Objective: To investigate the effect of danusertib (Danu), an inhibitor of Aurora kinase, on the proliferation, cell cycle, apoptosis, and autophagy of hepatocellular carcinoma HepG2 cells and explore the underlying mechanisms.
Methods: MTT assay was used to examine the effect of Danu on the viability of HepG2 cells to determine the IC50 of Danu. The effect of Danu on cell cycle distribution, apoptosis and autophagy were determined using flow cytometry. Western blotting was used to detect the expressions of the proteins related to cell cycle, apoptosis and autophagy. Chloroquine was used to suppress Danuinduced autophagy to test the apoptosis-inducing effect of Danu.
Results: Danu significantly inhibited the proliferation of HepG2 cells with IC50 of 39.4 μmol and 14.4 μmol at 24 h and 48 h, respectively. Danu caused cell cycle arrest in G2/M phase in HepG2 cells and led to polyploidy accumulation via up-regulating the expressions of p53 and p21 and down-regulating the expressions of cyclin B1 and DC2. Danu also caused apoptosis of HepG2 cells through up-regulating the expressions of Bax, Puma, cleaved caspase-3, cleaved caspase-9, cleaved PARP and cytochrome C and down-regulating the expressions of Bcl-xl and Bcl-2. Danu induced autophagy via activating AMPK signaling and inhibiting PI3K/PTEN/AKT/mTOR axis, and inhibition of Danu-induced autophagy with chloroquine enhanced the pro-apoptotic effect of Danu.
Conclusions: Danu inhibits cell proliferation and induces cell cycle arrest in G2/M phase, apoptosis and cytoprotective autophagy in HepG2 cells.
目的: 探讨极光激酶抑制剂Danusertib（Danu）对肝癌HepG2细胞株增殖、细胞周期、凋亡和自噬的影响及其作用机制。
方法: 采用四甲基偶氮唑蓝（MTT）法检测Danu对各组细胞的增殖抑制率，确定Danu的半数抑制浓度（IC50）。采用流式细胞仪检测Danu对HepG2细胞周期阻滞、凋亡及自噬的影响。采用Western blot检测细胞周期阻滞、凋亡及自噬相关效应蛋白和通路蛋白的表达。采用氯喹抑制自噬以明确其所起作用。
结果: Danu能有效抑制HepG2细胞增殖，24 h和48 h的IC50分别为39.4 μmol和14.4 μmol。Danu通过上调p53及p21的表达和下调Cyclin B1及CDC2的表达引起HepG2细胞出现G2/M周期阻滞和异倍体，通过上调Bax、Puma、cleaved caspase-3、cleaved caspase-9、cleaved PARP及细胞色素C的表达和下调Bcl-xl及Bcl-2的表达引起HepG2细胞凋亡，通过激活上调AMPK信号通路和和抑制PI3K/PTEN/AKT/mTOR信号轴引起HepG2细胞出现自噬。采用氯喹抑制自噬后，能够增强Danu对HepG2细胞的促凋亡作用。
结论: Danu能够有效抑制肝癌HepG2细胞增殖，引起G2/M细胞周期阻滞，导致细胞凋亡，具有良好的体外抗肿瘤效果。Danu能够引起细胞保护性自噬。
Keywords: Aurora kinase; apoptosis; autophagy; cell cycle arrest; danusertib; hepatocellular carcinoma.