| Objective: Salivary adenoid cystic carcinoma (SACC) is one of the most common malignant tumors of salivary gland. It is characterized by strong invasiveness, infiltrating growth and remarkable recurrence and metastasis, which can be accounted for histology by its infiltrating capacity and distinct propensity for invasions along nerves and vessels. At present, the main clinical treatment for SACC is surgery which is hard to achieve the most desirable effects for the lesion can not be removed thoroughly. And this lead to the high incidence of the postoperative recurrence, poor long-term effect, insensitive to radiotherapy and no significant effect to the traditional chemotherapeutic drugs. Therefore, to explore the new treatment methods become a focus. And one of the hot points is to extract the effective component from plants to inhibit the proliferation or induce the apoptosis of tumor cells. Evodiamine is the mainly active component from dry seeds of rueaceae plant fructuc evodlae. Recent researches show that evodiamine plays a role in inhabiting tumor cells'metastasis and invasion. Different concentrations of Evodiamine were be used in this research to treated the salivary adenoid cystic carcinoma ACC-M cells, and effect of evodiamine on the invasive behaviors of human salivary adenoid cystic carcinoma ACC-M cells was to be explored. The aim of this study was to provide test basis for the prevention and treatment of SACC metastasis and invasion by means of the traditional chinese medicine preparation in clinical application.Materials:1 Materials(1) Cell line: highly-metastatic ACC-M salivary adenoid cystic carcinoma cell line was purchased from the laboratory of oral and maxillofacial surgery of shanghaijiaotong university school of medicine, which was established in 1995. (2) Evodiamine: reference substance, provided by the National Institute for the Control of Pharmaceutical and Biological Products. (3) Matrigel. (4) transwell cell culture chambers, (filters of 8um pore size). 2. Methods: (1) preparation of agent: Evodiamine was dissolved in dimethyl sulfoxide (DMSO) (20mg/ml), it was diluted before used(The final concentration of DMSO less than 0.1%, this concentration had no effect on cells). (2) Cell Culture: ACC-M cells were cultured in RPMI 1640 medium containing 10% (v/v) fetal calf serum and streptomycin (100μg/ml) and penicillin (100U/ml). It was incubated at 37℃in 5% CO2 gas incubator with saturation humidity. A new generation was formed after 24~48 hours in the mixed liquid containing 0.25% trypsin and 0.04% EDTA (1:1). (3) Put 10% Cell culture medium (200ul) which contain HGF (30ng) into the underside of the transwell cell culture chamber (30μg/200μl), the membrane of each upper transwell cell culture chamber coated with Matrigel (50μl). (4) add ACC-M Cell suspension(2×105/200μl,200μl) which pretreated with various concentrations of evodiamine(0μg/ml, 0.01μg/ml, 0.1μg/ml, 1μg/ml, 10μg/ml, 100μg/ml) for 20 minutes at the degree of 37℃; incubated under the conditions of 37℃5% CO2. The cells of membrane's upper surface were wiped with cotton swab after reaction for 2h,4h,6h,8h,10h. Then after fixation with formaldehyde (10%) for 30 minutes, swiss-ji staining was done. (5) The entire membrane was observed, which was divided into 9 check as the Chinese character"井", under the 400x microscope. Randomly one visual field at the center check was selected, and another 4 visual fields at 4 checks surrounding the central one were choose, then the total number of the cells passed through the microporous membrane was counted in view of the 5 checks. Invasion inhibitory rate formula is as follows: (control groups'mean value of Transmembrane cells - treat groups'mean value of Transmembrane cells) / control groups'mean value of Transmembrane cells×100%. (6) Statistical analyses was performed by SPSS13.0 using ANOVA of repeated measurement .Results: Compared with the control group, the number of the cell through the upper chamber in the evodiamine- treated group was smaller.The cells number presented a gradually decreasing trend with higher concentration and longer time. The invasion inhibition increased with the increasing of the concentration and the time. Moreover, the invasion inhibition was 59.99% with the concentration of 100μg/ml at 10 hour. According to statistic analysis, there was no difference at different time (F=7.050, P>0.05) while the number changed in different concentrations (F=7.050, P<0.05). But time groups and concentration groups had no interaction effect on each other(F=1.426,P>0.05).Conclusion:1 Compared with the control group, the tumor cell invasion could be significantly inhibited in the group treated with evodiamine, there was difference significantly between experiment group and control group.2 There was the dependence between Evodiamine inhibiting human salivary adenoid cystic carcinoma ACC-M cell invasion and the Evodiamine concentration. The invasion inhibition increased with the increasing of the concentration. There was no the dependence between the inhibition rate and time. There was no interaction effect between the time group and concentration group.
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