and Technology, Wuhan, People’s Republic of China (C.C., G.L., W.L., J.W., L.L., D.M., J.Z., D.W.W.);
and Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health,
Research Triangle Park, North Carolina (R.H.E., M.L.E., D.C.Z.)
Received February 10, 2009; accepted March 12, 2009

Downloaded from jpet.aspetjournals.org at ASPET Journals on March 6, 2015

ABSTRACT
The cytochrome P450 epoxygenase, CYP2J2, converts arachi- reduced their ability to adhere, invade, and migrate, and atten-
donic acid to four regioisomeric epoxyeicosatrienoic acids uated activation of epithelial growth factor receptor signal and
(EETs). We found recently that this enzyme is dramatically kinases and phosphatidylinositol 3 kinase/Akt pathways. Inhi-
up-regulated in a variety of established human carcinoma cell bition of CYP2J2 also significantly potentiated human tumor
lines and in human cancerous tissue and promotes the neo- cell apoptosis and caused a corresponding increase in
plastic phenotype. In the present study, we tested the hypoth- caspase-3 activity and change in expression of apoptosis-
esis that specific inhibitors of CYP2J2 related to the drug related proteins Bax and Bcl-2. In murine xenograft models
terfenadine are effective antitumor agents. Four of these inhib- using MDA-MB-435 cells, treatment with compound 26 signif-
itors (compounds 4, 5, 11, and 26) were tested for effectiveness icantly repressed tumor growth, decreased lung metastasis,
in vitro and in vivo. In Tca-8113 cells, the CYP2J2 inhibitors and was associated with increased expression of the antican-
decreased EET production by approximately 60%, whereas cer genes CD82 and nm23, without causing toxicity. These
they had no effect on CYP2J2 mRNA or protein expression. data suggest that CYP2J2 inhibitors hold significant promise
Compound 26 inhibited the proliferation of human tumor cells, for use in treatment of neoplastic diseases.

Cytochrome P450 (P450) epoxygenases actively metabolize 2001; Kroetz and Zeldin, 2002). Most of the arachidonic acid
arachidonic acid to four regioisomers of cis-epoxyeicosatrie- epoxygenases are members of the CYP2 family and include
noic acid (5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic members of the CYP2B, CYP2C, and CYP2J subfamilies.
acids) (EETs). Differences in the catalytic efficiencies of in- Although expressed primarily in the liver, many P450 en-
dividual P450 isoforms result in different regio- and stereo- zymes are expressed in extrahepatic organs, including lung,
selective EET product profiles (Capdevila et al., 2000; Zeldin, kidney, and gastrointestinal tissues (Wu et al., 1996; Enay-
etallah et al., 2004). CYP2J2 is a major enzyme found in
This work was supported in part by the China Natural Science Foundation extrahepatic tissue, with predominant expression in the car-
Committee [Grants 30540087, 30430320]; the International Collaboration diovascular system, including endothelial cells (Node et al.,
Project [Grant 2005DFA30880]; the 973 Program [Grants 2007CB512004,
2002CB513107]; and by the Intramural Research program of the National 1999) and cardiomyocytes (Wu et al., 1996).
Institutes of Health National Institute of Environmental Health Sciences Each P450 epoxygenase isozyme produces all four EET
[Grant Z01 ES025034].
C.C., G.L., and W.L. contributed equally to this work.
regioisomers, but one or two usually are the predominant
Article, publication date, and citation information can be found at products (Karara et al., 1993; Wu et al., 1997). 11,12- and
http://jpet.aspetjournals.org. 14,15-Regioisomers are the predominant EETs produced by
doi:10.1124/jpet.109.152017.
□S The online version of this article (available at http://jpet.aspetjournals.org)
many different cells and tissues and account for 67 to 80% of
contains supplemental material. total EETs produced by five purified and reconstituted rat

ABBREVIATIONS: P450, cytochrome P450; EET, epoxyeicosatrienoic acid; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine
serum; RT, reverse transcriptase; PCR, polymerase chain reaction; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; EGFR, epithelial
growth factor receptor; PI3, phosphatidylinositol 3; HEK, human embryonic kidney; BAEC, bovine aortic endothelial cell; C26, compound 26;
compound 11, 1-(4-bromophenyl)-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-1-butanone; GAPDH, glyceraldehyde-3-phosphate dehydroge-
nase; 14,15-DHET, 14,15-dihydroxyeicosatrienoic acid; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; 20-HETE,
20-hydroxyeicosatetraenoic acid; HPF, high-power field; FITC, fluorescein isothiocyanate; TUNEL, terminal deoxynucleotidyl transferase dUTP
nick-end labeling.

908
 CYP2J2 Inhibition and Cancer Therapy 909
epoxygenases (Capdevila et al., 2000). Until now, little evi- terfenadine derivative as a hydrochloride salt. We subse-
dence shows that there are different biological properties quently investigated the potential effects of these compounds
among different EETs. on a host of processes related to cancer cell behavior and
A myriad of studies have documented the role of EETs in tumor pathogenesis. These findings suggest that selective
the maintenance of cardiovascular health. In the heart, EETs inhibitors of CYP2J2 markedly attenuate the neoplastic phe-
activate K⫹ channels, serve as endogenous regulators of car- notypes of carcinoma cells and may represent a novel class of
diac electrical excitability, shorten the cardiac action poten- therapeutic agents for the treatment of human cancers.
tial, and confer cardioprotection after ischemia/reperfusion
(Seubert et al., 2004). EETs are potent vasodilators and
mediate the vasodilator responses to acetylcholine and bra- Materials and Methods
dykinin (Roman, 2002). In addition to their vasodilatory ef- Chemicals. TRIzol and cell culture medium and reagents, includ-
fects, EETs possess potent anti-inflammatory effects and de- ing Dulbecco’s modified Eagle’s medium (DMEM), RPMI 1640 me-
crease the expression of vascular cell adhesion molecule-1 dium, trypsin, and fetal bovine serum (FBS), were purchased from
and E-selectin in cultured endothelial cells (Node et al., Life Technologies (Carlsbad, CA). Reverse transcriptase (RT)-poly-
1999). EETs are also potent inducers of angiogenesis (Wang merase chain reaction (PCR) kit was from Takara (Kyoto, Japan).
et al., 2005). 11,12-EET, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
(MTT), and collagen IV were purchased from Sigma-Aldrich (St.
Although angiogenesis is pivotal in the processes of wound
Louis, MO). Matrigel was purchased from BD Biosciences (San Jose,
healing and tissue regeneration, it is also implicated in the CA). Antibodies against epithelial growth factor receptor (EGFR),

Downloaded from jpet.aspetjournals.org at ASPET Journals on March 6, 2015

pathological growth of neoplastic tumors. In recent publica- p-EGFR, phosphatidylinositol 3 (PI3) kinase, Bcl-2, Bax, CD82,
tions, we have highlighted a pivotal role for EETs in promot- nm23, and ␤-actin were purchased from Santa Cruz Biotechnology,
ing the cancer phenotype of carcinoma cells. We have shown Inc. (Santa Cruz, CA). Antibodies against AKT and p-AKT were
that addition of exogenous EETs or recombinant adeno-asso- purchased from Cell Signaling Technology Inc. (Danvers, MA).
ciated viral vector-mediated delivery of P450 epoxygenases Horseradish peroxidase-conjugated secondary antibodies (goat anti-
markedly enhanced the proliferation of cancer cells in vitro mouse IgG and goat anti-rabbit IgG) were purchased from KPL, Inc.
and in vivo (Jiang et al., 2005). EETs also inhibited carci- (Gaithersburg, MA). Enhanced chemiluminescence reagents were
noma cell apoptosis through up-regulation of the antiapop- purchased from Pierce Chemical (Rockford, IL); polyvinylidene diflu-
oride membranes, prestained protein markers, and SDS-polyacryl-
totic proteins Bcl-2 and Bcl-XL and down-regulation of the
amide gel electrophoresis gels were from Bio-Rad (Hercules, CA). All
proapoptotic protein Bax (Jiang et al., 2005). In addition to other reagents were purchased from standard commercial suppliers
inducing tumor cell proliferation, both endogenously formed unless otherwise indicated.
and exogenously applied EETs increased cancer cell motility, Cell Lines. The Tca-8113 human tongue squamous cell carcinoma
adhesion, invasion, prometastatic gene expression, and xeno- line, A549 human alveolar basal epithelial carcinomic cell line,
graft metastasis to the lungs. In contrast, antisense to HepG2 human hepatoma liver cell line, HeLa human cervix carci-
CYP2J2 or nonspecific P450 inhibitors significantly attenu- noma cell line, HEK293 human kidney epithelial cell line, and H9c2
ated these neoplastic phenotypes (Jiang et al., 2005, 2007). rat myocardium cell line were obtained from the American Type
All the findings suggested that inhibition of CYP2J2-medi- Culture Collection (Manassas, VA) and maintained as recommended
ated EET biosynthesis may represent a novel approach for by the source. Cells were cultured in DMEM, adjusted to contain 4
mM L-glutamine, 1.5 g/l sodium bicarbonate, 4.5 g/l glucose, 10%
the treatment of human cancers (Jiang et al., 2005).
FBS, 100 units/ml penicillin, and 65 units/ml streptomycin. The
H1 histamine receptor antagonists are classified as specific TC-1 mouse lung epithelial carcinomic cell line and MDA-MB-435
antagonists, such as terfenadine and astemizole, and nonspe- human breast carcinoma cell line were also obtained from the Amer-
cific antagonists, such as diphenhydramine and triprolidine ican Type Culture Collection and were maintained in RPMI 1640
(Simons, 2004). The antiinflammatory activities of H1-anti- medium, supplemented with 4 mM L-glutamine, 1.5 g/l sodium bi-
histamines occur through a variety of mechanisms (Ciprandi carbonate, 4.5 g/l glucose, 10% FBS, 100 units/ml penicillin, and 65
et al., 1999; Triggiani et al., 2001). Antiinflammatory effects units/ml streptomycin. All cell lines were maintained at 37°C in a
such as the inhibition of the expression of cell adhesion humidified incubator containing 5% CO2.
molecules and the chemotaxis of eosinophils and other cells Isolation and Culture of Bovine Aortic Endothelial Cells.
may involve down-regulation of the H1-receptor-activated Fresh bovine thoracic aortas were obtained from a local slaughter-
house, and bovine aortic endothelial cells (BAECs) were harvested
nuclear factor-␬B, a ubiquitous transcription factor that
using trypsin (0.25%) and cultured as described previously (Wang et
binds to the promoter and enhancer regions of many genes al., 2005). In brief, BAECs were grown to confluence in DMEM
that regulate the production of proinflammatory cytokines supplemented with 4 mM L-glutamine, 1.5 g/l sodium bicarbonate,
and adhesion proteins (Bakker et al., 2001). Terfenadines, 1.0 g/l glucose, 15% FBS, and an antibiotic mixture of 100 units/ml
which are associated with cardiac toxic effects, are no longer penicillin and 100 ␮g/ml streptomycin. Purity of the BAEC prepara-
approved for use (Leurs et al., 2002). However, terfenadine tion was determined by cell morphology using phase-contrast micros-
may induce DNA damage and apoptosis in human melanoma copy and by immunofluorescent staining for CD31 (⬃90%). Cells
cell lines through Ca2⫹ homeostasis modulation and tyrosine were maintained at 37°C in a humidified incubator containing 5%
kinase activity, independently of H1 histamine receptors CO2. All passages were performed using 0.05% trypsin and 0.02%
EDTA. Only cells passaged less than five times were used for
(Jangi et al., 2006, 2008).
experiments.
Derivatives of the drug terfenadine were shown recently to
Synthesis of Terfenadone Derivatives. The design and syn-
be selective, high-affinity inhibitors of human CYP2J2 thesis of high-affinity and selective CYP2J2 inhibitors derived from
(Lafite et al., 2006, 2007). However, the role of these inhibi- terfenadone, a derivative of the drug terfenadine, have been de-
tors in ameliorating EET-mediated promotion of the neoplas- scribed in detail by Lafite et al. (2007). We synthesized three of these
tic phenotype has yet to be examined. In the present study, compounds (compounds 4, 5, and 11), and an additional novel com-
we utilized some of these inhibitors and synthesized a novel pound, which we have labeled compound 26 (C26), as hydrochloride
910 Chen et al.

salts. A schematic description for the synthesis of these compounds solution turned black (1–2 h). After cooling to room temperature, 5
is shown in Scheme 1. In brief, substituted benzene derivatives and ml of H2O was added. The resulting mixture was extracted with
4-chlorobutanoyl chloride were added progressively to a cold solution ether and washed with water. The combined extract was dried with
of CH2Cl2 and AlCl3. After stirring at 0°C for 2 h, the mixture was Na2SO4. After evaporation of the solvent, the crude product was
poured into ice water and stirred. After complete melting of the ice purified by distillation. The resulting oil was dissolved in ether, and
water, CH2Cl2 was again added to the solution. The organic phase hydrochloric acid was added to precipitate the product. The precip-
was then separated, and the aqueous phase was extracted with itate was filtered and washed with ethyl acetate.
CH2Cl2. The extract was dried (NaSO4), and the solvent was evap- Analysis of CYP2J2 Expression by RT-PCR. Total RNAs were
orated under vacuum. The resulting 4-chloro-1-butanone derivatives isolated from cell cultures 24 h after treatment with 10 ␮M CYP2J2
were added to a dry flask with ␣,␣-diphenyl-4-piperidinomethanol, inhibitors using TRIzol reagent. Semiquantitative analysis of the
KHCO3, KI, and toluene. The mixture was refluxed for 72 h, after expression of CYP2J2 mRNA was done using a multiplex RT-PCR
which the solvent was evaporated under heating, and the residue technique. Expression of glyceraldehyde-3-phosphate dehydrogenase
was washed with water. The organic layers were then concentrated (GAPDH) mRNA was used as an internal standard. RNA was re-
under vacuum. verse-transcribed using the Takara Bio RT-PCR kit (Takara Bio
Synthesis of C26. Chloromethylbis(triphenylphosphine) palla- USA, Madison, WI), according to the manufacturer’s protocol. The
dium and vinyltributyltin were added to a mixture of compound 11 in PCR reaction mixture contained 5 ␮l of cDNA, 1⫻ PCR buffer, 1.5
a solution of hexamethylphosphoramide. The yellow solution was mM MgCl2, 0.8 mM deoxynucleotide triphosphates, 1 unit of Taq
heated to 65°C with constant stirring in a sealed tube until the DNA polymerase, and 100 nM each primer for CYP2J2 (sense

Downloaded from jpet.aspetjournals.org at ASPET Journals on March 6, 2015

Scheme 1. Synthesis of terfenadone derivatives.
The schematic shows synthesis and chemical
structures of compounds 4, 5, 11, and 26.
 CYP2J2 Inhibition and Cancer Therapy 911
primer, 5⬘-CTCCTACTGGGCACTGTCGC-3⬘; antisense primer, 5⬘- medium at 37°C for 24 h to allow for synchronization. Cells were then
TGGGCCTCCTCCTGAAT-3⬘) or for GAPDH (sense primer, 5⬘-AC- treated with CYP2J2 inhibitors (10 ␮M) for 24 h and then fixed with
CACAGTCCATGCCATCAC- 3⬘; antisense primer, 5⬘-TCCACCAC- formaldehyde. After Cytonin antigen retrieval, slides were incubated
CCTGTTGCTGTA-3⬘). After 35 cycles, PCR products were resolved with the Ki67 antibody for 16 h at 4°C. Slides were washed thrice (15
in 1% agarose gels stained with ethidium bromide. The relative min each) with PBS at room temperature and then incubated with a
intensity of CYP2J2 compared with GAPDH was calculated for each secondary antibody for 1 h at room temperature. After three more
sample by densitometry.
Analysis of Protein Expression and Phosphorylation by
Western Blotting. Proteins from cell lysates (20 ␮g) were separated A
by 8% SDS-polyacrylamide gel electrophoresis and transferred to a
polyvinylidene difluoride membrane. After blocking in 5% nonfat Cyp2J2
milk, protein blots were incubated with a specific antibody followed
by incubation with a peroxidase-conjugated secondary antibody in
blocking buffer. The bands were visualized with the enhanced chemi- GAPDH
luminescence method according to manufacturer’s instructions
(Pierce Chemical). Polyclonal antibodies against CYP2J2 were de-
veloped as described previously (Wu et al., 1996) and showed no Control DMSO C4 C5 C11 C26
cross-reactivity with other P450 isoforms.
Determination of 14,15-Dihydroxyeicosatrienoic Acid Lev- B
els. For the measurement of the stable EET metabolite 14,15-dihy- CYP2J2

Downloaded from jpet.aspetjournals.org at ASPET Journals on March 6, 2015

droxyeicosatrienoic acid (14,15-DHET) in cultured cells, cells were
scraped in ice-cold PBS, pH 7.2, with triphenylphosphine after wash-
ing with PBS and then homogenized and sonicated over ice, as β-actin
described previously (Jiang et al., 2005; Yang et al., 2007). For the
measurement of 14,15-DHET in urine of nude mice, the urine was Control DMSO 5 10
preserved with triphenylphosphine and stored at ⫺80°C until use.
Eicosanoids were extracted from the cell homogenates and urine C26 (uM)
thrice with ethyl acetate after acidification with acetic acid. After
evaporation, the samples were dissolved in N,N-dimethylformamide C 200 14,15-EET
(AMRESCO Inc., Solon, OH), and the concentration of 14,15-DHET 20-HETE
(pg/250 µg protein)

was determined by an ELISA kit (R&D Systems, Minneapolis, MN),

according to the manufacturer’s instructions.
150 *
Eicosanoid

Determination of 20-Hydroxyeicosatetraenoic Acid Levels. *

For measurement of 20-hydroxyeicosatetraenoic acid (20-HETE) in 100 * *
cultured cells, cells were scraped in cold PBS, pH 7.2, with tri-
phenylphosphine after washing with PBS and then homogenized and
sonicated over ice. 20-HETE was also measured in the urine of nude 50
mice, which was preserved with triphenylphosphine and stored at
⫺80°C until use. The concentration of 20-HETE was determined by 0
an ELISA kit (R&D Systems) according to the manufacturer’s 0 6 12 18 24
instructions.
Growth Inhibition Assays. Growth inhibition studies in Tca- Time (hrs)
8113, HeLa, A549, HepG2, MDA-MB-435, TC-1, and HEK293 cell
lines, primary BAECs, and neonatal rat primary cardiomyocytes D Contol
were performed using the MTT assay as described previously (Jiang 300
5 µM C26
(pg/250 µg protein)

et al., 2005). This assay measures the conversion of MTT to formazan

14,15-DHET

crystals by enzymes in the mitochondria of metabolically active cells.

For these studies, cells seeded at 1 ⫻ 104 cells/well (quintuplicate 200
repeats in 96-well plates) were allowed to attach for 24 h. At 60%
confluence, the medium was removed, and the cells were washed *
thrice with PBS and incubated with serum-free medium at 37°C for 100
24 h to allow for synchronization. Cells were treated with CYP2J2
inhibitors (10 ␮M) for various time periods from 4 to 24 h every 4 h.
After treatment with MTT, the formed crystals were dissolved in 0
dimethyl sulfoxide, and the intensity of the color in each well was Tca-8113 BAEC H9c2
measured at a wavelength of 490 nm using a microplate reader. Fig. 1. Effect of terfenadone derivatives on CYP2J2 expression and
Before these experiments, we tested doses of CYP2J2 inhibitor C26 activity. A, CYP2J2 mRNA levels. Total RNAs were isolated from Tca-
ranging from 10 nM to 100 ␮M in these cell lines to define the proper 8113 cells treated with compounds 4, 5, 11, and 26 (10 ␮M each) for 24 h.
dose by antiproliferation effect. Results showed that C26 dose-de- Semiquantitative analysis of the expression of CYP2J2 mRNA was done
using a multiplex RT-PCR technique as described under Materials and
pendently inhibited human carcinoma cell growth from 0.5 to 20 ␮M, Methods. B, CYP2J2 protein levels. Tca-8113 cells were treated with 5 or
and 5 and 10 ␮M C26 showed the best cell growth inhibition effect 10 ␮M C26 for 24 h. Proteins from cell lysates were then subject to
(Supplemental Fig. 1) without effect on CYP2J2-negative cells, in- Western blot analysis as described under Materials and Methods. C,
cluding HEK293 cells and TC-1 (mouse lung cancer cells). Therefore, 14,15-DHET and 20-HETE levels in Tca-8113 cells. Levels of 14,15-
these doses were chosen for further experiments. DHET and 20-HETE were determined as described under Materials and
Methods over a 24-h period after treatment of cells with C26 (10 ␮M). D,
Ki67 Immunohistochemistry. Cells were loaded onto slides,
14,15-DHET levels in BAECs, H9C2 and Tca-8113 cells. 14,15-DHET
plated in six-well plates at 1 ⫻ 106 cells/well, and allowed to attach for levels in cells treated with C26 (5 ␮M) for 24 h were determined. Results
24 h. After growth to 60% confluence, the medium was removed, and shown are mean ⫾ S.E. (n ⫽ 5); ⴱ, p ⬍ 0.05 versus control, representative
the cells were washed thrice with PBS and incubated with serum-free of three independent experiments.
912 Chen et al.

washes with PBS (15 min each) at room temperature, immunostaining for 24 h. At 60% confluence, the medium was removed, and the cells
was visualized with diaminobenzidine (Sigma-Aldrich). Slides were were washed thrice with PBS and incubated with serum-free me-
scanned under an inverted microscope (Nikon TE 2000; Nikon, Tokyo, dium at 37°C for 24 h to allow for synchronization. After treatment
Japan) equipped with digital imaging. For each treatment, 10 high- with the C26 (10 ␮M) for 6, 12, 18, and 24 h, cells were harvested
power field (HPF) images were captured. with trypsin/EDTA, resuspended in binding buffer, and incubated
Colorimetric Assay for the Measurement of Caspase-3 Ac- with FITC-conjugated annexin V and propidium iodide according to
tivity. Caspase-3 activity was measured using a colorimetric assay the manufacturer’s protocol (Annexin V-FITC kit; Bender MedSys-
kit according to manufacturer’s instructions (R&D Systems). In tems Inc., Burlingame, CA). Cells were then analyzed with a FAC-
brief, treated cells were collected and lysed by the addition of lysis Star-Plus flow cytometer (BD Biosciences, Franklin Lakes, NJ).
buffer. After centrifugation, a caspase-specific peptide that was con- TUNEL Assay. Cells were loaded onto slides, plated in six-well
jugated to a color reporter molecule and reaction buffer were added plates at 1 ⫻ 106 cells/well, and allowed to attach for 24 h. After
to the supernatant and incubated at 37°C for 2 h in the dark. Release growth to 60% confluence, the medium was removed, and the cells
of the chromophore by the caspase enzymatic activity was quantified were washed thrice with PBS and incubated with serum-free me-
spectrophotometrically at a wavelength of 405 nm. dium at 37°C for 24 h to allow for synchronization. Cells were then
Determination of Apoptosis by Flow Cytometry. Cells were treated with compounds 4, 5, 11, and 26 for 24 h (10 ␮M) and then
seeded into six-well plates at 1 ⫻ 106 cells/well and allowed to attach fixed with fresh 4% paraformaldehyde at room temperature for 10

A 125 Tca-8113 D 125

Hela

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Number of cells

100 A549
MDA-MB-435 100
(% control)

Number of cells
*

(% control)
75 * * * 75
* *
50
*
*
50 * *
25 25

0
0
0 4 8 12 16 20 24
Control DMSO C4 C5 C11 C26
Time (hrs)

B 125 E
100
Number of cells
(% control)

75

50 * *
5 μM C26
25
10 μM C26

0
0 6 12 18 24
Time (hrs)

C 125

100
Number of cells
(% control)

75

50
HEK293
BAEC
25
TC-1

0
0 2 4 6 8 10
C26 (μM)
Fig. 2. Effect of CYP2J2 inhibitors on cell proliferation. A, effect of C26 (10 ␮M) on number of Tca-8113, HeLa, A549 and MDA-MB-435 tumor cells.
B, effect of C26 on number of HepG2 cells. C, effect of C26 (10 ␮M) on number of HEK293 cells, BAECs, and TC-1 cells. D, effect of CYP2J2 inhibitors
on number of Ki67 immunopositive Tca-8113 cells. Cells were treated with vehicle and one of the CYP2J2 inhibitors (10 ␮M each), and the numbers
of Ki67-positive cells per HPF were determined as described under Materials and Methods. E, effect of 11,12-EET (200 nM) on the antiproliferative
effect of C26 (10 ␮M) in Tca-8113 cells. Growth inhibition studies for A, B, C, and E were performed using the MTT assay, as described under Materials
and Methods. Data are expressed as percentage of untreated controls, which is set at 100% ⫾ S.E. (n ⫽ 5); ⴱ, p ⬍ 0.05 versus control; #, p ⬍ 0.05 versus
C26.
 CYP2J2 Inhibition and Cancer Therapy 913
min. The In Situ Apoptosis Detection Kit was used according to the proper dose by antitumor growth. Results showed that 0.25 mg/kg/
manufacturer’s instructions (R&D Systems). The slides were day C26 had the significant effect (data not shown), and this dose
scanned under an inverted microscope (Nikon TE 2000) equipped was chosen for the further in vivo experiment.
with digital imaging. For each treatment, 10 HPF images were Statistics. Data are presented as mean ⫾ S.E. The Wilcoxon test,
captured. Student’s t test, or analysis of variance was performed to determine
Fibronectin Adhesion Assay. Adhesion of cells to fibronectin statistical significance of differences among treatment groups, as
was carried out as described previously (Zipin et al., 2004). In brief, appropriate. In all cases, statistical significance was defined as p ⬍
nontissue culture 96-well plates were coated with 100 ␮l/well of 10 0.05.
␮g/ml fibronectin for 60 min at 37°C and blocked twice with 200 ␮l of
1% bovine serum albumin in phenolsulfonphthalein-free medium for Results
30 min at 37°C. Cells (1 ⫻ 104), in 100 ␮l of phenolsulfonphthalein-
free medium containing 0.1% bovine serum albumin, treated with or Design and Synthesis of Selective CYP2J2 Inhibi-
without C26 (10 ␮M), were added to fibronectin-coated wells for 60 tors. We synthesized a series of small-molecule, high-affin-
min at 37°C. After three washes with medium to remove nonadhered ity, selective inhibitors of CYP2J2 based on previously pub-
cells, the cells were covered with 100 ␮l of phenolsulfonphthalein- lished methods (Lafite et al., 2006, 2007). The compounds
free medium, and 10 ␮l of MTT solution (5 mg/ml) was added for cell were related to terfenadone, a derivative of the drug terfena-
count determination (Jiang et al., 2005). Absorbance values at 490 dine, and synthesized as hydrochloride salts for ease of ad-
nm reflect the proportional number of cells adhering to fibronectin. ministration (Scheme 1). Specifically, we chose compounds 4
Cell Migration and Invasion Assays. Chemotactic migration (-CH2-CH2-CH3), 5 (-CH2-CH ⫽ CH2), and 11 (-Br) because of
was evaluated using a modified Boyden chamber as described pre-
their low IC50 values for CYP2J2 (⬃0.4 ␮M), which were 14-

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viously (Fishman et al., 2001). Porous filters (8-␮m pores) were
to 20-fold higher than for other P450s (Lafite et al., 2007). In
coated by passive adsorption for 24 h with type IV collagen (10 ␮g/ml
collagen in coating buffer; Sigma-Aldrich). Cells (1 ⫻ 106) were then addition to these three compounds, we designed and synthe-
plated in the upper chamber, whereas 5% FBS-supplemented me- sized a novel inhibitor, C26, for which we used a vinyl moiety
dium was added to the bottom chamber to act as a chemoattractant. (-CH ⫽ CH2) as the substitute group. The chemical struc-
Subsequently, serum-free medium with compounds 4, 5, 11, or 26 (10 tures of all four compounds were established from their 1H
␮M) was added to the upper chamber, and cells were allowed to NMR and mass spectra; 1H NMR spectroscopy analyses in
migrate for up to 4 h. Nonmigrating cells were removed from the the presence of an internal standard showed that all four
upper chamber, with a cotton swab and filters were stained with compounds were greater than 95% pure.
DiffQuik stain. Migrating cells adherent to the underside of the filter Terfenadone Derivatives Inhibit the Activity of
were counted with an ocular micrometer, counting a minimum of 10 CYP2J2 but Do Not Affect Its Expression. To verify that
HPFs. To study cell invasiveness, cells (1 ⫻ 106) were plated onto the synthesized terfenadone derivatives inhibit the activity
Boyden chambers coated with Matrigel (500 ␮g/filter) and incubated
of CYP2J2, but not its expression, we examined mRNA and
for 4 h. Chambers were stained as described above and invasiveness
protein levels of CYP2J2 in Tca-8113 cells after treatment
was evaluated by quantifying the number of cells invading the Ma-
trigel assay and reaching the opposite side of the filter. All the data with the four compounds for 24 h (10 ␮M). As expected, none
are expressed as relative migration (number of cells per field) and of the inhibitors altered mRNA or protein expression of
represent the mean ⫾ S.E. of quadruplicate experiments. CYP2J2 (Fig. 1, A and B). Similar results were obtained with
In Vivo Antitumor Activity. Athymic BALB/c mice, 4 weeks of all six tumor cell lines and three nontumor cells (data not
age, were housed in a 12-h light/dark cycle in a pathogen-free envi- shown). Lafite et al. (2006, 2007) previously have established
ronment and allowed ad libitum access to food and water. All animal the specificity of terfenadone derivatives for inhibition of
studies were approved by the Animal Research Committee of Tongji CYP2J2. To confirm inhibition of P450 epoxygenase but not
Medical College and done according to the NIH Guide for the Care ␻-hydroxylase activity by these compounds, we measured the
and Use of Laboratory Animals (National Research Council, 1996). levels of the stable EET metabolite 14,15-DHET and the
MDA-MB-435 cells, cultured in log-phase growth, were harvested on ␻-hydroxylase metabolite 20-HETE in cell homogenates.
the day of use and inoculated into the right flank of male athymic
14,15-DHET levels, but not 20-HETE levels, were signifi-
BALB/c mice. After inoculation of tumor cells, mice were monitored
cantly decreased in Tca-8113 cells after treatment with C26
daily and weighed weekly. Digital caliper measurements were begun
when tumors became visible. When tumors had grown to approxi-
in a time-dependent fashion (Fig. 1C). In cells with non-
mately 40 mm3, approximately 14 days after implantation, tumor- CYP2J2 expression (e.g., BAECs, H9c2 cells), 14,15-DHET
bearing mice were randomized into control and drug treatment levels were not significantly altered in the presence of C26
groups (n ⫽ 6). C26 dissolved in dimethyl sulfoxide was administered (10 ␮M) for 24 h, verifying the specificity of this inhibitor
orally for 30 days at a dose of 0.25 mg/kg/day. At the end of the toward CYP2J2 activity (Fig. 1D).
experiment, mice were sacrificed by sodium pentobarbital overdose CYP2J2 Inhibitors Decrease Human Tumor Cell Pro-
(90 mg/kg i.p.), and primary tumors were removed and weighed. liferation. As a first step in establishing the effect of
Tumor volumes were calculated as length ⫻ width2 ⫻ ␲/6 as de- CYP2J2 inhibitors, we determined the effect of C26 (10
scribed previously (Bajo et al., 2002). Lungs were also removed for ␮M) on proliferation of cells in vitro over a 24-h time
quantification of white metastatic colonies on the lung surfaces. period. C26 caused a significant, time-dependent decrease
The effect of C26 on lung metastasis and survival was also as-
in the number of viable Tca-8113, HeLa, A549, and MDA-
sessed. Cells were harvested and inoculated into the right flank of
MB-435 cells, as determined by the MTT assay (Fig. 2A).
male athymic BALB/c mice as described above. C26 or vehicle was
administrated orally for up to 11 weeks at a dose of 0.25 mg/kg/day.
On the other hand, in HepG2 cells, which express lower
Lungs were removed for quantification of white metastatic colonies levels of CYP2J2 than other human tumor cells, inhibition
on the lung surfaces and primary tumors were removed, weighed, of CYP2J2 by C26 caused a significant, albeit less pro-
and analyzed by immunoblotting. found, decrease in cell number (Fig. 2B). In contrast, in
Before these experiments, we tested doses of C26 from 0.05 to 1 human nontumor cells (HEK293) and in nonhuman tumor
mg/kg/day in tumor-bearing athymic BALB/c mice to define the and nontumor cells (TC-1, BAECs) that do not express
914 Chen et al.

CYP2J2, increasing doses of C26 had no significant effect (10 ␮M) increased the number of annexin V-positive cells
on cell number (Fig. 2C). Furthermore, all four inhibitors over a 24-h period (Fig. 3C).
(10 ␮M) decreased the number of Ki67-positive Tca-8113 CYP2J2 Inhibitors Reduce Human Tumor Cell Adhe-
cells (Fig. 2D). It is not surprising that addition of exoge- sion, Migration, and Invasion. The effect of CYP2J2 in-
nous 11,12-EET (200 nM) attenuated the antiproliferative hibitors on metastatic potential of cancer cells was assessed
effect of C26 (10 ␮M), confirming the role of EETs in in vitro by examining adherence to fibronectin, migration,
promoting cell proliferation (Fig. 2E). and invasion potential. The effect of C26 on the adhesion of
CYP2J2 Inhibitors Activate Caspase-3 and Enhance Tca-8113 cells to fibronectin was assessed using the MTT
Human Tumor Cell Apoptosis. Given the observed effects assay. As shown in Fig. 4A, C26 (5 and 10 ␮M) significantly
on tumor cell proliferation, it was of interest to determine the reduced the number of cells adhering to fibronectin after 2 h
effect of CYP2J2 inhibitors on the apoptosis of cancer cells. of treatment. Similar results were obtained when cells were
Using a colorimetric assay, we determined the effect of C26 (5 treated with 10 ␮M of compounds 4, 5, or 11 (Fig. 4B).
and 10 ␮M) on the activity of caspase-3, an intracellular The effect of CYP2J2 inhibitors on cell migration was as-
cysteine protease that exists as a proenzyme and becomes sessed in two cancer cell lines. C26 inhibited cell migration in
activated during the cascade of events associated with apo- a dose- and time-dependent manner (Fig. 4, C and D). In
ptosis (Roy et al., 2001). C26 significantly increased addition, the presence of 11,12-EET (200 nM) attenuated the
caspase-3 activity in Tca-8113 cells in a dose-dependent man- inhibitory effect of C26 (10 ␮M) on cell migration, confirming
ner (Fig. 3A). Apoptotic Tca-8113 cells were then identified the role of EETs in promoting the metastatic phenotype of

Downloaded from jpet.aspetjournals.org at ASPET Journals on March 6, 2015

by TUNEL staining after treatment with compounds 4, 5, 11, cancer cells and verifying that the antineoplastic properties
and 26 (10 ␮M) for 24 h. As shown in Fig. 3B, the four of C26 stem from its inhibitory effect on CYP2J2 (Fig. 4E). In
inhibitors significantly increased the number of TUNEL- agreement with the results of migration assays, inhibition of
positive cells, although the increase was more pronounced in CYP2J2 by the inhibitors significantly decreased the number
cells treated with compounds 4, 5, and 26. In addition, C26 of cells invading into Matrigel assay (Fig. 4F).

A C
250 *
200
Caspase activity

*
(% control)

150 Control DMSO 6 hrs

100

50

0
Control DMSO C26 C26
(5 μM) (10 μM)
12 hrs 18 hrs 24 hrs
Annexin V positive cells (%)

75
*
B 40
*
TUNEL positive cells (%)

50
30

* * *
20 * 25 *
10 *
0
Control DMSO 6 12 18 24
0
Control DMSO C4 C5 C11 C26 Hours
Fig. 3. C26 increases human tumor cell apoptosis. A, caspase-3 activity in Tca-8113 cells. Cells were treated with 5 and 10 ␮M C26 for 18 h, lysed,
and analyzed spectrophotometrically for caspase activity. Data are reported as mean absorption relative to control ⫾ S.E. (n ⫽ 3). B, identification of
apoptotic cells by TUNEL staining. Tca-8113 cells were treated with compounds 4, 5, 11, and 26 for 24 h and then fixed with paraformaldehyde. DNA
fragments and double-strand breaks in apoptotic cells were labeled with biotinylated nucleotides and detected using the In Situ Apoptosis Detection
Kit. Apoptotic cells with their characteristic fragmented chromatin exhibit a dark-blue nuclear staining. Graph represents quantification of the
percentage of cells that were TUNEL positive. Data are reported as mean ⫾ S.E. (n ⫽ 5). C, analysis by flow cytometry of Tca-8113 cells treated with
C26 (10 ␮M) using annexin V-FITC and propidium iodide. The lower left quadrant represents nonapoptotic cells, the lower right quadrant is
representative of early apoptotic cells (annexin positive, propidium iodide negative), and the upper right quadrant represents annexin V and
propidium iodide-positive late apoptotic or necrotic cells. Graph represents the mean number of annexin-positive Tca-8113 cells expressed as
percentage of control untreated cells ⫾ S.E. (n ⫽ 3); ⴱ, p ⬍ 0.05 versus control.
 CYP2J2 Inhibition and Cancer Therapy 915

Fig. 4. CYP2J2 inhibitors decrease

the adhesion, migration, and invasion
of Tca-8113 to fibronectin. A, number
of cells adhering to fibronectin after
treatment with 5 and 10 ␮M C26 for
30 min to 2 h. B, number of cells ad-
hering to fibronectin 2 h after treat-

Downloaded from jpet.aspetjournals.org at ASPET Journals on March 6, 2015

ment with 10 ␮M compounds 4, 5, 11,
or 26. Fibronectin adhesion assay was
performed as described under Materi-
als and Methods. C, migration of Tca-
8113 cells treated with 5 and 10 ␮M
C26 for 4 h. D, migration of MDA-MB-
435 cells treated with 10 ␮M C26 over
a 4-h time period. E, effect of 11,12-
EET (200 nM) on the antimigratory
effect of C26 (10 ␮M) in Tca-8113 cells.
The Boyden chamber migration assay
was performed as described under
Materials and Methods. F, cells were
treated with 10 ␮M compounds 4, 5,
11, or 26 for 4 h. Thereafter, invasive-
ness was evaluated by quantifying the
number of cells invading the Matrigel
in a Boyden chamber as shown. Data
are expressed as percentage of un-
treated controls, which is set at
100% ⫾ S.E. (n ⫽ 5); ⴱ, p ⬍ 0.05 versus
control; #, p ⬍ 0.05 versus C26.

CYP2J2 Inhibitors Depress Murine Xenograft Tu- paralleled by a decrease in tumor growth over a 28-day
mor Growth and Metastasis. After our in vitro assessment period (Fig. 5C). The difference in tumor volume between the
of effects of CYP2J2 inhibitors, we examined the effect of control and treatment groups was apparent within 7 days
CYP2J2 inhibitors on tumor growth in an in vivo MDA-MB- after randomization of the animals but only became signifi-
435 cell murine xenograft model. Athymic BALB/c mice were cant after 21 days. At the end of the treatment period, C26
injected subcutaneously with 2 ⫻ 106 MDA-MB-435 cells/ was associated with decreased tumor weight, without change
mouse, and the tumor was allowed to grow for 14 days before in body weight (Fig. 5D).
randomization of the animals to control or CYP2J2 inhibitor To confirm the effect of C26 on lung metastases and sur-
treatment groups (n ⫽ 6 for each group). During the treat- vival, we performed additional experiments. In the survival
ment period, 14,15-DHET and 20-HETE urine levels were study, the control group died significantly earlier than the
measured. As expected, C26 decreased 14,15-DHET levels treatment group (n ⫽ 10 for each group) (Fig. 5E). In the
but had no significant effect on 20-HETE levels (Fig. 5, A and metastasis experiment, all animals were sacrificed at 8
B). The decrease in EET levels in the treatment group was weeks, and lungs were removed to count metastatic tumor
916 Chen et al.

A Control
D 30.0 Control
600 C26
C26
25.0
14,15-DHET (ng/ml)

500
20.0
*

Weight (g)
400
*
300

200 1.0
*
100 0.5

0 0
0 14 28 Tumor Body
Time (Days)

B E 1.0

0.8 Control (n=10)

1000

Cumulative Survival
Control

Downloaded from jpet.aspetjournals.org at ASPET Journals on March 6, 2015

20-HETE (ng/ml)

C26 C26 (n=10)

800
0.6
600

400 0.4

200
0.2
0
0 14 28
0
Time (Days) 0 20 40 60 80
Days

C F
1400 30
Control
Tumor volume (mm3)

Control
1200 C26 25 C26
Volume (mm3)

1000
20
800 * *
15
600 *
* 10
400
200 5 *
0 0
0 7 14 21 28 Lung node Right flank Left flank
Time (Days) lymph node lymph node
Fig. 5. Effect of C26 on tumor growth and metastasis. Athymic mice were inoculated with MDA-MB-435 cells, and the tumors were allowed to grow
to approximately 40 mm3. The mice were then randomized to control versus C26 treatment. Mice were dosed orally with vehicle or C26 (0.25
mg/kg/day) for 30 days. A, 14,15-DHET levels in the urine of nude mice during treatment with C26 or vehicle control (n ⫽ 6). B, 20-HETE levels in
the urine of nude mice during treatment with C26 or vehicle control. 14,15-DHET and 20-HETE were detected by ELISA according to the
manufacturer’s instructions (n ⫽ 6). C, tumor volume as measured weekly in control and C26-treated mice. Tumor volume was monitored by digital
caliper on a weekly basis and calculated as length ⫻ width2 ⫻ ␲/6 (n ⫽ 6). D, tumor and body weight of mice 30 days after randomization into control
and C26 treatment groups (n ⫽ 6). E, cumulative survival curve of control and C26-treated mice (n ⫽ 10). F, average volume of lung metastases for
each group (n ⫽ 6). Results shown are mean ⫾ S.E. (n ⫽ 6); ⴱ, p ⬍ 0.05 versus control.

colonies. It is interesting that we observed a significant de- reveal any significant toxicity in control or C26-treated ani-
crease of number of metastases in mice treated with C26 mals. C26 treatment did not increase activity of liver en-
compared with mice treated with vehicle (n ⫽ 6 for each zymes (alanine aminotransferase and aspartate aminotrans-
group). Metastatic colonies were observed in the lungs from ferase) or elevate serum urea nitrogen levels (Table 1). No
all 10 control mice but observed in only two of the C26- obvious abnormalities were found in control or C26-treated
treated mice. Moreover, the average volume of lung metas- liver, heart, aorta, or kidney tissues stained with hematoxy-
tases in C26-treated mice was significantly lower than in lin and eosin (data not shown). Furthermore, hemodynamic
control mice (Fig. 5F). Together, these data indicate that C26 monitoring with an indwelling Milar catheter showed that
treatment can inhibit lung metastasis and prolong survival C26 treatment did not have any inhibitory effects on systolic
of tumor-bearing mice. and diastolic heart function as measured by dP/dt max and
Histological, biochemical, and enzymatic assays did not dP/dt min (Table 1). These results suggest that C26 de-
 CYP2J2 Inhibition and Cancer Therapy 917
TABLE 1 2005, 2007). Thus, C26 treatment attenuated prometastatic
Liver, kidney, and cardiac toxicity in control and C-26-treated mice signaling and antiapoptotic protein expression in cancer
(n ⫽ 6) cells.
Liver Function
Kidney Function: Heart Function
Urea Nitrogen (⫻1000)
ALT AST Discussion
U/l mM dP/dt The pathological consequences of cancer are mainly related
Control 56.6 ⫾ 15.6 239.8 ⫾ 31.3 11.1 ⫾ 2.3 3.53 ⫾ 0.40 to uncontrolled tumor growth and metastasis, both of which
C26 55.6 ⫾ 17.3 233.4 ⫾ 45.7 11.7 ⫾ 2.2 3.54 ⫾ 0.24
are a consequence of abnormal tumor cell proliferation,
adhesion, invasion, and migration. Our previous studies
presses growth and metastasis of breast carcinoma cells in demonstrated a role for CYP2J2-derived EETs in promot-
vivo and extends survival time without significant end-organ ing the neoplastic phenotype of cancer cells; increased
toxicity. CYP2J2 expression or activity was associated with in-
C26 Attenuates the Activation of Growth Factor Sig- creased tumor growth and lung metastasis (Jiang et al.,
naling Pathways. Western blot analysis showed that 11,12- 2007). Given that CYP2J2 is up-regulated in human car-
EET (200 nM) addition up-regulated PI3 kinase and in- cinoma cell lines (Jiang et al., 2005), we hypothesized that
creased the phosphorylation level of the EGFR and Akt in inhibition of EET production would reduce tumor growth
MDA-MB-235 cells. Treatment with C26 (10 ␮M) had the by modifying the neoplastic phenotype of cancer cells.
opposite effects, which could be rescued by exogenous EET Herein, we have shown that a novel class of selective

Downloaded from jpet.aspetjournals.org at ASPET Journals on March 6, 2015

addition (Fig. 6, A–C). These data are consistent with the inhibitors of CYP2J2 has potent antitumor activity in vitro
inhibitory effects of C26 on growth factor signaling pathways and in vivo. These terfenadone derivatives reduced human
in cancer cells. C26 treatment also decreased levels of the cancer cell proliferation and promoted apoptosis. They also
antiapoptotic protein Bcl-2 and increased levels of the pro- inhibited the adhesion, migration, and invasion of cancer
apoptotic protein Bax (Fig. 6D), consistent with the proapop- cells in vitro. More importantly, selective CYP2J2 inhibi-
totic effect of C26. Furthermore increased expression of the tors markedly attenuated the growth of a murine xeno-
antimetastatic proteins CD82 and nm-23 were found in C26- graft tumor induced by MDA-MB-435 cells in athymic
treated cells (Fig. 6E). Numerous signal transduction path- BALB/c mice.
ways have been shown to regulate tumor cell biology (Anag- Our previous study showed that in addition to inducing
nostopoulos et al., 2008; Kroemer and Pouyssegur, 2008). As tumor cell proliferation, both endogenously formed and exog-
reported previously, EETs and P450 epoxygenases induce enously applied EETs enhanced tumor cell motility, invasion,
signaling and gene expression changes that promote tumor- adhesion, prometastatic gene expression, and xenograft me-
igenic and malignant phenotypes through multiple path- tastasis to lungs (Jiang et al., 2007). In the current study, we
ways, including Bcl-2/Bax and CD82/nm-23 (Jiang et al., showed that selective inhibition of CYP2J2 reduces adhesion

A D
pEGFR Bax

Bcl-2
EGFR

control DMSO 11,12-EET C26 C26/

β-actin Fig. 6. Effect of C26 treatment on cell
11,12-EET
signaling pathways. MDA-MB-435
cells were treated with 200 nM EET
control DMSO 11,12-EET C26 C26/ and/or 10 ␮M C26 for 24 h and then
11,12-EET
analyzed for protein expression and
phosphorylation. A, decreased phos-
B E phorylation of the EGFR in MDA-MB-
PI3K CD82 435 cells after C26 treatment is res-
cued by EETs. B, decreased PI3
kinase protein expression in MDA-
β-actin nm-23 MB-435 cells after C26 treatment is
rescued by EETs. C, decreased phos-
phorylation of the Akt in MDA-MB-
control DMSO 11,12-EET C26 C26/ 435 cells after C26 treatment is res-
11,12-EET β-actin cued by EETs. D, effects of EET and
C26 treatments on apoptosis-related
control DMSO 11,12-EET C26 C26/ proteins in MDA-MB-435 cells. E, ef-
11,12-EET fects of EET and C26 treatments on
metastasis-related proteins in MDA-
C MB-435 cells.
AKT

pAKT

control DMSO 11,12-EET C26 C26/

11,12-EET
918 Chen et al.

and invasion of human tumor cells. It is important that promotes matrix metalloproteinase (MMP) activation and MMP-dependent inva-
sion in ovarian cancer cells. Cancer Res 61:3194 –3199.
addition of exogenous EET partially reverses these effects. Jangi SM, Díaz-Pérez JL, Ochoa-Lizarralde B, Martín-Ruiz I, Asumendi A, Pérez-
Furthermore, CYP2J2 inhibition mitigated the resistance of Yarza G, Gardeazabal J, Díaz-Ramón JL, and Boyano MD (2006) H1 histamine
receptor antagonists induce genotoxic and caspase-2-dependent apoptosis in hu-
cancer cells to apoptosis. Although not examined in this man melanoma cells. Carcinogenesis 27:1787–1796.
work, it is likely that the inhibition of EET production im- Jangi SM, Ruiz-Larrea MB, Nicolau-Galmés F, Andollo N, Arroyo-Berdugo Y, Or-
tega-Martínez I, Díaz-Pérez JL, and Boyano MD (2008) Terfenadine-induced
pedes the expression of the neoplastic phenotype of cells apoptosis in human melanoma cells is mediated through Ca2⫹ homeostasis mod-
through several mechanisms. We reported previously that ulation and tyrosine kinase activity, independently of H1 histamine receptors.
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