Inhibitory Effect Of Topical Application Of COX-2 Inhibitor Celecoxib On DMBA Induced Rat Tongue Carcinoma

Oral squamous cell carcinoma (OSCC) is one of high morbidity and mortality of head and neck cancers, which accounts for about 80% of oral and maxillofacial malignant tumors and occurs cervical lymph node metastasis at an early stage. Currently approved treatments of OSCC are more based on surgery combined with radiotherapy and/or chemotherapy. Although the diagnosis and treatment has greatly improved, but because of the biological behavior of OSCC and specific oral maxillofacial anatomical structure, the recurrence rate and metastasis rate did not reduced significantly, the clinical outcome and survival rate are still not very satisfied.Epidemiological survey found that long-term use of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) treatment of other diseases, can significantly reduce the incidence of colorectal cancer, reduce colon polyps sizes and numbers, and can reduce the risk of malignant colon polyps. Additionaly, the research and clinical observations show NSAIDs can also be used to prevent prostate cancer, bladder cancer, esophageal cancer, gastric cancer, breast cancer and head and neck squamous cell carcinoma. NSAIDs’role in tumor prevention and treatment by inhibiting cyclooxygenase-2 (COX-2) activity become a hot spot in prevention and treatment of malignant tumors. COX-2, an inducible enzyme, was found high overpression in many precancerous lesions and malignant tumors, such as gastrointestinal metaplasia, esophagus, stomach, colon, liver, breast, bladder, non-small cell lung cancer, prostate cancer, nasopharyngeal cancer and oral cancer. COX-2 induced prostaglandin could promote cell proliferation, inhibit apoptosis and stimulate angiogenesis and other functions, so, the inhibition of COX-2 activity may be play a crucial role in prevention of tongue carcinomaRecent studies have confirmed that topically administration of NSAIDs could avoid liver first-pass effect and metabolic side effects associated with gastrointestinal tract administration and gained more and more attention. Oral mucosa (sublingual mucosa and buccal mucosa) route of administration has a good compliance and rich vascular, the advantages of relatively fixed position, more importantly, some fat-soluble drugs through the oral mucosa cells can be the class of lipid components into the tissue and play a role in drug blood.In this study, Celecoxib, a selective COX-2 inhibitor, was applied topically onto SD rat tongue mucosa to explore the possibility of inhibiting DMBA induced tongue carcinogenase. The absorption of Celecoxib in SD rat tongue tissue was detected with high performance liquid chromatography (HPLC). Histological analysis was used to observe pathological changes, Immunohistochemistry staining and Western blot technique were employed for COX-2 and VEGF-C protein expression in tongue tissue. Fluorescent quantitive real-time RT-PCR was used to detect COX-2 and VEGF-C mRNA expression. ELISA was used to measure PGE2 production in tissue during DMBA induced tongue carcigenasis.ChapterⅠThe absorption of topical application of COX -2 inhibitor Celecoxib in the SD rat tongue tissue 35 4-week-old SD rats, were divided randomly into 7 groups (5 rats each).When rats were anesthetized with ether,6%(w/w) Celecoxib paste was applied to the tongue mucosa of left edge. After 5,10,15,30,60,90 and 120 min, the rats were sacrificed respectively. The tongue tissue was resected, ultrasonic rinsed and homogenized, then, certain volume of ethyl acetate was added and mixed with vortex and centrifuged. The supernatant was evaporated to dryness at 60℃instrument on the nitrogen evaporated to dryness, residue dissolved in mobile phase, with 0.22μm microporous membrane filter backward kind of vacuum. Using HPLC in VP-ODS reverse column (250 mm×4.6 mm,5μm) as the stationary phase, methanol:water (85:15) as mobile phase, flow rate:1.0 ml/min. Column temperature: 40℃. UV detection wavelength was 254 nm. The absorption of Celecoxib in SD rat tissue was detected at different time by comparing the rat tongue tissue absorption.The data were analysed with SPSS 13.0 package. In this system, the retention time of Celecoxib was 4.4 min. Celecoxib concentration presented a good linear range of 25～800μg·L-1, R2=0.989 4 (n=6), the detection limit for Celecoxib was 10μg·L-1 (S/N=3). The extraction and method recoveries for Celecoxib were 84.75%～90.21% and 93.73%～98.85%, respectively. The inter-day RSDs were 0.35%, 5.21% and 3.73%, and intra-day RSDs were 1.44%,4.33% and 4.40%, respectively. The concentration of Celecoxib in rats tongue tissue within first 120 min were ranged from (0.804±0.056)μg·g-1 to (3.382±0.167)μg·g-1, and reached its peak at 15 min. The results suggest that Celecoxib can be rapidly absorbed through tongue mucosa and reach a higher concentration in tongue tissue, which will be helpful for the local application of COX-2 inhibitors in prevention of oral cancer.ChapterⅡInhibitory effect of topical application of COX-2 inhibitor Celecoxib on DMBA induced rat tongue carcinoma 80 SD rats were randomly divided into control and Celecoxib treated groups (Celecoxib group). In both groups, the 1% DMBA acetone solution was applied to the left border of tongue combined with local trauma (scratched by rough sand paper) to induce tongue carcinoma thrice a week. In Celecoxib treated group, the 6%(w/w) Celecoxib paste was painted in situ,5 times a week. At 8,12,16 and 20 weeks,10 animals in each group were sacrificed and tongue tissue were resected for pathological observation, immunohistochemical staining Western blot, RT-PCR and ELISA studies. All data were processed with SPSS 13.0 software package, the results show that there were no findings of canceration in both groups at 8 weeks, whereas, at 12,16 and 20 weeks, the cancerous rates in control group were higher than that in Celecoxib treated group. Immunohistochemistrical staining and Western blot result showed that the expression of COX-2, VEGF-C protein in Celecoxib group at 8,12, 16 and 20 weeks. The result also showed a positive correlation between the expression level of COX-2 and VEGF-C. Their correlation coefficient (rs) was 0.724, 0.672,0.629 and 0.850 at 8,12,16 and 20 weeks, respectively (all P<0.05). RT-qPCR results showed that the COX-2 mRNA expression was not significantly affected by Celecoxib, whereas the expression of VEGF-C mRNA was significantly down-regulated by the Celecoxib in a time-dependent manner. ELISA results:PGE2 standard curve is 7=0.273 4Ln (X)+0.1853, R2=0.9947. And it suggested that the anti-cancer mechanism of Celecoxib was correlated with its inhibitory effect on decreasing PGE2 production. At 16 and 20 weeks, the PGE2 content in control group was significantly higher than that in Celecoxib group (P<0.05). But at 8,12 weeks, there were no significant difference between two groups (P>0.05).In summary, we confirmed that topically administration of Celecoxib paste could inhibit DMBA-induced SD rat tongue tissue epithelial dysplasia and canceration. The mechanism may be correlated with the inhibition of rat tongue activity of COX-2 and VEGF-C as well as decrease the production of PGE2. These findings are helpful for the application of COX-2 inhibitors in prevention of oral cancer.