胡桃醌通过PI3K/AKT途径促进前列腺癌LNCaP细胞氧化应激

Juglone promote oxidative stress through PI3K/AKT pathway in prostate cancer cell

目的探讨胡桃醌对人前列腺癌LNCa P细胞的杀伤活性与其诱导氧化应激反应的相关性及机制。方法采用0、6.25、12.5、25、50和100μmol/L胡桃醌作用前列腺癌LNCa P细胞,MTT法检测不同剂量胡桃醌对LNCa P细胞生长抑制影响;胡桃醌联合抗氧化剂NAC作用细胞后,DCFHDA作为荧光探针,用荧光酶标法检测活性氧(ROS);胡桃醌联合PI3K/AKT通路抑制LY294002作用细胞后,Western blot法检测p-AKT、AKT和Nrf2蛋白表达变化。结果与阴性对照组比较,胡桃醌浓度在12.5μmol/L或以上显著抑制前列腺癌细胞生长(P<0.05,P<0.01)。胡桃醌能够促进细胞ROS含量(P<0.01);胡桃醌联合抗氧化剂N-乙酰半胱氨酸(NAC)导致ROS含量下降(P<0.05),细胞存活率升高(P<0.01)。胡桃醌抑制p-AKT的表达,NAC能够恢复胡桃醌抑制的p-AKT表达。胡桃醌能够抑制细胞核Nrf2的表达,与PI3K/AKT抑制剂LY294002联合使用进一步抑制了Nrf2的表达。结论胡桃醌能够抑制前列腺癌LNCa P细胞增殖,作用的机制可能与促进ROS产生从而抑制PI3K/AKT途径,导致Nrf2表达下降有密切关系。

Objective To investigate the killing activity of Juglone in human prostate cancer LNCaP cell and the correlation between oxidative stress and its mechanism. Methods LNCaP cells were treated with 0, 6. 25, 12. 5, 25, 50 and 100 μmol/L Juglone. MTT assay was used to determine the growth of LNCaP cells treated with different concentrations of Juglone. Reactive oxygen species （ROS） was detected using fluorescence microplate reader with 2＇ ,7＇-dichlorofluoresceindiacetate （DCFH-DA） as fluorescent probe after treating LNCaP cells with Juglone and NAC. The expression levels of p-AKT, AKT and Nrf2 proteins were examined in LNCaP cells that were cultured with Juglone and LY294002 using Western blot. Results Compared with the control group, the concentration of Juglonein more than 12.5 μmol/L could significantly inhibit the growth of LNCaP cells （ P 〈 0.05, P 〈 0. 01 ）. The ROS levels were increased in Juglone group compared with control group （P 〈0.01 ）. Compared with the Juglone group, ROS levels was inhibited （P 〈 0. 05 ） and increased the cell proliferation rates by combination use with NAC （P 〈 0.01 ）. Juglone inhibited expression of p-AKT and addition of NAC resorted the Juglone inhibition of p-AKT expression. Nrf2 expression were down-regulated in cell nucleus treated with Juglone and then inhibition of PI3K/AKT with LY294002 augmented Juglone-mediated function. Conclusion Juglone could significantly in- hibit the growth of LNCaP cells and the effect may be related to promoted ROS levels through PI3K/AKT pathway and then inhibit the Nrf2 expression.