| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Carcinogenesis |
Department of Biotechnological Science, Faculty of Biology-Oriented Science and Technology, Kinki University, Wakayama 649-6493 [A. M., T. K., K. K.]; Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502 [Y. N., K. T., H. O.]; First Department of Pathology, Kanazawa Medical University, Ishikawa 920-0293 [T. T.]; Research and Development Division, Wakayama Agricultural Processing Research Corporation, Nagagun, Wakayama 649-6112 [S. K., Y. T.]; Department of Citriculture, Fruit Tree Research Station, Shizuoka 424-0292 [K. O., M. Y.]; Department of Biochemistry, Kyoto Prefecture University of Medicine, Kyoto 602-0841 [H. T., H. N.]; Laboratory of Medicinal Plant Science, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Tokyo 192-0392 [Y. M., Y. S.]; and Laboratory of Pharmacognosy, College of Pharmacy, Nihon University Chiba 274–8555 [S. K.], Japan
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
in mouse macrophage RAW 264.7 cells,
and by 12-O-tetradecanoylphorbol-13-acetate (TPA) in
differentiated human promyelocyte HL-60, respectively. Expression of
inducible NO synthase and cyclooxygenase 2 proteins were
detected by Western blotting. The in vivo
anti-inflammatory and antitumor promoting activities were evaluated by
topical TPA application to ICR mouse skin with measurement of edema
formation, epidermal thickness, leukocyte infiltration, hydrogen
peroxide production, and the rate of proliferating cell nuclear
antigen-stained cells. As a result, nobiletin, a polymethoxyflavonoid,
was identified as an inhibitor of both NO and
O2- generation. Nobiletin significantly
inhibited two distinct stages of skin inflammation induced by double
TPA application [first stage priming (leukocyte infiltration) and
second stage activation (oxidative insult by leukocytes)] by
decreasing the inflammatory parameters. It also suppressed the
expression of cyclooxygenase-2 and inducible NO synthase proteins and
prostaglandin E2 release. Nobiletin inhibited
dimethylbenz[a]anthracene (0.19 µmol)/TPA (1.6
nmol)-induced skin tumor formation at doses of 160 and 320 nmol by
reducing the number of tumors per mouse by 61.2%
(P < 0.001) and 75.7%
(P < 0.001), respectively. The present
study suggests that nobiletin is a functionally novel and possible
chemopreventive agent in inflammation-associated tumorigenesis. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Nitric oxide (NO), a gaseous free radical, is released by a family of enzymes, constitutive NO synthase and an inducible one (iNOS; Ref. 14 ) with the formation of stoichiometric amounts of L-citrulline from L-arginine (15) . iNOS-mediated excessive and prolonged NO generation has attracted attention because of its relevance to epithelial carcinogenesis (16, 17, 18) . NO has been reported to cause mutagenesis (19) and deamination of DNA bases (20) and to form carcinogenic N-nitrosoamines (21) . With respect to the role of NO in the postinitiation phase, it should be noted that it plays important roles in such inflammatory responses as edema formation and hyperplasia, as well as in papilloma development in mouse skin (22 , 23) . NO is also involved in the production of VEGF (24) , the overexpression of which induces angiogenesis, vascular hyperpermeability, and accelerated tumor development (25) . Topical application of TPA to mice led to edema and papilloma formation by enhancing COX-2 protein expression. Specific COX-2 inhibitors (26 , 27) could counteract these biological events. Collectively, suppression of enzyme induction and the activities of iNOS/COX-2 is a new paradigm for the prevention of carcinogenesis in several organs, including the stomach and colon. In fact, some dietary compounds, including chemopreventive agents (28, 29, 30, 31) and the extracts from edible plants (32) , have recently been reported to suppress iNOS-mediated NO generation.
The present study was undertaken to search for NO generation inhibitors
in Citrus unshiu (satsuma mandarin), the most popular citrus
fruit in Japan, by activity-guiding fractionation and purification,
using a combined LPS- and IFN--induced NO generation test in
mouse macrophage RAW 264.7 cells. As a result, we identified a
polymethoxyflavonoid, nobiletin, to be a dual inhibitor of both NO and
O2- generation in leukocytes.
Wei et al. (33)
reported that double
applications of TPA to mouse skin led to excessive ROS production. Ji
and Marnett (34)
termed each application as "priming"
(the first stage illustrated by leukocyte recruitment, maturation, and
infiltration of inflammatory leukocytes such as polymorphonuclear
leukocytes and macrophages into inflamed lesions) and
"activation" (the second stage illustrated by ROS production from
accumulated leukocytes). Our research addressed whether nobiletin
inhibits the priming and/or activation stages in this double
application model. In addition, the inhibitory effect of nobiletin on
mouse skin tumor promotion using a two-stage carcinogenesis model was
investigated.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
from Genzyme
(Cambridge, MA). Cytochrome c was obtained from Sigma
(St. Louis, MO. Luteolin was obtained from Extrasynthèse
(Genay, France). All other chemicals were purchased from Wako Pure
Chemical Industries, Co., Ltd. (Osaka, Japan). HL-60 cells were
purchased from American Type Culture Collection (Manassas, VA).
RAW 264.7 cells were kindly donated by Ohtsuka Pharmaceutical Co. Ltd.
(Ohtsu, Japan). Female ICR mice, ages 6–7 weeks, were obtained from
Japan SLC (Shizuoka, Japan).
Isolation and Identification of Nobiletin.
Whole fruit of C. unshiu (10-kg fresh weight), harvested in
Wakayama Prefecture, Japan, were processed by an FMC in-line
citrus juice extractor (FMC Corporation, Chicago, IL) to produce
cold-pressed oil (150 ml). Most of the monoterpenes,
d-limonene being the major constituent (
90%), were
removed from the oil by evaporation under reduced pressure at 80°C.
Then, the terpene-less oil was separated into useful fractions by
monitoring the inhibition of NO generation. The active fraction was
separated by silica gel column chromatography
(n-hexane/ethyl acetate, stepwise method), then by
preparative TLC (n-hexane/ethyl acetate, 1:1), and finally
by HPLC (column: µBondasphere C18; Waters,
Milord, MA; 19 x 150 mm; elute: 70% methanol in water;
flow rate: 7.0 ml/min; detection: UV254 nm) to
produce nobiletin (84 mg; retention time at 16.9 min; purity >98%).
The chemical identity was confirmed by comparison of its spectral data
with those reported previously (35)
.
NO Generation Test.
Murine macrophage cell line RAW 264.7 cells grown confluent in 2 ml of
DMEM on a 60-mm dish were treated with LPS (100 ng/ml),
tetrahydrobiopterin (10 mg/ml), IFN- (100 units/ml),
L-arginine (2 mM), and a test compound (0, 25,
50, or 100 µM). After 18 h, the levels of
NO2- were measured by Griess
assay. Cytotoxicity was measured by a
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. Each experiment was done independently in duplicate twice, and
the data are expressed as mean ± standard
deviation (SD) values.
Western Blotting.
Confluent RAW 264.7 cells were stimulated and incubated in the same
manner as described above. After the cells were washed, a boiling lysis
solution was added to the cells, which were then scraped from the dish,
sonicated, and boiled for 10 min. Protein concentrations were
determined using a DC Protein Assay kit (Bio-Rad Laboratories,
Hercules, CA), and BSA was used as the standard. Ten-µg proteins were
separated on 10% polyacrylamide gels and electrophoretically
transferred onto polyvinylidene difluoride membranes (Millipore, MA).
After blocking, the membranes were incubated with a primary antibody
(antimouse iNOS, 1:1000 dilution; Affinity Bioreagents, Inc.,
Golden, CO), and then with a secondary antibody (peroxidase-conjugated
swine antirabbit IgG, 1:1000 dilution; Dako, Glostrup, Denmark). The
blots were developed using an ECL detection kit (Amersham Life
Science, Buckinghamshire, United Kingdom). The antibodies were
stripped and the blots were successively reprobed with each primary
antibody. The first incubation was with: (a) goat
antirat COX-2 antibody [cross-reacts with mouse counterparts, 1:1000
dilution (Santa Cruz Biotechnology, Inc., Santa Cruz, CA)]; or
(b) rabbit polyclonal anti-ß-actin antibody (1:1000
dilution; Biochemical Technologies, Stoughton, MA). Each membrane was
then incubated with a corresponding secondary antibody: (a)
peroxidase-conjugated rabbit antigoat IgG (1:1000 dilution; Dako); and
(b) peroxidase-conjugated swine antirabbit IgG (1:1000
dilution; Dako). The levels of iNOS or COX-2 bands were corrected using
those of ß-actin as an internal standard. Each experiment was done
independently in duplicate twice, and the data are expressed as
mean ± SD values.
Double TPA Treatment of Mouse Skin.
The double TPA treatment experiment was performed as reported
previously (36)
. Briefly, the back of each mouse was
shaved using surgical clippers 2 days before each experiment; each
experimental group consisted of five mice. Nobiletin (810 nmol in 100
µl of acetone) was applied topically to the shaved area of
the dorsal skin 30 min before application of a TPA solution (8.1 nmol
in 100 µl of acetone). In the double-treatment protocol, the
same doses of TPA and the test compounds or acetone were applied twice
at an interval of 24 h. We divided the mice into five groups as
follows: group 1 (acetone x 2/acetone x 2); group 2 (acetone-TPA/acetone-TPA); group 3
(nobiletin-TPA/nobiletin-TPA); group 4 [nobiletin
(priming)-TPA/acetone-TPA]; and group 5 [acetone-TPA/nobiletin
(activation)-TPA].
Measurement of H2O2 and Edema Formation
in Mouse Skin.
Measurement of the levels of
H2O2 and edema formation
was done as reported previously (36)
. In brief, mice
treated by the double-treatment protocol were sacrificed 1 h after
the second TPA treatment. Skin punches (epidermis and dermis) were
obtained with an 8-mm-diameter cork borer and were weighed with
an analytical balance. The skin punches were minced in 3 ml of 50
mM phosphate buffer (pH 7.4) containing 5 mM
sodium azide and then homogenized at 4°C for 30 s twice. The
homogenate was centrifuged at 10,000 x g for
20 min at 4°C. The H2O2
content was determined by the phenol red-horseradish peroxidase method.
Histological Examination.
Histological examinations were done as reported previously
(36)
. Briefly, excised skin was fixed in 10% buffered
formalin and then embedded in paraffin. For each skin section (3 µm)
stained with H&E, the thickness of the epidermis from the basal layer
to the stratum corneum was measured at five equidistant interfollicular
sites using a Leica Q500IW-EX image analysis system (Leica, Co. Ltd.,
Tokyo, Japan) with a Leica DMRE HC microscope (Leica, Co. Ltd.). The
number of infiltrating leukocytes was counted at five different areas.
PCNA Immunohistochemistry.
A PCNA immunohistochemistry was done as reported previously
(36)
. Briefly, skin sections were treated with 1.2%
H2O2 in absolute methanol
for 30 min and were stained by the indirect abidin-biotin-horseradish
peroxidase method (ABC standard; Vector laboratories, Burlingame, CA).
Color development with diaminobenzidine (Vector laboratories) was
monitored by the appearance of normal PCNA brown staining in the normal
epidermis. The primary antibody to PCNA (PC10; Boehringer Mannheim,
Mannheim, Germany) was applied at a 1:300 concentration overnight at
4°C. The PCNA index was counted at six different areas of each
section using the image analysis system and was expressed as the number
of positive squamous cells divided by the total number of squamous
cells x 100.
O2- Generation in Differentiated HL-60
Cells.
An inhibitory test of TPA-induced
O2- generation was
performed as reported previously (36)
. Briefly,
differentiated HL-60 cells were treated with 100 nM TPA,
appropriate concentrations of the test compounds, and 1 mg/ml
cytochrome c. After incubation at 37°C for 30 min, the
level of extracellular O2- was
measured by cytochrome c reduction. Each experiment was done
in duplicate independently twice, and the data are
expressed as mean ± SD values.
Cellular Uptake.
Differentiated HL-60 cells were incubated and treated in the same
manner as described above. For extracellular measurement, after the
cell suspension was centrifuged, supernatant was collected, and the
cells were washed with media three times to combine the media used for
washing with the supernatant. The combined solution (4 ml) was
partitioned between distilled water (1 ml) and ethyl acetate (5 ml).
For intracellular measurement, cells were suspended in iced water and
ethyl acetate (5 ml each) and were sonicated for 10 s twice on
ice. The ethyl acetate fractions from the intracellular and
extracellular samples were separately concentrated in vacuo
and subjected to HPLC analysis [column: YMC-Pack ODS H-80 J'sphere
(4.6 x 150 mm; Yamamuta Chemical Co. Ltd.,
Kyoto, Japan); detection: UV280 nm at 45°C with
a flow rate of 1.0 ml/min]. Nobiletin and luteolin were detected using
40% acetonitrile in water as an elute at 8.1 min and 25% acetonitrile
in water at 3.9 min, respectively. Their incorporation efficiencies
were determined by the intracellular:extracellular ratio. Each
experiment was done in duplicate independently twice, and the data are
expressed as mean ± SD values.
PGE2 Determination.
RAW 264.7 cells, grown confluent in 2 ml of DMEM on a 60-mm dish, were
treated with LPS (100 ng/ml), tetrahydrobiopterin (10 mg/ml), IFN-
(100 units/ml), L-arginine (2 mM), and a test
compound (0, 25, 50, or 100 µM) dissolved in DMSO (0.5%,
v/v). After 18 h, the concentrations of
PGE2 in the media were measured using a
commercial experimental kit (Cayman, Ann Arbor, MI), and protein
concentrations were determined using a DC Protein Assay kit (Bio-Rad
Laboratories). BSA was used as the standard. Each experiment was done
in duplicate independently twice, and the data are expressed as
mean ± SD values.
Two-Stage Carcinogenesis Experiment in Mouse Skin.
The antitumor promoting activity of nobiletin was examined by a
standard initiation-promotion protocol with DMBA and TPA, as reported
previously (10)
. One group was composed of 15–17 female
ICR mice. These mice were given commercial rodent pellets and fresh tap
water ad libitum, both of which were freshly exchanged twice
a week. The back of each mouse was shaved with a surgical clipper 2
days before initiation. Mice at 6 weeks old were initiated with DMBA
(0.19 µmol in 100 µl of acetone). One week after
initiation, the mice were promoted with TPA (1.6 nmol in 100 µl of
acetone) twice a week for 20 weeks. In the other four groups,
the mice were treated with nobiletin (40, 80, 160, or 320 nmol in 100
µl of acetone) 40 min before each TPA treatment. The
antitumor-promoting activity was evaluated by both the ratio of
tumor-bearing mice and the number of tumors that measured more than 1
mm in diameter, per mouse. The data were statistically analyzed using
Welch’s t test for the average number of tumors per mouse
and by the 
2 test for the rates of
tumor-bearing mice.
Statistical Analysis.
The statistical significance of differences between groups in each
assay was assessed by Welch’s t test, Student’s
t test, or 2 test.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, the chemical identity of which was elucidated by comparison of the
spectral data with those reported previously (35)
.
|
for 18 h resulted
in NO generation and then nitrite
(NO2-) accumulation in the
media (40.3 ± 7.3 µM). Nobiletin, at a
concentration range of 25–100 µM,
concentration-dependently suppressed
NO2- production by 18.6–58.1%
(Fig. 2)
. The inhibitory effect of a red wine polyphenol, resveratrol
(37, 38, 39)
, was also examined for comparison of activity,
and its inhibitory potency was found to be similar to that of
nobiletin. iNOS protein expression was then detected using Western
blotting. As also shown in Fig. 2
, although being hardly detectable in
the cellular lysate without stimulation, iNOS protein was remarkably
up-regulated 25-fold 18 h after stimulation. Whereas nobiletin
showed a dose-dependent suppression at a concentration range of 25–100
µM (IRs, 32.7–82.0%), resveratrol showed marked
inhibition only at a concentration of 100 µM, and
activity drastically declined at the concentrations of 50 and 25
µM. Notable cytotoxicity was not observed in any of the
experiments (data not shown).
|
, double TPA applications, at a dose of 8.1 nmol each with a 24-h
interval, led to notable edema formation (42.3 ± 2.6
mg/punch in group 2) as compared with the control (24.7
± 4.5 mg/punch in group 1; P < 0.001
versus group 2). Double pretreatment with nobiletin (group
3) 30 min prior to each TPA application suppressed edema formation by
54.5% (P < 0.05). Pretreatment in the
priming phase (group 4: IR, 72.1%; P < 0.01) was found to be more effective for edema suppression than in the
activation phase (group 5: IR, 29.5%; P < 0.001) and was comparable or higher in double pretreatment. In
accordance with the TPA-induced enhancement of edema formation, an
increase in epidermal thickness was observed with double TPA
applications (Table 1
and Fig. 3, A and B
; 12.2 ± 1.6 µm in
group 1 versus 29. 1 ± 3.3 µm in group 2;
P < 0.001). Pretreatment with nobiletin in
the priming stage (Table 1
and Fig. 3
D; IR, 42.0%;
P < 0.001 in group 4) was also more
suppressive than that in the activation stage (Table 1
and Fig. 3
E; IR, 59.2%; P < 0.001 in
group 5). Double nobiletin pretreatment showed the highest inhibition
(Table 1
and Fig. 3
C; IR, 81.7%; P
< 0.001 in group 3).
|
|
and Fig. 3
, A and B, a
greater number of leukocytes were found to have infiltrated the dermis
by double TPA application as compared with the control (26.2
± 8.7/mm2 in group 1 versus
396.3 ± 9.9/mm2 in group 2;
P < 0.001; 15-fold). Double pretreatment
with nobiletin-inhibited leukocyte infiltration by 76.9%
(P < 0.001; Table 1
and Fig. 3
C)
and a single pretreatment in either the priming or activation phase
also suppressed infiltration by 58.0 and 47.2%, respectively (Table 1
and Fig. 3
, D and E).
Notably, the double TPA application dramatically increased the level of
H2O2 in the mouse epidermis
and dermis 7.5-fold (Table 1
; 0.73 ± 0.15 nmol/punch in
group 1 versus 5.47 ± 0.28 nmol/punch in
group 2; P < 0.001). A higher inhibition by
nobiletin was again observed when applied in the priming stage (IR,
57.2% in group 4; P < 0.001) than in the
activation phase (IR 44.5% in group 4; P < 0.001). Double pretreatment with nobiletin showed the highest
inhibition of H2O2
production among the three inhibitory groups (IR, 69.1% in group 3;
P < 0.001).
The PCNA-labeling index, a marker for cell proliferation, in the
epidermis of the group 2 mice increased by 3.3-fold over that of group
1 (Table 1
and Fig. 4, A and B
; 15.7 ± 2.4% in group 1
versus 52.0 ± 4.1% in group 2;
P < 0.001). Pretreatment with either a
single or double nobiletin application significantly reduced the
PCNA-labeling indices (IRs, 57.0–73.6%; Table 1
and Fig. 4
,
C–E).
|
, TPA stimulation of differentiated HL-60 cells for 30 min
led to marked extracellular O2-
generation (27.9 ± 3.4 µM in
the media). Nobiletin demonstrated a much higher level of inhibition
than did luteolin toward O2-
generation accompanied by a complete suppression at concentrations of
20 and 100 µM. The IC50
value of nobiletin (IC50, 1.2
µM) was approximately eight times lower than
that of luteolin (IC50, 9.8
µM). No cytotoxicity was detected in any
experiments (data not shown). As shown in Fig. 5B
, the
intracellular and extracellular ratios of nobiletin and luteolin, at a
100 µM concentration 45 min after incubation,
were found to be 0.108 ± 0.032 and 0.048
± 0.010 (intracellular:extracellular), respectively. The
intracellular luteolin was not detected at a concentration of 20
µM.
|
for 18 h led to
PGE2 production in the media (15.1 ng/ml/mg
protein; Fig. 6
). Nobiletin suppressed PGE2 production at the
concentrations of 50 and 100 µM (IRs, 35.8 and 41.2%,
respectively), whereas resveratrol exhibited higher suppressive rates
at the 25–100 µM concentrations (IRs, 48.6%–100%).
Stimulation of RAW 264.7 cells also led to a dramatic up-regulation of
COX-2 protein expression by 90.9-fold (Fig. 6)
, which was hardly
detectable in the control. Whereas nobiletin showed a dose-dependent
suppression at a concentration range of 25–100 µM (IRs,
50.7–90.4%), resveratrol showed marked inhibition only at a
concentration of 100 µM, and its activity declined at a
concentration range of 50 and 25 µM. Notable cytotoxicity
was not observed in any of the experiments (data not shown).
|
, the tumor incidence in the control group was 47.1% 20 weeks after
promotion. In the four nobiletin-pretreated groups, the incidence was
reduced by 33.5–43.3% (statistically not significant). The average
number of tumors per mouse in the control was 2.06 ± 1.00, and pretreatment with nobiletin at 160 and 320 nmol
dose-dependently reduced them by 61.2% (P < 0.01) and 75.7% (P < 0.01), respectively.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-induced
NO2- production in a
dose-dependent manner (Fig. 2)
. Whereas resveratrol was reported to
suppress iNOS expression through down-regulation of nuclear factor-
B
activity (37)
, the action mechanism of nobiletin for iNOS
suppression is unclear. In addition, issues of whether nobiletin
directly inhibits iNOS activity and affects both neural and endothelial
NOS functions remain to be addressed.
As summarized in Table 1
, nobiletin inhibited
double-TPA-application-induced biological and histological parameters
relating to oxidative damage and inflammation. Nobiletin notably
suppressed all of the parameters in both the priming and the activation
stages. The inhibitory effects of the double nobiletin pretreatment
appear to be caused by the additive effects of each application in both
stages because the double pretreatments were most suppressive.
TPA has recently been reported to produce NO and VEGF in human
polymorphonuclear leukocytes (24
, 46, 47, 48)
. Overexpressed
VEGF leads to the induction of vascular hyperpermeability
(25)
. On the other hand, PGE2 is well known to
increase vascular permeability. Nobiletin inhibited the expression of
COX-2 protein, which is an inducible and rate-limiting enzyme for PG
synthesis (Fig. 6)
. Accordingly, nobiletin inhibited the release of
PGE2 from RAW 264.7 cells (Fig. 6)
and also from
rabbit synovial cells (44)
. Although we have not examined
the effects of nobiletin on vascular permeability, the inhibition by
nobiletin of edema formation and the reduction of epidermal thickness
(Table 1
and Fig. 3
, C--E) may be partly
attributable to the suppression of NO, VEGF, and
PGE2 production.
As shown in Table 1
and Fig. 3
, leukocyte infiltration, a critical step
in the priming stage, was successfully suppressed by nobiletin in both
stages. After TPA application to mouse skin, an increased release of
IL-1 (a cytokine involved in immune and inflammatory events) becomes
predominant (49)
. There are two distinct forms of IL-1,
namely, IL-1
and IL-1ß. Of these cytokines, IL-1 is especially
noted as an important chemotactic factor released from keratinocytes in
the skin carcinogenesis model because its release involves cell
proliferation and hyperplasia in the tumor-promoting stage
via autocrine regulation (49)
. On the other
hand, LPS-stimulated RAW 264.7 cells release TNF-, IL-1ß, and IL-6
(50)
, and, recently, we found that nobiletin inhibited the
release of both LPS-induced and IFN--induced TNF- and IL-1ß
from RAW 264.7 cells (data not shown). Additional studies on the
influence of nobiletin on cytokine regulations may elucidate the action
mechanism(s) by which nobiletin suppresses leukocyte infiltration.
Because nobiletin did not significantly scavenge
O2- generated from the
xanthine/xanthine oxidase system nor inhibit xanthine oxidase activity
up to a concentration of 500 µM (data not shown), it may
inhibit the assembly or activity of a multicomponent NADPH oxidase
system in differentiated HL-60 cells (Fig. 5A)
. This
inhibitory mode, i.e., not radical scavenging but generation
inhibition, is also found in NO generation. A chemical characteristic
of nobiletin, which bears none of the hydroxyl groups that are
indispensable for O2-
scavenging activity, explains such an inhibitory mode.
Possessing no hydroxyl groups may occasionally give some advantages to
nobiletin in terms of antioxidation because, although being reported to
exhibit antioxidative activities in rat liver microsomes, some
polyhydroxyflavonoids change to become pro-oxidants to generate
H2O2 (51)
.
Nobiletin possesses a much higher ability to inhibit
O2- generation than does
luteolin (Fig. 5A)
. A predominance of polymethoxyflavonoids
over polyhydoxyflavonoids was also observed in antitumor activities
using a cell culture system (42)
. Differences in their
activities may be derived from the relatively greater membrane uptake
efficiencies of polymethoxyflavonoids than those of
polyhydoxyflavonoids because methoxylation of the phenolic hydroxyl
groups, in general, increases the molecular hydrophorbicity that
regulate incorporation rates. In fact, intracellular content of
nobiletin was approximately two times higher than that of luteolin in
differentiated HL-60 cells when incubated at a concentration of 100
µM (Fig. 5B)
.
Double-TPA-application-induced
H2O2 production in
vivo was markedly inhibited by the pretreatment(s) of nobiletin
(Table 1)
. Decreased levels of
H2O2 may be attributable to
the inhibition of O2-
generation because H2O2 is
mostly derived from O2- as a
function of O2- dismutase or
nonenzymatically. Nobiletin, a dual inhibitor of both
O2- and NO radical generation,
can be recognized as a potent, naturally occurring anti-inflammatory
agent, because peroxynitrite, a coupling product of
O2- and NO, enhances COX-2
activity (52)
, involving inflammatory processes and
thereby leading to carcinogenesis. By contrast, resveratrol was a
selective inhibitor of NO generation (Fig. 2)
.
Certain ROS, such as H2O2,
and proinflammatory cytokines including TNF- are reported to
cause cell death, which is occasionally attributable to apoptosis
(53)
. Conversely, cell injury and cell death are manifest
in TPA-applied mouse skin. Compensatory growth is subsequently
necessary and is induced by growth factors such as transforming growth
factor and ß and keratinocyte growth factor, all of which are
inducible by TPA (54, 55, 56)
. Double TPA applications
promoted cell growth, as revealed by increased PCNA-labeling indices,
and resulted in epidermal hyperplasia and edema formation (Table 1
, and
Fig. 4
, A and B). Pretreatment with nobiletin
decreased cell growth rates (Table 1
, and Fig. 4
,
C–E), possibly because of the escaping of
epidermal cells from oxidative cellular death, which diminished the
growth factor production in surrounding cells.
As predicted by the suppressive efficacies of biochemical markers
relating to oxidative stress and inflammation (Figs. 2
3
4
5
6)
, topical
application of nobiletin at doses of 160 and 320 nmol inhibited the
multiplicity of skin tumors in a dose-dependent manner (Table 2)
. The
chemopreventive efficacy of nobiletin in the mouse skin model may be
superior to that of resveratrol, which inhibited tumor formation at the
dose range of 1–25 µmol although experimental conditions were
different (39)
. In any case, this is the first report
demonstrating the chemopreventive ability of nobiletin in an animal
model.
In conclusion, our approach, i.e., searching for iNOS induction inhibitors by activity-guiding separation, may be a promising strategy for discovering effective chemopreventive agents. Citrus flavonoid nobiletin was found to be a functionally novel antitumor promoter by working in both the priming and activation stages in mouse skin. It is also notable that it exclusively occurs in citrus fruits, is chemically stable, and is suitable for mass preparation for a variety of rodent studies. Nobiletin is currently being investigated by our collaborative research group for its chemopreventive efficacies in other inflammation-associated carcinogenesis models.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported by the Grant-in-Aid for the Japan
Society for the Promotion of Science (to Y. N.); the Program for
Promotion of Basic Research Activities for Innovative Biosciences (to
A. M., K. O., M. Y., H. T., H. N., K. K., H. O.); the 2nd Term
for a Comprehensive 10-year Strategy for Cancer Control from the
Ministry of Health and Welfare of Japan, Scientific Research (Nos.
10671782 and 11138255) from the Ministry of Education, Science, Sports
and Culture of Japan; and by Grant HS-52260 for a comprehensive
research project on health sciences focusing on drug innovation from
the Japan Health Sciences Foundation (to. T. T.). 
2 To whom requests for reprints should be
addressed, at Division of Applied Life Sciences, Graduate School of
Agriculture, Kyoto University, Kyoto 606-8502, Japan. Phone:
81-75-753-6281; Fax: 81-75-753-6284; E-mail: ohigashi{at}kais.kyoto-u.ac.jp
3 The abbreviations used are: TPA,
12-O-tetradecanoylphorbol-13-acetate; COX,
cyclooxygenase; DMBA, 7,12-dimethylbenz[a]anthracene;
HPLC, high-performance liquid chromatography; IL, interleukin; iNOS,
inducible NO synthase; IR, inhibitory rate; LPS, lipopolysaccharide;
O2-, superoxide; PCNA, proliferating cell
nuclear antigen; PG, prostaglandin; ROS, reactive oxygen species; TNF,
tumor necrosis factor; VEGF, vascular endothelial growth factor.
Received 1/18/00. Accepted 7/13/00.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
3 polyunsaturated fatty acids. Jpn. J. Cancer Res., 88: 234-237, 1997.[Medline]
-induced nitric oxide production through suppression of inducible nitric oxide synthase gene expression in RAW264 cells. Carcinogenesis (Lond.), 19: 1007-1012, 1998.B in macrophages by resveratrol. Br. J. Pharmacol., 126: 673-680, 1999.[Medline]
in mouse skin, pp. 161–177. In: R. M. McClain, T. J. Slaga, LeBoeuf, and H. Pitot (eds.), Growth Factors and Tumor Promotion: Implications for Risk Assessment. New York: Wiley-Liss, Inc., 1995.
, IL-1, and IL-6 by bovine monocytes and the murine macrophage cell line RAW 264.7. Microb. Pathog., 16: 401-411, 1994.[Medline]
during the process of skin tumor promotion. Mol. Carcinog., 4: 52-60, 1991.[Medline]
This article has been cited by other articles:
|
|
 |
|
  S. Rahman, R. A. Ansari, H. Rehman, S. Parvez, and S. Raisuddin Nordihydroguaiaretic Acid from Creosote Bush (Larrea tridentata) Mitigates 12-O-Tetradecanoylphorbol-13-Acetate-induced Inflammatory and Oxidative Stress Responses of Tumor Promotion Cascade in Mouse Skin Evid. Based Complement. Altern. Med., October 27, 2009; (2009) nep076v1. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Yasuda, T. Nishizawa, H. Ohigashi, T. Tanaka, D.-X. Hou, N. H. Colburn, and A. Murakami Linoleic acid metabolite suppresses skin inflammation and tumor promotion in mice: possible roles of programmed cell death 4 induction Carcinogenesis, July 1, 2009; 30(7): 1209 - 1216. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-S. Lai, S. Li, C.-Y. Chai, C.-Y. Lo, S. Dushenkov, C.-T. Ho, M.-H. Pan, and Y.-J. Wang Anti-inflammatory and antitumor promotional effects of a novel urinary metabolite, 3',4'-didemethylnobiletin, derived from nobiletin Carcinogenesis, December 1, 2008; 29(12): 2415 - 2424. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Onozuka, A. Nakajima, K. Matsuzaki, R.-W. Shin, K. Ogino, D. Saigusa, N. Tetsu, A. Yokosuka, Y. Sashida, Y. Mimaki, et al. Nobiletin, a Citrus Flavonoid, Improves Memory Impairment and A{beta} Pathology in a Transgenic Mouse Model of Alzheimer's Disease J. Pharmacol. Exp. Ther., September 1, 2008; 326(3): 739 - 744. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Miyamoto, Y. Yasui, T. Tanaka, H. Ohigashi, and A. Murakami Suppressive effects of nobiletin on hyperleptinemia and colitis-related colon carcinogenesis in male ICR mice Carcinogenesis, May 1, 2008; 29(5): 1057 - 1063. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-S. Lai, S. Li, C.-Y. Chai, C.-Y. Lo, C.-T. Ho, Y.-J. Wang, and M.-H. Pan Inhibitory effect of citrus 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone on 12-O-tetradecanoylphorbol 13-acetate-induced skin inflammation and tumor promotion in mice Carcinogenesis, December 1, 2007; 28(12): 2581 - 2588. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. P. Datla, V. Zbarsky, D. Rai, S. Parkar, N. Osakabe, O. I. Aruoma, and D. T. Dexter Short-Term Supplementation with Plant Extracts Rich in Flavonoids Protect Nigrostriatal Dopaminergic Neurons in a Rat Model of Parkinson's Disease J. Am. Coll. Nutr., August 1, 2007; 26(4): 341 - 349. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Y. Chung, J. H. Park, M. J. Kim, H. O. Kim, J. K. Hwang, S. K. Lee, and K. K. Park Xanthorrhizol inhibits 12-O-tetradecanoylphorbol-13-acetate-induced acute inflammation and two-stage mouse skin carcinogenesis by blocking the expression of ornithine decarboxylase, cyclooxygenase-2 and inducible nitric oxide synthase through mitogen-activated protein kinases and/or the nuclear factor-{kappa}B Carcinogenesis, June 1, 2007; 28(6): 1224 - 1231. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J. Roberts, B. Y. Ng, R. B. Filler, J. Lewis, E. J. Glusac, A. C. Hayday, R. E. Tigelaar, and M. Girardi Characterizing tumor-promoting T cells in chemically induced cutaneous carcinogenesis PNAS, April 17, 2007; 104(16): 6770 - 6775. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Murakami, T. Shigemori, and H. Ohigashi Zingiberaceous and Citrus Constituents, 1'-Acetoxychavicol Acetate, Zerumbone, Auraptene, and Nobiletin, Suppress Lipopolysaccharide-Induced Cyclooxygenase-2 Expression in RAW264.7 Murine Macrophages through Different Modes of Action J. Nutr., December 1, 2005; 135(12): 2987S - 2992S. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. M. Sung, G. He, and S. M. Fischer Lack of Expression of the EP2 but not EP3 Receptor for Prostaglandin E2 Results in Suppression of Skin Tumor Development Cancer Res., October 15, 2005; 65(20): 9304 - 9311. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Osakabe, A. Yasuda, M. Natsume, and T. Yoshikawa Rosmarinic acid inhibits epidermal inflammatory responses: anticarcinogenic effect of Perilla frutescens extract in the murine two-stage skin model Carcinogenesis, April 1, 2004; 25(4): 549 - 557. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Naasani, F. Oh-hashi, T. Oh-hara, W. Y. Feng, J. Johnston, K. Chan, and T. Tsuruo Blocking Telomerase by Dietary Polyphenols Is a Major Mechanism for Limiting the Growth of Human Cancer Cells in Vitro and in Vivo Cancer Res., February 15, 2003; 63(4): 824 - 830. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. W. Kim, A. Murakami, M. V. Williams, and H. Ohigashi Mutagenicity of reactive oxygen and nitrogen species as detected by co-culture of activated inflammatory leukocytes and AS52 cells Carcinogenesis, February 1, 2003; 24(2): 235 - 241. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M.-H. Pan, W.-J. Chen, S.-Y. Lin-Shiau, C.-T. Ho, and J.-K. Lin Tangeretin induces cell-cycle G1 arrest through inhibiting cyclin-dependent kinases 2 and 4 activities as well as elevating Cdk inhibitors p21 and p27 in human colorectal carcinoma cells Carcinogenesis, October 1, 2002; 23(10): 1677 - 1684. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Sato, L. Koike, Y. Miyata, M. Hirata, Y. Mimaki, Y. Sashida, M. Yano, and A. Ito Inhibition of Activator Protein-1 Binding Activity and Phosphatidylinositol 3-Kinase Pathway by Nobiletin, a Polymethoxy Flavonoid, Results in Augmentation of Tissue Inhibitor of Metalloproteinases-1 Production and Suppression of Production of Matrix Metalloproteinases-1 and -9 in Human Fibrosarcoma HT-1080 Cells Cancer Res., February 1, 2002; 62(4): 1025 - 1029. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Nakamura, Q. Feng, T. Kumagai, K. Torikai, H. Ohigashi, T. Osawa, N. Noguchi, E. Niki, and K. Uchida Ebselen, a Glutathione Peroxidase Mimetic Seleno-organic Compound, as a Multifunctional Antioxidant. IMPLICATION FOR INFLAMMATION-ASSOCIATED CARCINOGENESIS J. Biol. Chem., January 18, 2002; 277(4): 2687 - 2694. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
, luteolin. *1,
P < 0.001; *2,
P < 0.01 versus the group
treated only with TPA in Student’s t test. In
B, nobiletin (
) or luteolin (