The Experimental Study Of The Effect Of Periplocin On The Proliferation Of MDA-MB-231 And Its Anti-tumor Mechanism

Objective: The novel anti-tumor agent, periplocin is one active component of Chinese herbal drug cortex periplocase. My study is to observe the apoptotic effect on MDA-MB-231 cell lines, and inhibit Stat3 transcriptional activity and regulate the expression of downstream anti-apoptotic genes. The goal is to primarily evaluate the anti-tumor mechanism of periplocin.Methods:1 The inhibitory effect on cell growth of MDA-MB-231 was measured by MTT assay in 10 treated or untreated groups (0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10, 20μg/ml periplocin and control) for three different treatment times (24h, 48h and 72h);2 Wright and Giemse's staining, DNA ladder assay and flow cytometry (FCM) are carried out for observing the inducible apoptosis by periplocin in MDA-MB-231 cells. FCM measured apoptosis of four experimental groups (2.5μg/ml periplocin treatment for 0h, 12, 24h and 48h);3 Western blot analyzed the expression of Stat3 in nucleus and cytoplasm of MDA-MB-231 cells untreated or treated with 0.625μg/ml, 2.5μg/ml periplocin for 24h. Additionally the level of tyrosine705-phopholated Stat3 in the MDA-MB-231 cells was measured in the same way after treated by periplocin (0μg/ml, 0.625μg/ml, 2.5μg/ml) for 24 hours;4 Bonding activity of Stat3 in nucleus and Stat3-specific DNA was measured by electrophoretic mobility shift assay (EMSA) after MDA-MB-231 cells were treated by 0.625μg/ml, 2.5μg/ml periplocin for 24 hours;5 Expression of anti-apoptotic genes including bcl-2, mcl-1 and survivin in cells of four experimental groups (2.5μg/ml periplocin for 0h, 6h, 12, 24h), were observed by revers transcription PCR (RT-PCR);6 Treatment with 2.5μg/ml periplocin affected the concentration of intracellular Ca2+ and expression of Bax and Stat3 in MDA-MB-231 cells observed by laser scanning microscopes (LSM).Results:1 Periplocin (0.078~20μg/ml) can significantly inhibit the growth of MDA-MB-231 cells in vitro (p<0.05). The growth inhibition is in dose-dependent and time-dependent manner. The greatest inhibit rate is 65.32% observed in the treatment group of MDA-MB-231 cells by 20μg/ml periplocin for 72 hours;2 Wright and Giemsa's staining reveals MDA-MB-231 cells treated by periplocin had proapoptotic morphology. DNA ladder was observed in MDA-MB-231 cells (treated with 2.5μg/ml periplocin for 48 hours). This suggests that periplocin can induce apoptosis of MDA-MB-231 cells. The inducible cell apoptosis is in time-dependent manner by 2.5μg/ml periplocin (p<0.05). The apoptosis rate of cells treated for 48 hours is as high as 28.66%;3 The results by Western blotting reveals that periplocin downregulated the level of tyrosine705-phopholated Stat3 and expression of Stat3 in nucleus and cytoplasm of MDA-MB-231 cells in treated group with 0.625μg/ml, 2.5μg/ml periplocin for 24 hours, compared with the control group (untreated) (p<0.05);4 The results by EMSA reveals that binding activity of Stat3 in nucleus to its specific DNA was repressed after MDA-MB-231 cells treated by 0.625μg/ml, 2.5μg/ml periplocin for 24 hours, compared with the untreated cells;5 Periplocin inhibits the mRNA expression of the anti-apoptotic genes bcl-2, mcl-1 and survivin in MDA-MB-231 cells treated by 2.5μg/ml periplocin for 6h, 12h, 24h in time-dependent manner, compared with untreated cells (p<0.05);6 LSM images of MDA-MB-231 cells in the different treatment times (0 min,5 min and 15 min) reveals that Ca2+ concentration and Bax protein in the cytoplasm increased rapidly after periplocin treatment (p<0.05). Expression of Stat3 in MDA-MB-231 cells was repressed after treated by 2.5μg/ml periplocin for 5, 15 minutes (p<0.05).Conclusion:1 Periplocin inhibits significantly proliferation of the human breast cancer MDA-MB-231 cells in vitro in dose-dependent and time-dependent manner;2 The treatment of periplocin induced apoptosis of MDA-MB-231 cells in vitro;3 In the MDA-MB-231 cells, periplocin represses the Stat3 signaling pathway through downregulating the expression of Stat3, blocking its translocation into nucleus and inhibiting the binding activity of Stat3 to its specific DNA motif;4 Periplocin can inhibit significantly the expression of anti-apoptotic genes including bcl-2, mcl-1 and survivin;5. Expression of Bax protein and Ca2+ concentration in the cytoplasm increases rapidly caused by periplocin treatment. The increase of Ca2+ and Bax is associated with the apoptosis induced by periplocin in the MDA-MB-231 cells;6 The study provides the new sight on the molecular mechanism of the anti-tumor effect of periplocin, which can inhibit Stat3 signaling pathway, and induce apoptosis of MDA-MB-231 cells. These suggests that periplocin would be furthor a novel therapeutic agents against the invasive breast cancer.