| 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 onset 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 - evodiae, which belongs to rutacese, is the dry and nearly repeningfruits. Evodiamine is the main active component, which has effects of coldwarming the middle warmer, removing cold, dispersing stagnated hepatoqi to stop pain, and often be applied to cure psychroalgia in stomach, emesia, diarrhea, headache, stomachache, menalgia and so on. Current medicine proves that evodiamine has pharmacologic actions of relieving pain, sedation , antibiosis, decreasing blood pressure, antitumor. Nevertheless, whether the evodiamine could induce the apoptosis and necrosis of SACC has not been reported yet. The aim of study was to observe the effect of evodiamine of different concention on SACC ACC-M at different time in vitro, to explore the possible mechanism and to provide theoretical basis for clinical application of evodiamine in treating SACC.Materials: 1 Materials: (1)Cell line ACC-M was purchased from the laboratory of oral and maxillofacial surgery of Shanghai jiaotong University,which was established in 1995; (2) Evodiamine was obtained from national institute for the control of pharmaceutical and biological products.2 Methods: (1)Preparation of physic liquor: Evodiamine was dissolved in dimethyl sulfoxide(DMSO) and saved on 20mg/ml concentration, which was diluted before used(The finial concentration of dimethyl sulfoxide is less than 0.001% and has no effect on cells). (2)Cell culture: ACC-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 in the mixed liquid containing 0.25% trypsin and 0.04% EDTA (1:1). (3)MTT assay: 2×107?L-1 Cell suspension in logarithmic phase were seeded into 96-well plates, 100μl tissue culture medium every well. After 24h incubating, 100μl evodiamine solution was added into each well, and the final concentrations were 0.01, 0.1, 1, 10, 100 ug/ml respectively. After cultured for 24, 48 and 72h, 20μl MTT solution (5mg/mL in PBS) was added to every well. Then an additional 4h incubation was carried on, supernatant was abandoned 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 50ml culture flask, the growth status of cells in control group and different concentration groups (0, 0.01, 0.1, 1,10,100ug/ml) were observed at different time(24, 48, 72h).(5) Observation under the light microscope: Cells were inoculated into into 6 well plate with vitreous flake in every well. The culture medium with and without evodiamine were added after the elementary adherence and cultured for different time(24, 48, 72h). After Wright-Giemsa staining, cells were observed under the microscope.(6) Flow cytometric analyses (FCM) of apoptotic ACC-M: After cells were incubated with and without evodiamine at different concentrations (0, 0.01, 0.1, 1,10, 100ug/ml) for different time (24, 48, 72h). Doubly stained by Annexin-V-FITC/propidiumiodide (PI), the apoptosis of cell was analyzed by FCM. 3 Statistical analyses: SNK-q test, correlation analysis,χ2–test were performed by SPSS10.0 software.Results: 1 MTT assay: Evodiamine could inhibit the growth of ACC-M obviously(>1ug/ml), and IC50 are 6.5ug/ml,4.5ug/ml,0.85ug/ml at diffrent time (24h, 48h and 72h) respedtively. The highest inhibition rate could reach 89%. By statistics analysis, the absorbance is significantly different among groups at different concentrations at the same time (p<0.01), and the inhibition rate of growth is significantly different among groups at different time at the same concentration (p<0.01). The inhibitory effect of evodiamine on ACC-M is positive correlated with the concentration and time.2 Observation under the inversion phase contrast microscope: the control cell was flat and polygon, growed quickly and adhered the wall like slabstone, the cytoplasm was full and the rounded nucleus was in the center of cells. The growth of treated cells got slower obviously. Although a few treated cells desquamated from the wall to the medium, majority of the cells still adhered the wall, the shape became round and smaller than the control, the nucleus were into shade, the refraction of cells were reinforcing.3 Observation under the light microscope: The proportion of nucleu to cytoplasm of the treated cells increased in comparison with the control. The cytoplasm condensed and the nucleolus reduced or disappeared, the chromatins clustered under the nuclear membrane or 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, especially when the concentration reached 1ug/ml, the rate went up sharply. The difference between groups was significant by Statistical analysis (p<0.01).Conclusion:This study suggested that evodiamine could inhibit proliferation and induce apoptosis in salivary gland adenoid cystic carcinoma cell line ACC-M, which was time and dose-dependent.
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