环巴胺对人前列腺癌LNCaP细胞增殖和凋亡作用及对PCA3基因表达的影响

Effects of cyclopamine on the proliferation and apoptosis of LNCaP cells and expression of the PCA3 gene in human prostate cancer

目的:探讨环巴胺对人前列腺癌LNCaP细胞增殖和凋亡的作用及对PCA3基因表达的影响。方法:不同浓度环巴胺(1、5、10、15μmol/L)干预LNCaP细胞,以只加入RPMI 1640培养液为空白对照组,分别在不同作用时间(24、48、72h),用MTT法检测其对细胞增殖的抑制、流式细胞术观察细胞凋亡率变化、Hoechst染色观察凋亡细胞形态变化、实时荧光定量逆转录聚合酶链反应(FQ-RT-PCR)观察对PCA3基因表达的影响。结果:5、10、15μmol/L环巴胺对LNCaP细胞增殖均有显著抑制作用,与空白对照组相比差异有显著性(P<0.01),10μmol/L组于48h达到半数抑制量(IC50);10、15μmol/L组24、48、72hLNCaP细胞的凋亡率分别为37.21%、57.38%、57.98%和21.16%、71.31%、72.90%,与空白对照组相比差异均有显著性(P<0.01)。随着环巴胺浓度增大和作用时间的延长,LNCaP细胞凋亡显著增加。PCA3基因的表达随着环巴胺浓度的上升呈现明显的递减趋势,较空白对照组显著降低(P<0.01)。浓度为10μmol/L时,不同作用时间PCA3基因的表达均极低。结论:环巴胺浓度为10、15μmol/L作用48、72h时能够明显抑制LNCaP细胞的增殖,诱导细胞凋亡,并显著下调LNCaP细胞PCA3基因的表达。

Objective ： To explore the effects of cyclopamine on the proliferation and apoptosis of LNCaP cells and the expres- sion of the PCA3 gene in human prostate cancer in vitro. Methods ： LNCaP ceils were treated with cyclopamine at the concentrations of 1, 5, 10 and 15 μmol/L for 24, 48 and 72 hours. The inhibitory effects of cyclopamine on the proliferation and apoptosis of the LNCaP cells were detected by MrIT and flow cytometry respectively, the morphological changes of the cells observed by Hoechst 33258 staining, and the expression of the PCA3 gene determined by real-time fluorescence quantitative reverse transcriptase polymerase chain reaction （FQ-RT-PCR）. Results ： Compared with the blank control group, cyclopamine significantly inhibited the proliferation of the LNCaP cells at 5, 10 and 15 μmol/L （P 〈0.01 ）, reaching IC50 at 10 μmol/L at 48 hours. The apoptosis rates of the LNCaP cells at 24, 48 and 72 hours were 37.21%, 57.38% and 57.98% in the 10 μmol/L group and 21.16%, 71.31% and 72.90% in the 15 μmol/L group, significantly different from those in the control （ P 〈 0.01 ）. The cell apoptosis showed a rising trend with the in-crease of cyclopamine concentration and acting-time, while the expression of the PCA3 gene was decreasing with the increased concen- tration of cyclopamine, significantly lower than that of the blank control group （P 〈 0.01 ）, and extremely low in the 10 ~mol/L group at all time points. Conclusion ： Cyclopamine intervention at 10 and 15 ~mol/L for 48 and 72 hours could significantly inhibit the pro- liferation and induce the apoptosis of LNCaP cells and reduce the expression level of PCA3. Natl J Androl, 2014, 20 （3） ： 213 - 217