ICANCERRESEARCH56. 1023-1030. MarchI.

19961

Inhibition of Tumor Promotion in SENCAR Mouse Skin by Ethanol Extract of

Zingiber officinale Rhizome1
Santosh K. Katiyar, Rajesh Agarwal, and Hasan MUkhtar@
Depara@mentof Dermatology, Skin Diseases Research Center, University Hospitals of Cleveland, Case Western Reserve University, Cleveland. Ohio 44106

ABSTRACT identify anti-tumor-promoting agents present in diets consumed by the

human population.
Thereis considerableemphasison Identifyingpotentialchemopreven Ginger rhizome (Zingiber officinale), commonly known as ginger,
five agents present in food consumed by the human population. Ginger
is consumed worldwide as a spice and a flavoring agent. Limited in
rhizome (Zingiber officinale), known commonly as ginger, Is consumed
vitro studies have shown that water and organic solvent extracts of
worldwide in cookeries as a spice and a flavoring agent. In prior in vitro
studies, it has been shown that the water or organic solvent extract of ginger as well as some constituents isolated from ginger possess
ginger possesses antioxidative and antlinflammatory properties. In this antioxidant properties (6—10). Zingerone, a compound isolated from
study, we evaluated whether ethanol ginger, has been shown to inhibit nitro blue tetrazolium reduction in
extract of ginger (GE) possesses
anti-tumor.promoting effects In a mouse skin tumorigenesis model. Be. a xanthine-xanthine oxidase system, providing the evidence that it
cause skin tumor promoters induced epidermal ornithine decarboxylase scavenges superoxide anions (7). Reddy and Lokesh (8), using liver
(ODC), cyclooxygenase, and lipoxygenase activities, and edema and hy microsomes, reported that zingerone also inhibits lipid peroxidation,
perplasia are conventionally used markers of skin tumor promotion, first, suggesting its antioxidant properties. Similar results were also re
we assessed the effect of GE on these parameters. Preapplication of GE ported by Jitoe et a!. (9) using different ginger extracts. Oral con
onto the skin of SENCAR mice resulted in significant inhibition of 12-0- sumption of dried, powdered ginger for 3 months—2.5years by pa
tetradecanoylphorbol-13-acetate (TPA)-caused induction of epidermal tients with rheumatoid arthritis, osteoarthritis, or muscular discomfort
ODC, cyclooxygenase, and lipoxygenase activities and ODC mRNA ex has been shown to result in relief of pain and swelling (10, 11). The
pression In a dose-dependent manner. Preapplication of GE to mouse skin
investigators of these studies suggested that the mechanism of ame
also afforded significant inhibition of TPA-caused epidermal edema
(56%) and hyperplasia (44%). In long-term tumor studies, topical appli
liorative effects of ginger could be related to the inhibition of PG@and
cation of GE 30 mm prior to that of each TPA application to 7,12- leukotriene biosynthesis (10, 11). In other studies, ginger oil, obtained
dimethylbenz(a)anthracene-initiated SENCAR mice resulted in a highly by steam distillation of dried ginger, has been shown to be an inhibitor
significant protection against skin tumor incidence and Its subsequent of both cyclooxygenase and lipoxygenase activities (12, 13). Re
multiplicity. The animals pretreated with GE showed substantially lower cently, Sharma et a!. (14) reported that ginger oil (33 mg/kg), given
tumor body burdens compared with non-GE-treated controls. The results orally for 26 days to male Sprague-Dawley rats, caused a significant
of our study,for the first time,provideclearevidencethat GE possesses suppression of both paw and joint swelling. This finding suggested
anti-skin tumor-promoting effects, and that the mechanism ofsuch effects that ginger oil possesses antiinflammatory properties.
may involve inhibition of tumor promoter-caused cellular, biochemical, In view of the antiinflammatory and antioxidative activities of
and molecular changes in mouse skin. ginger extract, as well as its inhibitory potential against cyclooxyge
nase and lipoxygenase activities (6—13),we considered that ginger
extract may possess significant anti-tumor-promoting potential. In this
INTRODUCTION study, we assessed the anti-tumor-promoting effect of ginger extract
To reduce the occurrence of cancer, one promising approach is its on SENCAR mouse skin and delineated the mechanism of such an
prevention, specifically by chemical intervention through minor non effect.
nutrient dietary constituents. Chemoprevention, therefore, is the
means of cancer control in which the occurrence of this disease, as a MATERIALS AND METHODS
consequence of exposure to carcinogenic agents, can be slowed,
blocked, or reversed by the administration of one or more naturally Chemicals
occurring and synthetic compounds (1—4and references therein). TPA, mezerein, (—)-indolactam V, n-dodecane, anthralin, formamide, SSC
Chemoprevention also deals with the chemotherapy of precancerous [1 X SSC = 0.15 M NaCI, 0.015 M Na citrate (pH 7.0)1, and D,L-ornithine were
lesions, which are called preinvasive neoplasia, dysplasia, or intraepi purchased from Sigma Chemical Co. (St. Louis, MO). DMBA and benzoyl
thelial neoplasia, depending on the organ system (2). Chemopreven peroxide were purchased from Aldrich Chemical Co. (Milwaukee, WI).
tive agents can be targeted for intervention at either the initiation, Arachidonic acid, 5-HETh, 8-HETh, 12-HETE, 15-HETh, PGE2, POD2, and
PGF20 were purchased from Biomol Research Laboratories, Inc. (Plymouth,
promotion, or progression stage of multistage carcinogenesis (1—4
PA). [l-'4C]arachidonic acid (52 mCi/mmol) was purchased from DuPont
and references therein). The intervention of cancer at the promotion
New England Nuclear (Boston, MA). D,L-[l-['4Cjornithine (58 Ci/mmol) was
stage, however, seems to be most appropriate and practical. The major purchased from Amersham Searle (Chicago, IL). [a-32P]dCTP (6000 CL'
reason for that relates to the fact that tumor promotion is a reversible mmol) and a GeneScreen nitrocellulose membrane were purchased from
event at least in early stages and requires repeated and prolonged DuPont-New England Nuclear. cDNA of G3PDH was obtained from Strat
exposure of a promoting agent (5). For this reason, it is important to agene (La Jolla, CA). Guanidine thiocyanate was a product of Fluka
(Ronkonkoma, NY), and agarose was obtained from Fisher Scientific (Spring

Received 9/20/95; accepted 1/2/96. field, NJ). The chemicals used for RNA isolation and Northern blot analysis
The costs of publication of this article were defrayed in part by the payment of page were of molecular biology grade and RNase free. The mouse ODC cDNA
charges. This article must therefore be hereby marked advertisement in accordance with probe (15) was a generous gift from Ajit K. Verma (University of Wisconsin
18 U.S.C. Section 1734 solely to indicate this fact.
I This work was supported by USPHS Grant ES-l900, American Institute for Cancer

Research Grant 92B35, and Skin Diseases Research Center Core Grant P-30-AR-39750. 3 The abbreviations used are: PG. prostaglandin; DMBA, 7,12-dimethylbenz(a)anthra

2 To whom requests for reprints should be addressed, at Department of Dermatology, cene; TPA. l2-O-tetradecanoylphorbol-13-acetate; GE, ethanol extract of ginger root;
University Hospitals of Cleveland, Case Western Reserve University. 2074 Abingron ODC, ornithine decarboxylase; G3PDH, glyceraldehyde-3-phosphate dehydrogenase;
Road, Cleveland, OH 44106. Phone: (216) 368-1 127; Fax: (216) 368-0212. HETh, hydroxyeicosatetraenoic acid.

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 INHIBITION OF SKIN TUMORIGENESIS BY GINGER

Comprehensive Cancer Center, Madison, WI). All other chemicals were ob radioactivity was determined in each fraction, and the radioactive profile was
mined in the purest forms commercially available. Fresh ginger was purchased compared with the 236-nm UV absorption profile of authentic HE'FEs sub
from a local vegetable market. jected to high-pressure liquid chromatography under the identical conditions.

Aalmals Edema and Hyperplasia

Six-to-i-week-old female SENCAR mice were obtained from Harlan-Spra To assess the inhibitory effect of preapplication of GE on TPA-induced
gue-Dawley (Indianapolis, IN). These mice were housed four or five per cage edema, 1-cm-diameter punches of skin from vehicle-, TPA- or GE- and
and were acclimatized for 1 week before use, subjected to a 12-h light/12-h TPA-treated animals were removed, made free of fat pads, and weighed
dark cycle, and housed at 24 ±2°Cand 50 ±10% relative humidity. Animals quickly. After drying for 24 h at 50°C, the skin punches were reweighed, and
were fed a Purina chow diet and water ad libitum. the loss of water content was determined. The difference in the amount of
water gain between the control vehicle and TPA represented the extent of
Isolation of GE edema induced by TPA, whereas that between the control vehicle and GE plus
TPA represented the inhibitory effect of GE. For the hyperplasia study, skin
Fresh ginger was cut into small pieces, crashed, maccrated with ethanol, and was removed, fixed in 10% formalin, and embedded in paraffin. Vertical
filtered through muslin cloth. The filtrate thus obtained was centrifugedat 2500 sections (5 @m)were cut, mounted on a glass slide, and stained with hema
rpm for 15 ruin at 4°C,and the supematant was collected. This extraction toxylin and eosin. For each section of the skin, the thickness of the epidermis
procedure was repeated three times, and all the supematants were pooled and from the basal layer to the stratum comeum was measured at five equidistant
evaporated to dryness in a rotary vacuum evaporator. A viscous, oily mass thus interfollicular sites using an Olympus (Palo Alto, CA) light microscope
obtainedwas designatedGE. This extractwas dissolvedin methanol:acetone(1:1, equipped with an ocular micrometer.
v/v) and used in all the experiments.The total extractableethanol solublefraction
was 3.6% offresh gmger(w/w). It is useful to mention here that other investigators Isolation of Total RNA and Northern Blot Analysis
(12, 13)used ginger oil that was extractedby steam distillation,and this oil was a
mixture of camphen, limonen,linalool,citral, and bomel compounds. The treated area of the dorsal skin was removed. The epidermis was
separated and homogenized using a tissue homogenizer (Tissuemizer; Tekmar,
Treatment of Animals for Short-Term, in Vivo Studies Cincinnati, OH) in 4 Mguanidine thiocyanate lysis buffer, and total RNA was
extracted by the method of Chomczynski and Sacchi (22) with some modifi
The animals were shaved on the dorsal side of the skin, divided into three cation as described earlier (23). The concentration of total RNA was deter
groups of eight each, and treated topically on the shaved area with either 0.2 mined by measuring the absorbance at 260 ma in a Beckman DU 640
ml vehicle (methanol:acetone, 1:1, vlv), TPA (5 pg/animal), or GE (2 or 4 spectrophotometer. Total RNA (20 @xg)was electrophoresed through a 1.2%
mg/animal), followed 30 mm later with TPA (5 gig/animal). All the test agarose gel containing 6% formaldehyde and transferred onto a mtrocellulose
compounds were applied in 0.2 ml vehicle. Animals were sacrificed either 6 h membrane. The membrane was UV cross-linked (1200 millijoules/cm2), vac
after the TPA treatment (for ODC activity and ODC mRNA expression) or uum dried at 80°Cfor 2 h, prehybridized at 42°Cfor 2—3
h, and hybridized to
after 24 h (for cyclooxygenase and lipoxygenase activities), and the treated the 32P-labeledODC cDNA probe pOD48 (specific activity, 1.5 X 10@cpm/
area of the skin was used for the determination of enzyme activities. The ml) overnight at 42°C.The ODC cDNA, a fragment of about 2.1 kb, was
epidermis was separated from the whole skin and homogenized in 0.1 M labeled with [a-32P]dCTP using a random primer DNA-labeling kit (GIBCO
Tris-HC1 buffer (pH 7.2) using a Polytron tissue homogenizer (Brinkmann BRL, Gaithersburg, MD). After hybridization, the nitrocellulose membrane
instruments, Westbury, NY) and 100,000 X g supernatant, and microsomal was washed twice for 15 mm each time in low-stringency buffer (2X SSC and
fractions were prepared as described earlier (16). 1.0% SDS) at room temperature and once in high-stringency buffer (1X SSC
and 0.5% SDS) at 45—50°C. The membrane was autoradiographed using
Assays of Enzyme Activities KOdakXAR-5 film with intensifying screens at —70°C. After stripping, the
same membrane was rehybridized to 32P-labeled G3PDH cDNA to verify
ODC Activity. ODC activity was determined using 0.4 ml epidermal
equal loading of RNA onto the gel. The autoradiographs were scanned with a
100,000 x g supernatant fraction/assay tube by measuring the release of
densitometer (Microtek International Inc., Taiwan, Republic of China), and the
14C02 from the D,L-['4C]ornithine by the method of O'Brien et a!. (17) as
results were integrated and normalized to the value for G3PDH.
described by Verma et a!. (18). The details of the assay procedure were
described earlier (19). Enzyme activity is expressed as pmol CO2 released/ Skin Tumorigenesis
h/mg protein. Protein concentration was determined by the method of
Bradford (20). Female SENCAR mice were used in DMBA- and TPA-induced, two-stage
Cyclooxygenase Activity. Epidermal microsomal cyclooxygenase activity skin tumorigenesis protocol (21). The dorsal side of the skin was shaved using
was determined by the method described earlier (21). In brief, 150 @.d
reaction electric clippers, and the mice with hair cycles in the resting phase were used
mixture contained 12 @M [‘4C]arachidonic acid (—400,000dpm), 1 mM for tumor studies. In each group, 20 animals were used. Tumorigenesis was
epinephrine, 1 IBM glutathione and —10 @igepidermal microsomal protein in initiated in the animals by a single topical application of 12.5 nmol DMBA in
50 mMpotassium phosphate buffer (pH 7.4). After incubation at 37°Cfor 15 0.2 ml vehicle on the dorsal shaved skin, and 1 week later, the tumor growth
rain, the reaction was terminated by the addition of 50 @.d0.2 M HC1. was promoted with twice-weekly applications of 4 nmol TPA in 0.2 ml
The radioactive prostaglandin metabolites (PGE@,PGF2a, and POD2) of vehicle. To assess the anti-skin tumor-promoting effect of GE, various doses of
[14C]arachidonicacid were detected by TLC as described earlier (21). The GE (1, 2, or 4 mg/animal), which produced significant inhibition against
radioactivity profile of each metabolite was determined by counting the sam TPA-caused induction of ODC, cyclooxygenase, and lipoxygenase activities,
ples in a Beckman LS-6K SC liquid scintillation counter (Beckman Instru were applied topically 30 mm prior to each TPA application in different
ments, Fullerton, CA) equipped with automatic external standardization. groups. Treatment with TPA alone or various doses of GE plus TPA was
Lipoxygenase Activity. Epidermal cytosolic lipoxygenase activity was repeated twice weekly up to the termination of the experiments at 20 weeks.
determined by the method described earlier (21). In brief, 150 @dreaction One group of animals was treated with 0.2 ml vehicle alone and served as a
mixture contained [‘4C]arachidonic acid (5 nmol, —460,000 dpm), 2 mM negative control to assess spontaneous tumor induction. To test whether GE
calcium chloride, and —400 @&g
epidermal 100,000 X g cytosolic protein in itself possesses tumor-promoting effects, a group of animals was initiated with
100 mM Tris-HC1 (pH 7.2). After incubation for 30 mm at 37°C,the reaction the 12.5-nmol dose of DMBA and 1 week later was treated with GE (4
was terminated by the addition of 10 ,.d 0.2 MHC1.The HETE metabolites of mg/animal) twice weekly up to the end of the experiment. Animals in all the
arachidonic acid were extracted as described elsewhere (21) and subjected to groups were watched for any apparent signs of toxicity, such as weight loss or
normal-phase, high-pressure liquid chromatography using a 3.9 x 300-mm mortality, during the entire period of study. Skin tumor formation was recorded
,.L-PORASILcolumn (Waters Associates, Milford, MA) at a flow rate of 0.7 weekly, and tumors larger than 1 mm in diameter were included in the
ml/min. The column eluate was monitored at 236 nm using a Shimadzu cumulative number if they persisted for 2 weeks or more. The tumors were
(Columbia, MD) variable length detector; and 0.1-mi fractions were collected, diagnosed histologically at the termination of the experiment.
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 INHififflON OF SKIN TIJMORIGENESIS BY GINGER

Statistical Analysis @ei@

Student's t test was used in enzymatic studies. In tumorigenesis experi

E-@ E-
ments, the statistical significance of difference in terms of tumor incidence and
+.@ +@ei@+E
multiplicity between the TPA and GE-plus-TPA groups was evaluated by the
Wilcoxon rank sum and x@tests. An advantage of the Wilcoxon rank sum test
is that its validity does not depend on any assumption about the shape of the
distribution of tumor multiplicities, whereas the Cochran-Mantel-Haenzel sta
@‘@x1
C,E-—c-Si
tistical test was used to evaluate the statistical significance of the decreasing
number of tumors with increasing doses of GE.
@ ODC .@* .@ . — .• , . 2.1kb
RESULTS

Inhibitory Effect of GE on TPA-caused Induction of Epidermal

ODC Activity. To determinethe optimaleffective dose ofGE against G3PDH 1.4 kb
the TPA-caused induction of epidermal ODC activity in SENCAR
mice, groups of animals were treated topically with varying doses of
GE (1, 2, or 4 mg GElanimal) 30 mm prior to topical application of
TPA (2.5 pg/animal). As shown in Fig. 1A, pretreatment of animals
100

A
50

Ca 0
Ca Fig. 2. Inhibitory effect of GE on TPA-induced epidermal ODC mRNA expression in
E SENCAR mice. Six animals in each group were treated topically with vehicle or I, 2, or
ca GE(1 mg}+TPA 4 mg GE 30 mm prior to that of 5.0 @g TPA, as described in Fig. IA, and animals were
Ca killed 6 h after the TPA treatment. In each case, three epidermis samples were pooled, and
5-. the total RNA was isolated. Fractionation of RNA by agarose gel electrophoresis,
I- Northem blotting, and hybridization to the 32P-labeledODC cDNA probe are described
GE(2mg)+TPA
in “Materialsand Methods.―

GE(4 mg)+TPA

0 300 600 900 1200 1500 18 0 1 with GE resulted in a dose-dependent inhibition of the TPA-caused
ODC Activity (pmol/h/mg protein) induction of epidermal ODC activity. At the maximum dose of GE (4
mg) used in this study, 67% inhibition (P < 0.0005) was observed
@uuu@ (Fig. lA ). Similarly, at lower doses, GE also resulted in a significant
B@Ca inhibition (46—55%;P < 0.005). Topical application of GE alone of
0 [ —.-.TPA
5-.
0.
up to 4 mg/animal was without any effect on basal enzyme activity
@ 1500 S GE+TPA
Dl and did not cause any induction of epidermal ODC activity.
The effect of preapplication of GE on TPA-caused induction of
epidermal ODC activity was also studied as a function of time after
<U) 1000 the TPA application. Consistent with published studies (23—25),the
maximum induction of epidermal ODC activity after the single topical
application of TPA was observed at 6 h, which started declining after
0 500
that period and reached an almost basal level by 16 h. When GE (4
0
0 mg/animal) was applied 30 mm prior to each topical application of
E TPA, significant inhibition was observed at all the time points studied,
0.
0 1 2 4 6 8 10 12 18 24 with a similar pattern as observed with TPA alone (Fig. 1B).
Time (hrs) Inhibition of TPA-caused Induction of Epidermal ODC mRNA
Expression by GE. In the next series of experiments, we assessed the
Fig. 1.A, inhibitory effect of GE on TPA-caused induction of epidermal ODC activity
in SENCAR mice. Eight animals in each group were treated topically with either 0.2 ml effect of skin application of GE on TPA-caused, enhanced expression
vehiclealoneor indicateddosesof GE (1, 2, or 4 mg/animal)in 0.2 ml vehicle30 mm of ODC mRNA in the epidermis. Northern blot analysis revealed that
prior to that of 2.5 @&g
TPA in 0.2 mi vehicle. Animals were killed by cervical dislocation
6 h aftertheTPAtreatment,andODCactivitywasdeterminedasdescribedin “Materialstopical application of TPA (5 @g)resulted in a marked increase in the
and Methods.―Data represent the mean ±SEM of four values; epidermis from two concentration of epidermal ODC mRNA. Skin application of GE prior
animals was pooled for each determination. P < 0.005-0.0005 in the case of TPA alone to that of TPA resulted in a significant inhibition against TPA-caused
versus 1, 2, or 4 mg GE plus TPA. B, time-dependent inhibitory effect of GE on
TPA-induced epidermal ODC activity in SENCAR mice. Eight animals in each group induction of epidermal ODC mRNA expression in a dose-dependent
were treated topically with vehicle or 4 mg GE 30 mm prior to that of 5.0 @gTPA, as manner (Fig. 2, top panel). Densitometric scanning of these blots
described in “Materials and Methods,―and ODC activity was determined. Positive
controls included animals treated topically with vehicle and 30 mm later with the same indicated that, under the experimental conditions used, the inhibition
dose ofTPA alone. Each data point represents the mean ±SEM of four values; epidermis varied from 40—70% in GE-pretreated animals (Fig. 2, bottom panel).
from two animals was pooled for each determination. SEM values, not evident in some
cases, were smaller than the symbols. P < 0.005 in the case of TPA alone versus GE plus Fig. 2, middle panel, shows the equal loading of RNA samples in each
TPA at the 2—12-h time points. lane.
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 INHIBITION OF SKIN TUMORIGENESIS BY GINGER

activitiesCyclooxygenase Table I Inhibitory effect of topically applied GE on TPA-induced epidermal cyclooxygenase and lipoxygenase
activity was determined in epidermal microsomes using the TLC procedure, whereas lipoxygenase activity was determined in epidennal cytosols using the
normal-phase, high-pressure liquid chromatography procedure as described in “Materials
and Methods.―Values in parentheses represent
GE.Lipoxygenase
percentage protection by

activity (pmol HETE

protein)Treatmentsa Cyclooxygenase activity (pmol PG metabolites/l5 mn/mg protein) metabolitesl30 mn/mg

POE2 PGF2@ PGD2 84IETE 541ETE

Vehicle 972 ±31b 619 ±23 537 ±20 126 ±17 129 ±19
GE 960±26 596±23 561±17 121±18 133±22
TPA 2665±52 1293±41 839±21 547±33 271±21
GE (2 mg) + TPA 1875 ±19―
(47) 995 ±17―
(42) 685 ±I5d (5 1) 387 ±23c (38) 172 ±I 3C(69)
GE (4 mg) + TPA 1310 ±l9―(80) 800 ±15d(72) f,44@
±11d(@) 244 ±17d(72) 140 ±13―(92)
@ a were treated once with 0.2 ml vehicle, 4 mg GE in 0.2 ml vehicle, 5 @gTPA in 0.2 ml vehicle, or 2 or 4 mg GE in 0.2 ml vehicle followed 30 mm later by 5 @gTPA
in 0.2 ml vehicle. Twenty-four h after the TPA treatment, animals were killed, and skin was used for enzymatic studies as described in “Materials
and Methods.―
M@ ±SE of four individual values;the epidermisfrom two animals was pooled for each determination.
C Highiy significant versus TPA alone; P < 0.005.

d Highly significant versus TPA alone; P < 0.0005.

Inhibitory Effects of GE on TPA-induced Epidermal Cyclooxy skin affords protection against TPA-caused edema and hyperplasia.
genase and Lipoxygenase Activities. The effect of preapplication of As determined by the weight of the 1-cm-diameter punch of dorsal
GE on TPA-caused inductionof epidermalcyclooxygenase activity is skin, the application of TPA to SENCAR mouse skin showed a
shown in Table 1. As quantitated by the formation of PGE@, PGF2a, significant edema (Table 2). The skin application of GE, 30 mm prior
and POD2, the application of TPA to the SENCAR mouse skin to that of TPA, showed 56% inhibition (P < 0.0005) against TPA
resulted in a significant induction in epidermal cyclooxygenase activ caused edema.
ity. The application of GE prior to that ofTPA resulted in a significant The effect of preapplication of GE on TPA-caused induction of
inhibition of TPA-induced epidermal cyclooxygenase activity, as cv epidermal hyperplasia is shown in Fig. 3. To determine the induction
ident by the quantitative analysis of PG metabolite formation. Com in mean epidermal thickness by TPA and inhibition by GE, epidermal
pared with TPA application alone, preapplication of 2 mg GE to thickness was measured at five equidistant points between interfol
mouse skin resulted in 47, 42, and 51% inhibition (P < 0.0005) of licular spaces along the length of the epidermis from the dermoepi
PGE2, PGF2a, and PGD2 formation, respectively. Preapplication of 4 dermal junction to the top of the stratum corneum, and all five values
mg GE to that of TPA was found to result in 80, 72, and 66% were averaged and reported as the mean epidermal thickness in tam.
(P < 0.0005) inhibition of PGE2, PGF2a, and PGD2 formation, Similarly, cell layers were also counted from the dermoepidermal
respectively (Table 1). junction to the bottom of the stratum corneum to determine the mean
The effect of preapplication of GE on TPA-caused induction of vertical thickness in terms of cell layers in the epidermis. As shown in
epidermal lipoxygenase activity is shown in Table 1. As quantitated Fig. 3, application of TPA (5 j.@g),followed 24 h later by the killing
by the formation of 8-HETh and 5-HETh, the application of TPA to of the animals (Fig. 3, middle panel), resulted in a significant increase
the SENCAR mouse skin resulted in significant inductions in 8- and in mean epidermal thickness (72.1 ±5.1 tam) and mean vertical
5-lipoxygenase activity in the epidermis. The application of 2 or 4 mg thickness in terms of epidermal cell layers (5.9 ±0.2) when compared
GE priorto that of TPA was found to result in significant inhibition with the skin of only vehicle-treated animals (15.5 ± 0.8 and
of TPA-induced epidermal lipoxygenase activity, as evident by the 1.8 ±0.2 @m,respectively; Fig. 3, top panel). However, the preap
quantitative analysis of different HETh metabolite formations from plication of GE to that of TPA resulted in 41% (P < 0.005) inhibition
arachidonic acid. Compared with TPA alone, prior application of 2 or in the induction of epidermal thickness (48.8 ±3.2 @m)and 44%
4 mg GE showed 38—72%(P < 0.005) or 69—92%(P < 0.0005) (P < 0.005) inhibition in terms of vertical cell layers (4.1 ±0.2 sm),
inhibition in 8-HETh and 5-HETh formation, respectively, as shown
as shown in Fig. 3, bottom panel. The application ofTPA also resulted
in Table 1. The application of GE alone at the dose of 4 mg did not in mixed cell infiltration in the dermis, which comprised mostly
produce any change in epidermal cyclooxygenase and lipoxygenase
neutrophils with some mononuclear cells admixed (Fig. 3, middle
activities when compared with only vehicle-treated control animals
panel); this effect of TPA in the dermis was also inhibited by the
(Table 1).
preapplication of GE (Fig. 3, bottom panel). The application of GE
Inhibitory Effects of GE on TPA-caused Epidermal Edema and
alone, however, did not induce epidermal edema or hyperplasia in
Hyperplasia. Because the dose of 4 mg GEfanimal applied topically
these experiments, because the mean epidermal thickness (16 tim) and
afforded optimum and significant inhibition against TPA-caused in
mean vertical thickness in terms of epidermal cell layers (2.1 p@m)
duction of ODC, cyclooxygenase, and lipoxygenase activities, this
were comparable with those observed in vehicle-treated animals (data
dose of GE was selected to determine whether its application on the
not shown).
Additionally, microscopic examination of the sections of skin bi
Table
edemaEdema
2 Inhibitory effect of topically applied GE on TPA-induced cutaneous opsies also revealed that interstitial cell spaces were larger, and cells
circlepunchresponse of the skin was determined by weighing the 1-cm-diameter were spongiotic in nature in the TPA-treated group of animals com
Methods.―Treatments@'
of treated skin, as described in “Materialsand
pared with the vehicle- and GE-plus-TPA-treated groups of animals.
inhibitionVehicle Skin punch weight (mg) % This shows that preapplication of GE to that of TPA inhibited TPA
0.5―GE 22. 1 ± caused infusion of edema in animal skin. It is also noteworthy to
0.4TPA 22.2±
29.1±0.6GE+TPA
mention that, in the microscopic observations of the skin sections, the
56a 25.2±0.4c number of cells in the mitotic phase was 10 when counted in each of
onlyone
The details of the treatments are the same as described in Table 1, except that three skin samples of 1-cm length, whereas under similar conditions,
dose of GE (4 mg/animal) was used in this experiment. only 2 and 5 cells in this phase were observed in the vehicle- and
fromeach
b Mean ±SE of four individual values, and four skin punch biopsies were taken
animals.C
of the four individual GE-plus-TPA-treated groups, respectively. This observation demon
Significant versus TPA alone; P < 0.0005. strates further that skin application of ginger 30 mm prior to TPA
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 INHIBITION OF sKIN TUMORIGENESIS BY GINGER

application inhibits TPA-induced stimulation of cell proliferation

through mitosis.
Anti-Skin Tumor-promoting Effects of GE. As shown by data in
Fig. 4, topical application of GE prior to that of TPA in DMBA
initiated SENCAR mouse skin resulted in a dose-dependent inhi
bition of skin tumongenesis. This inhibition was evident when
tumor data were considered as the percentage of mice with tumors
(Fig. 4, top panel) and the number of tumors per mouse (Fig. 4,
bottom panel). At the termination of the experiment at 20 weeks,
compared with 100% animals with skin tumors in non-GE-treated
group, 90, 65, and 50% of the animals exhibited the appearance of
@ ‘4 4/ skin tumors in the 1, 2, and 4 mg GE-treated groups of animals,
respectively. The tumor incidence data at the dose of 1 mg GE
lJ' p., were not significantly different from those of the non-GE-treated
control group (f test). However, at the 2- and 4-mg doses of GE
individually, they were significantly different compared with those

‘i of the non-GE-treated group (P < 0.05 or 0.005; x@test). At the

termination of the experiment, compared with a total of 330 tumors
in the non-GE-treated group of animals, 195, 92, and 74 tumors per
group in the 1, 2, and 4 mg GE-treated groups, respectively, were
recorded. Compared with the non-GE-treated group, such de
creases in the total numbers of tumors in the GE-treated groups
corresponded to 40, 72, and 77% inhibition, respectively. In addi
tion, when tumor multiplicity data were evaluated using Cochran
Mantel-Haenzel statistical test, a linear trend of decrease in tumor
incidence was evident with increasing doses of GE (P < 0.01).
When these tumor data were considered in terms of tumor volume
per mouse, at the termination of the experiment at 20 weeks,
preapplication of 1, 2, and 4 mg GE resulted in 54, 85, and 91%
inhibition, respectively (P < 0.03—0.0001) in the sizes of devel
oping papillomas (Table 3).
,d,

‘4 DISCUSSION

. Cancer chemoprevention has become an important area of cancer

9 research, which, in addition to providing a practical approach to
identifying potentially useful inhibitors of cancer development, also
affords opportunities to study the mechanisms of carcinogenesis (1—
4). Limited preliminary studies (6—13)provide evidence that water or
organic solvent extracts of ginger possess antioxidant and antiinflam
matory properties. This study was designed to provide a substantial
amount of mechanistic data to show the chemopreventive potential of
GE by using carcinogenesis-associatedbiochemical end points in a
mouse skin tumorigenesis model.
ODC is the rate-limiting enzyme in the biosynthesis of poly
amines, which appears to be a prerequisite for cell proliferation,
differentiation, and neoplastic transformation. The induction of
ODC has been suggested to play a significant role in tumor
promotion. Initial studies with the mouse skin model (25, 26) and
subsequently with other organ systems (27, 28) showed an excel
lent correlation between the induction of ODC activity and the
tumor-promoting ability of a variety of substances. Because tumor
formation can be prevented by the agents that block induction of
ODC or by inhibitors of arachidonic acid metabolism, including
indomethacin (29, 18), ODC inhibition was shown to be a prom
Fig. 3. Inhibitory effect of skin application of GE against TPA-caused epidermal
hyperplasia in SENCAR mouse. The single dose of TPA and GE (4 mg) plus TPA were
ising tool for screening inhibitors of tumongenesis (29, 18). In the
applied topically on the shaved dorsal skin of SENCAR mice. The animals were killed by present study, topical application of GE prior to that of TPA
cervical dislocation 24 h after TPA application, and the skin was excised. Skin biopsies resulted in a significant inhibition of TPA-induced epidermal ODC
wereexaminedhistologicallyasdescribedin“Materials
andMethods.―
Toppanel,vehicle
alone; middle panel, TPA alone in vehicle; bottom panel, GE applied topically 30 mm activity (Fig. 1). It is reasonable to believe that GE application
prior to the application of TPA in vehicle. Sections were photographed by using a Zeiss inhibited the action of the tumor promoter and/or the enzymatic
(Oberkochen,Germany)Axioskopmicroscopeand a Sony(Tokyo,Japan)colorvideo pathway(s) that regulates the ODC induction rather than interact
printer. Original magnification, X 440.
ing directly with the enzyme. In addition, our data obtained from
Northern blot analysis demonstrate that prior application of GE to
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Research.
 INHIBITION OF SKIN TUMORIGENESIS BY GINGER

Further research is needed to evaluate a possible effect of GE on

—U-. DMBA+TPA the translation of ODC mRNA. Furthermore, the inhibition of
—.— DMBA + (1 mg GE + TPA)
TPA-induced epidermal ODC activity by GE at each time point
studied suggests that GE does not delay the peak of TPA-induced
—*.. DMBA+(2mgGE+TPA) ODC activity.
The topical application of the phorbol ester TPA to mouse skin or
—.— DMBA+(4mgGE+TPA)
its treatment in certain epidermal cells is known to result in a number
of biochemical alterations, changes in cellular functions, and histo
logical changes leading to skin tumor promotion (30—32).The induc
tion of inflammation in skin mediated by TPA is believed to be
governed by cyclooxygenase- and lipoxygenase-catalyzed metabolites
of arachidonic acid, specifically, PGs and HEThs, respectively (30—
0 32). Such an assumption is supported strongly by the studies showing
E that inhibitors of various parts of these metabolic pathways inhibit
I- skin tumor promotion (32—35).These inhibitory studies support the
.@ involvement of arachidonic acid metabolism pathways in skin tumor
promotion (32—35).
Cyclooxygenases, the microsomal enzymes, play an important
role in cutaneous inflammation, cell proliferation, and skin tumor
promotion (32, 35, 36, and references therein). Arachidonic acid,
0 released predominantly from skin phosphatidylcholine by the ac
tivation of skin phospholipase A2 due to a variety of mechanical,
chemical, or hormonal stimuli, undergoes oxidative metabolism
via the cyclooxygenase pathway, resulting in the formation of PGs
such as PGE2, PGF2a, and PGD2 (37 and references therein). In
addition to dominant inflammatory effects, TPA application also
produces epidermal hyperproliferation, including ODC induction
(25), associated with epidermal hyperplasia. Although the results
of the present study show the inhibitory effects of GE against
TPA-caused induction of epidermal cyclooxygenase activity in the
SENCAR mouse (Table 1), it also correlates with the inhibitory
effect of GE against TPA-caused induction of skin edema (Table 2)
Cl) and hyperplasia (Fig. 3).
0 Arachidonic acid is released from membrane phospholipids by
phospholipase activity in the skin by a number of stimuli, and
5-.
0 phorbol ester tumor promoters are some of them (38). Depending
0. on their nature, different lipoxygenases catalyze the metabolism of
Cl)
5-. arachidonic acid, resulting in the formation of HETEs (32, 36, 39,
0 and references therein). Lipoxygenase-catalyzed metabolites of
E
arachidonic acid have been considered to play an important role in
many physiological and pathophysiological events, including in
flammation and growth regulation; the presence of these metabo
lites in the mammalian epidermis has been reported by several
investigators (35, 36, 38, 40, 41). Although 12-lipoxygenase is
characterized predominantly in the normal murine epidermis (42,
10
43) along with small amounts of 5-, 8-, 12-, and 15-lipoxygenases
Weeks on Test (21, 44), treatment of a mammalian skin or cell-free extract with
Fig. 4. Inhibitory effect of topical application of GE (1, 2, or 4 mg/application/animal) TPA results in the induction of 8-lipoxygenase, identified by the
on TPA-caused skin tumor promotion in DMBA-initiated SENCAR mice. The doses of
DMBA and TPA used were 12.5 and 4.0 nmol, respectively. The percentage of mice with
generation of 8-HETE (2 1, 45).
tumors (top panel) and the number of tumors per mouse (bottom panel) were plotted as
a function of the number of weeks of the test. Details are provided in “Materials
and
Methods.― thetermination
Table 3 Inhibitory effect of topical application of GE on tumor size (mm3) at
miceTumor of TPA-caused skin tumor promotion in DMBA-iniriated SENCAR
theexperiment
volumes in the different groups were determined at the termination of
that of TPA showed an inhibitory effect of GE against TPA at 20 weeks.AverageTotal
induced increases in the levels of ODC mRNA in the mouse
epidermis (Fig. 2). The magnitude of the inhibitory effect of inhibitionvolume/
tumor Tumor tumor %
(volume/Treatments volume/ volumel
topical application of GE on TPA-induced increases in ODC mouse)DMBA group mouse tumor
m.RNA seems to be similar to that for inhibition of TPA-induced
11DMBA+ TPA I1,506 575±125 35 ±
increases in ODC enzyme activity (Figs. 1 and 2). These observa + I mg GE + TPA 5,250 263 ±56a 27 ±4 54
tions indicate that GE inhibits TPA-induced increases in ODC DMBA + 2 mg GE + TPA
85DMBA 1,670 84 ±19b 18 ±4
+ 4 mg GE + TPA 983 49 ±9C 13 ±3 91
enzyme activity by inhibiting the formation and/or enhancing the
a Significant versus TPA alone; P < 0.03.
breakdown of ODC mRNA. It is also possible that the application b Highly significant versus TPA alone; P < 0.001.
of GE may inhibit the translation of ODC mRNA to ODC protein. C Highly significant versus TPA alone; P < 0.0001.

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 INHIBITION OF SKIN TUMORIGENESIS BY GINGER
As observed in the present study, applications of TPA on SEN
CAR mouse skin resulted in the highly significant induction of
8-lipoxygenase activity (Table 1). The results of several studies14. Sharma, J. N., Srivastava, K. C., and Gan, E. K. Suppressive effects of eugenol and
(45 andreferences therein)suggest that8-lipoxygenase-catalyzed ginger oil on arthritic rats. Pharmacology (Basel), 49: 314-318, 1994.
15. McConlogne, L., Gupta, L., and Coffino, P. Molecular cloning and expression of
arachidonic acid metabolites, specifically 8-HETE, play an impor the mouse ornithine decarboxylase gene. Proc. NatI. Acad. Sci. USA, 81: 540—
tant role in skin tumor promotion, at least in cases of phorbol
ester-type skin tumor promoters (45). In the present study, skin
application of GE also showed significant inhibition against TPA
caused induction of epidermal lipoxygenase activity. Such an
inhibitory effect of GE may be responsible for the TPA-caused
induction of epidermal ODC and epidermal edema and hyperpla
sia, as observed in the present study. Moreover, the application of
TPA to mouse skin results in the rapid accumulation of inflamma
tory cells such as neutrophils and macrophages (46), stimulates the
release and metabolism of arachidonic acid (47), and increase the
formation of reactive oxygen species (48). Studies on possible
mechanisms of GE action indicate that GE is a potent inhibitor of
arachidonic acid metabolism via the lipoxygenase and cyclooxy
genase pathway and that GE is an antioxidant and scavenger of free
radicals.
The results in Fig. 4 and Table 3 show the protective effects of
skin application of GE on TPA-caused tumor promotion in DMBA
initiated SENCAR mouse skin. The preapplication of GE to that of
TPA showed protective effects when the tumor data were consid
ered as the total number of tumors or tumors per mouse (Fig. 4) and
the tumor volume per mouse or average volume per tumor (Table
3). These chemopreventive and anti-tumor promotion observations
in murine skin by GE can be explained by the biochemical mech
anisms studied in the present study. Additionally, the present study
showed that the inhibitory effects of GE on TPA-induced increases
in the levels of ODC mRNA in the mouse epidermis provide an
explanation for the inhibitory effect of GE on TPA-induced ODC
enzyme activity and suggest additional evidence that GE inhibits
the expression of genes that play a role in tumor promotion. These
data provide highly useful information for further study to eluci
date the exact nature of the compounds present in ginger respon
sible for chemopreventive effects.
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Inhibition of Tumor Promotion in SENCAR Mouse Skin by
Ethanol Extract of Zingiber officinale Rhizome
Santosh K. Katiyar, Rajesh Agarwal and Hasan Mukhtar

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