Skip Navigation


Carcinogenesis Advance Access originally published online on April 21, 2006
Carcinogenesis 2006 27(9):1902-1908; doi:10.1093/carcin/bgl039
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
27/9/1902    most recent
bgl039v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (4)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Sun, Z.
Right arrow Articles by Chen, X.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sun, Z.
Right arrow Articles by Chen, X.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Involvement of the 5-lipoxygenase/leukotriene A4 hydrolase pathway in 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral carcinogenesis in hamster cheek pouch, and inhibition of carcinogenesis by its inhibitors

Zheng Sun1,2,{dagger}, Sandeep Sood3,{dagger}, Ning Li4, Divya Ramji3, Peiying Yang5, Robert A. Newman5, Chung S. Yang3 and Xiaoxin Chen1,2,3,*

1 Cancer Research Program, Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University NC 27707, USA
2 Faculty of Stomatology, Capital University of Medical Sciences Beijing 100050, China
3 Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, State University of New Jersey 164 Frelinghuysen Road, Piscataway, NJ 08854, USA
4 Division of Food Toxicology, Institute of Nutrition and Food Hygiene, Chinese Center for Disease Control Beijing 100050, China
5 Pharmaceutical Development Center, UT M.D. Anderson Cancer Center 8000 El Rio, Houston, TX 77054, USA

*To whom correspondence and requests for reprints should be addressed at: Cancer Research Program, Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, 700 George Street, Durham, NC 27707, USA. Tel: +1 919 530 6425; Fax: +1 919 530 7998; Email: lchen{at}nccu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Previous studies have shown that aberrant arachidonic acid (AA) metabolism, especially cyclooxygenase-2 (Cox-2) and 5-lipoxygenase (5-Lox) pathways, are activated during oral carcinogenesis, and can be targeted for cancer prevention. This study was designed to investigate the importance of 5-Lox/leukotriene A4 hydrolase (LTA4H) pathway of AA metabolism in 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster cheek pouch carcinogenesis. In a short-term study, topical application of DMBA for 3 weeks induced severe inflammation and aberrant AA metabolism. Subsequent topical treatment with zileuton, celecoxib, or their combination for 1 week significantly suppressed aberrant AA metabolism and cell proliferation in the oral epithelium. Interestingly, zileuton was effective in inhibiting biosynthesis of multiple AA metabolites, including leukotriene B4 (LTB4), 5-, 12-, 15-hydroxyeicosatetraenoic acid and prostaglandin E2 (PGE2), while celecoxib only suppressed PGE2 biosynthesis significantly at a high dose. In a long-term carcinogenesis study topical application of LTB4 or PGE2 enhanced oral carcinogenesis by increasing the incidence and volume of visible tumors, and the incidence of squamous cell carcinoma (SCC). To further examine the role of LTB4 in oral carcinogenesis, two LTA4H inhibitors, bestatin and SA6541, were evaluated in a long-term chemoprevention experiment. Both agents significantly inhibited SCC, and such an inhibition correlated with reduced levels of LTB4 in hamster cheek pouch. In summary, our studies have demonstrated that 5-Lox/LTA4H pathway is one of the major AA-metabolizing pathways involved in DMBA-induced oral carcinogenesis in hamsters, and may be targeted for chemoprevention.

Abbreviations: AA, arachidonic acid; BrdU, bromodeoxyuridine; Cox, cyclooxygenase; DMBA, 7,12-dimethylbenz[a]anthracene; HETE, hydroxyeicosatetraenoic acid; LC/MS/MS, high performance liquid chromatography/electrospray ionization tandem mass spectrometry; LTA4H, leukotriene A4 hydrolase; LTB4, leukotriene B4; LTC4S, leukotriene C4 synthase; 5-Lox, 5-lipoxygenase; PGE2, prostaglandin E2; SCC, squamous cell carcinoma


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Oral cancer is a common neoplasm worldwide, especially in developing countries such as Sri Lanka, India, Brazil and Philippines, where it constitutes up to 25% of all kinds of cancer (1). In the United States ~29 370 new cases and 7320 deaths were expected in 2005 (2). Despite improvements in radiotherapy and chemotherapy, the 5-year survival rate has not improved significantly over the past decade and remains ~50% (3,4). Hence it is important to understand the pathogenesis of oral cancer in order to design new and effective chemopreventive strategies.

Aberrant AA metabolism has been suggested to play an important role in the development of human oral cancer, with the involvement of both cyclooxygenase (Cox) and lipoxygenase (Lox) pathways (5). During carcinogenesis the dynamic equilibrium between the Lox pathways may be disturbed and shift from the anti-carcinogenic (15-Lox) towards the pro-carcinogenic (5-, 8- and 12-Lox) pathways (6). The 5-Lox pathway has been well studied in inflammation and can lead to the formation of several groups of major metabolites: LTA4H dependent biosynthesis of LTB4, leukotriene C4 synthase (LTC4S) dependent biosynthesis of cysteinyl leukotrienes (LTC4, LTD4 and LTE4), 5-hydroxyeicosatetraenoic acid (5-HETE) and 5-oxoETEs (7). These metabolites have been shown to recruit and activate inflammatory cells, increase vascular permeability and induce smooth muscles contraction (8). In addition, 5-Lox knockout mice were found to be more resistant to inflammation or certain inflammation-associated diseases (e.g. atherosclerosis), but more susceptible to infections, than wild-type mice (9).

Recently, 5-Lox has been shown to be overexpressed in prostrate (10), pancreatic (11), colon (12), bladder (13), testicular (14), and esophageal cancers (15). In colon cancer, 5-Lox overexpression was also negatively associated with clinical prognosis (12). In pancreatic cancer cells, 5-Lox played an important role in mediating oxidative stress and cell survival induced by extracellular matrix (16). Inhibitors of the 5-Lox pathway were shown to possess anti-proliferative and pro-apoptotic activities in various cancer cells (17–21). In vivo, these inhibitors suppressed carcinogenesis in animal models of lung (22), skin (23), pancreatic (24,25) and esophageal cancers (15). In the oral cavity, LTB4 was found to be 10- to 30-fold higher in hamster and human squamous cell carcinoma (SCC) than in normal tissues (26). We have shown previously that 5-Lox was overexpressed in hamster and human oral cancer, and zileuton, a specific 5-Lox inhibitor, was able to suppress the development of 7,12-dimethylbenz[a]anthracene (DMBA)-induced SCC in hamster cheek pouch (27). The above data suggested an important role of the 5-Lox/LTA4H pathway in the pathogenesis of oral cancer.

The major goals of this study were to investigate the importance of 5-Lox/LTA4H pathway in DMBA-induced carcinogenesis in hamster cheek pouch, and to evaluate the chemopreventive activity of their respective inhibitors. Initially, zileuton was evaluated in a short-term experiment for its effects on AA metabolism and cell proliferation in hamster oral epithelium. The promoting effect of LTB4 on carcinogenesis was then examined, and finally two LTA4H inhibitors (bestatin and SA6541) were tested for their chemopreventive effects.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Chemicals
LTB4, PGE2 (16,16-dimethyl prostaglandin E2; PGE2) and bestatin were obtained from Cayman Chemical (Ann Arbor, MI). They were dissolved in mineral oil (Sigma Chemical Company, St Louis, MO) at appropriate concentrations, aliquoted and stored at –20°C. SA6541, S-(4-dimethyl-aminobenzyl)-N-[(2S)-3-mercapto-2-methylpropionyl]-L-cysteine, was obtained as a gift from Dr Fumio Tsuji at Santen Pharmaceutical Inc. (Osaka, Japan) (28,29).

Short-term effects of topical zileuton and celecoxib on aberrant AA metabolism and cell proliferation in DMBA-treated hamster cheek pouch
This study was conducted at Rutgers University under Protocol number 91-024 as per the experimental scheme shown in Figure 1A. Male Syrian golden hamsters (6 weeks old) weighing 60–80 g were purchased from Harlan (Indianapolis, IN) and housed 4 per cage in a room with controlled temperature and humidity with 12 h light/dark cycles. All animals were given AIN-93M diet (Research Diets, New Brunswick, NJ) and water ad libitum. After 1 week of acclimatization, the animals were divided into two groups, with Group 1A serving as the negative control (9 animals). The left cheek pouch of the remaining 42 hamsters was topically treated with 0.5% DMBA in 100 µl mineral oil using a paintbrush three times per week for 3 weeks. They were then randomly divided into six groups with Group 1B (12 animals) serving as the positive control and receiving no further treatment. Groups 1C (3% zileuton), 1D (6% zileuton), 1E (3% celecoxib), 1F (6% celecoxib) and 1G (3% zileuton and 3% celecoxib) were treated with the respective chemopreventive agents topically in 100 µl mineral oil three times per week for 1 week (6 animals each). Six animals were sacrificed from the negative control (Group 1A) and the positive control (Group 1B) at the end of DMBA treatment (Week 3). The rest of the animals were sacrificed at the end of the experiment (Week 4), 6 h after the last treatment. The animals were injected with bromodeoxyuridine (BrdU) i.p. at 50 mg/kg body wt 2 h prior to sacrificing. The cheek pouch was harvested, one half being snap frozen in liquid nitrogen for analysis of AA metabolites, and the other half being fixed in 10% phosphate-buffered saline (PBS)-buffered formalin for histopathology.


Figure 1
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Experimental schemes of DMBA-induced oral carcinogenesis in hamster cheek pouch. (A) short-term study on the effects of zileuton and celecoxib on AA metabolism and cell proliferation in DMBA-treated hamster cheek pouch; (B) long-term effects of LTB4 and PGE2 on oral carcinogenesis; (C) long-term chemopreventive effects of LTA4H inhibitors, bestatin and SA6541, on oral carcinogenesis.

 
Effects of topical LTB4 and PGE2 application on DMBA-induced carcinogenesis in hamster cheek pouch
The animals were housed under the same conditions as mentioned above. They were randomly divided into six groups according to the experimental plan shown in Figure 1B. Group 2A was the negative control while animals in Groups 2B, 2C and 2D were treated with topical DMBA (0.5%, 100 µl, 35 animals each) three times per week for 3 weeks. In addition, Groups 2C and 2D were further treated with topical LTB4 (2 µM) or PGE2 (100 µM) three times per week for another 21 weeks, respectively. At Week 24, all the animals were sacrificed and the left cheek pouch harvested and fixed in 10% PBS-buffered formalin for histopathology.

Effects of LTA4H inhibitors (bestatin and SA6541) on DMBA-induced carcinogenesis in hamster cheek pouch
The animals were housed in the same conditions as mentioned above. The experimental plan is shown in Figure 1C. Group 3A served as the negative control while animals in rest of the groups were topically treated with 100 µl of 0.5% DMBA in mineral oil using a paintbrush three times per week for 6 weeks, and then randomly divided into four groups. Group 3B received no further treatment. Groups 3C and 3D received 20 mM and 40 mM bestatin respectively, while Group 3E received 1 mM of SA6541, three times per week for an additional 18 weeks. At the end of Week 24, all the animals were sacrificed and the left hamster cheek pouch was harvested, one half of which snap frozen in liquid nitrogen and the other half fixed in 10% PBS-buffered formalin for histopathology.

Histopathological analyses
The whole cheek pouch was excised and flattened on a transparency plate for counting the number of visible tumors. The length, width and height of each tumor were measured with a caliper and the tumor volume calculated using the formula: volume = 4/3{pi}r3 (where r was the average radius of the three diameter measurements in mm). Formalin-fixed pouches were cut into 4–6 pieces of approximately equal width, Swiss-rolled, processed and embedded in paraffin. Thirty sections (5 µm) of each sample were cut and the 1st, 15th and 30th slides were stained with hematoxylin and eosin for histopathological analysis. Basal cell hyperplasia, dysplasia, SCC and papillomas were diagnosed with established criteria (30,31).

Cell proliferation analysis
To assess cell proliferation in the squamous epithelium of hamster oral mucosa, bromodeoxyuridine (BrdU) immunostaining was performed on formalin-fixed, paraffin-embedded tissue sections. The avidin–biotin peroxidase method was used with a rat monoclonal antibody (Serotec, Raleigh, NC) at a concentration of 5 µg/ml. Three noncontiguous, randomly selected fields under x400 were photographed per sample, and the sum of all positive cells was divided by total number of cells. Image-Pro Plus 4.5 software (Media Cybernetics, Silver Spring, MD) was used for cell counting.

Profiling of AA metabolites with LC/MS/MS
After homogenization in a buffer containing 10 µM zileuton and indomethacin, tissues were extracted with hexane:ethyl acetate under reduced light conditions after the addition of an internal standard (PGE2-d4). The samples were dried under nitrogen, reconstituted in methanol:2mM ammonium acetate, and analyzed using the established method (32). The levels of LTB4, PGE2, 5-HETE, 12-HETE, and 15-HETE were determined and expressed as nanograms per milligram protein.

Enzyme immunoassay for LTB4
Frozen samples of the hamster oral mucosa were analyzed immediately after being taken out of –80°C freezer. After pulverization and homogenization in a buffer containing 10 µM of zileuton, a part of the sample was used for analyzing the protein concentration while the other was extracted with an organic solvent. The organic extract was dried under nitrogen and reconstituted in the enzyme immunoassay buffer for analysis with a kit according to the manufacturer's instructions (Cayman Chemical). The tissue levels of LTB4 were expressed as picogram per milligram protein.

Statistical analysis
The tumor incidence was compared by the {chi}2-test. One-way ANOVA test was used to compare body weight, number of visible tumors, and number of oral lesions. The tumor volume was analyzed using the Wilcoxon signed rank test. The statistical significance between the levels of AA metabolites among the various groups was analyzed using the Student's t-test.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
The DMBA-induced hamster cheek pouch model is one of the most widely used oral cancer models due to its similarity in histopathological, molecular and biochemical alterations to human oral cancer. Inflammation is an early pathological change in hamster oral epithelium (33,34). Our short-term study mimics the inflammatory response of hamster oral epithelium to carcinogen treatment (Figure 1A). To study the cancer-promoting effects of LTB4 and PGE2, carcinogenesis in hamster oral epithelium was initiated with DMBA for 3 weeks, and then promoted with LTB4 or PGE2 for 21 weeks (Figure 1B). In the long-term chemoprevention model, topical application of DMBA (0.5%, three times per week, 6 weeks) led to the development of hyperplasia and dysplasia in 100 and 75% of the animals, respectively. Without further DMBA treatment, 77% of the animals developed SCC at Week 24 (Figure 1C) (35). This post-initiation model mimics human patients with precancerous lesions, e.g. former and current smokers.

Overall the animals appeared healthy throughout the three studies. However, DMBA-treated animals weighed less than untreated animals by 4–8% probably due to the inflammation or irritation in the cheek pouch. The body weight of the animals was monitored once a week and increased steadily with less than 10% variation among various groups (data not shown).

Short-term effects of topical zileuton and celecoxib on aberrant AA metabolism and cell proliferation in DMBA-treated hamster cheek pouch
The aim of this study was to examine the effects of 5-Lox and Cox-2 inhibitors, zileuton and celecoxib, on DMBA-induced aberrations of AA metabolism in hamster cheek pouch. DMBA-treated hamster cheek pouch appeared reddish, hyperkeratotic and inflamed. The tissue levels of both LTB4 and PGE2 were significantly higher after DMBA treatment for 3 weeks (Figure 2A and B). The level of LTB4 increased by ~58 fold as compared to the negative control (Group 1A), while the level of PGE2 increased by only ~3 fold. Even 1 week after DMBA was discontinued, the levels of LTB4 and PGE2 were still much higher than the control group. This suggests that the 5-Lox pathway may be the major AA-metabolizing pathway activated by DMBA.


Figure 2
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Effects of topical zileuton and celecoxib on AA metabolism and cell proliferation in DMBA-treated hamster cheek pouch. The concentrations of AA metabolites were expressed as ng/mg protein. At the end of DMBA treatment (Week 3) Group 1A was compared with Group 1B. At the end of the experiment (Week 4), Group 1A (negative control), Group 1C (3% zileuton), Group 1D (6% zileuton), Group 1E (3% celecoxib), Group 1F (6% celecoxib) and Group 1G (3% zileuton and 3% celecoxib) were compared with Group 1B (positive control), respectively. Topical treatment with zileuton (3 and 6% in Groups 1C and 1D, respectively) significantly reduced the levels of LTB4 (A), PGE2 (B), 5-HETE (C), 12-HETE (D) and 15-HETE (E); whereas, celecoxib (only 6%) significantly reduced the level of PGE2 (B), but not those of other AA metabolites. DMBA treatment for 3 weeks produced hyperproliferation in hamster cheek pouch (F). Zileuton alone or in combination with celecoxib significantly suppressed cell proliferation. Student's t-test was used for the statistical analysis between the groups (*P < 0.05; **P < 0.01).

 
Topical treatment with zileuton (3 and 6% in Groups 1C and 1D, respectively) significantly reduced the levels of LTB4 (Figure 2A), PGE2 (Figure 2B), 5-HETE (Figure 2C), 12-HETE (Figure 2D) and 15-HETE (Figure 2E); whereas, celecoxib (only 6%) significantly reduced the level of PGE2 (Figure 2B), but not those of other AA metabolites. These results suggested that suppression of 5-Lox pathway by zileuton was effective in ameliorating the aberrant AA metabolism.

DMBA treatment for 3 weeks produced hyperproliferation in hamster cheek pouch (Figure 2F). One week after discontinuation of DMBA (Week 4), the rate of cell proliferation reduced dramatically. Treatment with zileuton (3 and 6%) for 1 week significantly inhibited cell proliferation. Combination of zileuton and celecoxib (3% each) had similar effects on AA metabolism and cell proliferation as zileuton alone. No synergy between zileuton and celecoxib could be demonstrated based on our data.

Effects of topical LTB4 and PGE2 application on DMBA-induced carcinogenesis in hamster cheek pouch
This long-term carcinogenesis study was undertaken to determine whether topical application of LTB4 and PGE2 would promote DMBA-induced carcinogenesis in hamster cheek pouch. Application of 0.5% DMBA three times per week for 3 weeks led to the development of visible tumors (45.7%, 16/35) and SCC (20%, 7/35) at the end of 24 weeks.

Topical treatment with either LTB4 (2 µm) or PGE2 (100 µm) for 21 weeks promoted carcinogenesis initiated by DMBA. LTB4 treatment increased the incidence, number and volume of visible tumors, as well as the number of hyperplasia, number of dysplasia, number and incidence of SCC. PGE2 treatment only increased the number of visible tumors and the incidence of SCC (Table I). LTB4 and PGE2 are not carcinogens by themselves since topical application of LTB4 or PGE2 alone for 21 weeks did not produce any visible tumors or SCC in hamster cheek pouch (data not shown).


View this table:
[in this window]
[in a new window]

  		Table I Effects of topical LTB4 and PGE2 on DMBA-induced carcinogenesis in hamster cheek pouch

 
Chemopreventive effects of bestatin and SA6541 on DMBA-induced carcinogenesis in hamster cheek pouch
This long-term study was undertaken to evaluate the effects of LTA4H inhibitors, bestatin and SA6541, on oral carcinogenesis in hamster cheek pouch. Application of DMBA three times per week for 6 weeks led to the development of both visible tumors and SCC in hamster cheek pouch at the end of 24 weeks.

Treatment with bestatin (20 or 40 mM) significantly inhibited the incidence, number and volume of visible tumors, as well as the numbers of hyperplasia, dysplasia and SCC (Table II). High-dose bestatin (40 mM) was more effective as it also significantly inhibited the incidence of SCC and the tissue level of LTB4.


View this table:
[in this window]
[in a new window]

  		Table II Chemopreventive effects of topical bestatin and SA6541 on DMBA-induced carcinogenesis in hamster cheek pouch

 
SA6541 exerted similar chemopreventive effects as 40 mM bestatin. It inhibited not only carcinogenesis, but also the tissue level of LTB4 (Table II). Inhibition of carcinogenesis by bestatin and SA6541 was associated with reduced biosynthesis of LTB4, suggesting that these compounds exerted their chemopreventive effects through inhibition of LTA4H.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Aberrant AA metabolism has been well established as an important factor in oral carcinogenesis. Previous studies have focused mainly on the Cox-2 pathway. Cox-2 was shown to be overexpressed in both premalignant and malignant lesions of the oral cavity with increasing levels of expression from hyperplasia to dysplasia and SCC (36). It has been shown previously that overexpression of Cox-2 is an early event in oral epithelial cells exposed to tobacco and areca nut constituents, and might occur prior to alterations of other biomarkers related to apoptosis and angiogenesis (37). Consistent with these observations, other studies have indicated that the PGE2 levels were higher in oral SCC tissue as compared to normal tissue (38). Cox-2 inhibitors have also been shown to retard the growth of human oral cancer cells in both PGE2-dependent and -independent manners (39), and suppressed 4-nitroquinoline-1-oxide-induced tongue cancer in rats (40), as well as DMBA-induced oral cancer in hamster cheek pouch (27).

The present study and previous studies have clearly shown that 5-Lox is also a critical AA metabolism pathway in oral carcinogenesis (15,26,27), based on the following evidence: (i) 5-Lox was overexpressed during oral carcinogenesis in hamsters and humans; (ii) The level of LTB4 was much higher in hamster and human oral SCC than in normal tissues; (iii) A 5-Lox inhibitor, zileuton, inhibited aberrant AA metabolism and cell hyperproliferation induced by DMBA in hamster cheek pouch; (iv) Chemical inhibitors of 5-Lox and LTA4H had chemopreventive effects in the DMBA model of oral carcinogenesis; (v) Modulation of the levels of LTB4 in hamster cheek pouch correlated with the chemopreventive effects of these chemical compounds; (vi) Exogenous LTB4 promoted DMBA-induced oral carcinogenesis in hamster cheek pouch.

Furthermore, a recent study has showed that 5-Lox is overexpressed in esophageal SCC, and its inhibitors suppressed proliferation and induced apoptosis of esophageal cancer cells (41). Esophageal SCC is known to share similar etiology and mechanism as oral SCC. According to our short-term study, zileuton was effective in modulating AA metabolism pathways and cell proliferation significantly. In our long-term study LTA4H inhibitors reduced the incidence of SCC and suppressed LTB4 biosynthesis in hamster cheek pouch. All these data supported importance of 5-Lox/LTA4H pathway in oral carcinogenesis.

LTB4 may promote carcinogenesis through multiple mechanisms. Both in vitro and in vivo studies have demonstrated that it stimulates cell proliferation (42–45), regulates chemotaxis of inflammatory cells, and promotes the formation of reactive oxygen species (46,47). It has also been shown to inhibit apoptosis in intestinal epithelial cells by regulating the expression of Cox-2, ß-catenin and Bcl-2 (48). According to an in vivo study on human skin, topical LTB4 quickly recruited polymorphonuclear leukocytes and T lymphocytes, while epidermal proliferation was stimulated thereafter (49). These observations suggested that the activated 5-Lox/LTA4H pathway might promote oral carcinogenesis by stimulating inflammation in oral epithelium and inducing hyperproliferation in DMBA-initiated cells. Similar to LTB4, topical application of PGE2 also promoted carcinogenesis in this study. This is in line with previous studies showing cancer promoting effects of exogenous PGE2 (50,51).

A 5-Lox inhibitor (zileuton) and two LTA4H inhibitors (bestatin and SA6541) were topically administered in this study. The purpose was to maximize local distribution in the oral mucosa, and minimize systemic side effects. For example, in the short-term study, 100 µl of 3 and 6% zileuton was equivalent to 20 and 40 p.p.m., respectively, if it was given in the diet ad libitum. Such low doses were not expected to produce significant side effects. Bestatin was chosen because it has demonstrated chemopreventive effects on gastric and esophageal cancer in animal models (52–54). A recent clinical trial showed that bestatin significantly improved postoperative survival of patients with completely resected stage I lung SCC (55). Another LTA4H inhibitor, SA6541, is known to inhibit edema and neutrophil influx in a mouse dermatitis model (28,29).

In summary, we show here that the 5-Lox/LTA4H pathway of AA metabolism is a major pathway involved in DMBA-induced oral carcinogenesis in hamster cheek pouch, and may be targeted for chemoprevention of oral cancer.


   Notes
 
{dagger}These authors contributed equally to this work. Back


   Acknowledgments
 
The following grant supports are acknowledged: NIH grants R01-CA101235, U56-CA092070; Beijing Natural Science Foundation No. 7032020; and National Natural Science Foundation of China No. 30271414.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Magrath I. and Litvak J. (1993) Cancer in developing countries: opportunity and challenge. J. Natl Cancer Inst. 85:862–874.[Abstract/Free Full Text]
  2. Jemal A., Murray T., Ward E., Samuels A., Tiwari R.C., Ghafoor A., Feuer E.J., Thun M.J. (2005) Cancer statistics, 2005. CA Cancer J. Clin. 55:10–30.[Abstract/Free Full Text]
  3. Jemal A., Tiwari R.C., Murray T., Ghafoor A., Samuels A., Ward E., Feuer E.J., Thun M.J. (2004) Cancer statistics, 2004. CA Cancer J. Clin. 54:8–29.[Abstract/Free Full Text]
  4. Funk G.F., Karnell L.H., Robinson R.A., Zhen W.K., Trask D.K., Hoffman H.T. (2002) Presentation, treatment, and outcome of oral cavity cancer: a National Cancer Data Base report. Head Neck 24:165–180.[CrossRef][ISI][Medline]
  5. el-Hakim I.E. and Langdon J.D. (1991) Arachidonic acid cascade and oral squamous cell carcinoma. Clin. Otolaryngol. Allied Sci. 16:563–573.[Medline]
  6. Shureiqi I. and Lippman S.M. (2001) Lipoxygenase modulation to reverse carcinogenesis. Cancer Res. 61:6307–6312.[Abstract/Free Full Text]
  7. Haeggstrom J.Z. and Wetterholm A. (2002) Enzymes and receptors in the leukotriene cascade. Cell Mol. Life Sci. 59:742–753.[CrossRef][ISI][Medline]
  8. Peters-Golden M. and Brock T.G. (2003) 5-lipoxygenase and FLAP. Prostaglandins Leukot Essent Fatty Acids 69:99–109.[CrossRef][ISI][Medline]
  9. Funk C.D., Chen X.S., Johnson E.N., Zhao L. (2002) Lipoxygenase genes and their targeted disruption. Prostaglandins Other Lipid Mediat 68–69:303–312.
  10. Gupta S., Srivastava M., Ahmad N., Sakamoto K., Bostwick D.G., Mukhtar H. (2001) Lipoxygenase-5 is overexpressed in prostate adenocarcinoma. Cancer 91:737–743.[CrossRef][ISI][Medline]
  11. Hennig R., Ding X.Z., Tong W.G., Schneider M.B., Standop J., Friess H., Buchler M.W., Pour P.M., Adrian T.E. (2002) 5-Lipoxygenase and leukotriene B (4) receptor are expressed in human pancreatic cancers but not in pancreatic ducts in normal tissue. Am. J. Pathol. 161:421–428.[Abstract/Free Full Text]
  12. Ohd J.F., Nielsen C.K., Campbell J., Landberg G., Lofberg H., Sjolander A. (2003) Expression of the leukotriene D4 receptor CysLT1, COX-2, and other cell survival factors in colorectal adenocarcinomas. Gastroenterology 124:57–70.[CrossRef][ISI]
  13. Yoshimura R., Matsuyama M., Tsuchida K., Kawahito Y., Sano H., Nakatani T. (2003) Expression of lipoxygenase in human bladder carcinoma and growth inhibition by its inhibitors. J. Urol. 170:1994–1999.[CrossRef][ISI][Medline]
  14. Yoshimura R., Matsuyama M., Mitsuhashi M., Takemoto Y., Tsuchida K., Kawahito Y., Sano H., Nakatani T. (2004) Relationship between lipoxygenase and human testicular cancer. Int. J. Mol. Med. 13:389–393.[ISI][Medline]
  15. Chen X., Wang S., Wu N., et al. (2004) Overexpression of 5-lipoxygenase in rat and human esophageal adenocarcinoma and inhibitory effects of zileuton and celecoxib on carcinogenesis. Clin. Cancer Res. 10:6703–9.[Abstract/Free Full Text]
  16. Edderkaoui M., Hong P., Vaquero E.C., Lee J.K., Fischer L., Friess H., Buchler M.W., Lerch M.M., Pandol S.J., Gukovskaya A.S. (2005) Extracellular matrix stimulates reactive oxygen species production and increases pancreatic cancer cell survival through 5-lipoxygenase and NADPH oxidase. Am. J. Physiol. Gastrointest. Liver Physiol. 289:G1137–G1147.[Abstract/Free Full Text]
  17. Anderson K.M., Alrefai W.A., Bonomi P.A., Anderson C.A., Dudeja P., Harris J.E. (2000) A genomic response of H-358 bronchiolar carcinoma cells to MK 886, an inhibitor of 5-lipoxygenase, assessed with a cDNA array. Anticancer Res. 20:2433–2439.[ISI][Medline]
  18. Avis I.M., Jett M., Boyle T., Vos M.D., Moody T., Treston A.M., Martinez A., Mulshine J.L. (1996) Growth control of lung cancer by interruption of 5-lipoxygenase-mediated growth factor signaling. J. Clin. Invest. 97:806–813.[ISI][Medline]
  19. Fan X.M., Tu S.P., Lam S.K., Wang W.P., Wu J., Wong W.M., Yuen M.F., Lin M.C., Kung H.F., Wong B.C. (2004) Five-lipoxygenase-activating protein inhibitor MK-886 induces apoptosis in gastric cancer through upregulation of p27kip1 and bax. J. Gastroenterol. Hepatol. 19:31–37.[CrossRef][ISI][Medline]
  20. Moody T.W., Leyton J., Martinez A., Hong S., Malkinson A., Mulshine J.L. (1998) Lipoxygenase inhibitors prevent lung carcinogenesis and inhibit non-small cell lung cancer growth. Exp. Lung Res. 24:617–628.[ISI][Medline]
  21. Tsukada T., Nakashima K., Shirakawa S. (1986) Arachidonate 5-lipoxygenase inhibitors show potent antiproliferative effects on human leukemia cell lines. Biochem. Biophys Res. Commun. 140:832–836.[CrossRef][ISI][Medline]
  22. Gunning W.T., Kramer P.M., Steele V.E., Pereira M.A. (2002) Chemoprevention by lipoxygenase and leukotriene pathway inhibitors of vinyl carbamate-induced lung tumors in mice. Cancer Res. 62:4199–4201.[Abstract/Free Full Text]
  23. Jiang H., Yamamoto S., Kato R. (1994) Inhibition of two-stage skin carcinogenesis as well as complete skin carcinogenesis by oral administration of TMK688, a potent lipoxygenase inhibitor. Carcinogenesis 15:807–812.[Abstract/Free Full Text]
  24. Wenger F.A., Kilian M., Achucarro P., Heinicken D., Schimke I., Guski H., Jacobi C.A., Muller J.M. (2002) Effects of Celebrex and Zyflo on BOP-induced pancreatic cancer in Syrian hamsters. Pancreatology 2:54–60.[CrossRef][ISI][Medline]
  25. Wenger F.A., Kilian M., Bisevac M., Khodadayan C., von Seebach M., Schimke I., Guski H., Muller J.M. (2002) Effects of Celebrex and Zyflo on liver metastasis and lipidperoxidation in pancreatic cancer in Syrian hamsters. Clin. Exp. Metastasis 19:681–687.[CrossRef][ISI][Medline]
  26. el-Hakim I.E., Langdon J.D., Zakrzewski J.T., Costello J.F. (1990) Leukotriene B4 and oral cancer. Br. J. Oral. Maxillofac. Surg. 28:155–159.[CrossRef][ISI][Medline]
  27. Li N., Sood S., Wang S., Fang M., Wang P., Sun Z., Yang C.S., Chen X. (2005) Overexpression of 5-lipoxygenase and cyclooxygenase 2 in hamster and human oral cancer and chemopreventive effects of zileuton and celecoxib. Clin. Cancer Res. 11:2089–2096.[Abstract/Free Full Text]
  28. Tsuji F., Miyake Y., Enomoto H., Horiuchi M., Mita S. (1998) Effects of SA6541, a leukotriene A4 hydrolase inhibitor, and indomethacin on carrageenan-induced murine dermatitis. Eur. J. Pharmacol. 346:81–85.[CrossRef][ISI][Medline]
  29. Tsuji F., Miyake Y., Horiuchi M., Mita S. (1998) Involvement of leukotriene B4 in murine dermatitis models. Biochem. Pharmacol. 55:297–304.[CrossRef][ISI][Medline]
  30. Leininger I. and KJokinen M. (1982) Tumors of the oral cavity, pharynx, oesophagus and stomach. In Turosov V. (Ed.). Pathology of Tumors in Laboratory Animals (IARC, Albany, NY) 3: pp. 167–169.
  31. Kramer I., Lucas R., Pindborg J., Sobin L. (1978) WHO, Collaborating center for oral precancerous lesions: definitions of leukoplakia and related lesions: an aid to studies on oral precancer. Oral Surg. Oral Med. Oral Pathol. 46:518–589.[ISI][Medline]
  32. Kempen E.C., Yang P., Felix E., Madden T., Newman R.A. (2001) Simultaneous quantification of arachidonic acid metabolites in cultured tumor cells using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry. Anal. Biochem. 297:183–190.[CrossRef][ISI][Medline]
  33. Gimenez-Conti I.B. and Slaga T.J. (1993) The hamster cheek pouch carcinogenesis model. J. Cell Biochem. 17F:Suppl., 83–90.
  34. Schwartz J. and Gu X. (2001) Hamster oral cancer model. In Teicher B. (Ed.). Tumor Models in Cancer Research (Humana Press Inc., Totowa, NJ) pp. 141–171.
  35. Li N., Chen X., Liao J., Yang G., Wang S., Josephson Y., Han C., Chen J., Huang M.T., Yang C.S. (2002) Inhibition of 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral carcinogenesis in hamsters by tea and curcumin. Carcinogenesis 23:1307–1313.[Abstract/Free Full Text]
  36. Minter H.A., Eveson J.W., Huntley S., Elder D.J., Hague A. (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. 9:1885–1897.[Abstract/Free Full Text]
  37. Jeng J.H., Wang Y.J., Chiang B.L., Lee P.H., Chan C.P., Ho Y.S., Wang T.M., Lee J.J., Hahn L.J., Chang M.C. (2003) Roles of keratinocyte inflammation in oral cancer: regulating the prostaglandin E2, interleukin-6 and TNF-alpha production of oral epithelial cells by areca nut extract and arecoline. Carcinogenesis 24:1301–1315.[Abstract/Free Full Text]
  38. Karmali R.A., Wustrow T., Thaler H.T., Strong E.W. (1984) Prostaglandins in carcinomas of the head and neck. Cancer Lett. 22:333–336.[CrossRef][ISI][Medline]
  39. Yang C.Y., Meng C.L., Liao C.L., Wong P.Y. (2003) Regulation of cell growth by selective COX-2 inhibitors in oral carcinoma cell lines. Prostaglandins Other Lipid Mediat. 72:115–130.[CrossRef][ISI][Medline]
  40. Shiotani H., Denda A., Yamamoto K., Kitayama W., Endoh T., Sasaki Y., Tsutsumi N., Sugimura M., Konishi Y. (2001) Increased expression of cyclooxygenase-2 protein in 4-nitroquinoline-1-oxide-induced rat tongue carcinomas and chemopreventive efficacy of a specific inhibitor, nimesulide. Cancer Res. 61:1451–1456.[Abstract/Free Full Text]
  41. Hoque A., Lippman S.M., Wu T.T., Xu Y., Liang Z.D., Swisher S., Zhang H., Cao L., Ajani J.A., Xu X.C. (2005) Increased 5-lipoxygenase expression and induction of apoptosis by its inhibitors in esophageal cancer: a potential target for prevention. Carcinogenesis 26:785–791.[Abstract/Free Full Text]
  42. Tong W.G., Ding X.Z., Hennig R., Witt R.C., Standop J., Pour P.M., Adrian T.E. (2002) Leukotriene B4 receptor antagonist LY293111 inhibits proliferation and induces apoptosis in human pancreatic cancer cells. Clin. Cancer Res. 8:3232–3242.[Abstract/Free Full Text]
  43. Qiao L., Kozoni V., Tsioulias G.J., Koutsos M.I., Hanif R., Shiff S.J., Rigas B. (1995) Selected eicosanoids increase the proliferation rate of human colon carcinoma cell lines and mouse colonocytes in vivo. Biochim Biophys Acta 1258:215–223.[Medline]
  44. Kragballe K., Desjarlais L., Voorhees J.J. (1985) Leukotrienes B4, C4 and D4 stimulate DNA synthesis in cultured human epidermal keratinocytes. Br. J. Dermatol. 113:43–52.[ISI][Medline]
  45. Bauer F.W., van de Kerkhof P.C., Maassen-de Grood R.M. (1986) Epidermal hyperproliferation following the induction of microabscesses by leukotriene B4. Br. J. Dermatol. 114:409–412.[CrossRef][ISI][Medline]
  46. Woo C.H., You H.J., Cho S.H., Eom Y.W., Chun J.S., Yoo Y.J., Kim J.H. (2002) Leukotriene B(4) stimulates Rac-ERK cascade to generate reactive oxygen species that mediates chemotaxis. J. Biol. Chem. 277:8572–8578.[Abstract/Free Full Text]
  47. Steiner D.R., Gonzalez N.C., Wood J.G. (2001) Leukotriene B(4) promotes reactive oxidant generation and leukocyte adherence during acute hypoxia. J. Appl. Physiol. 91:1160–1167.[Abstract/Free Full Text]
  48. Ohd J.F., Wikstrom K., Sjolander A. (2000) Leukotrienes induce cell-survival signaling in intestinal epithelial cells. Gastroenterology 119:1007–1018.[CrossRef][ISI][Medline]
  49. de Jong E.M., van Erp P.E., van Vlijmen I.M., van de Kerkhof P.C. (1992) The inter-relation between inflammation and epidermal proliferation in normal skin following epicutaneous application of leukotriene-B4--an immunohistochemical study. Clin. Exp. Dermatol 17:413–420.[CrossRef][ISI][Medline]
  50. Wang D., Buchanan F.G., Wang H., Dey S.K., DuBois R.N. (2005) Prostaglandin E2 enhances intestinal adenoma growth via activation of the Ras-mitogen-activated protein kinase cascade. Cancer Res. 65:1822–1829.[Abstract/Free Full Text]
  51. Kawamori T., Uchiya N., Sugimura T., Wakabayashi K. (2003) Enhancement of colon carcinogenesis by prostaglandin E2 administration. Carcinogenesis 24:985–990.[Abstract/Free Full Text]
  52. Ebihara K., Abe F., Yamashita T., Shibuya K., Hayashi E., Takahashi K., Horinishi H., Enomoto M., Ishizuka M., Umezawa H. (1986) The effect of ubenimex on N-methyl-N'-nitro-N-nitrosoguanidine-induced stomach tumor in rats. J. Antibiot. (Tokyo) 39:966–970.[Medline]
  53. Scornik O.A. and Botbol V. (2001) Bestatin as an experimental tool in mammals. Curr. Drug Metab. 2:67–85.[CrossRef][ISI][Medline]
  54. Chen X., Li N., Wang S., Wu N., Hong J., Jiao X., Krasna M.J., Beer D.G., Yang C.S. (2003) Leukotriene A4 hydrolase in rat and human esophageal adenocarcinomas and inhibitory effects of bestatin. J. Natl Cancer Inst. 95:1053–1061.[Abstract/Free Full Text]
  55. Ichinose Y., Genka K., Koike T., Kato H., Watanabe Y., Mori T., Iioka S., Sakuma A., Ohta M. (2003) Randomized double-blind placebo-controlled trial of bestatin in patients with resected stage I squamous-cell lung carcinoma. J. Natl Cancer Inst. 95:605–610.[Abstract/Free Full Text]
Received January 24, 2006; revised April 6, 2006; accepted April 9, 2006.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 NEJMHome page
M. Peters-Golden and W. R. Henderson Jr.
Leukotrienes
N. Engl. J. Med., November 1, 2007; 357(18): 1841 - 1854.
[Full Text] [PDF]

Home page
 Cancer Res.Home page
K. Srivastava, V. Kundumani-Sridharan, B. Zhang, A. K. Bajpai, and G. N. Rao
15(S)-Hydroxyeicosatetraenoic Acid-Induced Angiogenesis Requires STAT3-Dependent Expression of VEGF
Cancer Res., May 1, 2007; 67(9): 4328 - 4336.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
27/9/1902    most recent
bgl039v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (4)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Sun, Z.
Right arrow Articles by Chen, X.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sun, Z.
Right arrow Articles by Chen, X.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?