Carcinogenesis vol.29 no.11 pp.2182–2189, 2008
Advance Access publication August 6, 2008
Zyflamend? reduces LTB4formation and prevents oral carcinogenesis in a 7,12-
dimethylbenz[a]anthracene (DMBA)-induced hamster cheek pouch model
Peiying Yang, Zheng Sun1, Diana Chan, Carrie
A.Cartwright, Mary Vijjeswarapu, Jibin Ding, Xiaoxin
Chen2and Robert A.Newman?
Department of Experimental Therapeutics The University of Texas MD
Anderson Cancer Center, Houston, TX 77054, USA,1Department of Oral
Medicine Beijing Hospital for Stomatology, Capital Medical University,
Beijing 100050, People’s Republic of China and2Cancer Research Program,
Julius L.Chambers Biomedical/Biotechnology Research Institute, North
Carolina Central University, Durham, NC 27707, USA
?To whom correspondence should be addressed. Department of Experimental
Therapeutics, Unit 601, The University of Texas MDAnderson Cancer Center,
8000 El Rio Street, Houston, TX 77054, USA. Tel: þ1 713 563 7543;
Fax: þ1 713 563 9093;
Aberrant arachidonic acid metabolism, especially altered cyclo-
oxygenase and 5-lipoxygenase (LOX) activities, has been associated
with chronic inflammation as well as carcinogenesis in human
oral cavity tissues. Here, we examined the effect of Zyflamend?,
a product containing 10 concentrated herbal extracts, on devel-
opment of 7,12-dimethylbenz[a]anthracene (DMBA)-induced in-
flammation and oral squamous cell carcinoma (SCC). A hamster
cheek pouch model was used in which 0.5% DMBA was applied
topicallyonto the left cheek pouch of male Syrian golden hamsters
either three times per week for 3 weeks (short term) or 6 weeks
(long term). Zyflamend was then applied topically at one of three
different doses (25, 50 and 100 ml) onto the left cheek pouch three
times for 1 week (short-term study) or chronically for 18 weeks.
Zyflamend significantly reduced infiltration ofinflammatory cells,
incidence ofhyperplasia and dysplastic lesions, bromodeoxyuridine-
labeling index as well as number of SCC in a concentration-
dependent manner. Application of Zyflamend (100 ml) reduced
formation of leukotriene B4 (LTB4) by 50% compared with
DMBA-treated tissues. The reduction of LTB4was concentration
dependent. The effect of Zyflamend on inhibition of LTB4forma-
tion was further confirmed with in vitro cell-based assay. Adding
LTB4to RBL-1 cells, a rat leukemia cell line expressing high levels
of 5-LOX and LTA4hydrolase, partially blocked antiproliferative
effect of Zyflamend. This study demonstrates that Zyflamend in-
hibited LTB4formation and modulated adverse histopathological
changes in the DMBA-induced hamster cheek pouch model. The
study suggests that Zyflamend might prevent oral carcinogenesis
at the post-initiation stage.
Oral cancer is a common malignancy occurring in developing coun-
tries and remains problematic in that few effective therapies are avail-
able to treat it (1). Although both the incidence and prevalence of oral
cancer are relatively low in the USA compared with that of other
developing countries, there are still 35,310 new cases of this disease
and 7590 deaths expected in the USA in 2008 (2). Despite recent
advances in radiotherapy and chemotherapy, the survival of oral can-
cer patients has not improved significantly over the last couple of
decades (3). Oral leukoplakia is a premalignant lesion associated with
development of oral cancer, and statistically up to 20% of the patients
with this stage of oral disease will develop invasive carcinoma (4). It
has been established that oral leukoplakia is associated with chronic
inflammation in adjacent connective tissues (5). The association of
oral leukoplakia and oral cancer with inflammation has also been
shown to be associated with an increase of inducible nitric oxide
synthase in oral epithelium (6). These studies therefore suggest that
chronic inflammation may play a significant role in development of
oral cancer and, consequently, that anti-inflammatory agents may be
of value in the prevention and/or progression of premalignant to ma-
lignant stages of this disease.
Aberrant arachidonic acid (AA) metabolism, especially elevated
cyclooxygenase (COX) and 5-lipoxygenase (LOX) activities, has
been associated with chronic inflammation as well as carcinogenesis
in human oral cavity tissues (7,8). Modulation of AA metabolism by
inhibition of these enzymes has been considered to be an effective
mechanism for chemoprevention. For example, the use of the COX
inhibitor aspirin has been associated with a lower risk of cancer of the
upper aerodigestive tract, including that of oral cancer (9). COX-2
enzyme has been shown to be upregulated in oral tissues exhibiting
hyperplasia, dysplasia and squamous cell carcinoma (SCC) while
remaining barely detectable in normal epithelium (10,11). Addition-
ally, 5-LOX enzyme activity is also elevated in human oral cancer.
to be 10- to 30-fold higher in hamster and human SCC than in normal
tissues (12). Furthermore, 5-LOX and its metabolites, such as 5-
hydroeicosatetraenoic acid (HETE) and LTB4, are known to recruit and
activate inflammatory cells, increasevascular permeability and induce
contraction of smooth muscles (13,14). Recently, Li et al. reported
that the combination of two prescription medicines, celecoxib
(a COX-2 inhibitor) and Zileuton (a 5-LOX inhibitor), additively in-
hibited the incidence of SCC in the 7,12-dimethylbenz[a]anthracene
(DMBA)-induced hamster cheek pouch model (15). Inhibition of AA
metabolism (COX-2 and 5-LOX) by curcumin has also been sug-
gested as a key mechanism of its anticarcinogenic action in DMBA-
mediated hamster oral carcinoma (16). Finally, we have demonstrated
previously that LTB4promotes oral cacinogenesis, whereas multiple
5-LOX inhibitors have chemopreventive effects on cancer develop-
ment in the same animal model system (17).
There is increasingevidence thatnatural products might offer apro-
tective effect against oral cancer. For example, an association between
a high consumption of fruits and vegetables and reduced risk of oral
cancer has been reported (18,19). Additionally, the combination of
green tea and curcumin has been shown to inhibit oral carcinogenesis
at the post-initiation stage (16). Zyflamend? is a combination of 10
concentrated herbal extracts [rosemary, turmeric, ginger, holy basil,
green tea, hu zhang, Chinese goldthread, barberry, oregano and
Scutellaria baicalensis (skullcap)] each with potent anti-inflamma-
tory activity. We and other investigators have reported that Zyflamend
inhibits the proliferation of prostate cancer cells through induction of
apoptosis and inhibition of AA pathways, especially 5-LOX and 12-
LOX (20,21). Inhibition of both COX-1 and COX-2 activities by
Zyflamend was also observed (20,21). Additionally, Zyflamend’s
anti-inflammatory activity involves downregulation of nuclear fac-
tor-jb-associated genes involved with cancer cell invasion, metasta-
ses and angiogenesis (22). Since this product has shown inhibitory
activity against COX-1 and COX-2, as well as 5-LOX enzymes, it was
of interest to investigate the effect of Zyflamend on the development
of DMBA-induced oral inflammation that is a precursor of SCC.
The DMBA-treated hamster cheek pouch oral carcinoma model
was used in this study because it provides one of the most widely
accepted experimental models for oral carcinogenesis. Our objective
was to study the effect of Zyflamend on the development of DMBA-
induced oral inflammation and SCC and to investigate specific aspects
Abbreviations: AA, arachidonic acid; COX, cyclooxygenase; DMBA, 7,12-
dimethylbenz[a]anthracenene; HETE, hydroeicosatetraenoic acid; 13-HODE,
13-hydroxyoctadeca-9Z, 11E-dienoic acid; LOX, lipoxygenase; LTB4, leuko-
triene B4; MS, mass spectrometry; PBS, phosphate-buffered saline; PGE2,
prostaglandin E2; SCC, squamous cell carcinoma.
? The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: firstname.lastname@example.org 2182
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of its anti-inflammatory mechanisms in this disease. We show here
that Zyflamend prevents DMBA-induced oral carcinogenesis in the
hamster cheek pouch at the post-initiation stage and therefore may
represent a readily available product for clinical trial against this
Materials and methods
Zyflamend was provided by the manufacturer (NewChapter, Brattleboro, VT)
in a defined olive oil-based suspension of 10 concentrated anti-inflammatory
herbs (22). For all in vitro experiments, the product was mixed with dimethyl
sulfoxide at a 1:1 dilution and then further diluted (1:1000) in tissue culture
medium. Concentrations of this liquid herbal product are described as micro-
liter of Zyflamend per milliliter of tissue culture media. The eicosanoids
[Prostaglandin E2 (PGE2), LTB4, 5-HETE, 12-HETE, 15-HETE and 13-
standards were purchased from Cayman Chemical Company (Ann Arbor, MI).
DMBA was obtained from Sigma (St Louis, MO). All high-pressure liquid
chromatography-grade solvents used for analyses of eicosanoids by liquid
chromatography and tandem mass spectrometry (MS) were purchased from
Fisher Scientific Co. (Fair Lawn, NJ).
Anti-COX-2 and anti-LTA4hydrolase antibodies were obtained from Cayman
Chemical Company, anti-5-LOX antibody was purchased from Research
Diagnostics (Flanders, NJ) and anti-b-actin antibody was purchased from
Sigma (St Louis, MO).
Chemoprevention of hamster oral carcinogenesis by zyflamend
The animal study was approved by the Animal Care Committee of North
Carolina Central University. In order to test the anti-inflammatory and antitu-
mor effect of Zyflamend, short- and long-term treatments with Zyflamend in
a DMBA-induced hamster cheek pouch model were carried out.
For the short-term experiment (Experiment 1), male Syrian golden hamsters
(6 weeks old; Harlan, Indianapolis, IN) were housed four per cage. All animals
were given lab chow and tap water ad libitum. After 1 week of acclimatization,
the animals were divided into two groups, with Group A serving as the un-
treated negative control (three animals). The left cheek pouch of the remaining
24 hamsters was topically treated with 0.5% DMBA in 100 ll mineral oil using
a paintbrush three times a week for 3 weeks. They were then randomized to
four groups with Group B (six animals) serving as a positive control and re-
ceiving no further treatment. Groups C–E (six animal per group) were treated
topically with 25, 50 or 100 ll Zyflamend three times per week for 1 week. The
animals were injected with bromodeoxyuridine intraperitoneally at 50 mg/kg 2
h prior to killing. Six hours after the last treatment, animals were killed and
cheek pouch tissue was harvested. One half of the tissue was snap frozen in
liquid nitrogen for analysis of AA metabolites, and the other half was fixed in
10% phosphate-buffered saline (PBS)–buffered formalin for histopathological
For the long-term study (Experiment 2), animals were housed under the
same conditions as described above. Except for 30 hamsters serving as nega-
tive control (Group A), the remaining 86 animals were topically treated with
0.5% DMBA in 100 ll of mineral oil three times a week for 6 weeks. The
animals were then randomly divided into three groups with Group B receiving
no further treatment (30 animals). Group C and D (28 animal each) were
treated with 50 or 100 ll Zyflamend, respectively, three times per week for
another 18 weeks. At the end of 24 weeks, all animals were killed and the left
cheek pouch was harvested.
Thewhole cheek pouchwas excised and flattenedon a transparency platefor
visual determination of the number of tumors. The length, width and height of
each tumor were measured with calipers and tumor volume was calculated
using the formula: volume 5 4/3 pr3(where ‘r’ was the average radius of the
three diameter measurements in millimeter). An aliquot of tissue was frozen
immediately in liquid nitrogen for analysis of eicosanoid profiles. The remain-
ing tissue was cut and fixed in 10% buffered formalin for further histopatho-
logic analysis as described previously (15). Basal cell hyperplasia, dysplasia
and SCC were diagnosed using established criteria (15,23).
Since aberrant AA metabolism has been suggested to play an important role in
human oral carcinogenesis (15), and Zyflamend has been reported to inhibit key
AA pathways (21), Zyflamend-mediated alteration of eicosanoids in both the in
vivo DMBA-induced hamster cheek pouch carcinogenesis model and in vitro
cancer cells was examined. The effect of Zyflamend on the relative formation of
PGE2, LTB4, 5-HETE, 15-HETE and 13-HODE in RBL-1 cells or hamster
cheek pouch tissues was determined according to the method of Yang et al.
(24,25). For determination of eicosanoid metabolism in hamster cheek pouch
tissues, frozen tissue (25–50 mg) was ground to a fine powder using a liquid
nitrogen-cooled mortar (Fisher Scientific Co.). Samples were then transferred to
sealed microcentrifuge tubes, and three times the volume of ice-cold PBS buffer
containing 0.1% butylated hydroxytoluene and 1 mM ethylenediaminetetraace-
tic acid was added. The sample was then homogenized by an Ultrasonic
Processor (Misonix, Farmingdale, NJ) at 0?C for 3 min. A 100 ll aliquot of
the homogenate was transferred to a glass tube (13 ? 100 mm) and subjected to
extraction of eicosanoids using the procedure described previously (25).
For in vitro experiments, RBL-1 cells (5 ? 106) were harvested, washed with
2 ml of PBS and then resuspended in 0.5 ml of PBS containing 1 mM CaCl2.
Samples were then incubated with Zyflamend or individual concentrated
herbal components of Zyflamend (0.125 to 1 ll/ml) at 37?C for 10 min fol-
lowed by addition of 2.5 ll of calcium ionophore A23187 (1 mM). An aliquot
(2.5 ll) of a solution containing AA (10 mM) was then added, and samples
were incubated for an additional 10 min. The reaction was terminated by
addition of aliquots of 1 N citric acid (40 ll) and of 10% butylated hydrox-
ytoluene (5 ll). An aliquot (10 ll) of the deuterated relevant eicosanoids (i.e.
PGE2-d4, LTB4-d4, 15-HETE-d8, 12-HETE-d8and 5-HETE-d8; 100 ng/ml) as
internal standards was then added to the reaction mixtures. Eicosanoids were
extracted with 2 ml of hexane:ethyl acetate (1:1; vol/vol) three times. The
upper organic phases were pooled and evaporated to dryness under a stream
of nitrogen at room temperature. All extraction procedures were performed
underconditionsof minimal light.Sampleswerethenreconstitutedin 200 ll of
methanol/10 mM ammonium acetate buffer (70:30; vol/vol), pH 8.5, before
analysis by liquid chromatography and tandem MS.
Reverse-phase high-pressure liquid chromatography electrospray ionization
MS was used to determine eicosanoid levels in cells or tissues using a pre-
viously published method reported by our laboratory (25). A Micromass Quat-
tro Ultima Tandem Mass Spectrometer (Waters Corp., Milford, MA) was
equipped with an Agilent 1100 HP binary pump high-pressure liquid chro-
matograph inlet for use in these studies. Eicosanoids were separated using a
Luna 3 l phenyl–hexyl (2 ? 150 mm) LC column (Phenomenex, Torrence,
CA). The mobile phase consisted of 10 mM ammonium acetate (pH 8.5) and
methanol; the flow rate was 250 ll/min with a column temperature of 50?C.
The sample injection volume was 25 ll. Samples were kept at 4?C in an
autosampler prior to injection onto the analytical column.
The mass spectrometer was operated in the electrospray negative ion mode
with a cone voltage of 100 V, a cone gas flow rate of 117 l/h and a devolution
gas flow rate of 998 l/h. The temperature of the desolvation region was 400?C,
and the temperature of the source region was 120?C. Fragmentation of all
compounds was performed using argon as the collision gas at a cell pressure
of 2.1 ? 10?3Torr. The collision energy was 19 V. All eicosanoids were
detected using negative ionization and multiple-reaction monitoring of the
transition ions for eicosanoid products and their internal standards.
RBL-1 rat leukemia cells were obtained from the American Type Culture
Collection (Manassas, VA) and maintained in a humidified atmosphere con-
taining 5% CO2at 37?C. Cells were routinely cultured in modified Eagle’s
medium (Invitrogen Corp., Grand Island, NY) supplemented with 10% heat-
inactivated fetal bovine serum (Hyclone Laboratories, Logan, UT), 50 IU/ml
penicillin and 50 lg/ml streptomycin and 2 mM L-glutamine from GIBCO
Cells were grown in modified Eagle’s medium with 10% fetal bovine serum at
a density of 1 ? 104cells/well. After a 24 h incubation period, cells were
treated with various concentrations of Zyflamend (0.03–2.0 ll/ml). After an
additional 72 h, inhibition of cellular proliferation was assessed by 3-(4,5-
dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide assay (26). In brief,
3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (Sigma Chem-
ical) was added (0.3 mg/ml) and further incubated for 2 h. Then, medium was
removed without disturbing the cells, and the resulting blue formazan crystals
were dissolved in 50 ll dimethyl sulfoxide. Absorbance was read at a wave-
length of 570 nm with a referencewavelength of 650 nm using a V-Max Micro-
plate Reader by Molecular Devices (Sunnyvale, CA).
Western blot analysis
RBL-1 cells were treated in serum-free conditions with 1 ll/ml Zyflamend for
4, 8, 16 and 24 h. Cells were washed with cold PBS and lysed in a buffer
containing 20 mM 3-(N-morpholino)propane sulfonic acid, 2 mM ethylene-
glycol-bis(aminoethylether)-tetraacetic acid,5 mMethylenediaminetetraacetic
acid, 30 mM NaF, 40 mM b-glycerophosphate, 20 mM sodium pyruvate, 0.5%
Triton X-100 and 1 mM sodium orthovanadate with protease inhibitor cocktail
(Sigma). Cell lysates were then sonicated on ice for 3 min, further incubated
for 10 min on ice and then centrifuged at 14, 000rpm for 10 min at 4?C. Protein
Chemopreventive effect of Zyflamend against oral cancer
by guest on June 1, 2013
levelswerequantifiedusingthe Bio-RadDc ProteinAssay(Bio-Rad,Hercules,
CA). Equal levels of protein (50 lg) were fractionated on precast polyacryl-
amide gels (Bio-Rad) and then transferred onto polyvinylidene diflouride
membranes, according to standard methods. Following a 1–2 h incubation
period in 5% non-fat dry milk blocking buffer prepared in Tris-buffered saline
with 0.1% Tween 20, membranes were then probed with primary antibodies
diluted 1:2000 in blocking buffer. Protein bands were visualized via chemilu-
Piscataway, NJ). Equal loading of samples was illustrated by western blotting
for the presence of b-actin.
For determination of the effect of PGE2, LTB4and 5-HETE on Zyflamend-
induced cell growth inhibition, RBL-1 cells (1 ? 104) were plated on 96-well
plates. At 24 h, serum-free medium was added. Cells were then pretreated with
30 nM of above eicosanoids for 1 h, followed by addition of Zyflamend (0.5 or
1 ll/ml). Twenty-four hours after drug treatment,1 lM calcien AM (Molecular
Probes, Eugene, OR) was added, and samples were incubated at 25?C for 15
min. Fluorescence intensity was then read with an FLX 800 Fluorescence Plate
Reader (Bio-Tek Instruments, Winooski, VT) with excitation and emission
wavelengths of 485 and 528 nm, respectively.
Data involving tumor incidencewere comparedby the v2test. One-way analysis
of variance test was used to compare body weight, number of visible tumors, the
number of infiltrating inflammatory cells and number of oral lesions using SAS
software. Tumor volume was compared between a treatment group and positive
control group with Wilcoxon signed-rank test. Student’s t-test was used to
determine the statistical differences between treatment and control groups in
the in vitro experiments; a value of P , 0.05 was considered to be significant.
Inhibition of DMBA-induced hamster cheek pouch inflammation and
proliferation by Zyflamend—Experiment 1
To determine acute anti-inflammatory changes, DMBAwas applied to
hamster cheek pouch three times per week for 3 weeks followed by
1 week of topical application of Zyflamend. At the end of experimental
period, the buccal pouches of DMBA-treated hamsters contained nu-
merous areas exhibiting hyperplasia (Figure 1B) and dysplasia (Fig-
ure 1C) in comparison with that of right cheek pouch of the same
animals (Figure 1A). Zyflamend (100 ll) inhibited DMBA-induced
inflammation evidenced by the reduction of hyperplasia (Figure 1E)
compared with that of DMBA treatment alone (Figure 1D). Quanti-
tatively, after 1 week of treatment, Zyflamend (50 ll) significantly
reduced the number of inflammatory cells (P , 0.005), whereas at
the dose of 100 ll Zyflamend significantly inhibited the number of
inflammatory cells, hyperplasia and dysplasia by 70.7% (P , 0.01),
84.4% (P , 0.05) and 70.3% (P , 0.05), respectively (Figure 2A–C).
No SCC was observed in these animals. Cell proliferation was also
well correlated with histopathological observation. The proliferation
index increased remarkably from normal epithelium (0.7%) to
DMBA-treated area (6.7%). Zyflamend (50 and 100 ll) significantly
reduced the proliferation index to 3.4 ± 1.3% (P , 0.008) and
1.8 ± 1.3% (P , 0.001), respectively (Figure 2D). The inhibition of
cell proliferation by Zyflamend was concentration dependent.
Chemoprevention of oral carcinogenesis by Zyflamend in hamster
cheek pouch—Experiment 2
The effect of Zyflamend on prevention of development of oral carci-
noma in DMBA-induced hamster cheek pouch model was then eval-
uated. As shown in Table I, Zyflamend (50 and 100 ll) (Groups C and
D) significantly decreased the visible oral tumor incidence to 53.6%
(15/28) and 50% (14/28) from 86.7% (26/28) in the positive group
(Group B) (P , 0.01). Similarly, the average number of tumors in
Groups C and D was significantly decreased by 35.1% (P , 0.05)and
52.4% (P , 0.01), respectively, compared with that of Group B.
Histologically, in comparison with Group B, the average number of
hyperplasia lesions in Group D was significantly reduced by 47.5%
(P , 0.01). The average incidence of hyperplasia in Group C was
also decreased compared with that of group B, but the difference was
not statistically significant. While the average number of mild and
moderate dysplastic lesions per animal in Group C and D was signif-
icantly reduced by 37.2 and 41.9% (P , 0.01), respectively, com-
pared with that of Group B, the average number of severe
dysplastic lesions in Groups C and D was decreased by 38.8 and
59.2% (P , 0.05), respectively. Additionally, when compared with
Group B, the average number of SCC per animal in Groups C and D
was reduced to 1.82 ± 1.58 and 1.59 ± 1.31 (P , 0.05), respectively,
from 2.62 ± 2.00 in Group B.
Similar to the results of the short-term study, cell proliferation in
animals treated with Zyflamend for 18 weeks was also inhibited. The
proliferation index increased along with progression of the stage of
Fig. 1. DMBA-induced oral lesions of hamster cheek pouch after 3 weeks treatment with 0.5% DMBA (A–C) indicating the development of hyperplasia (B) and
dysplasia (C) comparing with normal epithelial (A). The DMBA-induced inflammation was inhibited by Zyflamend (100 ll) (E) compared with that of DMBA
alone treated group (D). The magnification was ?400.
P.Yang et al.
by guest on June 1, 2013
disease from normal epithelium (1.93 ± 1.80%), basal cell hyperplasia
(4.82 ± 3.34%) and dysplasia (4.82 ± 3.34) to SCC (13.01 ± 10.18%)
(supplementary Table I is available at Carcinogenesis Online).
Zyflamend (100 ll) significantly lowered the proliferation index in
normal epithelial, basal cell hyperplastic and dysplastic lesions by
60.1, 56.0 and 44.0%, respectively (P , 0.01). The proliferation of
SCC was also inhibited by Zyflamend (100 ll) (43.5%).
Effect of Zyflamend on eicosanoid metabolism—LTB4formation
The effect of Zyflamend on eicosanoid metabolism in DMBA-
treated hamster cheek pouch tissues was examined using liquid
chromatography and tandem MS method. As shown in Figure 3,
the level of LTB4 in the hamster oral tissues after 3 weeks
of DMBA treatment was increased by 2-fold (P , 0.01) compared
with that of the negative control group. Zyflamend, at a dose of 100 ll,
significantly decreased the tissue level of LTB4 compared with
that of the DMBA-treated group (P , 0.05). The inhibitory effect
of LTB4 formation by Zyflamend was concentration dependent.
Other eicosanoids, such as PGE2, 15-HETE, 5-HETE and 13-HODE
in tissues from animals in the acute study, were also examined.
Interestingly, Zyflamend did not reduce the formation of PGE2
(data not shown) and level of 5-HETE in the hamster cheek pouch.
No. of Inflammatory cells/mm2
0 DMBA 2510050
0 DMBA 2510050
0 DMBA25 10050
Fig. 2. The effect of Zyflamend on DMBA-induced inflammation (A), hyperplasia score (B), dysplasia score (C) and proliferation (D). The proliferation index (%)
was calculated as the total number of the positive staining nuclei divided by total number of epithelial cells in each lesion evaluated (?400). The DMBA-induced
hyperplasia and dysplasia were inhibited by Zyflamend in a concentration-dependent manner. Data are presented as the mean ± SD (n 5 6).?P , 0.05,
??P , 0.01, and????P , 0.001 represent significant differences versus positive control.
Table I. Chemopreventive effects of topical Zyflamend on DMBA-induced oral carcinogenensis in hamster cheek pouch (Experiment 2) (mean ± SD)
Group No.Treatment Macroscopic observationMicroscopic observation
Dysplasia no. (mild
Zyflamend 50 ll
Zyflamend 100 ll
000 0.45 ± 0.37??
3.03 ± 1.64
2.48 ± 1.45
1.59 ± 1.33??
0.10 ± 0.21??
5.17 ± 2.75
3.25 ± 2.46??
3.05 ± 1.67??
1.43 ± 1.07
0.93 ± 1.25?
0.68 ± 0.86??
57.8 ± 135.7
81 ± 331.9
4.5 ± 16.8??
1.57 ± 1.38
0.96 ± 1.82?
0.64 ± 0.64?
2.62 ± 2.00
1.82 ± 1.58
1.59 ± 1.31?
Group A served as non-treated control. Statistical analysis were based on the comparison with Group B.?P , 0.05,??P , 0.01.
Chemopreventive effect of Zyflamend against oral cancer
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In contrast, the levels of 15-HETE and 13-HODE in Zyflamend-
treated tissues were increased compared with that of DMBA
treatment alone; the changes, however, were not statistically signifi-
Inhibitory effect of Zyflamend on LTB4formation in RBL-1 cells
In order to further delineate the specific role of Zyflamend on inhibi-
tion of LTB4formation, the effects of Zyflamend on AA metabolism
in RBL-1 were examined. Because RBL-1 cells express relatively
high levels of 5-LOX enzyme, this cell line was chosen as an appro-
priate in vitro model to determine the effect of Zyflamend on the
5-LOX pathway. As shown in Figure 4, the effect of Zyflamend
and 13-HODE was examined. Intriguingly, only levels of LTB4and 5-
HETE in Zyflamend-treated RBL-1 cells were significantly reduced
by 67% (P , 0.05) and 77% (P , 0.01), respectively, compared with
that of vehicle-treated RBL-1 cells (Figure 4A). The inhibitory effect
of Zyflamend on LTB4formation in RBL-1 cells was concentration
dependent (Figure 4B). Furthermore, the protein expression of 5-LOX
was downregulated by Zyflamend (1 ll/ml) as early as 4 h after
treatment and this inhibitory effect continued up to 24 h (Figure
4C). However, the expression of LTA4hydrolase itself was not af-
fected by Zyflamend, suggesting that this multi-herb product selec-
tively inhibited 5-LOX enzyme expression and activity in this cell
The effect of specific herbal components of Zyflamend on inhibition of
growth of RBL-1 cells and eicosanoid metabolism
Since Zyflamend is composed of 10 different herbs, we explored the
relative effects of specific components of this multi-herb product that
might be responsible in whole or in part for Zyflamend’s inhibitory
effect on the 5-LOX pathway. First, the effect of Zyflamend and its
individual components on the proliferation of RBL-1 cells was com-
pared. Among the 10 herbs within Zyflamend, rosemary and Chinese
goldthread inhibited 50% of cell growth at concentrations of 4.6 and
6.0 lg/ml, respectively (supplementary Table II is available at
Carcinogenesis Online). Interestingly, only rosemary markedly re-
duced the formation of LTB4by 81% when the RBL-1 cells were
treated with the concentrated form of the individual herbs at concen-
trations equivalent to 0.5 ll/ml of Zyflamend. Zyflamend itself
(0.5 ll/ml), however, still had the strongest effect on inhibition of
LTB4in RBL-1 cells compared with that of individual 10 herbs tested
(Figure 5A). The inhibitory effect of rosemary on LTB4formation was
concentration dependent (Figure 5B).
Effect of LTB4‘add-back’ on Zyflamend-mediated inhibition of cell
Addition of PGE2and 5-HETE to RBL-1 cells failed to counteract or
block the inhibition of cell proliferation produced by Zyflamend. In
contrast, when 30 nM LTB4was added to cells treated with 1 ll/ml of
Zyflamend, a concentration that inhibited proliferation of RBL-1 cells
by 50%, a near doubling of cell proliferation occurred (Figure 5C).
15-HETE (ng/mg protein)
13-HODE (ng/mg protein)
LTB4 (ng/mg protein)
5-HETE (ng/mg protein)
Fig. 3. Eicosanoidmetabolismin Zyflamend-treated DMBA-inducedhamstercheek pouch. Zyflamend significantly inhibitedLTB4formationcomparedwith that
of control (A), whereas no changes were observed in the level of 5-HETE (B). Interestingly, the levels of 15-HETE and 13-HODE were increased in Zyflamend-
treated cheek pouch compared with that of control (C and D), but the differences were not significant. Data are presented as mean ± SD (n 5 10).aP , 0.01,
significant versus negative control;?P , 0.05 significant versus positive control by Student’s t-test.
P.Yang et al.
by guest on June 1, 2013
These results point out the importance of this particular eicosanoid in
Zyflamend-mediated inhibition of RBL-1 cell proliferation.
induced inflammation but also prevents the further development of
oral carcinogenesis as evidenced by reductions in skin tissue exhibit-
ing areas of hyperplasia, dysplasia as well as SCC. This preventive
effect of Zyflamend on the development of oral carcinoma in this
particular hamster model might be associated with alteration of AA
metabolism, especially reduction of LTB4formation.
Fig. 4. The effect of Zyflamend on eicosanoid metabolism and enzyme
expression in RBL-1 cells. (A) Zyflamend significantly inhibited the
production of both LTB4and 5-HETE in RBL-1 cells. (B) The inhibitory
effect of Zyflamend on formation of LTB4was in a concentration-dependent
manner. (C) Expression of 5-LOX was inhibited by Zyflamend in a time-
dependent manner. Data are presented as the means ± SDs of three separate
experiments.?P , 0.05,??P , 0.01,???P , 0.005 and????P , 0.001
significant versus control.
Fig. 5. Comparative study of Zyflamend and its individual herbs on LTB4
formation in RBL-1 cells. (A) Among 10 herbs tested, rosemary markedly
inhibited the formation of LTB4in RBL-1 cells. However, Zyflamend
inhibited formation of LTB4in RBL-1 cells more strongly than that of
rosemary alone. 1, control; 2, rosemary; 3, turmeric; 4, ginger; 5, holy basil;
6, green tea; 7, hu zhang; 8, Chinese goldthread; 9, barberry; 10, oregano; 11,
scutallaria and 12, Zyflamend. (B) Concentration-dependent inhibition of
LTB4productionby rosemary in RBL-1 cells was observed. (C) The effect of
LTB4, PGE2and 5-HETE on Zyflamend-induced inhibition of proliferation
of RBL-1 cells after 48 h treatment. Addition of Zyflamend produced
a concentration-dependent inhibition of RBL-1 cell proliferation, whereas
addition of PGE2, or 5-HETE (30 nM) only slightly stimulated cell growth.
Addition of LTB4with Zyflamend (1 ll/ml), however, partially reversed
Zyflamend-mediated inhibition of cell proliferation.?P , 0.05,??P , 0.01;
????P , 0.001 significantly different from control. Data are presented as the
mean ± SD of three separate experiments.
Chemopreventive effect of Zyflamend against oral cancer
by guest on June 1, 2013
The development of oral cancer is a multistep process requiring ini-
tiation, promotion and progression. The hamster cheek pouch model
is easily developed and can provide one of the most widely accepted
experimental models for oral carcinogenesis (27). Despite the exis-
tence of anatomic and histologic variations between hamster pouch
mucosa and human buccal tissue, the DMBA-treated hamster cheek
pouch model is able to produce premalignant changes and carcinomas
that are similar to the development of disease in human oral mucosa
(28). The progression of premalignant to malignant changes in ham-
ster cheek pouch was dependent on the duration of topical application
of DMBA. Applying DMBA three times per week for 3 weeks to
hamster cheek pouch usually induces chronic inflammation and hy-
perplasia in the epithelium. In comparison, treating hamster cheek
pouch for 6 weeks (three times per week) leads to the development
of SCC, which represents a premalignant stage of this disease (27).
Here, we examined the effect of Zyflamend in both a 3-week and a 6-
week treatment model in order to explore the potential role of Zy-
flamend in prevention of oral cancer development. Intriguingly,
Zyflamend (100 ll) consistently inhibited the development of hyper-
plasia and dysplasia in both short-term (3-week treatment) and long-
term experiments (6-week treatment) by .50%. In contrast, Zyfla-
mend was less potent in suppression of SCC. The incidence of SCC in
DMBA-treated group was not significantly inhibited by Zyflamend,
even at dose of 100 ll (data not shown). Li et al. (16) have reported
that Zileuton, a 5-LOX inhibitor, and celecoxib, a selective COX-2
inhibitor, effectively blocked the development of DMBA-induced oral
carcinogenesis. The inhibitory effect of celecoxib on the development
of oral carcinogenesis was also found in a similar DMBA hamster
cheek pouch model (29). Given the fact that the toxicity of Zileuton
has been associated with hepatic injury, Zyflamend, a natural herbal
supplement containing 10 herbs with no reported toxicities, represents
what may be considered as a good choice as a cancer-preventive
Many mechanisms have been proposed as etiologic factors in de-
velopment of DMBA hamster oral carcinoma, such as p53 and K-ras
mutations and overexpression of inducible nitric oxide synthase (30–
33). In addition, dysregulated bioactive lipid metabolism is also be-
lieved to be important. As an important AA-metabolizing enzyme,
5-LOX has been found to be markedly upregulated in stromal inflam-
matory cells and epithelial cells at the early stage of human oral SCC
(15). The downstream 5-LOX product, LTB4, was remarkably ele-
vated in DMBA-induced hamster oral carcinoma compared with
PGE2levels in this model, suggesting that the 5-LOX pathway might
play a critical role in human oral carcinogenesis. We have found
previously that Zyflamend inhibited 5-LOX activity much stronger
than COX-2 activity in human prostate cancer cells (21). The finding
from this present study further confirms that the level of LTB4was
elevated in DMBA-induced hamster cheek pouch. Our study failed to
find an elevation of PGE2in the DMBA-treated hamster cheek pouch
model (data not shown). Interestingly, the level of LTB4was signif-
icantly reduced in Zyflamend-treated cheek pouch, suggesting that the
preventive effect of Zyflamend on DMBA-induced inflammation and
oral carcinogenesis development might be mediated through reduc-
tion of 5-LOX expression as well as its enzymatic activity.
To further explore the effect of Zyflamend on 5-LOX activity, the
effect of this multi-herb agent on eicosanoid metabolism was exam-
ined in RBL-1 cells. These cells were chosen because they have a
relatively high expression of 5-LOX and have been widely used for
screening of selective 5-LOX inhibitors (30). Zyflamend significantly
inhibited the formation of both metabolites of 5-LOX, LTB4and
5-HETE, in RBL-1 cells. Adding LTB4back to Zyflamend-treated
RBL-1 cells partially blocked the antiproliferative effect of Zyfla-
mend, suggesting a strong link between LTB4presence and cell pro-
liferation. Interestingly, in comparing the inhibition of Zyflamend on
both LTB4and 5-HETE in RBL-1 cells, only LTB4levels but not
5-HETE levels were reduced in Zyflamend-treated DMBA hamster
pouch. Taken together, these results suggest that Zyflamend, a multi-
herb product, has an ability to inhibit the predominant AA metabolism
in different in vitro or in vivo settings.
Because Zyflamend contains 10 different herbs, it was of interest to
find out which component in Zyflamend might best explain its in-
hibitory action on 5-LOX. Among the 10 herbs tested, rosemary, at
a comparable level to that contained in Zyflamend, markedly reduced
formation of LTB4as well as inhibited the proliferation of RBL-1
cells, suggesting that rosemary might be an important component
responsible for the observed antiproliferative effect of Zyflamend.
At concentrations normalized to their respective levels in Zyflamend,
none of the other herbs showed a stronger effect either on the re-
duction of LTB4formation or inhibition on RBL-1 proliferation in
comparison with the combined multi-herb Zyflamend product. These
data suggest that components in Zyflamend in addition to rosemary
might also contribute to the chemopreventive effect of this product
through mechanisms not directly associated with AA metabolism.
Even though holy basil, turmeric, ginger, oregano and green tea did
not significantly reduce LTB4formation in the RBL-1 cell assay, these
agents have previously been reported to exhibit chemopreventive ac-
tivity in the hamster cheek pouch or mouse skin models. For example,
both green tea and curcumin (a major component of turmeric) in
Zyflamend have been found to markedly inhibit the post-initiation
stage of DMBA hamster cheek oral carcinoma (16). Ginger has also
been reported to induce apoptosis in human oral cancer cells (34).
Berberine, a component of barberry, has been found to modulate
apoptosis pathways and inhibit Mcl-1 expression in oral cancer cells
(35) and has been proposed itself as a chemopreventive agent for oral
cancer (36). Extracts of holy basil significantly reduced tumor forma-
tion when it was given orally or topically through enhanced expression
of O6-methylguanine-DNA methyltransferase repair enzymes (37).
The documented anticancer activities of these herbs within Zyflamend
may therefore be expected to contribute to the chemopreventive effect
of Zyflamend on DMBA-induced hamster oral carcinomas and most
probably through different mechanisms not directly linked AA me-
tabolism. Given the sparse therapeutic options for this devastating
human cancer, the use of Zyflamend as a single agent or as adjuvant
therapy for this disease deserves consideration.
In conclusion, our results demonstrated that Zyflamend modulated
histopathological progress in the DMBA-induced hamster cheek
pouch model. It suggested that Zyflamend might prevent oral carci-
nogenesis at the post-initiation stage. The chemopreventive effect of
Zyflamend might be, at least partially, associated with its anti-inflam-
matory properties, especially on reduction of the proinflammatory
mediator, LTB4. Because Zyflamend is an herbal supplement and has
been studied clinically in prostate cancer patients with very limited
toxicity, this agent might be explored as a chemopreventive agent for
humans having chronic inflammatory lesions, which might in turn
place them at risk for development of oral cancer.
Supplementary Tables I and II can be found at http://carcin.
NewChapter (Brattleboro, VT) LS2005-13655WC.
Conflict of Interest Statement: None declared.
1.Magrath,I. et al. (1993) Cancer in development countries: opportunity and
challenge. J. Natl Cancer Inst., 85, 862–874.
2.Jemal,A. et al. (2008) Cancer statistics. CA Cancer J. Clin., 58, 71–96.
3.Funk,G.F. et al. (2002) Presentation, treatment and outcome of oral cavity
cancer: a National Cancer Data Base report. Head Neck, 24, 165–180.
P.Yang et al.
by guest on June 1, 2013
4.Lodi,G. et al. (2006) Intervention of treating oral leukoplakia. Cochrane Download full-text
Database Syst. Rev., 18: CD001829.
5.Ali,A.A. et al. (2006) Histopathological changes in oral mucosa due to
takhzeen al-qat: a study of 70 biopsies. J. Oral Pathol. Med., 35, 81–85.
6.Kawanishi,S. et al. (2006) Oxidative and nitrative DNA damage in animals
and patients with inflammatory diseases in relation to inflammation-related
carcinogenesis. Biol. Chem., 387, 365–372.
7.Banerjee,A.G. et al. (2005) Identification of genes and molecular pathways
involved in the progression of premalignant oral epithelia. Mol. Cancer
Ther., 4, 865–875.
8.Metzger,K. et al. (1995) Lipoxygenase products in human saliva: patients
withoralcancercomparedto controls.Free Radic.Biol.Med.,18,185–194.
9.Bosetti,C. et al. (2003) Aspirin use and cancers of the upper aerodigestive
tract. Br. J. Cancer, 88, 672–674.
10.Shibata,M. et al. (2005) Cyclo-oxygenase-1 and -2 expression in human
oral mucosa, dysplasias and squamous cell carcinomas and their patholog-
ical significance. Oral Oncol., 41, 304–312.
11.Minter,H.A. et al. (2003) The cyclooxygenase-2 selective inhibitor NS398
inhibits proliferation of oral carcinoma cell lines by mechanisms dependent
and independent of reduced prostaglandin E2 synthesis. Clin. Cancer Res.,
12.El-Hakim,I.E. et al. (1990) Leukotriene B4 and oral cancer. Br. J. Oral
Maxillofac. Surg., 28, 155–159.
13.Chen,X. et al. (2004) Leukotriene A4 hydrolase as target for cancer pre-
vention and therapy. Curr. Cancer Drug Targets, 4, 267–283. Review.
14.Peters-Golden,M. et al. (2003) 5-Lipoxygenase and FLAP. Prostaglandins
Leukot. Essent. Fatty Acids, 69, 99–109.
15.Sood,S. et al. (2005) Overexpression of 5-lipoxygenase and cyclooxyge-
nase 2 in hamster and human oral cancer and chemopreventive effects of
Zileuton and celecoxib. Clin. Cancer Res., 11, 2089–2096.
16.Li,N. et al. (2002) Inhibition of 7,12-dimethylbenz[a]anthracene (DMBA)-
induced oral carcinogenesis in hamsters by tea and curcumin. Carcinogen-
esis, 23, 1307–1313.
17.Sun,Z. et al. (2006) Involvement of 5-lipoxygenase/leukotriene A4 hydro-
lase pathway in 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral
carcinogenesis in hamster cheek pouch and inhibition of carcinogenesis
by its inhibitors. Carcinogenesis, 27, 1902–1908.
18.Conway,D.I. (2007) Each portion of fruit or vegetable consumed halves the
risk of oral cancer. Evid. Based Dent., 8, 19–20.
19.Maserejian,N.N. et al. (2006) Prospective study offruitsand vegetables and
risk of oral premalignant lesions in men. Am. J. Epidemiol., 164, 556–566.
20.Bemis,D.L. et al. (2005) Zyflamend, a unique herbal preparation with non-
selective COX inhibitor activity, induces apoptosis of prostate cancer cells
that lack COX-2 expression. Nutr. Cancer, 52, 202–212.
21.Yang,P. et al. (2007) Zyflamend-mediated inhibition of human prostate
cancer PC3 cell proliferation: effects on 12-LOX and Rb protein phosphor-
ylation. Cancer Biol. Ther., 6, 228–236.
22.Sandu,S.K. et al. (2007) Zyflamend, a polyherbal preparation, inhibits in-
vasion, suppressions osteoclastogenesis, and potentiates apoptosis through
products. Nutr. Cancer, 57, 78–87.
23.Pingdborg,J. et al. (1997) World Health Organization. International Histo-
logical Classification of Tumors: Histological Typing of Cancer and Pre-
cancer of Oral Mucosa, 2nd edn. Springer, Berlin, Germany, 2nd edn1–40.
24.Kempen,E.C. et al. (2001) Simultaneous quantification of arachidonic acid
metabolites in cultured tumor cells using high-performance liquid chroma-
tography/electrospray ionization tandem mass spectrometry. Anal. Bio-
chem., 297, 183–190.
25.Yang,P. et al. (2006) Determination of endogenous tissue inflammation
profiles by LC/MS/MS: COX- and LOX-derived bioactive lipids. Prosta-
glandins Leukot. Essent. Fatty Acids, 75, 385–395.
26.Mosmann,T. (1983) Rapid colorimetric assay for cellular growth and sur-
vival: application to proliferation and cytotoxic assays. J. Immunol. Meth-
ods, 65, 55–56.
27.Gimenez-Conti,I.B. et al. (1993) The hamster cheek pouch carcinogenesis
model. J. Cell Biochem., 53 (suppl.): 83–90.
28.Morris,A.L. (1961) Factors influencing experimental carcinogenesis in the
hamster cheek pouch. J. Dent. Res., 40, 3–15.
29.Feng,L. et al. (2006) Chemopreventive effect of celecoxib in oral pre-
cancers and cancers. Laryngoscope, 116, 1842–1845.
30.Glimenez-Conti,I.B. et al. (1996) p53 alterations in chemically induced
hamster cheek-pouch lesions. Mol. Carcinog., 16, 197–202.
31.Chen,Y.K. et al. (2003) Correlation between inducible nitric oxide synthase
and p53 expression for DMBA-induced hamster buccal-pouch carcinomas.
Oral Dis., 9, 227–234.
32.Chen,Y.K. et al. (2000) Immunohistochemical expression of inducible ni-
tric oxide synthase in DMBA-induced hamster buccal pouch carcinogene-
sis. Oral Oncol., 36, 221–224.
33.Chen,Y.K. et al. (2002) Increased expression of inducible nitric oxide syn-
thase in human oral submucous fibrosis, verrucous hyperplasia, and verru-
cous carcinoma. Int. J. Oral Maxillofac. Surg., 31, 419–422.
34.Hsu,S. et al. (2004) Induction of apoptosis in oral cancer cells: agents and
mechanisms for potential therapy and prevention. Oral Oncol., 40,
35.Kou,C.L. et al. (2005) Modulation of apoptosis by berberine through in-
hibition of cyclooxygenase-2 and Mcl-1 expression in oral cancer cells.
In Vivo, 19, 247–252.
36.Kou,C.L. et al. (2004) The anti-inflammatory potential of berberine invitro
and in vivo. Cancer Lett., 203, 127–137.
37.Karthikeyan,K. et al. (2007) Chemopreventive effect of Ocimum sanctum
on DMBA-induced hamster buccal pouch carcinogenesis. Oral Oncol., 35,
Received March 6, 2008; revised August 1, 2008; accepted August 1, 2008
Chemopreventive effect of Zyflamend against oral cancer
by guest on June 1, 2013