The Effect Of Trichosanthin To Inhibit The Proliferation And Reverse Methylation On MDA-MB-231 Cell Line

Objective: To observe the effect of Trichosanthin(TCS) on the apoptosis of MDA-MB-231 cell line and demethylated effect on CpG island at the promoter region of Syk gene to find a novel demethylated medicine.Methods:1 The inhibitory effect of TCS on cells MDA- MB- 231 were detected by MTT assay in 0,10, 20, 40, 80 and 100μg/ml TCS treatment groups at three different treatment time points (24h, 48h and 72h);2 Giemsa staining, DNA ladder assay and flow cytometry (FCM) were carried out for observing the inducible apoptosis by TCS in MDA-MB-231 cells. FCM measured apoptosis of cells in four treatment experimental groups (0,20,40,80μg /ml TCS treatment for 48h);3 Methylation status of syk was detected by methylation- specific PCR (MSP);4 RT-PCR was used to measure the expression of methyl- transferases genes including dnmt 1, dnmt 3a, dnmt 3b and demethylation gene mbd2 in cells of experimental group (20μg/ml TCS treatment for 48h); 5 The changes of the activity of DNA methyltransferase in MDA-MB-231 cells were measured by DNMTs detecting kit after being treated by Trichosanthin.Results:1 TCS (10~100μg/ml) can significantly inhibit the growth of MDA-MB-231 cells in vitro (p<0.05). The growth inhibition is both in dose-dependent and time-dependent manners. The greatest inhibitory rate is 77.74±0.539% observed in treatment groups of MDA-MB-231 cells treated by 100μg/ml TCS for 72 hours;2 Giemsa staining reveals that TCS can induce typical morphological changes of apoptosis in MDA-MB-231 cells, such as cell body shrink, condensed cytoplasm, vacuole degeneration, some cells karyopyknosis, nuclear fragmentation. DNA ladder is observed in MDA-MB-231 cells treated by 20μg/ml TCS for 48 hours. This suggests that TCS can induce apoptosis of MDA-MB-231 cells. The inducible cell apoptosis is in a dose-dependent manner for 48 hour (p<0.05). The apoptotic rate of cells treated with 80μg/ml TCS is as high as 40.7% and the cells are arrested at S phase;3 Demethylation of syk gene occurs after treatment with 20μg/ml TCS for 48 hours;At mRNA level, there is no significant effect on the expression of methyltransferases including dnmt 1, dnmt 3a, dnmt 3b and demethylation gene mbd2 in cells of experimental group (20μg/ml TCS treatment for 48h) (p<0.05); 4 The activity of DNA methyltransferases in MDA-MB-231 cells is depressed after treated by 20μg/ml TCS for 48 hours.Conclusion:1 Trichosanthin (TCS) can significantly inhibit the proliferation of human breast cancer cells MDA-MB-231 in vitro both in dose-dependent and time-dependent manners;2. The effect of growth suppression induced by TCS on the human breast cancer cell line MDA-MB-231 may be related to apoptosis and cell cycle arrest, TCS enable MDA-MB-231 growth cycle arrest at S phase.3 Trichosanthin can reverse the hypermethylation of the promoter region of the syk gene.4 The effect of demethylation induced by TCS may result from depressing the activity of DNA methyltransferases。...