| Objective:Deguelin inhibits proliferation and induces apoptosis in a variety of cancer cells, which correlates with inhibiting the phosphorylation of protein kinase B/Akt. Deguelin's anti-cancer mechanism may be related to the phosphatidylinositol-3 kinase /protein kinase B(PI3K/Akt)signaling pathway.In this study,the effects of deguelin on proliferation and apoptosis in human breast cancer cell line MCF7 were observed. The mechanism of deguelin regulation of the PI3K/Akt signaling pathway was also explored.Methods:After treatment with 0,1,5,10,15 and 20μmol/L deguelin for 24,48 and 72 hours,the proliferation inhibition rate of MCF7 cells was measured by the CCK8 assay.After treatment with 0,1,5,10,15 and 20μmol/L deguelin for 6 hours,the apoptotic rate of MCF7 cells was detected with Annexin V/PI double staining by flow cytometry and the morphologic change of MCF7 cells was observed using the transmission electron microscopy.After treatment respectively with 0,1 and 5μmol/L deguelin for 6 hours,the total protein was extracted from MCF7 cells,then the levels of phosphorylated PTEN(Ser380),phosphorylated PDKl(Ser241),total Akt,phosphorylated Akt(Thr308),phosphorylated GSK-3p(Ser9)and phosphorylated c-Raf(Ser259) protein expression were examined by western blot analysis.Results:Effects of deguelin on the proliferation of MCF7 cellsAfter treatment with 1,5,10,15 and 20umol/L deguelin for 24 hours,the proliferation inhibition rate of MCF7 cells was(0.77±0.32)%,(6.87±0.50)%, (11.97±1.50)%,(15.87±1.15)%and(18.23±1.80)%,respectively.There was no statistical difference(p=0.408)between the lumol/L treatment group and the control group.However,there was a significant difference(p＜0.001)in the proliferation inhibition rate in each of the 5,10,15 and 20μmol/L groups compared with the control group.Furthermore,the proliferation inhibition rate increased with increasing concentration,and there were statistical differences(p＜0.05)among the 5,10,15 and 20umol/L groups.Similar differences were observed after 48 and 72 hours of deguelin.Again,there was no statistical difference in the lumol/L group but significant differences were found in each of the 5,10,15 and 20μmol/L groups versus control as well as among these groups.At 24,48 and 72 hours,the proliferation inhibition rate increased with the longer treatment time in the 5,10,15 and 20umol/L groups,and there were statistically significant differences(p＜0.01).However,1μmol/L deguelin inhibited the proliferation of MCF7 cells by(0.77±0.32)%,(1.00±0.36)%and(0.10±0.95)%at 24, 48 and 72 hours,and there was no statistical difference among these data(p=0.257).In short,5,10,15 and 20umol / L deguelin inhibited the proliferation of MCF7 cells at 24,48 and 72 hours.The inhibitory effect was more marked with increasing concentration and longer duration of treatment.However,1μmol/L deguelin had no significant effect.Effects of deguelin on the apoptosis of MCF7 cellsAfter treatment with 5,10,15 and 20μmol/L deguelin for 6 hours,the total apoptotic rate of MCF7 cells was(11.76±1.42)%,(15.73±0.84)%,(24.10±1.30)% and(29.83±1.66)%,respectively.There were significant differences(p＜0.001) between each treatment group and the control group.The total apoptotic rate increased with increasing concentration and there were significant differences among the 5,10,15 and 20μmol/L groups(p＜0.01).Similar trends were found in the rates of early apoptosis and last apoptosis.After treatment with 1 umol/L deguelin for 6 hours,the rate of early apoptosis, last apoptosis and total apoptosis of MCF7 cells was(1.47±0.66)%,(2.73±0.45)%and (4.20±0.61)%,respectively.Compared with the control group,the p value was 0.799,0.623 and 0.512.In summary,5,10,15 and 20μmol/L deguelin induced apoptosis of MCF7 cells, and the apoptotic effect was more apparent with increasing concentration. Furthermore,the typical apoptotic MCF7 cells were observed under the transmission electron microscopy.But 1μmol/L deguelin had no apparent effect on inducing apoptosis of MCF7 cells.Effects of deguelin on the expression of 6 proteins involved in the PI3K/Akt signaling pathwayAfter treatment with 5μmol/L deguelin for 6 hours,the expression of pPTEN (Ser380),pPDK1(Ser241),pAkt(Thr308)and pGSK-3p(Ser9)proteins was significantly reduced in MCF7 cells,while there was no significant change in the expression of total Akt and pc-Raf(Ser259)proteins.However,after treatment with 1μmol/L deguelin for 6 hours,there was no apparent change in the expression of these 6 proteins.Conclusion:Deguelin inhibited the proliferation of MCF7 cells.Deguelin induced apoptosis of MCF7 cells.Deguelin down-regulated the expression of pPTEN(Ser380),pPDK1(Ser241), pAkt(Thr308)and pGSK-3β(Ser9)protein,while it had no significant effect on the expression of total Akt and pc-Raf(Ser259)protein.Deguelin probably inhibited the phosphorylation of Akt(Thr308)and indirectly inhibited the phosphorylation of GSK-3β(Ser9)via inhibition of the phosphorylation of PTEN(Ser380)and PDKl(Ser241),thus inducing apoptosis and inhibiting the proliferation of MCF7 cells.
|
PDF Full Text Request