摘要
目的探讨阿伐他汀对耐顺铂的人慢性髓系白血病耐药株K562/DDP化疗增敏的作用及机制。方法取对数生长期耐顺铂的白血病细胞K562/DDP,设对照组、顺铂(5μmol/L)组、阿托伐他汀(10μmol/L)组、阿伐他汀(10μmol/L)+顺铂(5μmol/L)组。采用CCK8法测定细胞增殖活性;流式细胞术检测细胞周期和细胞凋亡作用,Westernblot方法检测羟甲基戊二酰辅酶A还原酶(HMGCR)、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、半胱氨酸天冬氨酸蛋白水解酶-3(caspase-3)、磷酸化腺苷酸活化蛋白激酶(p-AMPK)、磷酸化丝氨酸-苏氨酸蛋白激酶(p-Akt)、磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)的表达。将p-AMPK-siRNA转染至K562/DDP细胞后,进一步采用上述方法检测p-AMPK低表达对阿伐他汀调节K562/DDP细胞凋亡及p-Akt/p-mTOR信号通路的影响。结果CCK结果表明,随着顺铂浓度的增加,K562/DDP和K562细胞生存率逐渐降低。K562/DDP细胞的半数抑制浓度IC50值明显高于K562细胞(t=4.376,P<0.05)。流式结果显示,与单用顺铂组比较,阿伐他汀联合顺铂处理后G1期细胞比例明显升高(t=4.617,P<0.05),S期细胞比例明显降低(t=7.277,P<0.05);阿伐他汀联合顺铂处理后显著促进K562/DDP细胞凋亡(t=5.871,P<0.05)。Western blot结果表明,与单用顺铂组比较,阿伐他汀联合顺铂处理后显著抑制HMGCR蛋白的表达(t=4.929,P<0.05),显著下调Bcl-2蛋白的表达(t=3.899,P<0.05),上调Bax和caspase-3蛋白的表达(t=5.325、7.533,P均<0.05);阿伐他汀联合顺铂处理后显著促进p-AMPK蛋白的表达(t=5.178,P<0.05),抑制p-Akt和p-mTOR蛋白的表达(t=7.236、6.317,P均<0.05)。进一步将p-AMPK-siRNA转染至K562/DDP细胞后,结果显示低表达p-AMPK减弱了阿伐他汀联合顺铂对K562/DDP细胞p-Akt/p-mTOR信号途径的抑制作用,减弱了对K562/DDP细胞凋亡的促进作用。结论阿伐他汀具有增强慢性髓性白血病细胞株K562/DDP对顺铂化疗敏感性的作用,阻滞细胞在G1期,诱导细胞凋亡,推测机制可能通过抑制HMGCR表达,促进pAMPK激活,抑制p-Akt/p-mTOR信号途径有关。
Objective To investigate the chemosensitization effect and mechanism of atorvastatin to cisplatin resistant human chronic myeloid leukemia K562/DDP.Methods K562/DDP leukemia cells in logarithmic growth stage were selected and divided into control group,cisplatin group,atorvastatin group and atorvastatin combined with cisplatin group.CCK8 method was used to detect the cell proliferation activity.Flow cytometry was used to test the cell cycle and apoptosis.The protein expressions of HMGCR,Bcl-2,Bax,caspase-3,p-AMPK,p-Akt and p-mTOR were detected by Western blot.After p-AMPK-siRNA transfected into K562/DDP cells,it was investigated the effects of low expression of p-AMPK on the apoptosis and p-Akt/p-mTOR signaling pathway in K562/DDP cells regulated by atorvastatin.Results CCK results showed that the survival rates of K562/DDP and K562 cells were decreased gradually with the increasing concentration of cisplatin.IC50value of K562/DDP cells was significantly higher than that of K562 cells(t=4.376,P<0.05).Flow cytometry showed that compared with cisplatin group,the proportion of G1 phase cells were increased(t=4.617,P<0.05)and the proportion of S phase cells were decreased(t=7.277,P<0.05)in atorvastatin combined with cisplatin group.When treated with atorvastatin combined with cisplatin,it could significantly promote the apoptosis of K562/DDP cells(t=5.871,P<0.05).Western blot showed that when treated with atorvastatin combined with cisplatin,it could inhibit the protein expression of HMGCR(t=4.929,P<0.05),decreased the protein expression of Bcl-2(t=3.899,P<0.05),and increased the protein expression of Bax and caspase-3(t=5.325,7.533,P all<0.05).It might also promote the expression of p-AMPK protein(t=5.178,P<0.05)and inhibit the protein expression of p-Akt and p-mTOR(t=7.236,6.317,P all<0.05)in atorvastatin combined with cisplatin group.After transfected with p-AMPK-siRNA into K562/DDP cells,the results showed that low expression of p-AMPK weakened the inhibitory effect of p-Akt/p-mTOR signal pathway and attenuated the promotion of apoptosis in K562/DDP cells.Conclusions Atorvastatin could enhance the chemosensitivity to cisplatin in chronic myeloid leukemia cell line K562/DDP cells,block cells in G1 phase and induce apoptosis.Its mechanism might be related to decreasing the expression of HMGCR,promoting p-AMPK activation and inhibiting p-Akt/p-mTOR signal pathway.
作者
刘苗
周攀
LIU Miao;ZHOU Pan(Department of Paediatrics,Renmin Hospital,Wuhan University,Wuhan,Hubei 430060;Hubei Medical Devices Quality Supervision and Test Institute,Wuhan,Hubei 430075,China)
出处
《热带医学杂志》
CAS
2022年第4期518-524,共7页
Journal of Tropical Medicine
基金
湖北省自然科学基金(2020CFB611)
作者简介
刘苗(1982-),女,博士,副主任医师,主要从事小儿血液肿瘤方面临床及基础研究工作,E⁃mail:liumiao915@163.com
