阿伐他汀通过磷脂酰肌醇3-激酶／丝氨酸苏氨酸蛋白激酶／哺乳动物雷帕霉素靶蛋白途径调节HL-60白血病细胞凋亡的作用

Effect of Atorvastatin on apoptosis of HL -60 leukemic cells via the regulation of the phosphatidylinositol 3 - kinase/serine/threonine protein kinase/mammalian target of rapamycin signaling pathway

目的观察阿伐他汀对白血病细胞HL-60增殖和凋亡的影响，探讨磷脂酰肌醇3-激酶／丝氨酸苏氨酸蛋白激酶／哺乳动物雷帕霉素靶蛋白（PI3K／AKT／mTOR）信号途径在此过程中的作用。方法取对数生长期细胞，分为阴性对照组和实验组（阿伐他汀干预水平分别为1、5、10μmol/L），培养12、24、48h，采用四甲基偶氮唑盐（MTT）法检测白血病细胞的增殖能力；培养48h后，流式细胞术检测白血病细胞凋亡变化，反转录-聚合酶链反应（RT—PCR）和Western blot方法检测PI3K、AKT和mTOR基因表达的变化。结果阿伐他汀对白血病细胞HL-60有明显抑制增殖和促凋亡作用。10μmoL／L阿伐他汀对HL—60细胞干预48h后对细胞增殖的抑制作用最强，抑制率达到（39．77±3．01）％，与阴性对照组相比，差异有统计学意义（t=4．016，P〈0．01），同时对细胞凋亡的诱导作用最强，凋亡率达到（43．29±3．91）％，与阴性对照组相比，差异有统计学意义（t=3．625，P〈0．05）。此外，PI3K、AKT和mTOR在白血病细胞HL-60均有基础表达，10μmol/L阿伐他汀对HL-600细胞干预48h后，PI3K、AKT和mTOR基因mRNA表达水平下调最为明显，分别下降了（37．05±4．11）％、（53．79±3．27）％、（40．63±2．42）％，与阴性对照组比较差异均有统计学意义（t=4．805、3．799、4．312，P均〈0．05），同时对PI3K、AKT和mTOR蛋白表达水平下调最为明显，分别下降了（41．09±3．17）％、（45．67±2．92）％和（63．41±3．59）％，与阴性对照组比较差异均有统计学意义（t=3．576、4．727、4．902，P均〈0．05）。结论阿伐他汀可能通过PI3K／AKT／mTOR信号通路抑制白血病细胞增殖并诱导细胞凋亡。

Objective To investigate the effect of Atorvastatin on the proliferation and apoptosis of leukemic HL - 60 - cell line, and to explore the possible role of phosphatidylinositol 3 - kinase/serine/threonine protein kinase / mammalian target of rapamycin （PI3K/AKT/mTOR） signaling pathway in this process. Methods HL -60 cells were incubated with different concentrations of Atorvastatin （1,5,10 μmol/L）, and HL -60 ceils without any treatment were used as controls. The proliferation of HL - 60 which was investigated by four methyl thiazolyl tetrazolium assay when cells were cultured for 12,24,48 hours. The apoptosis was detected by flow cytometry after cells were incubated for 48 hours. The mRNA and protein expressions of AKT, PI3K and mTOR were detected by reverse transcription -polymerase chain reaction and Western blot methods, respectively. Results The results indicated that Atorvastatin could inhibit the proliferation and induce the apoptosis of HL -60 cells. When treated with 10 μmol/L Atorvastatin after 48 h ,the proliferation inhibition of HL - 60 was observed most obviously, with a high rate of （ 39.77 ± 3.01 ） % , compared with the control group, it had statistical significance （ t = 4.016, P 〈 0.01 ）, meanwhile, the apoptosis of HL - 60 was most notable, at a rate of （43.29 ± 3.91 ） %, compared with the control group, it had statistical significance （ t = 3. 625, P 〈 0. 05 ）. There were basal expression of AKT,PI3K and mTOR in the control group. When treated with 10 μmol/L Atorvastatin after 48 h,the mRNA expression of PI3K,AKT and mTOR were down -regulated most obviously,at a decrease of （37.05 ±4.11）% ,（53.79 ±3.27）%, （40.63 ±2.42）% （t =4.805,3. 799,4.312,all P 〈0.05） ,respectively, in comparison with the control group. At the same condition, the protein expression of PI3K, AKT and roTOR were decreased most visibly,with a decline of （41.09 ±3.17）%, （45.67 ± 2.92）%, （63.41 ±3.59）% （t =3. 576, 4. 727,4. 902 ,all P 〈 0.05 ）respectively in comparison with the control group. Conclusions Atorvastatin can inhibit the proliferation and induce the apoptosis of leukemic cell HL -60, and the mechanism may be associated with the PI3K/ AKT/mTOR signal pathway.