去氢表雄酮通过Akt信号通路诱导肝癌细胞凋亡和细胞周期阻滞

Molecular mechanisms of DHEA and DHEAs on apoptosis and cell cycle arrest via Akt pathway in hepatoma cell lines

目的 研究去氢表雄酮（DHEA）及其代谢物去氢表雄酮硫酸酯（DHEAs）对HepG2和HT-29细胞抑制增殖、促进凋亡及诱导细胞周期阻滞的作用。方法 应用MTT比色法检测不同浓度（1、10、50、100、200μmol／L）的DHEA或DHEAs与HepG2和HT-29细胞孵育8、24、48、72 h对两种细胞系的生长抑制作用;应用流式细胞术检测不同浓度的DHEA或DHEAs对细胞凋亡及细胞周期的变化;采用Western blot检测细胞内磷酸化Akt（Ser473,Thr308）蛋白的水平。结果 （1）不同浓度DHEA作用细胞24h时,HepG2的存活率24h时分别为92．7％±0．9%、84．7％±1．2％、62．4％±0．8％、49．5％±0．8％和50．7％±0．3％,HT-29细胞的存活率分别为92．5％±0.4％、89．5％±0．7％、80．5％±1．1％、67.5％±1.5％和70．6％±0．6％,与对照组相比,DHEA明显抑制HepG2和HT-29两种细胞的生长。在浓度为100μmol／L作用24 h时作用明显,而DHEAs对HepG2及HT-29细胞的增殖无明显影响。（2）100μmol／L的DHEA显著抑制两种细胞周期进程, HepG2细胞G0／G1期细胞比例显著升高,可以达到68．4％±2．0％,而对照组为48．6％±1．2％。HT-29细胞在100μmol／L时的G0／G1期的比率为90．3％±2．7％,而对照组仅为59．0％±1．2％,S及G2／M期细胞明显减少。（3）100μmol／L DHEA作用24 h时能显著诱导HepG2细胞凋亡（凋亡率为18．6％±2．2％）,而DHEAs却无此作用。（4）HepG2细胞在100μmol／L和200μmol／L DHEA作用24 h后,磷酸化Akt（Thr^308）、磷酸化Akt（Set^473）蛋白表达显著降低,这种作用在应用PI3K抑制剂和PI3K激活剂后分别被增强和消除。结论 DHEA对HepG2和HT-29两种肿瘤细胞系均具有较强的抗增殖作用。而对于不同的肿瘤细胞系,DHEA可能通过调节Akt的信号通路来诱导细胞凋亡,还可能通过阻滞细胞周期,使其阻滞在G0／G1期。DHEAs对细胞的生长没有明显的作用。

Objective To evaluate the anti-proliferation effects of dehydroepiandrosterone （DHEA） and DHEA sulfate （DHEAs） on tumor cells. Methods Human hepatoblastoma cells （HepG2） and colon adenocarcinoma cells （HT-29） were treated with DHEA and DHEAs of various concentrations. The cells were incubated for 8, 24, 48, and 72 hours, and the proliferation, apoptosis, cell cycle and the expression of phosphorylated Akt （Thr308 and Ser473） were analyzed using MTT assay, flow cytometry, and Western blotting at different time points. The influences of an inhibitor （LY294002） and an activator （hepatic growth factor; HGF） of PI3K on the effectiveness of DHEA were determined in HepG2 cells. Results By increasing the concentrations of DHEA （1, 10, 50, 100, 200 μmol/L）, the percentages of HepG2 and HT-29 survival cells treated with DHEA at 24 h were 92.7% ± 0.9%, 84.7% ± 1.2%, 62.4% ±0.8%, 49.5% ± 0.8%, 50.7% ± 0.3% and 92.5% ± 0.4%, 89.5% ± 0.7%, 80.5% ± 1.1%, 67.5% ± 1.5%, 70.6% ± 0.6%, respectively. Proliferations of HepG2 and HT-29 cells were significantly inhibited after 24 hours of being incubated with 100μmol/L DHEA treatment; the inhibition effect was stronger on HepG2 cells than on HT-29 cells. The effect of DHEAs on both cell lines on cell proliferation was weaker than that of the DHEA. In the cell cycle assay, DHEA treatment induced cell arrest in G0/G1 phase in both cell lines. Apoptosis of HepG2 cells was significantly triggered （18.6% ± 2.2%） by 100μmol/L DHEA treatment for 24 hours, but not by DHEAs. In addition, 100 and 200 lamol/L DHEA treatments for 24 hours markedly inhibited phosphorylations of Akt （Thr^308 and Ser^473） in HepG2 cells, and these effects were enhanced by exposing them to LY294002 and stopped by exposing them to HGF. The anti-proliferative effects of DHEA on tumor cell lines were much stronger than those of DHEAs, and they were even stronger in HepG2 cells than in HT-29 cells. Conclusion Our results suggest that the induction of apoptosis through the inhibition of Akt signaling pathway is one of the anti-proliferative mechanisms of DHEA in certain tumors, but DHEA also promotes cell-cycle arrest.