Effect Of Glycyrrhizic Acid On Proliferation And Apoptosis In Salivary Adenoid Cystic Carcinoma Cell Line SACC-M In Vitro

Objective: Salivary adenoid cystic carcinoma (SACC) is one of the most common malignant tumors in salivary glands, and ranks the second place in oral and maxillofacial tumors, which is characterized by infiltrating growth and high metastasis rate. Local recurrence and even distant metastasis often occurs after surgery, and the metastasis rate of SACC is the highest among all oral and maxillofacial tumors. However, its mechanism of morbidity and metastasis is unclear now, it is very necessary to explore new therapies for SACC in order to improve the curative effect, decrease local recurrence and distant metastasis, and improve the patients' quality of life. Traditional Chinese medicine: Liquorice, which belongs to dicotyledonous pulse family perennial herb, is a well-known traditional Chinese medicine and most frequently used in clinical work. Glycyrrhizic acid (GA), is the major active component of licorice root. Liquorice was extensively used in anti-inflammatory, antivirus, allergy relief, immunoregulation, treatment of peptic ulcer and neuroprotection in present times. And more and more studies showed that licorice and its major active ingredients had the effect of anti-tumor. Nevertheless, whether the GA can induce the apoptosis and necrosis of SACC has not been reported yet. The aim of this study is to observe the effect of Glycyrrhizic Acid on proliferation and apoptosis in salivary adenoid cystic carcinoma cell line SACC-M in vitro at different times and concentrations in vitro, to explore the possible mechanism and to provide theoretical basis for clinical application of GA in treating SACC.Methods:1 Materials:(1)Cell line SACC-M was purchased from the laboratory of oral and maxillofacial surgery of Shanghai Jiao Tong University , which was established in 1995; (2) Glycyrrhizic acid(GA) was obtained from Tokyo Chemical Industry(TCI), purity(neutralization titration) is 97%(HPLC).2 Methods:(1) Preparation of physic liquor: GA was dissolved with RPMI 1640 medium into 25mmol/L and stored in 4°C saved , which was diluted with RPMI 1640 before each assay.(2)Cell culture: SACC-M cells were cultured in RPMI 1640 medium containing 10 %( v/v) fetal calf serum, streptomycin (100μg/ml) and penicillin (100U/ml), then incubated at 37℃in 5% CO2 gas incubator with saturation humidity. A new generation was formed after 24~48 hours the cells were harvested with the mixed liquid containing 0.25% trypsin and 0.04% EDTA (1:1).(3)MTT assay: 2×10~5/ml cell suspension in logarithmic phase were seeded into 96-well plates at 100μl tissue culture medium every well. After 24h incubating, 100μl GA solution was added into each well, the final concentrations were 2.5,5.0,7.5,10.0,12.5mmol/L, respectively. Blank and control were set. Sextuplicate were done in each experimental group.After cultured for 24, 48 and 72h, 20μl MTT solution (5mg/mL in PBS) was added to each well. Then additional 4h incubation was carried on, supernatant was sucked out and 200μl DMSO was added to solubilize crystallize with concussion sufficiently. The absorbance was measured with ELIASA, the inhibition rate of growth was calculated, and IC50 at different treat time was described as graphics.(4)Observation under the inversion phase contrast microscope: Cells were inoculated into 25ml culture flask, the growth status of cells in control group and different concentration groups (2.5,5.0,7.5,10.0,12.5mmol/L) were observed at different times(24, 48, 72h).(5) Observation under the light microscope: Cells were seeded into 6 well plate, and grown on coverslips and allowed to attach by overnight incubation. The culture medium with and without GA were added and cultured for different time (24, 48, 72h). After Giemsa staining, cells were observed under the microscope.(6) Flow cytometric analyses (FCM) of apoptotic SACC-M: After cells were treated with and without GA at different concentrations (2.5,5.0,7.5,10.0,12.5mmol/L) for different time (24,48,72h). The apoptosis of cells were determined by FCM with double staining of Annexin-V-FITC/PI.3 Statistical analyses: Variance analysis, correlation analysis, u–test were performed by SPSS10.0 software.Results:1 MTT assay: GA could inhibit the growth of SACC-M obviously at different times (24h, 48h and 72h) respectively. The highest inhibition rate could reach 80.4%. By statistics analysis, the inhibition rate of growth is significantly different among groups at different times at the same concentration (P <0.01). The inhibitory effect of GA on SACC-M is positive correlated with the concentration and time.2 Observation under the inversion phase contrast microscope: the control cells were flat and polymorphic, grew quickly and attached in the wall, the cytoplasm was full and the rounded nucleus was in the center of cells. The growth of treated cells got slower obviously. Also a few treated cells desquamated from the wall to the medium, majority of the cells still attached in the wall, the shape became round, and were smaller than the control, the nucleus were into shade, the refraction of cells were reinforced.3 Observation under the light microscope: The proportion of nucleus to cytoplasm of the treated cells decreased in comparison with the control group, and there were multiple nucleoli in control group. The cytoplasm condensed and the nucleolus reduced or disappeared with treated cells, the chromatins clustered under the nuclear membrane and nuclear fragmentation was observed.4 FCM assay: Although apoptosis could be found in the control cells, the rate of apoptosis was very low. The apoptosis cells were obviously observed in the treated cells, and the rate ascended depending on the dose and time. The difference between groups was significant by statistical analysis (P <0.01). Conclusion: This study suggested that GA could inhibit proliferation and induce apoptosis in salivary gland adenoid cystic carcinoma cell line SACC-M, which was time and dose-dependent.