【作者】 初钊辉；

【导师】 梁晓华； 周鑫莉； 黄若凡；

【作者基本信息】 复旦大学 ， 肿瘤学， 2009， 硕士

【摘要】 背景和目的:鱼藤素可抑制多种肿瘤细胞的增殖并诱导其凋亡,而且与它能抑制Akt的磷酸化相关,因此它的抗肿瘤机制可能与磷脂酰肌醇-3激酶/蛋白激酶B(phosphatidylinositol 3-kinase/protein knase B,PI3K/Akt)信号通路有关。本研究以人乳腺癌细胞株MCF7为对象,观察鱼藤素对MCF7细胞增殖的影响和能否诱导其凋亡,并研究鱼藤素调控PI3K/Akt信号通路的机制,进一步阐明它的抗肿瘤机制。方法:1.CCK8法检测0、1、5、10、15和20μmol/L鱼藤素作用24、48、72小时后MCF7细胞的增殖抑制率。2.流式细胞仪Annexin V/PI双染法检测0、1、5、10、15和20μmol/L鱼藤素作用6小时后MCF7细胞的凋亡率。3.透射电镜观察0、1、5、10、15和20μmol/L鱼藤素作用6小时后MCF7细胞的形态。4.western blot法检测0、1和5μmol/L鱼藤素作用6小时后MCF7细胞内PI3K/Akt通路相关分子:pPTEN(Ser380)、pPDK1(Ser241)、总Akt、pAkt(Thr308)、pGSK-3β(Ser9)和pc-Raf(Ser259)的蛋白量表达。结果:1.鱼藤素对MCF7细胞增殖的影响:1、5、10、15和20μmol/L鱼藤素作用24小时后MCF7细胞的增殖抑制率分别为(0.77+0.32)%、(6.87+0.50)%、(11.97±1.50)%、(15.87+1.15)%和(18.23±1.80)%,鱼藤素1μmol/L组与对照组比较差异无统计学意义(p=0.408);其余各组与对照组比较差异均有显著的统计学意义(p＜0.001),抑制率随着浓度的增高而增加,各组间两两比较差异有统计学意义(p＜0.05)。48小时和72小时也出现了相似的差别。同一浓度下,5、10、15和20μmol/L鱼藤素作用24、48和72小时后MCF7细胞的增殖抑制率均随着时间的延长而增加,各组间两两比较差异有显著的统计??学意义(p＜0.01);而1μmol/L组鱼藤素作用24、48和72小时后MCF7细胞的增殖抑制率分别为(0.77±0.32)%、(1.00±0.36)%和(0.10±0.95)%,组间比较差异无统计学意义(p=0.257)。72小时内,5、10、15和20μmol/L鱼藤素对MCF7细胞的增殖呈抑制作用。并且随着浓度升高和时间延长其抑制作用愈加明显;1μmol/L鱼藤素对MCF7细胞的增殖无明显影响。2.鱼藤素对MCF7细胞凋亡的诱导作用:5、10、15和20μmol/L鱼藤素作用6小时后MCF7细胞的总凋亡率分别为(11.76±1.42)%、(15.73±0.84)%、(24.10±1.30)%和(29.83±1.66)%,各组与对照组比较差异均有显著的统计学意义(p＜0.001),随着浓度的增高其总凋亡率增加,各组间两两比较差异有显著的统计学意义(p＜0.01)。早期凋亡率和晚期凋亡率出现类似的趋势。1μmol/L鱼藤素作用6小时后MCF7细胞的早期、晚期和总凋亡率分别为(1.47±0.66)%、(2.73±0.45)%和(4.20±0.61)%,与对照组比较P值依次为0.799、0.623和0.512,差异均无统计学意义(p＞0.05)。5、10、15和20μmol/L鱼藤素能诱导MCF7细胞凋亡,凋亡诱导作用随着浓度的升高而愈加明显,并且透射电镜下可观察到典型的凋亡细胞形态。而1μmol/L鱼藤素对MCF7细胞的凋亡诱导作用不明显。3.鱼藤素对MCF7细胞的PI3K/Akt信号通路相关蛋白的影响:与对照组相比,5μmol/L鱼藤素作用6小时后,MCF7细胞内pPTEN(Ser380)、pPDK1(Ser241)、pAkt(Thr308)和pGSK-3β(Ser9)蛋白的表达量明显减少,而总Akt和pc-Raf(Ser259)蛋白的表达量则无明显变化,而1μmol/L鱼藤素作用6小时后,细胞内上述所有蛋白的表达量均无明显变化。结论:1.鱼藤素能抑制MCF7细胞增殖。2.鱼藤素能诱导MCF7细胞凋亡。3.鱼藤素能下调MCF7细胞内pPTEN(Ser380)、pPDK1(Ser241)、pAkt(Thr308)和pGSK-3β(Ser9)蛋白的表达水平,而对总Akt和pc-Raf(Ser259)蛋白的表达量无明显影响。鱼藤素可能通过抑制PTEN(Ser380)和PDK1(Ser241)蛋白的磷酸化,进而抑制Akt(Thr308)的磷酸化,间接抑制其下游GSK-3β(Ser9)的磷酸化,最终诱导MCF7细胞凋亡和抑制其增殖。更多还原

【Abstract】 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.更多还原