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Objective: salivary adenoid cystic carcinoma (salivary adenoid cystic carcinoma, SACC) is one of the most common malignant tumors in the oral and maxillofacial malignancies in second place. Highly invasive and easy to distant metastasis is an important feature of adenoid cystic carcinoma. Even after surgical resection often local recurrence and distant metastasis, the rate of distant metastasis in oral and maxillofacial first place. Salivary adenoid cystic carcinoma incidence and transfer mechanism is unclear. Therefore it is necessary to explore new treatments to improve the efficacy of adenoid cystic carcinoma, reduce local recurrence and distant metastasis, improve the patient's quality of life. Glycyrrhiza, the dicots leguminous perennial herb, is a famous traditional Chinese medicine, the most widely used clinical, licorice acid (Glycyrrhizic Acid, GA) is one of its main active ingredient. Today, licorice widely used in anti-inflammatory, anti-viral, anti-allergic, immune regulation, the treatment of peptic ulcer and the protection of the nerve, one after another in recent years, studies have shown that licorice and its major components have anti-tumor effect, but the licorice acid adenoid cystic carcinoma apoptosis induced by role at home and abroad has not been reported. In this study, the effect of different concentrations of cultured human salivary adenoid cystic carcinoma SACC-M cells were treated with different concentrations of different role Glycyrrhizinate different time licorice human salivary adenoid cystic carcinoma SACC- M cells inhibit proliferation and induce apoptosis, to explore its possible mechanism, and provide a theoretical basis for the clinical use of licorice acid treatment of salivary adenoid cystic carcinoma. Materials and Methods: 1 Materials SACC-M: Shanghai Jiao Tong University Oral and Maxillofacial Surgery Laboratory, established in 1995; glycyrrhizic acid: Tokyo Kasei Kogyo (TCI), a purity of 97% (HPLC) method (1) The liquid preparation of glycyrrhizic acid with RPMI1640 culture medium formulated as 25mmol / L storage solution 1640 diluted before use. (2) Cell Culture: RPMI1640 culture medium with 10% fetal calf serum and penicillin (100U/ml), streptomycin (100ug/ml), at 37 ° C, 5% CO 2 incubator within a culture saturated humidity 24 to 48 hours of passage 1, digestion and passage to 0.04? TA and 0.25% trypsin (1:1) mixture. (3) MTT colorimetric test: Take the logarithmic growth phase cells made of 2.0 × 10 5 / mL cell suspension were seeded in 96-well plates, each 1:100, after 24h, were added licorice acid solution 100μl, so that the final concentration of 2.5,5.0,7.5,10.0,12.5 mmol / L, and the control group and zero hole. 24,48,72 h after culture, each well was added 5 g / L MTT solution 20 ul for 4h to absorb all the supernatant, 200 ul of dimethyl sulfoxide is then added to each well, the oscillation shake, crystals fully dissolved After measuring the absorbance of each well microplate reader set, and calculate the rate of growth inhibition. (4) inverted phase contrast microscope: The cells were seeded in 25 ml culture flasks inverted microscope cells in the blank and drug-containing medium, the culture growth conditions in different time. (5) observed by light microscopy: coverslips placed in 6-well plates, the cells were seeded in which, the climbing film after the success with spaces or drug-containing medium, culture time, Giemsa staining, light microscopy cell morphology. (6) by flow cytometry: blank media and different concentrations of the drug-containing medium the cells were treated with different times, using Annexin-V-FITC and PI double labeling of living cells, cell apoptosis was detected by flow cytometry. 3 Statistical analysis SPSS10.0 statistical software, analysis of variance, correlation analysis, and u test rate of growth inhibition and apoptosis rate were statistically analyzed. Results: 1 MTT colorimetric test: glycyrrhizin on SACC-M cells with significant growth inhibition, the maximum growth inhibition rate of 80.4%. The statistical analysis, different concentrations of drug treatment group at different times between growth inhibition rate (P lt; 0.01), has a significant positive correlation between efficacy and drug concentration and duration of action. 2 inverted microscope: the control group of cells adherent growth, fast growth was flat polygonal plump cytoplasm, close to the cells at the center of a round nucleus; treatment group cell growth has slowed significantly, and visible cells off the wall, adherent cell rounding, cytoplasmic shrinkage, smaller cells, connections between cells release nuclear deepen the color, refraction enhancement of the cell. Observed by light microscopy: a control group of cells, a large proportion of nucleus and cytoplasm, and visible nucleoli; cytoplasmic concentration of the medication group, reducing the proportion of nucleus and cytoplasm, the nucleolus reduce or even disappear, chromatin arranged along the inner nuclear membrane phenomenon, and visible nuclear fragmentation phenomenon. 4 by flow cytometry: the control group apoptosis, but the lower rate of apoptosis; treatment group cells apoptosis, and the apoptosis rate increased with the increase of drug concentration and treatment time, the statistical analysis, a significant difference (P lt; 0.01) between the concentration and time group. Conclusion: glycyrrhizic acid to inhibit salivary adenoid cystic carcinoma SACC-M cells proliferation and induce apoptosis, and was dose and time dependent. The main mechanism of this growth inhibition is due to the induction of apoptosis.
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