| Objective:Although the treatments, including surgery, radiotherapy and chemotherapy, are improved continuously, the five years survival rate of laryngeal squamous cell carcinoma is still low. The disease threatens the safety of life and health severely. In recent years, laryngeal squamous cell carcinoma incidence does not descent. On the contrary, it appears ascent and lower-age incidence tendency. So, to seek an active and effective prophylactic method and drugs become an urgent mission. To evaluate the effect of nimesulide, a selective cyclooxygenase-2(COX-2) inhibitor, on the tumorigenesis, the expression of COX-2, Survivin and proliferating cell nuclear antigen (PCNA) of human Hep-2 cell xenografts in nude mice and explore the antitumor effect and the possible mechanisms, to provide experimental proof for the use of NIM in human laryngeal squamous cell carcinoma prevention and treatment. Methods:Hep-2 cells of human laryngeal squamous cell carcinoma were cultured, and Hep-2 cells suspension were subcutaneously inoculated into the right flank of Balb/c-nu/nu nude mice to establish the transplanted model of human laryngeal squamous cell carcinoma, then the 12 mice were randomly divided into 2 groups:experimental group (NIM group, intraperitoneal injection nimeisulide 50mg/kg/2d,0.2ml) and control group (intraperitoneal injection normal saline,0.2ml). The weight of nude mice and the shortest and longest diameter of the tumor were measured with slide gaud at 2-day intervals, and tumor volume (mm3) was calculated using the following standard formula:(the longest diameter)×(the shortest diameter) 2/2, and the side effect of NIM was observed during treatment; The mice were sacrificed at day 28 after treatment, the tumor and main organs were also dissected, fixed in 10%formalin, stained with hematoxylin-eosin for histological examination. COX-2, Survivin and PCNA protein were assessed immunohistochemically, and the expression of them were observed microscopically. By image analysis system, the average optical density value of COX-2, Survivin and PCNA protein in xenografts were analyzed. The expression of COX-2, Survivin and PCNA mRNA were measured by reverse transcription-polymerase chain reaction (RT-PCR) in xenografts, the gray value was analyzed by Biod-rad softwares. Results:(1) The model of laryngeal squamous cell carcinoma xenografts in nude mice was successfully established, the tumorigenic rate in nude mice injected with Hep-2 cells was 100%. Two groups of mice all appear the growth of implanted tumors in situ. The tumor tissues were proved to be laryngeal squamous cell carcinoma by pathological section examination. Two groups of animals had been all survive during the experimental phase. (2) Food and drink of nude mice was normity, mental state of nude mice was good, without dying and the action was free in two groups. The weight and pathological section examination of heart, liver, spleen, lung, kidney had no obvious difference, and there was no stigmas in alimentary tract. (3)Nimesulide can significantly inhibit the tumor growth of human laryngeal squamous cell carcinoma Hep-2 cell xenografts in nude mice. The tumor volume of the NIM group was (172.67±115.25)mm3, significantly lower than that of the control group(478.69±341.84)mm3(P<0.05). No Significant difference was found in body weight of the nude mice in the NIM group (23.22±1.78) g, and control group (24.05±1.79)g (P>0.05). The weight of tumor in the NIM group was (0.40±0.33)g, significantly lower than that of the control group(0.83±0.27)g (P<0.05), the tumor growth inhibition ratio was 51.81%. (4)The results of immunohistochemically technique to determine the expression of COX-2, Survivin and PCNA protein in implanted tumor of mice shows that the expression of COX-2 protein in NIM group(38.12±15.56) droped dramatically, the difference between NIM group and control group(140.92±23.12) was statistically significant (P<0.05). The expression of survivin protein in NIM group(46.79±10.04) droped dramatically, the difference between NIM group and control group(133.41±20.01) was statistically significant(P<0.05). The expression of PCNA protein in NIM group(62.97±13.83) droped dramatically, the difference between NIM group and control group(110.23±13.00) was statistically significant(P<0.05). (5) The expression of COX-2 mRNA was determined by RT-PCR, which were (0.70±0.18) and (2.23±0.38) in NIM group and control group, respectively. The difference was significant(P<0.05). The expression of Survivin mRNA were (0.34±0.04) and (1.85±0.18) in NIM group and control group, respectively. The difference was significant(P<0.05). And the expression of PCNA mRNA were (0.45±0.05) and (1.91±0.70) in NIM group and control group, respectively. The difference was significant(P<0.05). Conclusions:(1)Selective cyclooxygenas-e-2 inhibitor NIM had obvious inhibitory effect on the growth of human laryngeal squamous cell carcinoma Hep-2 cell xenografts in nude mice. (2)NIM can inhibit the expression of COX-2 protein and mRNA, suggesting that NIM may inhibit the tumor growth in a COX-2 dependent manner. (3)NIM can inhibit the tumor cells proliferation and inducting apoptosis in protein and mRNA level. The growth inhibition may be via the inhibition of the expression of COX-2, Survivin and PCNA. (4) NIM had no obvious toxicity and side effect. (5) Selective cyclooxygenase-2 inhibitor may become one part of combined therapy of human laryngeal squamous cell carcinoma in the future, and the manufacture and exploitation of selective cyclooxygenase-2 inhibitor may supply original broad prospect for preventing and treating human laryngeal squamous cell carcinoma.
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