| Objective:Resveratrol is the anthraquinone terpenoids, a natural antioxidant.The effect of Resveratrol includes reducing blood viscosity; Inhibition of platelet aggregation and vasodilation; maintaining blood flow;preventing the occurrence and development of cancer; Anti-atherosclerosis and coronary heart disease,ischemic heart disease, preventing and treating high blood cholesterol.Studies have shown that Resveratrol has anti-cancer effect,in other words,it Can induce apoptosis or inhibit cancer cell growth. It has been reported Resveratrol had effects on inhabiting human liver cancer, breast cancer, bladder carcinoma cells, but it has been yet reported to inhabit human salivary adenoid cystic carcinoma. Salivary adenoid cystic carcinoma(SACC) is one of the most common malignant tumors in salivary glands and a strong aggressive tumor. Through the submucosal and fibrous tissue surrounding the tumor, it disseminates gradually and widely. At the same time, it gradually extends along the nerve.So rate of local recurrence is higher, particularly in the tumor with positive margin. Distant metastasis often occur. Death is mainly due to local failure or distant metastasis. To improve survival quality of the patients, it is necessary to find a way to treat the disease. The study is to observe the effect of resveratrol on human salivary adenoid cystic carcinoma cell line ACC-M.Materials: (1) ACC-M high metastatic salivary adenoid cystic carcinoma cell lines: provided by School of Stomatology, Shanghai Jiao Tong University (2)Resveratrol: provided by china pharmaceutical biological products analysis institute.Methods: (1)cell culture: RPMI1640 medium with 10% fetal bovine serum and penicillin (100U/ml), streptomycin (100μg/ml)was used, the cells were seeded in 50ml culture flasks at 37℃, 5% CO2 saturation temperature and humidity. The cell generation was at 24-48 hour. 0.04% diethylamine tetraacetic acid disodium (EDTA) and 0.25% trypsin mixture(1:1) was used in the Harvest cell.(2) Liquid preparation: 20mg Resveratrol was dissolved with dimethyl sulfoxide (DMSO) and treated as 20mg/ml stock solution,and before use, diluted with culture medium (DMSO concentrations was less than 0.001%, no effect on the cells).(3) MTT colorimetric test: logarithmic growth phase cells were suspended as 5×104·ml-1 cells, seeded in 96 well plates, each well 100μl, cultured for 24h, and resveratrol 100μl was added, the final concentrations were 12.5,25,50,100 and 200μmol / L respectively, the control group (with same DMSO), each with 6 wells. The cells were cultured for 24,48,72 h, 5g / L of MTT solution 20μl was added in each wells. after culturing for 4h,all the supernatant was absorbed carefully, then 200μl dimethyl sulfoxide was added into each well, and the crystal was dissolved completely.The absorbance (A) of the each well was measured on microplate reader set. According to the formula: inhibition rate = (1 - the average A value of the drug group/ the average A value of the control group)×100% ,growth inhibition rate was calculated.(4) Inverted microscope observation: The cell survival in 50ml culture flasks was observed in experimental groups and control group.(5) Light microscopy observation: The cells were seeded in 6-well plates, the cover slip was placed in the well. After the cells attached, the drug was added in the experiment groups and media without any drug was added in the control group.After culturing for the different time,Giemsa staining was performed,and nuclear morphology was observed by light microscopy.(6) Apoptosis detection:The apoptosis of the cells was detected with Annexin-V-FITC and PI double labeling by flow cytometry.3 Statistical analysis: The absorbance values, the growth inhibition rate and apoptosis rate were analyzed with SNK-q test, correlation analysis, u-test by SPSS13.0 statistical software.Results: 1 MTT assay: The growth of ACC-M cells was significantly inhibited in the Resveratrol 100μmol / L or more than the Resveratrol 100μmol / L, the highest inhibition rate was up to 96.1% at the Resveratrol 200μmol / L for 72 hours.The OD values in resveratrol 200μmol / L group for 72h had significant differences in comparing with the other groups. The proliferation inhibition of the resveratrol at different concentrations on the salivary adenoid cystic carcinoma cell line ACC-M was positively correlated, but the inhibitory effect was not significantly increased with the time.2 Inverted microscope: The cells grew well, the cells was irregular, arranged in single layer, dense cytoplasm rich in cytoplasm, rounded nuclei located near the center, showed that the phenomenon of cell division and proliferation in the control group. The cells grew slowly, had fewer smaller cell nuclei stained black, cytoplasm cytoplasmic condensation, loose connections between the cells in the treatment group. Especially in the resveratrol 200μmol / L group for 72h, some cells floated in medium, clear cell shrinkage and increased intracellular particles were observed.3 Light microscopy observation: In the control group, bulky, irregular cells were observed, which the membrane was integrity, the cytoplasm was rich, the nuclear plasma ratio was greater, the nucleus round and large, the multiple nucleoli could be seen. In the treatment group, the size of cells were smaller, the cell membrane was incomplete or even rupture, leakage of cytoplasm was seen. The nucleus becomes smaller, the nuclear cytoplasm ratio was smaller. The less nucleolus, chromatin condensation, nuclear stained, destruction of nuclear and fragmentation, chromatin condensation gathered around the nuclear membrane were also observed, which showed the apoptosis of the cells.4 Flow cytometry observation: The apoptosis cells in the control group belonged to the natural apoptosis. The apoptosis rate in the treatment group increased with the increased concentration and with the time. The apoptosis rate significantly increased at more than resveratrol 100μmol / L concentration. There was statistically difference between groups.Conclusion: The proliferation inhibition of the resveratrol at different concentrations on the salivary adenoid cystic carcinoma cell line ACC-M was positively correlated, but the inhibitory effect was not significantly increased with the time. The effect of induce apoptosis of resveratrol on salivary gland adenoid cystic carcinoma cell line ACC-M was time and dose-dependent.
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