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Vol. 29, Issue 4, Part 1, 389-393, April 2001
College of Pharmacy, Chungang University, Seoul, Korea (Y.J.C., M.Y.K.); Korea Research Institute of Chemical Technology, Taejon, Korea (S.Y.R.); and College of Pharmacy, Yeungnam University, Kyungsan, Korea (T.C.J.)
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
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Recently we reported that resveratrol
(trans-3,4',5-trihydroxystilbene) showed selective
inhibition of recombinant human cytochrome P450 (P450) 1A1 in a
concentration-dependent manner. The inhibition of recombinant
human P450 1A1, 1A2, or 1B1 by various hydroxystilbene compounds having
a similar structure to resveratrol was investigated using bacterial
membranes from a human P450/NADPH-P450 reductase bicistronic expression
system to find new candidates for cancer chemopreventive agents. Of
seven compounds tested, rhapontigenin (3,3',5-trihydroxy-4'-methoxystilbene) exhibited a potent and selective
inhibition of human P450 1A1 with an IC50 value of 0.4 µM. Rhapontigenin showed 400-fold selectivity for P450 1A1 over P450
1A2 and 23-fold selectivity for P450 1A1 over P450 1B1. Rhapontigenin did not show any significant inhibition of ethoxyresorufin
O-deethylation (EROD) activity in human liver
microsomes, the other human P450s such as P450 2E1, P450 3A4, P450 2D6,
P450 2C8, and P450 2C9, or human NADPH-P450 reductase. We have further
investigated the inhibition kinetics of P450 1A1 by rhapontigenin.
Rhapontigenin inhibited EROD activity of expressed human P450 1A1 in a
competitive manner. The loss of EROD activity was time- and
concentration-dependent. The values for Ki
and kinactivation were 0.09 µM and 0.06 min
1, respectively. The loss was not blocked by the
trapping agents glutathione, N-acetylcysteine, or
dithiothreitol. These results suggest that rhapontigenin is a potent
mechanism-based inactivator of human P450 1A1 and may be considered as
a good candidate for a cancer chemopreventive agent in humans.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Human
cytochrome P450 (P4501) 1A1 is a well known aryl
hydrocarbon hydroxylase and is involved in the metabolic activation of
procarcinogens of the polycyclic aromatic hydrocarbons. In humans, P450 1A1 shares 80% amino acid sequence identity with P450 1A2
and about 40% with P450 1B1 (Jaiswal et al., 1985
; Quattrochi et al.,
1985; Shimada et al., 1998b). The substrate specificities of these
enzymes can often overlap. Although P450 1A1 is mainly expressed in
human lung, placenta, and lymphocytes, P450 1A2 is one of the major
P450s in human liver. P450 1B1 is normally expressed in human
fibroblasts and steroidogenic tissues. To distinguish the activities of
these P450s and to determine the mechanism of reactions of the enzymes,
selective inhibitors are required. Selective inhibitors may be good
chemopreventive agent candidates for cancer because these P450s are
related to tumor initiation.
Resveratrol has been studied to determine its role as a cancer
chemopreventive agent and shown to inhibit tumor formation, acting
through cyclooxygenase inhibition, blockage of free-radical formation,
and induction of quinone reductase (Jang et al., 1999).
Recently we reported that resveratrol can inhibit human P450 1A1 in a
concentration-dependent manner (Chun et al., 1999). Resveratrol
showed 50-fold selectivity in its inhibition of P450 1A1 over P450 1A2.
However, the IC50 value for 1A1 inhibition (23 µM) by resveratrol is not very low compared with other known P450 1A
inhibitors such as 
-naphthoflavone and 7-hydroxyflavone (Shimada et
al., 1998b; Zhai et al., 1998). To find more potent and
selective P450 1A1 inhibitors among resveratrol analogs, several hydroxystilbene compounds obtained from natural sources were evaluated for selective inhibition of P450 1A1 activity (Shin et al., 1998). To
examine these compounds, recombinant systems in which human P450s 1A1,
1A2, or 1B1 was expressed along with human NADPH-P450 reductase in
bacterial membranes were used (Parikh et al., 1997; Josephy et al.,
1998; Shimada et al., 1998a). Of the compounds tested, rhapontigenin
showed potent and selective inhibition of P450 1A1 over 1A2 or 1B1.
Kinetic analyses were performed to determine the mechanism of P450 1A1
inhibition, and we suggest that rhapontigenin is a mechanism-based
inactivator of human P450 1A1.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Materials.
The hydroxystilbenes used were isolated from herbal extracts or
obtained from chemical modification (Table
1) (Ryu et al., 1988). Rhapontigenin,
rhaponticin, and 3,5-dihydroxy-4'-methoxystilbene were purified from
Rheum undulatum Linne. Oxyresveratrol was isolated from
Morus alba Linne. Resveratrol was isolated from
Veratrium album var. grandiflorum Maxim., and
3,4',5-trimethoxystilbene was obtained by methylation of resveratrol.
Piceid was isolated from Polygonum cuspidatum Sieb. et
Zucc., and 3,4'-dimethoxy-5-hydroxystilbene was obtained by methylation
of piceid followed by the acid hydrolysis. Human liver samples were
kindly provided by Dr. F. Peter Guengerich (Vanderbilt University,
Nashville, TN). Ethoxyresorufin, resorufin, ethoxycoumarin,
hydroxycoumarin, dimethyl sulfoxide, thiamine, IPTG, 
-aminolevulinic
acid, and NADPH were purchased from Sigma Chemical Co. (St. Louis, MO).
Nifedipine and nifedipine metabolite were from RBI/Sigma
(Natick, MA). Bactopeptone, yeast extract, and bacto-agar were obtained
from Difco Lab. (Detroit, MI). Human P450 2C8 microsomes were purchased
from GENTEST (Woburn, MA). Other chemicals were of the highest grade
commercially available.
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Recombinant Human P450s.
Coexpression (bicistronic) plasmids for human P450s (1A1, 1A2, 1B1,
2E1, 3A4, 2C9, or 2D6), and NADPH-P450 reductase were transformed into
Escherichia coli DH5 (Parikh et al., 1997). A single
ampicillin-resistant colony of transformed cells was selected and grown
in overnight culture to saturation at 37°C in Luria-Bertani medium
containing 100 µg of ampicillin ml1. A 10-ml
aliquot was used to inoculate each liter of Terrific Broth containing
0.2% bactopeptone (w/v), 100 µg of ampicillin ml1, 1.0 mM thiamine, trace elements, 0.5 mM
-aminolevulinic acid, and 1.0 mM IPTG. The cultures were grown at
30°C with shaking at 200 rpm for 24 h. Membrane fractions were
prepared by differential centrifugation from bacteria and suspended in
10 mM Tris-HCl buffer (pH 7.4) containing 1.0 mM EDTA and 20% (v/v)
glycerol (Guengerich et al., 1996).
Human Liver Microsomes.
Frozen human liver samples were thawed in 0.1 M Tris acetate buffer (pH
7.4) containing 0.1 M KCl, 1.0 mM EDTA, and 20 µM butylated
hydroxytoluene and homogenized in a Teflon-glass homogenizer. The homogenate was centrifuged at
104g for 20 min at 4°C, and the
resulting supernatant was centrifuged at
105g for 60 min at 4°C. The
microsomal pellets were resuspended in 10 mM Tris acetate buffer (pH
7.4) containing 1.0 mM EDTA and 20% (v/v) glycerol (Guengerich, 1994).
Protein concentrations were estimated using the bicinchoninic acid
method according to the supplier's recommendations (Pierce Chemical
Co., Rockford, IL) using bovine serum albumin as a standard. The
isolated microsomes were stored at 
80°C.
Enzyme Assays.
EROD activity was determined for the measurement of P450 1A1, 1A2, or
1B1 activities (Burke et al., 1985). The reaction mixture contained 0.1 M potassium phosphate buffer (pH 7.4), 2 mg of bovine serum albumin
ml1, 10 µM dicoumarol, human liver
microsomes, or E. coli membranes (5 nM P450 1A1, 10 nM P450
1A2, or 10 nM P450 1B1, respectively), and 2 µM ethoxyresorufin. The
reaction mixtures were preincubated at 37°C for 3 min, and the
reaction was initiated by addition of 120 µM NADPH. Incubations were
performed in a shaking water bath for 20 min at 37°C and terminated
by addition of 1 ml of methanol. The formation of resorufin was
determined fluorometrically with a PerkinElmer LS 5 spectrofluorometer
(with excitation and emission wavelengths of 550 and 585 nm,
respectively) (PerkinElmer, Norwalk, CT).
using an extinction coefficient of 91 mM1 cm1 with a Shimadzu
UV-160A spectrophotometer at ambient temperature (Shimadzu, Kyoto,
Japan). For the measurement of P450 2E1 activity, O-deethylation of 7-ethoxycoumarin was determined
fluorometrically according to methods described previously (Yamazaki et
al., 1996). Reaction mixtures contained 0.1 M potassium phosphate (pH
7.4), 2 mM ethoxycoumarin, E. coli membranes (40 nM P450
2E1), and 1 mM NADPH. Incubations were performed in a shaking water
bath for 20 min at 37°C and terminated by addition of 25 µl of 10%
(w/v) trichloroacetic acid and 1 ml of
CH2Cl2. The mixture was
vortex mixed and extracted with 30 mM sodium borate (pH 9.0).
Concentrations of hydroxycoumarin were determined by a PerkinElmer LS 5 spectrofluorometer with excitation and emission wavelengths of 370 and
450 nm, respectively.
Nifedipine oxidation was determined by high-performance liquid
chromatography as a measure of recombinant human P450 3A4 activity using previously described methodology (Guengerich et al., 1986). Reaction mixtures contained 50 mM HEPES (pH 7.4), 30 mM
MgCl2, 0.2 mM nifedipine, E. coli
membranes (40 nM P450 3A4), and 1 mM NADPH. Incubations were performed
in a shaking water bath for 20 min at 37°C and terminated by addition
of 2 ml of CH2Cl2 and 100 µl of 1 M Na2CO3 (pH
10.5) containing 2 M NaCl. The samples were centrifuged at
3000g for 10 min, and 1.4 ml of each of the lower organic
layers were dried under a N2 stream.
Determination of a nifedipine metabolite was performed using a 150- × 4.6-mm steel C18 Nucleosil column with
ultraviolet detection at 254 nm. The flow rate was 1.0 ml/min, and the
mobile phase was 64% methanol. Under these conditions, retention times
of nifedipine metabolite and nifedipine were 6.9 and 9.1 min,
respectively. The detection limit of a nifedipine metabolite was ~50
pmol with a signal-to-noise ratio of 3:1. Bufuralol 1'-hydroxylation
activity was determined as previously described (Yamazaki et al.,
1994). Incubation mixtures consisted of membranes (100 nM P450 2D6) and
bufuralol (0.4 mM) in a final volume of 0.1 ml of 100 mM potassium
phosphate buffer (pH 7.4) containing 1 mM NADPH. Incubations were
carried out at 37°C for 20 min and were terminated by addition of 10 µl of 60% (w/v) HClO4. The mixtures were
centrifuged at 3000g for 5 min, and aliquots of the
supernatant were separated using a 150- × 4.6-mm steel
C18 Nucleosil column eluted with 45% (v/v)
acetonitrile/55% 20 mM NaClO4, pH 2.5. The
hydroxylated metabolites of bufuralol were detected with a fluorescence
detector (excitation = 252 nm; emission = 302 nm).
Paclitaxel 6-hydroxylase activity was used as a catalytic marker for
recombinant human P450 2C8 (Rahman et al., 1994). Reaction mixtures
(0.2 ml of total volume) contained 100 mM potassium phosphate buffer
(pH 7.4), 10 µM paclitaxel, membranes (50 nM P450 2C8), and 1 mM
NADPH. Incubations were performed in a shaking water bath for 20 min at
37°C and terminated by addition of 50 µl of ice-cold acetonitrile.
The samples were centrifuged at 12,000g for 5 min, and
aliquots of the supernatant were analyzed using a 150- × 4.6-mm
steel C18 Nucleosil column with a linear gradient of 45% of 10% (v/v) methanol and 55% of 100% (v/v) methanol to 35%
of 10% (v/v) methanol and 65% of 100% (v/v) methanol over 20 min,
then for a further 5 min with 35% of 10% (v/v) methanol and 65% of
100% (v/v) methanol with ultraviolet detection at 254 nm.
Diclofenac 4'-hydroxylation was determined for the recombinant human
P450 2C9 specific activity (Leemann et al., 1992). Incubation mixture
(0.2 ml of total volume) consisted of 100 mM Tris-HCl buffer (pH 7.5),
100 µM diclofenac, membranes (50 nM P450 2C9), and 1 mM NADPH.
Incubations were carried out at 37°C for 20 min and were terminated
by addition of 50 µl of a mixture of 94% acetonitrile/6% acetic
acid. The reaction mixtures were centrifuged at 12,000g for
5 min and the supernatant was analyzed using a 150- × 4.6-mm steel C18 Nucleosil column with a linear gradient
from 70% of 30% (v/v) acetonitrile containing 2 mM perchloric acid
and 30% of 100% (v/v) methanol to 100% of 100% (v/v) methanol over
20 min. The flow rate was 1.0 ml/min, and the production of
4'-hydroxydiclofenac was measured with ultraviolet detection at 280 nm.
Rates of NADPH-cytochrome c reduction were measured using an
extinction coefficient of 21 mM1
cm1 (Yasukoshi and Masters, 1976). The intra-
and interday variation coefficients did not exceed 10% in any of the assays.
NADPH Dependence of Inhibition.
Bacterial membranes containing human P450 1A1 and NADPH-P450 reductase
were preincubated in 0.1 M potassium phosphate buffer (pH 7.4)
containing hydroxystilbene at 37°C for 10 min in the presence or
absence of 1 mM NADPH (Chun et al., 1999). At various times during the
preincubation, an aliquot of the preincubation mixture was diluted
10-fold into the reaction mixture containing 0.1 M potassium phosphate
(pH 7.4), 2 mg/ml bovine serum albumin, 0.2 µM ethoxyresorufin, and
120 µM NADPH. The mixtures were further incubated at 37°C for 20 min. The product of 7-ethoxyresorufin was monitored fluorometrically as
described above.
Data Analysis. Kinetic parameters from individual experiments were calculated using a nonlinear regression analysis program (Prism, GraphPad Software, San Diego, CA).
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Results |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Inhibition of EROD activity by Hydroxystilbenes. To examine their effects on the activity of human P450 1A1, 1A2, and 1B1, seven hydroxystilbene compounds were compared with respect to inhibition of EROD activities (Table 2). Rhapontigenin was found to be a potent inhibitor of P450 1A1, with an IC50 value of 0.4 µM (Fig. 1; Table 2). Similar studies showed that rhapontigenin also inhibits P450 1A2 and 1B1, but the IC50 values were much higher (i.e., 160 µM for P450 1A2 and 9 µM for P450 1B1). Thus, rhapontigenin exhibited 400-fold greater selective inhibition of P450 1A1 over 1A2 and 23-fold greater inhibition of P450 1A1 over 1B1. As shown in Table 2, 3,4'-dimethoxy-5-hydroxystilbene and 3,5-dihydroxy-4'-methoxystilbene were also more potent inhibitors of P450 1A1 than of P450 1A2, but the selectivity between P450 1A1 and 1B1 was much lower than that of rhapontigenin. The most potent P450 1A1 inhibitor of all hydroxystilbenes we examined was 3,4'-dimethoxy-5-hydroxystilbene. 3,4',5-Trimethoxystilbene also strongly inhibited P450 1A1, 1A2, and 1B1 with IC50 values of 0.6, 0.6, and 0.4 µM for 1A1, 1A2, and 1B1, respectively, but no selectivity was shown. The IC50 of oxyresveratrol for P450 1A1 was about 15 µM. The hydroxystilbenes containing a glucosyl moiety at the R1 position, such as piceid or rhaponticin, had little inhibitory effect on P450s. The bulky and polar glucosyl moiety may block the accessibility of the chemicals into the active sites of P450s.
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Mechanism of Inhibition by Rhapontigenin.
To examine the mechanism of inhibition by rhapontigenin, kinetic
studies were performed using recombinant human P450 1A1. Rhapontigenin
showed competitive inhibition of P450 1A1, with a
Ki of 0.21 µM (Fig.
3). Preincubation of P450 1A1 with
various concentrations of rhapontigenin in the presence of NADPH
resulted in a time- and concentration-dependent loss of EROD activity. The inactivation of P450 1A1 by rhapontigenin exhibited first order
kinetics when plotted as the log of percentage of activity remaining
versus time (Fig. 4A). A replot of the
data in Fig. 4A showed that Ki and
kinactivation were 0.09 µM and 0.06 min1, respectively (Fig. 4B). To determine the
possibility that the reactive species could escape from the P450 active
site and bind to nucleophilic sites in the vicinity of the active site,
the effect of trapping agents such as glutathione,
N-acetylcysteine, or dithiothreitol on the inactivation was
studied. Rhapontigenin did not protect against P450 1A1 inactivation in
the presence of 2 mM glutathione, N-acetylcysteine, or
dithiothreitol (Fig. 5).
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Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Tumor initiation begins when DNA in cells is damaged by exposure
to carcinogens. If this damage is not repaired, it can lead to genetic
mutations (Hurting et al., 1999). Metabolic activation of
procarcinogens is often catalyzed by P450 enzymes through oxidation. Enzymes such as P450 1A1, 1A2, or 1B1 of the human P450 1 subfamily are
responsible for much of the metabolism of procarcinogens and regarded
as the target enzymes for blocking tumor initiation (Shimada et al.,
1989, 1996; Guengerich and Shimada, 1991). Therefore, specific P450
inhibitors or inactivators could be beneficial for preventing tumor formation.
Recently, several hydroxystilbene compounds isolated from herbal
sources including Morus alba or Rheum undulatum
were identified as having various pharmacological effects. For example,
oxyresveratrol potently inhibited dopa oxidase activity of tyrosinase
(Shin et al., 1998a). Rhapontigenin significantly inhibited the release of 
-hexaminidase from cultured RBL-2H3 cells (Cheong et al., 1999).
These hydroxystilbenes also suppressed ovine cyclooxygenase-1 activity
(Shin et al., 1998b).
In this study we demonstrated that rhapontigenin is a potent mechanism-based inactivator of P450 1A1 as well as a competitive inhibitor. Rhapontigenin inactivation of P450 1A1 followed pseudo-first order kinetics, was time- and concentration-dependent, and required NADPH. Trapping agents such as glutathione, N-acetylcysteine, or dithiothreitol could not block the inactivation of P450 1A1 by rhapontigenin.
Several chemopreventive agents are known as a mechanism-based
inactivators of P450s, such as isothiocyanates (P450 2E1) and oltipraz
(P450 1A2) (Kent et al., 1998; Moreno et al., 1999; Langouët et
al., 2000). The two main mechanisms for the chemopreventive action are
1) inhibition of phase I enzymes such as the P450 enzymes and 2)
induction of phase II enzymes such as glutathione
S-transferase and quinone reductase. We have previously
demonstrated that human P450 1A1 activity was selectively inhibited by
resveratrol (Chun et al., 1999). Resveratrol seems to be a promising
cancer chemopreventive agent because it inhibits phase I enzymes and
induces phase II enzymes. However, the inhibition of P450 1A1 by
resveratrol is relatively weak and not mechanism-based. Furthermore,
resveratrol could not discriminate in inhibiting the activities of
P450s 1A1 and 1B1 (unpublished results). We suggest that rhapontigenin
is a better candidate for cancer chemopreventive agents because its inhibitory potential of P450 1A1 is much stronger, selective, and
mechanism-based. We are studying the effects of rhapontigenin on the
expression of phase II enzymes in cultured cells.
A number of the known inhibitors of the different P450 enzymes are
mechanism-based (Yun et al., 1992; Wrighton et al., 1993; Roberts et
al., 1995; Heyn et al., 1996; Hickman et al., 1998). Many selective
P450 1A1 inhibitors have been reported. For example, 2-(1-propynyl)phenanthrene showed 70-fold greater inhibition of P450
1A1 than of 1A2 (Shimada et al., 1998b), and 7-hydroxyflavone exhibited
6-fold greater selectivity in its inhibition of P450 1A1 over P450 1A2
(Zhai et al., 1998). As we showed previously, resveratrol is also
selective for inhibiting P450 1A1 (Chun et al., 1999). These inhibitors
are used for labeling the P450 active site and identifying critical
amino acid(s) of P450s. The 400-fold greater selectivity of
rhapontigenin in the inhibition of P450 1A1 over P450 1A2 is of
interest. Thus far, rhapontigenin is one of the most selective
inhibitors of P450 1A1 from natural sources. We propose that
rhapontigenin also will be a useful compound for characterizing P450
1A1 active sites because of its strong selectivity.
The precise mechanism by which rhapontigenin is transformed by P450 1A1
into an inactivating intermediate is not yet known. We have considered
the possibility that activated oxygen bound to heme may react with the
allyl moiety of the stilbene structure, and the intermediate species
could then react with protein or heme nucleophiles in a manner that
destroys heme function or binding (Ortiz de Montellano and Correia,
1983; Hammons et al., 1989; Guengerich, 1990).
In summary, we report for the first time that rhapontigenin is a potent mechanism-based inactivator of human P450 1A1. Because rhapontigenin is isolated from the oriental medical plant Rheum undulatum, its potent and selective inhibitory effect on P450 1A1 takes on additional advantages as a chemopreventive agent. Future studies will focus on the potential of rhapontigenin as a strong chemopreventive agent in vivo.
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Acknowledgment |
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We thank Dr. F. P. Guengerich for providing us with the bacterial bicistronic expression plasmids and for helpful suggestions.
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Footnotes |
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Received October 3, 2000; accepted December 13, 2000.
This work was supported by a research grant from Chungang University, Seoul, Korea (1999).
Send reprint requests to: Young Jin Chun, Ph.D., College of Pharmacy, Chungang University, 221, Huksuk-dong, Dongjak-gu, Seoul 156-756, Korea. E-mail: yjchun{at}chungang.edu
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Abbreviations |
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Abbreviations used are:
P450, cytochrome P450;
EROD, ethoxyresorufin O-deethylation;
IPTG, isopropyl-1-thio--D-galactopyranoside.
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References |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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), P450 1A2 (
), or 1B1
(
), 7-ethoxycoumarin O-deethylation by P450 2E1
(
), nifedipine oxidation by P450 3A4 (
), bufuralol
1'-hydroxylation by P450 2D6 (
), paclitaxel 6
), or diclofenac 4'-hydroxylation by P450 2C9
(
) in the presence of the indicated concentrations of rhapontigenin.
Each data point represents the mean ± S.E.M. of three
experiments.
