| Objective: The incidence of oral and maxillofacial cancer was 5-10 % accounts for systemic malignant tumor. Because oral and maxillofacial's special anatomical location, the 5-year survival rate of oral cancer is only about 50%. It is a serious threat to people's life and health. In this one, about about 90% of the oral cancer are epidermis(oral squamous carcinoma cell,OSCC)tongue cancer is the highest incidence of Oral and Maxillofacial squamous cell carcinoma of the site, accounting for more than 40%. Incidence of tongue cancer in men is about 0.5 0.6/10 million, about 0.4 0.5/10 million in women. In recent years, with the living environment of change, the incidence of human tongue cancer did not decrease , Anti showed a rising trend year by year, with the younger age increasingly. Currently the treatment of tongue cancer is still the preferred operation, supplemented by local radiotherapy and systemic chemotherapy. But the dysfunction caused by surgery and appearance deformity is unwilling to patients, while radiotherapy and chemotherapy in patients with the body there are also serious side effects. Therefore actively seeking one kind of looking for effective and low toxicity of therapeutic drugs to become an important task.Xanthorrhizol is a sesquiterpenoid from the rhizome of Curcuma xanthorrhiza. Xanthorrhizol have proven to be a variety of biological activities, such as antibacterial,antifungal activity, anti-inflammatory ,antioxidant and so on. The anti-tumor effect is one of the focus in recent years, Current studies have shown that Xanthorrhizol have a strong inhibition on a variety of cancer cell including breast, colon cancer cells, etc. It also can inhibit the expression of multiple tumor-associated proteins.COX-2 and iNOS are two kinds of protein molecules, both of them play a key role in cancer development. Present study found COX-2 and iNOS take a significant positive in cancer cell including tongue cancer, breast cancer, endometrial cancer cells, etc. Their co-expression is closely related to biological behavior togue squamous cell carcinorma.Tca8113 cells were treated by different concentrations of Xanthorrhizol to observe the inhibitory effects of Xanthorrhizol Tca8113 cell lines in vitro,and to explore the possible related mechanism of Xanthorrhizol on Tca8113 cells Preliminarily,in Order to provide new ideas and reference for clinical studies.Methods: After recovery, Tca8113 cells were 37℃, CO2 saturated 5% humidity, cultured under routine conditions, until after the cells entered the logarithmic growth phase for experiments.1 The growth inhibition and on protein expression in Tca8113 cell with the Xanthorrhizol intervention1.1 The assay of MTT show proliferation and inhibition: devide experimental groups, in experimental group,fill in different doses of Xanthorrhizol solution,with the final concentration of 10μmol / L, 20μmol / L, 40μmol / L, 80μmol / L,set a blank group,without inoculated cells, set control group, fill in equal amount of cell culture medium, take the plate into incubator cultured after 72h.Record OD value in 24h, 48h, 72h. Calculation the inhibition rate of cell growth.1.2 Flow cytometry (FCM) detection of COX-2,iNOS gene expression: The collection of Tca8113 cells effected by different concentrations of Xanthorrhizol in 0h, 12h, 24h, 48h, 72h and control cells, according to Conventional methods of fluorescent labeling, flow cytometry fluorescence intensity to the mean fluorescence intensity channel number indicates the amount of protein expression.2 The inhibitory effects of Xanthorrhizol combined fluorouracil on Tca81132.1 Take different time and different doses of fluorouracil solution intervention Tca8113 cells, with the final concentration of 5μmol/L, 10μmol/L, 20μmol/L, 40μmol/L, Measure the inhibition.at 24h, 48h, 72h.2.2 Fill 10μmol/L fluorouracil in Xanthorrhizol group, Test OD value of per cells well, calculation the inhibition rate of cell growth. 2.3 fill 20μmol/L Xanthorrhizol in fluorouracil group, Test OD value of per cells well, calculation the inhibition rate of cell growth.Reslut:1 The growth inhibition and protein expression in Tca8113 cell with the Xanthorrhizol intervention1.1 The growth inhibition in Tca8113 cell with the Xanthorrhizol intervention1.1.1 OD value:with different concentrations of Xanthorrhizol, there were significant differences between groups. The results show that the value of OD are closely relationship between time and concentration. Growth curve shows: In the same reaction time, the higher the concentration, the lower the growth curve; In the same drug concentration, with the time increases the growth curve is relatively flat,1.1.2 Inhibition rate: With the higher concentration of Xanthorrhizol, the cell growth inhibition rate increased gradually. In the same reaction time, the average inhibition rates were 11.04%,17.84%,26.92%,40.45% in 10μmol/L,20μmol/L,40μmol/L,80μmol/L.Increase with the concentration, the inhibition rate increased significantly. In the same drug concentration, the average inhibition rates were 14.57%,25.47%,32.14% in 24h,48h,72h,with time increases, there were significant statistical difference between the concentration groups(P<0.01),the same there were significant statistical difference between the time groups(P<0.01). The IC50 values were 85.11μmol / L, 60.26μmol / L, 48.99μmol / L,in24h, 48h, 72h, IC50 with the role of time increases significantly. The higher the concentration, the longer the intervention, the greater the inhibition, showing a significant concentration - time dependent manner.1.2 Flow cytometry (FCM) detection of COX-2,iNOS gene expression1.2.1 The expression of COX-2: In the time factor, the expression of each group were statistically significant difference(sP<0.01)This suggests that with the time increase , the expression of COX-2 protein continued to decrease. In the concentration factor, the expression of each group were statistically significant differences(P<0.01)This suggests that with the concentration increase , the expression of COX-2 protein continued to decrease. According to the linear relationship with concentration and time increases, the expression of COX-2 protein was decreased.1.2.2 The expression of iNOS: In the time factor, the expression of group were statistically significant differences(P<0.01)except 48h-72h group(P=0.076>0.05) This suggests that with the time increase , the expression of iNOS protein continued to decrease. In the concentration factor, the expression of each group were statistically significant differences(P<0.01)This suggests that with the concentration increase , the expression of iNOS protein continued to decrease. According to the linear relationship with concentration and time increases, the expression of iNOS protein was decreased.Growth inhibition test and COX-2, iNOS results show that Xanthorrhizol Tca8113 can inhibit Tca8ll3 cells.2 combined with fluorouracil on Tca8113 cell growth, proliferation.2.1 Different doses of 5-Fu effect on Tca8113 cell, with the time increase, the inhibition increase. It suggest that with the concentration and time increase the inhibition of growth rate in Tca8113 cells were enhanced with in 5-Fu alone.2.2 In the application of 10μmol/L fluorouracil, the IC50 value in Xanthorrhizol were 85.11μmol/L, 60.26μmol/L, 48.99μmol/L,in 24h, 48h, 72h, down to 46.22μmol/L,33.4μmol/L,26.71μmol/L,in combine group. The fluorouracil combined with different doses of Xanthorrhizol, Are all shown adding to and enhancing relationships, particularly evident in low concentrations.2.3 In the application of 20μmol/L Xanthorrhizol, the IC50 value in Xanthorrhizol were 39.04μmol/L,32.23μmol/L,28.68μmol/L,in 24h, 48h, 72h, down to 24.94μmol/L,17.06μmol/L,13.55μmol/L,in combine group. The Xanthorrhizol, combined with different doses of fluorouracil, Are all shown adding to and enhancing relationships, particularly evident in low concentrations.Conclusion:1 Xanthorrhizol can significantly inhibit Tca8ll3 cells growth and Proliferation,it is relied on concentrations and time.2 Xanthorrhizol can significantly inhibited the expression of COX-2 and iNOS protein in Tca8113 cell. The inhibitory effect of the two proteins are more closely related to the time and concentration.3 The expression of COX-2 and iNOS protein in Tca8113 cell. in Xanthorrhizol effects with the corresponding. It suggests that some relationship about mechanism between the two Protein.4 Xanthorrhizol combined with fluorouracil can significantly enhance their inhibitory effects on Tca8113, the two drugs in low concentration has a strong synergistic effect.
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