Effect of Topically Applied Celecoxib on Caspase-3 and Survivin Expression in DMBA Induced Rat Tongue Carcinogenesis
|School||Southern Medical University,|
|Keywords||COX-2 inhibitors Celecoxib caspase-3 survivin Chemoprevention|
Tongue cancer is common head and neck cancer, lymphatic, rich blood supply, the early incidence of lymph node metastasis, a lot of research for tongue cancer etiology, progression, clinical outcome, and treatment methods, tongue cancer the mechanism is not yet very clear, clinical outcomes remains to be further improved. Prevention methods, including tongue cancer, including head and neck cancer, explore treatment and improve the survival rate of patients has been the focus of the study. That the presence of tongue cancer occur with oral precancerous lesions and cancer is closely related to the common clinical precancerous lesions including oral leukoplakia, mucosal erythema and human papilloma and other related risk factors for oral cancer, their common feature pathological findings are associated with epithelial dysplasia. Oral tumor formation by a variety of genetic factors, such as changes in oncogenes or tumor suppressor genes. There are lot of factors are involved in the process of development to cancer from oral epithelial dysplasia, which include: abnormal regulation of apoptosis, and a large number of changes in the expression of tissue markers of abnormal DNA expression. The cyclooxygenase -2 (Cycloxygenas-2, COX-2) as found in recent years with the development of malignant tumors is closely related to the expression of abnormal play an important role, through the selective COX-2 inhibitors inhibit COX-2 activity, become the new hot spot oral cancer prevention and treatment. Experimental and clinical observations prove that COX-2 inhibitors as adjuvant therapy enhances tumor radiotherapy and chemotherapy sensitivity, and reduce the incidence of gastrointestinal complications, so prompt us to COX-2 inhibitors intervention of oral precancerous lesions leukoplakia malignant the possibility of prevention of oral cancer has an important role. In this study, for topical application of COX-2 and COX-2 and oral mucosa precancerous condition lesions and oral cancer, as well as easy to absorb the characteristics of the local administration of the oral mucosa, to establish SD rats by DMBA chemically induced tongue cancer model, inhibitor celecoxib celecoxib inhibit intervention research topical application of celecoxib to inhibit the role of oral epithelial dysplasia and malignant transformation occurs, the view of the the Chase celecoxib inhibit DMBA-induced tongue cancer formation process of apoptosis precursor protein caspase- 3, the impact of survivin expression, from the mechanisms of apoptosis in the COX-2 inhibitor anti-tumor effect and promote the relationship between apoptosis, the experimental evidence for the topical application of the COX-2 inhibitors prevent oral leukoplakia and malignant . 80 SD rats were randomly divided into two groups, the control group and the experimental group of 40 each. The control group (A) for the simple tongue cancer modeling group, divided into A1, A2, A3 and A4 group (n = 10). SD rats were anesthetized with sandpaper wipe the left margin of the tongue mucosa of rats induced tongue mucosa congestion, but not bleeding, then in the tongue edge smear 1% 7,12 - dimethyl benzene with ultra-fine scanning pen anthracene and acetone solution, three times weekly. The experimental group (B) for the plug celebrex layout Ministry medication group, divided into B1, B2, B3 and B4 group, n = 10, giving the same treatment as the Group A, to give 6% celecoxib paste ( Southern Medical University medical laboratories to assist in preparation), once a day. To observe the state of the survival of rats, changes in body weight and tumor formation rate. The rats were sacrificed at 8, 12, 16 and 20 weeks, respectively, for each group of 10 each. Were killed after cutting entire tongue organizations, and tongue tissue is divided into two parts, the first to take the part of the organization of fresh lesions 500 mg at -80 ℃ to save the rest of 10% neutral formalin solution and fixed. Using light microscopy tongue mucosa in SD rats DMBA induced carcinogenesis process changes, and analyzed using Image-pro plus software, cortical thickness measurement on the organization of the various specimens, epithelial take 10 points per sample measurements averaged; caspase-3 in the tongue tissues using immunohistochemical staining and Western blot method to detect the expression of survivin protein; caspase-3 mRNA expression was detected by RT-PCR, survivin mRNA levels of change. The results show that: in the 8 weeks of the experimental group and the control group had no tumor; at 12 weeks, 16 weeks and 20 weeks, the canceration rate of the control group was respectively 20%, 60% and 70%, while the experimental group only two rat tongue carcinogenesis in the 20 weeks, the cancer rate was 20%. The cancer was 37.5% of the control group throughout the experiment and the experimental group, 5%, and the difference was statistically significant (P = 0.000). The light microscope observations, the experimental group and control group of epithelial thickness gradually increases with increasing DMBA stimulation time. Epithelial average thickness of the test results show that the control group at 8,12 and 16 weeks of epithelial average thickness (103.40 ± 29.40) μm, (108.70 ± 33.80) μm, (105.30 ± 27.50) μm, in the 20-week rat samples all tongue mucosa lesions occur not epithelial thickness comparison, while the experimental group were 8, 12, 16 and 20 weeks the epithelial average thickness (77.30 ± 21.80) μm (63.80 ± 22.10) μm (88.30 ± 31.70) μm and (95.00 ± 36.20) μm. The entire experimental group and control group difference was statistically significant (P = 0.012); dysplasia lesions of epithelial thickness comparison, the dysplasia lesion in epithelial thickness comparison, the control group and the experimental group, no statistically significant difference (P = 0.052), the experimental drug intervention and control groups showed no impact dysplasia thickness. Comparison of the keratin layer thickness also experimental group than the control group, thickening the low level (P = 0.000); comparison of the degree of inflammatory infiltration in the control group increased With DMBA stimulus time, gradually increase the degree of inflammatory infiltration 8, 12, 16 and 20 weeks unit area (5184μm2) of the number of inflammatory cells, 6.77 ± 2.36,10.74 ± 10.02,17.68 ± 13.20 and 17.22 ± 13.88, while the experimental group, 8, 12, 16 and 20 weeks of unit area of ??inflammation cell count was 9.30 ± 2.59,10.04 ± 3.01,9.02 ± 2.08 and 9.70 ± 3.36, the degree of inflammatory infiltration of the experimental group compared with the control group low degree of inflammatory infiltration, and the difference was statistically significant (P = 0.047). Immunohistochemical detection results show that the control group, caspase-3 with the The DMBA stimulus time increased expression decreased in 8, 12, 16 and 20 weeks, the average rate of positive cells were (61.36 ± 6.16)%, (51.18 ± 10.97) %, (48.11 ± 9.57)% and (39.73 ± 11.07)%, the difference was statistically significant (p = 0.000), while the experimental group and caspase-3 does not appear significant change, 8, 12, 16 and 20 weeks, the average positive cell rate (75.23 ± 7.00)%, (76.75 ± 7.39)%, (79.83 ± 8.56)% and (71.66 ± 16.57)%, the difference was not statistically significant (P = 0.394). But the entire control group, caspase-3 expression of caspase-3-positive cells in the entire experimental group was significantly higher than the difference between the two groups was statistically significant (P = 0.000). Survivin positive cell rate of the control group gradually increased with the increase in the time of DMBA role in 8, 12, 16 and 20 weeks, the average rate of positive cells were (12.45 ± 6.07)%, (26.50 ± 20.16)%, (46.33 ± 24.97)% and (58.49 ± 35.94)%, the difference was statistically significant (P = 0.000), while the experimental group survivin expression did not show a significant change, in 8, 12, 16 and 20 weeks, the average rate of positive cells, respectively survivin positive cells in the experimental group (6.66 ± 3.88)% and (6.45 ± 3.45)% and (5.76 ± 3.87)% and (11.51 ± 12.20)%, the difference was not statistically significant (P = 0.242), lower than that of the control group survivin expression rate, the difference was statistically significant (P = 0.000). Western blot analysis also confirmed that the control group, caspase-3 expressed as DMBA stimulation time decreased expression of the different time points the difference was statistically significant (P = 0.000), while the experimental group of caspase-3 expression strength and extension of time due to DMBA role changes in the expression of the different time points the difference was not statistically significant (P = 0.05), but its expression was significantly higher, the difference between the two groups was statistically significant (P = 0.000). Western blot analysis also confirmed the control group survivin extension of time with DMBA role increase in the expression of the different time points difference was statistically significant (P = 0.000), while the experimental group survivin expression with in DMBA stimulation time increases, the period the difference was statistically significant (P = 0.049), but its expression was significantly lower than the control group, the difference was statistically significant (P = 0.000) between the two groups. RT-PCR results showed that caspase-3 mRNA expression levels of the control group decreased gradually extend the with DMBA role time and 8,12,16 and 20 weeks caspase-3/β-actin expression ratio were 0.73 ± 0.12, 0.68 0.18,0.48 ± 0.22 and 0.39 ± 0.22, at each time point difference was statistically significant (P = 0.000), while the experimental group caspase-3/β-actin expression ratio was no significant difference, 8, 12, 16 and 20 weeks of caspase -3/β-actin value 0.93 0.18,0.97 ± 0.07,0.88 ± 0.17 and 0.82 ± 0.19, respectively, at each time point the difference was not statistically significant P = 0.195). Caspase-3 mRNA expression level of the experimental group was significantly higher than that in the control group, the difference was statistically significant (P = 0.004). Control group in 8, 12, 16 and 20 weeks survivin / β-actin expression ratio of 0.57 ± 0.12,0.72 ± 0.17,0.77 0.15 and 0.80 ± 0.11, respectively, at each time point between the difference was statistically significant (P = 0.000) experimental group survivin / β-actin expression ratio was no significant difference in the average expression ratio of 8, 12, 16 and 20 weeks were 0.29 ± 0.10,0.26 ± 0.10,0.36 ± 0.09 and 0.39 ± 0.11, each time point between difference was not statistically significant (P = 0.125) and survivin mRNA expression level of the entire experimental group Survivin expression below the entire control group, the difference was statistically significant? (P = 0.000). Through this experiment, we think: Topical application of 6% COX-2 inhibitor celecoxib ointment can effectively suppress the occurrence of DMBA chemically induced SD rat tongue cancer, its more important role in addition to inhibit oral epithelial dysplasia related to regulation and apoptosis-related caspase-3 for further exploration of survivin protein and mRNA expression, which can be adjusted through on caspase-3 and down survivin protein and mRNA expression, which in turn affect cell apoptosis COX-2 inhibitors and the tongue cancer formation, as well as the application of COX-2 inhibitors provide experimental evidence for prevention of malignant transformation, and tongue cancer oral precancerous condition.