Effect And Mechanism Of Allicin Inhibition On Human Papillary Thyroid Carcinoma TPC-1 Cells

To investigate the function and mechanism of allicin in inhibiting the proliferation of human papillary thyroid carcinoma TPC-1 cells in vitro.Methods Take human papillary thyroid carcinoma TPC-1 cells in exponential phase,use Allicin interfering cells in different density(control group 0μg/ml; test groups10μg/ml 、20μg/ml 、 30μg/ml 、 40μg/ml 、 50μg/ml); Four methyl thiazolyl tetrazolium assay(MTT method) was used to observe the effects of Allicin on the growth of TPC-1cells at the same time point with of different concentrations and at the same concentration with different time point, and counting IC50; Application of inverted microscope and transmission electron microscope were used to observe the effect of Allicin on the cell morphology and ultrastructure; The scratch experiments was used to observe the effects of Allicin on TPC-1 cell migration; Flow cytometry(FCM) was used to detect the changes of cell percentage in each cell cycle phase in TPC-1 cell after the Allicin intervention. The expression of Bcl-2 and Bax protein changed in TPC-1 cells were detected by immunocytochemistry and Western blot. Real time fluorescence quantitative PCR was used to detect the expression Bcl-2 m RNA and Bax m RNA.Results MTT results showed that the different concentrations of Allicin inhibited TPC-1 cell proliferation, compared with the control group, the difference was statistically significant(P<0.05), and the inhibition of Allicin on cell proliferation rate was showed dose and time dependent manner; Inverted microscope and transmission electron microscope observation showed that the experimental group after 48 h culture, cell shrinkage smaller size, contour, the transmittance decreases cell surface microvilli decreased or even disappeared, nucleo cytoplasmic ratio decreased, the distribution of chromatin and irregular margination, pyknosis was changed, while the control group had a better cell status, no significant changes in cell device; Scratch test showed that, after48 h of culture in the control group the scratch distance was significantly smaller in the experimental group, with the increase of drug concentration, the migration rate decreased,compared with the control group P<0.05, Allicin showed a concentration dependent inhibition of the migration ability of TPC-1 cells(P<0.05); The proportion of cells in different cell cycle phases occurred after the intervention of different concentrations of Allicin, when the Allicin concentration is 10μg·m L-1 in each phase of the percentage ofcells compared with the control group, P>0.05, but with the increase of the concentration of Allicin the proportion of cells at G2/M phase obviously increased and the proportion of G0/G1 phase cells decreased, with the compared to the control group P<0.05. The difference was statistically significant;Immunocytochemistry and Western blot showed that the expression of group and experimental group, Bcl-2 protein and Bax protein of control group, but with the increase of the expression of Bcl-2 Allicin was decreased,whereas Bax expression increased, compared with the control group P<0.05, and in a concentration dependent manner; PCR experiments showed that the experimental group and the control group, the expression of Bcl-2 m RNA and Bax m RNA, but with the decreased expression of Bcl-2 m RNA Allicin concentration decreased, the expression of Bax in m RNA increased, compared with the control group P<0.05, and in a concentration dependent manner.Conclusion 1 Allicin can inhibit the proliferation of TPC-1 cells, the inhibitory effect in a dose and time-dependent manner; 2 Allicin can regulate the cell cycle of TPC-1 cells,mainly for the cell cycle arrest in G2/M phase; 3 Allicin can inhibit the migration and invasion ability of TPC-1 cells; 4 Allicin can induce TPC-1 cells apoptosis through upregulation of Bax and down-regulation of Bcl-2.