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Carcinogenesis. 2008 November; 29(11): 2182–2189.
Published online 2008 August 6. doi:  10.1093/carcin/bgn181
PMCID: PMC3697064

Zyflamend® reduces LTB4 formation and prevents oral carcinogenesis in a 7,12-dimethylbenz[α]anthracene (DMBA)-induced hamster cheek pouch model


Aberrant arachidonic acid metabolism, especially altered cyclooxygenase 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 development of 7,12-dimethylbenz[α]anthracene (DMBA)-induced inflammation and oral squamous cell carcinoma (SCC). A hamster cheek pouch model was used in which 0.5% DMBA was applied topically onto 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 μl) onto the left cheek pouch three times for 1 week (short-term study) or chronically for 18 weeks. Zyflamend significantly reduced infiltration of inflammatory cells, incidence of hyperplasia and dysplastic lesions, bromodeoxyuridine-labeling index as well as number of SCC in a concentration-dependent manner. Application of Zyflamend (100 μl) reduced formation of leukotriene B4 (LTB4) by 50% compared with DMBA-treated tissues. The reduction of LTB4 was concentration dependent. The effect of Zyflamend on inhibition of LTB4 formation was further confirmed with in vitro cell-based assay. Adding LTB4 to RBL-1 cells, a rat leukemia cell line expressing high levels of 5-LOX and LTA4 hydrolase, partially blocked antiproliferative effect of Zyflamend. This study demonstrates that Zyflamend inhibited LTB4 formation 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 countries and remains problematic in that few effective therapies are available 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 cancer 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 malignant 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). Additionally, 5-LOX enzyme activity is also elevated in human oral cancer. A downstream metabolite of 5-LOX, leukotriene B4 (LTB4), was shown 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, increase vascular 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 inhibited the incidence of SCC in the 7,12-dimethylbenz[α]anthracene (DMBA)-induced hamster cheek pouch model (15). Inhibition of AA metabolism (COX-2 and 5-LOX) by curcumin has also been suggested as a key mechanism of its anticarcinogenic action in DMBA-mediated hamster oral carcinoma (16). Finally, we have demonstrated previously that LTB4 promotes oral cacinogenesis, whereas multiple 5-LOX inhibitors have chemopreventive effects on cancer development in the same animal model system (17).

There is increasing evidence that natural products might offer a protective 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-inflammatory 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 factor-κβ-associated genes involved with cancer cell invasion, metastases 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 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 devastating disease.

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 microliter of Zyflamend per milliliter of tissue culture media. The eicosanoids [Prostaglandin E2 (PGE2), LTB4, 5-HETE, 12-HETE, 15-HETE and 13-hydroxyoctadeca-9Z, 11E-dienoic acid (13-HODE)] and deuterated eicosanoid 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-LTA4 hydrolase antibodies were obtained from Cayman Chemical Company, anti-5-LOX antibody was purchased from Research Diagnostics (Flanders, NJ) and anti-β-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 antitumor 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 untreated negative control (three animals). The left cheek pouch of the remaining 24 hamsters was topically treated with 0.5% DMBA in 100 μl 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 receiving no further treatment. Groups C–E (six animal per group) were treated topically with 25, 50 or 100 μl 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 examination.

For the long-term study (Experiment 2), animals were housed under the same conditions as described above. Except for 30 hamsters serving as negative control (Group A), the remaining 86 animals were topically treated with 0.5% DMBA in 100 μl 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 μl 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.

The whole cheek pouch was excised and flattened on a transparency plate for 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 = 4/3 πr3 (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 remaining tissue was cut and fixed in 10% buffered formalin for further histopathologic analysis as described previously (15). Basal cell hyperplasia, dysplasia and SCC were diagnosed using established criteria (15,23).

Eicosanoid analyses

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 ethylenediaminetetraacetic acid was added. The sample was then homogenized by an Ultrasonic Processor (Misonix, Farmingdale, NJ) at 0°C for 3 min. A 100 μl 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 μl/ml) at 37°C for 10 min followed by addition of 2.5 μl of calcium ionophore A23187 (1 mM). An aliquot (2.5 μl) 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 μl) and of 10% butylated hydroxytoluene (5 μl). An aliquot (10 μl) of the deuterated relevant eicosanoids (i.e. PGE2-d4, LTB4-d4, 15-HETE-d8, 12-HETE-d8 and 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 under conditions of minimal light. Samples were then reconstituted in 200 μl 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 previously published method reported by our laboratory (25). A Micromass Quattro Ultima Tandem Mass Spectrometer (Waters Corp., Milford, MA) was equipped with an Agilent 1100 HP binary pump high-pressure liquid chromatograph inlet for use in these studies. Eicosanoids were separated using a Luna 3 μ 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 μl/min with a column temperature of 50°C. The sample injection volume was 25 μl. 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 × 10−3 Torr. 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.

Cell line

RBL-1 rat leukemia cells were obtained from the American Type Culture Collection (Manassas, VA) and maintained in a humidified atmosphere containing 5% CO2 at 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 μg/ml streptomycin and 2 mM L-glutamine from GIBCO (Invitrogen Corp.).

Cytotoxicity determination

Cells were grown in modified Eagle’s medium with 10% fetal bovine serum at a density of 1 × 104 cells/well. After a 24 h incubation period, cells were treated with various concentrations of Zyflamend (0.03–2.0 μl/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 Chemical) 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 μl dimethyl sulfoxide. Absorbance was read at a wavelength of 570 nm with a reference wavelength 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 μl/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 ethyleneglycol-bis(aminoethylether)-tetraacetic acid, 5 mM ethylenediaminetetraacetic acid, 30 mM NaF, 40 mM β-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 levels were quantified using the Bio-Rad Dc Protein Assay (Bio-Rad, Hercules, CA). Equal levels of protein (50 μg) were fractionated on precast polyacrylamide 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 chemiluminesence, using the ECL+ detection kit and hyper-film (Amersham Biosciences, Piscataway, NJ). Equal loading of samples was illustrated by western blotting for the presence of β-actin.

For determination of the effect of PGE2, LTB4 and 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 μl/ml). Twenty-four hours after drug treatment, 1 μM 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.

Statistical analysis

Data involving tumor incidence were compared by the χ2 test. 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, DMBA was 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 numerous areas exhibiting hyperplasia (Figure 1B) and dysplasia (Figure 1C) in comparison with that of right cheek pouch of the same animals (Figure 1A). Zyflamend (100 μl) inhibited DMBA-induced inflammation evidenced by the reduction of hyperplasia (Figure 1E) compared with that of DMBA treatment alone (Figure 1D). Quantitatively, after 1 week of treatment, Zyflamend (50 μl) significantly reduced the number of inflammatory cells (P < 0.005), whereas at the dose of 100 μl 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 μl) 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.

Fig. 1.
DMBA-induced oral lesions of hamster cheek pouch after 3 weeks treatment with 0.5% DMBA (AC) indicating the development of hyperplasia (B) and dysplasia (C) comparing with normal epithelial (A). The DMBA-induced inflammation was inhibited by ...
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 ...

Chemoprevention of oral carcinogenesis by Zyflamend in hamster cheek pouch—Experiment 2

The effect of Zyflamend on prevention of development of oral carcinoma in DMBA-induced hamster cheek pouch model was then evaluated. As shown in Table I, Zyflamend (50 and 100 μl) (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 significantly reduced by 37.2 and 41.9% (P < 0.01), respectively, compared 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.

Table I.
Chemopreventive effects of topical Zyflamend on DMBA-induced oral carcinogenensis in hamster cheek pouch (Experiment 2) (mean ± SD)

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 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 μl) 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 μl) (43.5%).

Effect of Zyflamend on eicosanoid metabolism—LTB4 formation

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 μl, 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. 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 significant.

Fig. 3.
Eicosanoid metabolism in Zyflamend-treated DMBA-induced hamster cheek pouch. Zyflamend significantly inhibited LTB4 formation compared with that of control (A), whereas no changes were observed in the level of 5-HETE (B). Interestingly, the levels of ...

Inhibitory effect of Zyflamend on LTB4 formation in RBL-1 cells

In order to further delineate the specific role of Zyflamend on inhibition of LTB4 formation, 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 appropriate in vitro model to determine the effect of Zyflamend on the 5-LOX pathway. As shown in Figure 4, the effect of Zyflamend (1 μl/ml) on biosynthesis of PGE2, LTB4, 15-HETE, 12-HETE, 5-HETE and 13-HODE was examined. Intriguingly, only levels of LTB4 and 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 LTB4 formation in RBL-1 cells was concentration dependent (Figure 4B). Furthermore, the protein expression of 5-LOX was downregulated by Zyflamend (1 μl/ml) as early as 4 h after treatment and this inhibitory effect continued up to 24 h (Figure 4C). However, the expression of LTA4 hydrolase itself was not affected by Zyflamend, suggesting that this multi-herb product selectively inhibited 5-LOX enzyme expression and activity in this cell line.

Fig. 4.
The effect of Zyflamend on eicosanoid metabolism and enzyme expression in RBL-1 cells. (A) Zyflamend significantly inhibited the production of both LTB4 and 5-HETE in RBL-1 cells. (B) The inhibitory effect of Zyflamend on formation of LTB4 was in a concentration-dependent ...

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 compared. Among the 10 herbs within Zyflamend, rosemary and Chinese goldthread inhibited 50% of cell growth at concentrations of 4.6 and 6.0 μg/ml, respectively (supplementary Table II is available at Carcinogenesis Online). Interestingly, only rosemary markedly reduced the formation of LTB4 by 81% when the RBL-1 cells were treated with the concentrated form of the individual herbs at concentrations equivalent to 0.5 μl/ml of Zyflamend. Zyflamend itself (0.5 μl/ml), however, still had the strongest effect on inhibition of LTB4 in RBL-1 cells compared with that of individual 10 herbs tested (Figure 5A). The inhibitory effect of rosemary on LTB4 formation was concentration dependent (Figure 5B).

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 LTB4 in RBL-1 cells. However, Zyflamend inhibited formation of LTB4 in RBL-1 cells more strongly ...

Effect of LTB4 ‘add-back’ on Zyflamend-mediated inhibition of cell proliferation

Addition of PGE2 and 5-HETE to RBL-1 cells failed to counteract or block the inhibition of cell proliferation produced by Zyflamend. In contrast, when 30 nM LTB4 was added to cells treated with 1 μl/ml of Zyflamend, a concentration that inhibited proliferation of RBL-1 cells by 50%, a near doubling of cell proliferation occurred (Figure 5C). These results point out the importance of this particular eicosanoid in Zyflamend-mediated inhibition of RBL-1 cell proliferation.


This study clearly demonstrates that Zyflamend not only inhibits DMBA-induced inflammation but also prevents the further development of oral carcinogenesis as evidenced by reductions in skin tissue exhibiting 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 LTB4 formation.

The development of oral cancer is a multistep process requiring initiation, 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 existence 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 hamster 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 hyperplasia 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 Zyflamend in prevention of oral cancer development. Intriguingly, Zyflamend (100 μl) consistently inhibited the development of hyperplasia and dysplasia in both short-term (3-week treatment) and long-term experiments (6-week treatment) by >50%. In contrast, Zyflamend 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 μl (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 agent.

Many mechanisms have been proposed as etiologic factors in development of DMBA hamster oral carcinoma, such as p53 and K-ras mutations and overexpression of inducible nitric oxide synthase (3033). In addition, dysregulated bioactive lipid metabolism is also believed to be important. As an important AA-metabolizing enzyme, 5-LOX has been found to be markedly upregulated in stromal inflammatory cells and epithelial cells at the early stage of human oral SCC (15). The downstream 5-LOX product, LTB4, was remarkably elevated in DMBA-induced hamster oral carcinoma compared with PGE2 levels 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 LTB4 was elevated in DMBA-induced hamster cheek pouch. Our study failed to find an elevation of PGE2 in the DMBA-treated hamster cheek pouch model (data not shown). Interestingly, the level of LTB4 was significantly 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 reduction 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 examined 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, LTB4 and 5-HETE, in RBL-1 cells. Adding LTB4 back to Zyflamend-treated RBL-1 cells partially blocked the antiproliferative effect of Zyflamend, suggesting a strong link between LTB4 presence and cell proliferation. Interestingly, in comparing the inhibition of Zyflamend on both LTB4 and 5-HETE in RBL-1 cells, only LTB4 levels 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 inhibitory action on 5-LOX. Among the 10 herbs tested, rosemary, at a comparable level to that contained in Zyflamend, markedly reduced formation of LTB4 as 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 reduction of LTB4 formation 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 LTB4 formation in the RBL-1 cell assay, these agents have previously been reported to exhibit chemopreventive activity 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 formation 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 metabolism. 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 carcinogenesis at the post-initiation stage. The chemopreventive effect of Zyflamend might be, at least partially, associated with its anti-inflammatory 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 material

Supplementary Tables I and II can be found at


NewChapter (Brattleboro, VT) LS2005-13655WC.

Supplementary Material

Supplementary Data:


Conflict of Interest Statement: None declared.



arachidonic acid
hydroeicosatetraenoic acid
13-hydroxyoctadeca-9Z, 11E-dienoic acid
leukotriene B4
mass spectrometry
phosphate-buffered saline
prostaglandin E2
squamous cell carcinoma


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