达努塞替通过极光激酶B/核糖体p70S6蛋白激酶/核糖体蛋白15信号通路调控白血病细胞自噬

Danusertib induces autophagy of leukemia cells via Aurora kinase B/ribosomal protein p70S6 kinase/ribosomal protein 15 signaling pathway

目的探索达努塞替处理人白血病细胞后极光激酶B/核糖体p70S6蛋白激酶/核糖体蛋白15(AuroraB/p70S6K/RPL15)信号通路的变化及细胞自噬的发生情况,并研究其作用机制。方法选择髓系白血病细胞株THP-1和K562为研究对象。实验分为2部分,第1部分:分别采用0.1μmol/L、1.0μmol/L、5.0μmol/L浓度达努塞替共培养THP-1和K562细胞,对照组(0μmol/L达努塞替)加入体积分数为2mL/L的二甲基亚砜(DMSO),以上各组均作用24h;采用四唑盐比色法检测细胞生长的抑制作用,流式细胞术检测细胞自噬情况,Westernblot法检测p70S6K、AuroraB、磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(AKT)、哺乳动物雷帕霉素靶蛋白(mTOR)、微管相关蛋白(LC3)、Beclin1、核糖体蛋白15(RPL15)的表达水平。第2部分:分别下调THP-1和K562细胞中AuroraB和RPL15,采用DMSO溶解达努塞替(5.0μmol/L),实验分组:DMSO组(空白对照组)、达努塞替处理组、空载质粒组、小干扰RNA(siRNA)组、空载质粒+达努塞替处理组和siRNA+达努塞替处理组。检测自噬信号通路PI3K/AKT/mTOR中AuroraB、p70S6K、RPL15和自噬蛋白Beclin1、LC3的表达情况。结果1.达努塞替可抑制THP-1、K562细胞生长,半抑制浓度分别为26.9μmol/L和30.2μmol/L。2.在THP-1和K562细胞中,0.1μmol/L、1.0μmol/L、5.0μmol/L浓度的达努塞替处理细胞后,p-AuroraB/AuroraB、p-p70S6K/p70S6K、RPL15、p-mTOR/mTOR、p-AKT/AKT蛋白表达水平均较对照组降低,差异均有统计学意义(均P<0.05);自噬蛋白Beclin1、LC3表达量及细胞自噬率均升高,差异均有统计学意义(均P<0.05)。3.THP-1细胞中,下调AuroraB后,siRNA组及siRNA+达努塞替处理组p70S6K、RPL15分别低于空载质粒组22.1%、61.3%(F=18.1,P=0.001)和55.4%、56.1%(F=19.4,P=0.001);LC3表达量较空载质粒组升高13.6%、17.1%(F=15.4,P=0.001)。下调RPL15后,siRNA组及siRNA+达努塞替处理组Beclin1、LC3分别高于空载质粒组39.5%、92.3%(F=25.2,P=0.001)和40.2%、58.3%(F=23.9,P=0.001)。K562细胞,下调AuroraB后,siRNA组及siRNA+达努塞替处理组p70S6K、RPL15分别低于空载质粒组24.2%、62.7%(F=20.4,P=0.001)和57.2%、60.1%(F=23.9,P=0.001);LC3表达量较空载质粒组增加17.9%、56.7%(F=20.9,P=0.001)。下调RPL15后,siRNA组及siRNA+达努塞替处理组Beclin1、LC3分别高于空载质粒组20.6%、98.4%(F=22.4,P=0.001)和41.5%、70.1%(F=26.2,P=0.001)。结论达努塞替可能通过影响PI3K/AKT/mTOR自噬通路来抑制THP-1、K562白血病细胞的增殖,还可负性调控AuroraB/p70S6K/RPL15信号通路引起细胞发生程序性死亡,其中RPL15可能是AuroraB/p70S6K/RPL15信号通路中抑制肿瘤增殖作用的关键靶点。

Objective To investigate the effects of Danusertib on the changes in Aurora kinase B (Aurora B)/ ribosomal protein p70S6 kinase (p70S6K)/ribosomal protein 15 (RPL15) signaling pathway and autophagy in human leukemia cells and its mechanism. Methods Myeloid leukemia cell lines THP-1 and K562 were selected as the research subjects.The experiment was divided into 2 phases.Phase 1: each cell line was treated with the concentration of Danusertib in 0.1 μmol/L, 1.0 μmol/L and 5.0 μmol/L.In control group, 2 mL/L dimethyl sulfoxide (DMSO) was given.All the treated cells were cultured for 24 hours.The viability of each cell line was examined by methylthiazoletrazolium assay and the autophagy was assessed by flow cytometry.In addition, the protein levels of p70S6K, Aurora B, phosphatidylinositol 3-kinase (PI3K), AKT(phosphorylated protein kinase B), mammalian target of rapamycin (mTOR), microtubule-associated protein (LC3), Beclin1 and RPL15 were determined by using Western blot.Part 2: Aurora B and RPL15 were down-regulated in THP-1 and K562 cells, respectively.DMSO was used to dissolve Danusertib(5.0 μmol/L). The grouping was designed as following: DMSO group (blank control group), Danusertib-treated group, empty plasmid group, small interfering RNA(siRNA) group, empty plasmid + Danusertib-treated group and siRNA+ Danusertib treated group.The protein levels of Aurora B, p70S6K, RPL15, Beclin1 and LC3 were detected by using Western blot. Results (1)Danusertib decreased the viability of THP-1 and K562 cells and the half maximal inhibitory concentration values were 26.9 μmol/L and 30.2 μmol/L for THP-1 and K562 cells, respectively.(2)The protein levels of p-Aurora B/Aurora B, p-p70S6K/p70S6K, RPL15, p-mTOR/mTOR and p-AKT/AKT decreased compared with control cells after being treated with 0.1 μmol/L, 1.0 μmol/L and 5.0 μmol/L of Danusertib in THP-1 and K562 cells, and the differences were statistically significant(all P<0.05). (3)In THP-1 cells, compared with the empty plasmid group, the protein levels of p70S6K and RPL15 decreased by 22.1%, 61.3%(F=18.1, P=0.001) and 55.4%, 56.1%(F=19.4, P=0.001) in siRNA group and siRNA+ Danusertib-treated group after knockdown of Aurora B. In contrast, the protein levels of LC3 increased by 13.6% and 17.1%(F=15.4, P=0.001) compared with the empty plasmid group.In addition, the protein levels of Beclin1 and LC3 increased by 39.5%, 92.3%(F=25.2, P=0.001) and 40.2%, 58.3%(F=23.9, P=0.001) in siRNA group and siRNA + Danusertib-treated group, compared with the empty plasmid group after down-regulation of RPL15.In K562 cells, compared with the empty plasmid group, the protein levels of p70S6K and RPL15 decreased by 24.2%, 62.7%(F=20.4, P=0.001) and 57.2%, 60.1%(F=23.9, P=0.001) in siRNA group and siRNA + Danusertib treated group after downregulation of Aurora B. But the protein levels of LC3 increased by 17.9% and 56.7%(F=20.9, P=0.001) compared with the empty plasmid group.Moreover, the protein levels of Beclin1 and LC3 were increased by 20.6%, 98.4% (F=22.4, P=0.001) and 41.5%, 70.1%(F=26.2, P=0.001) in siRNA group and siRNA+ Danusertib-treated group, compared with the empty plasmid group after downregulation of RPL15. Conclusion Danusertib can induce autophagy via inhibition of the PI3K/AKT/mTOR signaling pathway and can negatively regulate Aurora B/p70S6K/RPL15 axis in THP-1 and K562 cells.In addition, RPL15 may be a key target of Aurora B/p70S6K/RPL15 signaling pathway in the inhibition of tumor proliferation.