原苏木素A通过调控Notch1信号通路增加前列腺癌细胞的放疗敏感性

Radiosensitivity of Prostate Cancer Cells Enhanced by Protosappanin A through Regulating Notch1 Signaling Pathway

目的:研究原苏木素A(PrA)对前列腺癌PC-3细胞放疗敏感性及的Notch1信号通路影响。方法:体外培养人前列腺癌PC-3细胞,分别用不同浓度PrA(100,200,400,800,1600μmol·L-1)处理对数生长期PC-3细胞,分别设为PrA-1组、PrA-2组、PrA-4组、PrA-8组、PrA-16组,另设无处理PC-3细胞为正常对照(NC)组,只进行放射处理的PC-3细胞为放射处理(RT)组。采用四甲基偶氮唑蓝(MTT)法检测各组前列腺癌PC-3细胞增殖情况;采用平板克隆试验法检测前列腺癌PC-3细胞增殖能力;采用流式细胞仪和AnnexinV-FITC/PI法体外检测前列腺癌PC-3细胞凋亡率;Western Blot检测前列腺癌PC-3细胞中凋亡相关半胱氨酸天冬氨酸蛋白酶3(cleaved-caspase3)、B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)蛋白表达水平及Notch1信号通路相关蛋白Jagged1、Notch1、Hes-1蛋白表达。结果:与NC组比较,RT组、PrA-1、PrA-2、PrA-4、PrA-8组前列腺癌PC-3细胞增值抑制率呈时间依赖性依次增加(P<0.05),PrA-8组48 h时,前列腺癌PC-3细胞抑制率为51.03%,与半数抑制浓度(IC50)接近,因此将800μmol·L-1作为PrA最佳浓度,48 h作为最佳作用时间用于下游实验。与NC组比较,RT组、PrA-4、PrA-8组列腺癌PC-3细胞平板克隆形成率、Bcl-2蛋白水平依次降低(P<0.05),细胞凋亡率、Bax、cleaved-caspase3、Jagged1、Notch1、Hes-1蛋白水平依次升高(P<0.05)。PrA-8和PrA-16组前列腺癌PC-3细胞增值抑制率、克隆形成率、凋亡率、Bcl-2、Bax、cleaved-caspase3、Jagged1、Notch1、Hes-1蛋白表达水平比较,差异均无统计学意义(P>0.05)。结论:原苏木素A可通过上调Bax、cleaved-caspase3,下调Bcl-2蛋白,激活Notch1信号通路增加前列腺癌PC-3细胞的放疗敏感性。

Objective:To study the effect of protosappanin A(PrA)on the radiosensitivity of prostate cancer PC-3 cells through regulating Notch1 signal pathway.Methods:Human prostate cancer PC-3 cells were cultured in vitro and treated with different concentrations of PrA(100,200,400,800 and 1600!mol·L-1)at logarithmic growth stage.PC-3 cells were divided into PrA-1 group,PrA-2 group,PrA-4 group,PrA-8 group and PrA-16 group.PC-3 cells without any treatment were also set as the normal control(NC)group,and those only treated with radiotherapy were used as the radiotherapy(RT)group.The proliferation of PC-3 cells in each group was detected by MTT assay,the proliferation ability of PC-3 cells was detected by plate cloning assay,the apoptotic rate of PC-3 cells was detected by flow cytometry and Annexin V-FITC/PI assay in vitro,and Western blot was used to detect apoptosis-related cleaved-caspase 3,B-cell lymphoma-2(Bcl-2)and Bax protein expressions and Notch1 signaling pathway related proteins Jagged1,Notch1 and Hes-1 protein expressions in PC-3 cells of prostate cancer.Results:Compared with that in NC group,the inhibition rates of PC-3 cell proliferation in RT,PrA-1,PrA-2,PrA-4 and PrA-8 groups increased in a time-dependent manner(P<0.05).At 48 h in PrA-8 group,the inhibition rate of PC-3 cells was 51.03%,which was close to the half inhibitory concentration(IC50),therefore,800!mol·L-1 was used as the optimum concentration of PrA and 48-hour as the optimum time for downstream test.Compared with those in NC group,the platelet cloning rates of PC-3 cells and Bcl-2 protein levels in RT group,PrA-4 group and PrA-8 group decreased in turn(P<0.05),and the apoptotic rates,and Bax,cleaved-caspase 3,Jagged 1,Notch1 and Hes-1 protein levels increased in turn(P<0.05).There were no significant differences in the cell proliferation inhibition rate,cloning formation rate,apoptosis rate,and Bcl-2,Bax,cleaved-Caspase 3,Jagged 1,Notch1 and Hes-1 protein expression levels between PrA-8 group and PrA-16 group(P>0.05).Conclusion:Protosappanin A can increase the radiosensitivity of PC-3 prostate cancer cells by up-regulating Bax and cleaved-caspase 3,down-regulating Bcl-2 protein and activating Notch1 signaling pathway.