摘要
目的探究狼毒大戟中二萜类化合物12-去氧佛波醇-13-棕榈酸酯(DP)能否通过磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)信号通路发挥抗白血病的作用,为将其开发成抗白血病新药提供实验依据。方法以LY294002(PI3K特异性抑制剂)为工具药,采用MTT、AnnexinⅤ-FITC/PI、AO-EB染色法观察并测定DP作用24 h后对人原髓细胞白血病细胞HL60增殖、凋亡的影响;采用ELISA法测定DP作用24 h后细胞培养液中乳酸脱氢酶(LDH)的释放量及细胞中胱天蛋白酶3(caspase-3)、caspase-9的活性。采用实时荧光定量-PCR法检测DP作用24 h后细胞中caspase-3、caspase-9、叉头框O3a(FoxO3a)、B细胞淋巴瘤2细胞死亡的相互作用介质(Bim)的mRNA转录水平;采用Western blot法检测细胞中磷酸化FoxO3a(p-FoxO3a)、磷酸化Akt(p-Akt)的蛋白表达水平,并采用免疫染色法在激光共聚焦显微镜下观察细胞中FoxO3a蛋白核转位情况。结果10μmol/L DP和10μmol/L DP+LY294002对HL60细胞有明显的增殖抑制及凋亡诱导作用(P<0.01);5、10、20μmol/L DP作用后的细胞均呈典型的凋亡形态学特征,细胞培养液中LDH释放量和细胞中caspase-3、caspase-9活性均显著升高(P<0.05或P<0.01),其作用具有一定的浓度依赖性趋势;10μmol/L DP和10μmol/L DP+LY294002作用后细胞中caspase-3、caspase-9和Bim mRNA转录水平均显著升高(P<0.05或P<0.01),FoxO3a mRNA转录水平和p-FoxO3a、p-Akt蛋白表达水平均显著降低(P<0.05或P<0.01);10μmol/L DP+LY294002组细胞可见FoxO3a蛋白的核转位变化,且该变化较LY294002组明显。结论DP可通过抑制PI3K/Akt信号通路抑制人白血病HL60细胞增殖并诱导其凋亡。
OBJECTIVE To explore whether diterpenoid 12-deoxyphorbol-13-palmitate(DP)from Euphorbia fischeriana can exert anti-leukemia effects through the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)signal pathway,and to provide experimental evidence for developing it into a new anti-leukemia drug.METHODS Using LY294002(PI3K specific inhibitor)as tool drug,the effects of 24 h DP treatment on the proliferation and apoptosis of human myeloid leukemia HL60 cells were detected by MTT method,AnnexinⅤ-FITC/PI staining and AO-EB staining.ELISA method was used to detect lactic dehydrogenase(LDH)release and the activities of cysteinyl aspartate specific proteinase 3(caspase-3)and caspase-9.The transcriptional level of caspase-3,caspase-9,forkhead box O3a(FoxO3a)and B cell lymphoma 2 interacting mediator of cell death(Bim)mRNA were detected by real-time quantitative polymerase chain reaction(qRT-PCR).The protein expression of phosphorylated FoxO3a(p-FoxO3a)and phosphorylated Akt(p-Akt)were detected by Western blot method.The nuclear translocation of FoxO3a protein was detected by immunostaining combined with laser confocal microscopy.RESULTS 10μmol/L DP and 10μmol/L DP+LY294002 could inhibit the proliferation and induce the apoptosis of HL60 cells(P<0.01).After treatment of 5,10,20μmol/L DP,HL60 cells showed typical morphological characteristics of apoptosis;DP could significantly increase the levels of LDH release and the activities of caspase-3 and caspase-9(P<0.05 or P<0.01),in dose-dependent manner.After treatment of 10μmol/L DP and 10μmol/L DP+LY294002,the transcriptional levels of caspase-3,caspase-9 and Bim mRNA were increased significantly(P<0.05 or P<0.01),and transcriptional level of FoxO3a mRNA and protein expressions of p-FoxO3a and p-Akt were decreased significantly(P<0.05 or P<0.01).Nuclear translocation changes were observed in FoxO3a protein in 10μmol/L DP+LY294002 group,and the change was more significant than that of LY294002 group.CONCLUSIONS DP can inhibit the proliferation and induce the apoptosis of HL60 cells via inhibiting PI3K/Akt signaling pathway.
作者
马立威
陈哲
王文豹
张金玲
张洪涛
葛鹏玲
刘吉成
MA Liwei;CHEN Zhe;WANG Wenbao;ZHANG Jinling;ZHANG Hongtao;GE Pengling;LIU Jicheng(Research Institute of Medicine and Pharmacy(Postdoctoral Workstation),Qiqihar Medical University,Heilongjiang Qiqihar 161006,China;Mobile Postdoctoral Station,Heilongjiang University of Chinese Medicine,Harbin 150040,China;School of Public Health,Qiqihar Medical University,Heilongjiang Qiqihar 161006,China;School of Pharmacy,Qiqihar Medical University,Heilongjiang Qiqihar 161006,China;Dept.of Obstetrics and Gynecology,the Third Affiliated Hospital of Qiqihar Medical University,Heilongjiang Qiqihar 161099,China)
出处
《中国药房》
CAS
北大核心
2023年第7期825-831,共7页
China Pharmacy
基金
黑龙江省中医药管理局科技计划项目(No.ZHY2020-179)
齐齐哈尔市科技计划联合引导项目(No.LHYD-202028)。
作者简介
第一作者:马立威,副研究员,博士。研究方向:天然药物抗肿瘤。Email:maliwei@qmu.edu.cn;通信作者:刘吉成,教授,博士生导师,博士。研究方向:天然药物抗肿瘤。E-mail:jcliu@qmu.edu.cn。
