Selective Cycloxygenase-2 Inhibitor Influent Lingual Squamous Cell Carcinoma Tca-8113 Cell Vascularization: An In Vivo And In Vitro Study

OBJECTIVE:5%malignant tumors of human are oral carcinoma.Oral carcinoma is one of the ten most common malignant tumors in the world.Above 90%of them is oral squamous cell carcinoma(OSCC)of epithelium origin.According to a incomplete survey,about 8000 patients die because of this disease each year in china.Although the treatments,including surgery, radiotherapy and chemotherapy,are improved continuously,the five year survival rate of OSCC patients is merely 50%or so.The disease threatens the safety of life and health severely.In recent years,OSCC incidence doesn't descent.On the contrary,it appears ascent and lower-age incidence tendency.So,to seek an active and effective prophylactic method and to seek effective drugs become two urgent missions.Vascularization is an important component for original carcinoma to grow,expand and infiltrate to normal tissue nearby.Experimental study and clinical data show that the growth and metastasis of tumors depend on vascularization.Compared to normal blood vessels,new born tumor blood vessels have winding structure,abnormal flow and increased permeability.Many studies show that the increased activity of vascularization correlates with bad prognosis of malignant tumors,including oral carcinoma.Vascularization is a complex procedure.It is the results of cooperation between vascularization promoter and stable factor.It correlates with many pathways,and it results in growth and differentiation of endoepithelial cells.At last,it forms a blood vessel net with function.It has been a new hot spot in study of tumor therapy to inhibit vasularization of tumor and prevent the growth of tumor.Cycloxygenase(COX)is an important rate-limiting enzyme in the procedure of catalysis arachidonic acid to synthesize prostaglandin.It has two isomers,COX-1 and COX-2.Recent study shows that COX-2 participates in generation and development of many tumors.The expression of COX-2 has been detected in many tumors including the carcinoma in head and neck.COX-2 inhibitor has the anti-carcinoma effect.Until now,we have poor knowledge of function of COX-2 and mechanism of vascularization in OSCC.And we don't know the relationship between expression of COX-2 and vasularization of carcinoma.This study aims to investigate the influence of Selective Cycloxygenase-2 Inhibitor,nimesulide,to Lingual Squamous Cell Carcinoma Tca-8113 cell to release PGE2.In the mean while,the inhibition effect of nimesulide to COX-2,VEGF,Ang-1 and Ang-2 is observed.So,we can investigate the influence of Selective Cycloxygenase-2 Inhibitor,nimesulide,to the vascularization of lingual squamous cell carcinoma.Methods:1.We use radioimmunity method to determine the influence of nimesulide to Lingual Squamous Cell Carcinoma Tca-8113 cell to release PGE₂ in vitro.2.We use RT-PCR technique to determine the expression of COX-2,VEGF,Ang-1 and Ang-2 mRNA after affected by drugs of different concentration.3.We use western-blotting method to determine the change of protein expression of COX-2, VEGF,Ang-1 and Ang-2 in the cell.4.We establish a tumor carried naked mice model with lingual squamous cell carcinoma Tca-8113 cell.We divide the animals into three groups.We lavage mice with nimesulide 30mg/kg after the day tumor is created every day in prophylactic group;we do the same to the mice after the day tumor is found every day in therapeutic group;and we lavage mice with normal sodium every day in control group,last for 42 days continuously.So,the influence of the drug to the mice and tumor is observed.5.We use RT-PCR technique to determine the expression of COX-2,VEGF,Ang-1 and Ang-2 mRNA in implanted tumor of mice after affected by the drug.6.We use western-blotting method to determine the change of protein expression of COX-2, VEGF,Ang-1 and Ang-2 in explanted tumor of mice.7.We use SABC immunohistochemistry technique to determine the expression of Ang-1,Ang-2 and Tie-2 in 34 OSCC tissue and 16 normal oral mucosa tissue.Results:1.The result of RIA method shows that,the PGE2 basal expression of cell culture supernatant in experimental group and control group is almost the same.The PGE2 level of supernatant descents after the addition of nimesulide.As the drug concentration get higher and the affecting time get longer,the PGE2 level descents more apparently.In a certain drug affecting time and does,it presents concentration-time dependence. 2.The result of RT-PCR technique and agarose gel electrophoresis to determine COX-2,VEGF, Ang-1 and Ang-2 mRNA show that,after 48 hours of nimesulide(NIM)of different concentration(25,50,100,200 umol/L)to deal with lingua squamous cell carcinoma Tca-8113 cell,compared with COX-2 level between NIM experimental group and control group,the difference of means is statistically significant(P＜0.01).Compared with VEGF level between NIM experimental group and control group,the difference of means is statistically significant (P＜0.01).Compared with Ang-1 level between NIM experimental group and control group,the difference of means is statistically significant(P＜0.01).Compared with Ang-2 level between NIM experimental group and control group,the difference of means is statistically significant (P＜0.01).3.The result of Western blot technique to determine protein expression of COX-2,VEGF,Ang-1 and Ang-2 shows that,after 48 hours of NIM of different concentration(25,50,100,200 umol/L) to deal with lingual squamous cell carcinoma Tca-8113 cell,compared with COX-2 protein between NIM experimental group and control group,by statistic analysis,the difference between each group and control group is statistically significant(P＜0.05),compared with the quantity of protein expression between 50umol/L group and 10umol/L group,it descents,but the difference is not statistically significant(P＞0.05),the descent of COX-2 protein expression is concentration dependent.Compared with VEGF level between NIM experimental group and control group,the difference between each group and control group is statistically significant(P＜0.05),compared with the quantity of protein expression between 50umol/L group and 100umol/L group,it descents,but the difference is not statistically significant(P＞0.05),the descent of VEGF protein expression is concentration dependent.Compared with Ang-1 level between NIM experimental group and control group,the difference of means is statistically significant(P＜0.05),compared with the quantity of protein expression between 25umol/L group and 50 umol/L group, 50umol/L group and 100 umol/L group,it descents,but the difference is not statistically significant(P＞0.05).Compared with Ang-2 level between NIM experimental group and control group,the difference of means is statistically significant(P＜0.05),compared with the quantity of protein expression between 25umol/L group and 50 umol/L group,it descents,but the difference is not statistically significant(P＞0.05).4.Three groups of animals are all survive during the experimental phase.Three groups of mice all appear the growth of implanted tumors in situ.The tumor tissues are proved to be lingual squamous cell carcinoma by pathological section examination.5.The tumor volume of control group is 4.2956±2.06 cm³,the tumor volume of therapeutic group is 2.2661±1.37 cm³,it is smaller than control group significantly(P＜0.05),the tumor growth inhibition ratio is 31.0%.The tumor volume of prophylactic group is 1.8705±0.87 cm³,it is smaller than control group significantly(P＜0.05),the tumor growth inhibition ratio is 76.2%. However,there is no statistically significance between the last two groups.6.The result of RT-PCR technique to determine the expression of COX-2,VEGF,Ang-1 and Ang-2 mRNA in implanted tumor of mice shows that,the COX-2 mRNA expression of NIM prophylactic group and therapeutic group drops dramatically,the difference between them and control group is statistically significant,but the difference between them is not statistically significant.The VEGF mRNA expression of NIM prophylactic group and therapeutic group drops dramatically,the difference between them and control group is statistically significant,but the difference between them is not statistically significant.The Ang-1 mRNA expression of NIM prophylactic group and therapeutic group drops,but the difference between them is not statistically significant.The Ang-2 mRNA expression of NIM prophylactic group and therapeutic group drops dramatically,but the difference between them is not statistically significant.7.The result of RT-PCR technique to determine the expression of COX-2,VEGF,Ang-1 and Ang-2 protein in implanted tumor of mice shows that,the COX-2 protein expression of NIM prophylactic group and therapeutic group drops dramatically,the difference between them and control group is statistically significant,but the difference between them is not statistically significant.The VEGF protein expression of NIM prophylactic group and therapeutic group drops dramatically,the difference between them and control group is statistically significant,but the difference between them is not statistically significant.The Ang-1 protein expression of NIM prophylactic group and therapeutic group drops,but the difference between them is not statistically significant.The Ang-2 protein expression of NIM prophylactic group and therapeutic group drops dramatically,but the difference between them is not statistically significant.8.The CD34 positive cell expression of OSCC patients is apparently higher than it of person with normal oral tissue.Compared with Ang-1 level between OSCC group and normal mucosa group,there is no significant difference(P＞0.05).The Ang-2 expression of OSCC group is dramatically higher than it of normal group(P＜0.001).Compared with each group after they are pathologically classified,the difference is significant and it is relevant with the quantity of vascularization positively.It indicates that the expression of Ang-2 ascent apparently in OSCC tissue,and it increases with the increase of malignance of tumors.The Tie-2 expression of OSCC group is significantly higher than it of normal group(P＜0.05),but the difference of its expression between each pathological classification is not significant(P＞0.05).Conclusions:1.In a certain drug affecting time and does,nimesulide inhibit PGE2 release of lingual squamous cell carcinoma Tca-8113 cell in vitro with the presence of concentration-time dependence.2.After affecting 48 hours,in the range of 25～200 mmol/L,nimesulide is able to inhibit the expression of COX-2,VEGF,Ang-1 and Ang-2 in Tca-8113 cell in mRNA and protein level with the presence of concentration dependence.3.We establish an animal model with human lingual squamous cell carcinoma Tca-8113 cell transplanted to naked mice successfully.4.During 42 days of therapeutic time,to lavage the mice with nimesulide 30mg/kg can inhibit the growth of transplanted tumor significantly.And it can inhibit the vasularization of lingual squamous cell carcinoma in mRNA and protein level.5.The inhibition function of nimesulide to vasularization of lingual squamous cell carcinoma is COX-2 dependent.6.It is the first time for us to find the prophylactic function of nimesulide to human lingual squamous cell carcinoma.7.The expression of Ang-2 ascent apparently in OSCC tissue,and its expressed level increase with the increase of malignance of tumors.It may be relevant with the vascularization of OSCC tissue as a marker of newborn tumor vessels.