Objective: To explore the inhibitory effect of polyphyllin Ⅰ (PPⅠ) on the proliferation of castration-resistant prostate cancer PC3 cells and its molecular mechanism.
Methods: We cultured human prostate cancer PC3 cells in vitro and treated them with PPⅠ at the concentrations of 0 (blank group), 0.4, 0.8, 1.2, 1.6, 2.0, and 2.4 μmol/L for 24, 48, and 72 hours, respectively. Then we detected the proliferation of the cells by MTT assay, measured their apoptosis by flow cytometry, and determined the expressions of p-ERK1/2, ERK1/2, NF-κB/p65 and DNMT1 proteins as well as the level of NF-κB/p65 in the cells additionally treated with the ERK1/2 inhibitor SP600125 by Western blot.
Results: Compared with the blank control group, the PPⅠ-treated PC3 cells showed a concentration- and time-dependent reduction of the survival rate (1.00 ± 0.00 vs 0.85 ± 0.05, P < 0.01) at 0.4 μmol/L after 48 hours of intervention, concentration-dependent early apoptosis at 0.8 μmol/L (4.83 ± 0.95 vs 13.83 ± 2.97, P < 0.01), time-dependent increase of the expressions of p-ERK1/2 (1.00 ± 0.00 vs 1.73 ± 0.17, P < 0.01) and ERK1/2 (1.00 ± 0.00 vs 1.36 ± 0.12, P < 0.01) at 2 hours, and concentration-dependent decrease of the expressions of NF-κB/p65 and DNMT1 at 1.2 μmol/L (1.00 ± 0.00 vs 0.78 ± 0.10 and 0.63 ± 0.06, P < 0.01) and 1.6 μmol/L (1.00 ± 0.00 vs 0.67 ± 0.11 and 0.52 ± 0.09, P<0.01). Inhibition of ERK1/2 phosphorylation with PD98059 markedly reversed PPⅠ-induced decrease of the NF-κB/p65 expression as compared with that in the PPⅠ group (0.86 ± 0.18 vs 0.43 ± 0.09, P < 0.05).
Conclusions: PPⅠ induces the early apoptosis and suppresses the proliferation of PC3 cells, probably by activating the ERK1/2 pathway and inhibiting the expressions of the NF-κB/p65 and DNMT1 proteins.
目的： 研究重楼皂苷Ⅰ(PPⅠ)对去势抵抗性人前列腺癌PC3细胞生长的抑制作用及其分子机制。方法： 体外培养PC3细胞，予不同浓度（0.4、0.8、1.2、1.6、2.0、2.4 μmol/L）的PPⅠ处理24、48、72 h，另设对照组，MTT法观察PPⅠ对PC3细胞增殖的影响；流式细胞术检测细胞凋亡率；Western印迹检测PPⅠ对磷酸化细胞外调节蛋白激酶1/2（p-ERK1/2）、细胞外调节蛋白激酶1/2（ERK1/2）、核转录因子kappa B（NF-κB）/p65以及DNA甲基转移酶1（DNMT1）蛋白表达的影响，并加入ERK1/2抑制剂（PD98059）后检测PPⅠ对NF-κB/p65表达的影响。结果： MTT结果显示，PPⅠ能抑制PC3细胞的体外增殖，与对照组相比，PC3细胞从给药浓度0.4 μmol/L（0.85±0.05 vs 1.00±0.00, P <0.01）开始明显下降，且呈时间和剂量依赖性。流式细胞术检测显示，PPⅠ能明显诱导PC3细胞早期凋亡，与对照组相比，PC3细胞早期凋亡率从给药浓度0.8 μmol/L（13.83±2.97 vs 4.83±0.95）开始明显增加（P均<0.01），且呈剂量依赖性；Western印迹显示，PPⅠ以时间依赖性上调p-ERK1/2和ERK1/2蛋白的激活与表达，与对照组相比，给药时间从2 h（1.73±0.17 vs 1.00±0.00，P <0.01）开始，p-ERK1/2明显被激活表达，PPⅠ以剂量依赖性下调NF-κB/p65、DNMT1蛋白的表达，与对照组相比，给药浓度分别从1.6 μmol/L（0.67±0.11 vs 1.00，P<0.01）与1.2 μmol/L（0.63±0.06 vs 1.00±0.00，P<0.01）开始，NF-κB/p65、DNMT1表达明显下调；抑制ERK1/2磷酸化能逆转重楼皂苷Ⅰ对NF-κB/p65蛋白表达的下调，与PPⅠ组相比，PD98059+PPⅠ组NF-κB/p65蛋白表达（0.86±0.18 vs 0.43±0.09，P<0.05）明显上调。结论： PPⅠ可能通过介导ERK1/2通路抑制NF-κB/p65和 DNMT1蛋白表达，诱导PC3细胞早期凋亡，进而抑制细胞增殖。.
Keywords: DNMT1; ERK1/2; NF-κB/p65; apoptosis; polyphyllin Ⅰ; proliferation; prostate cancer.