亚硒酸钠抑制PI3K/AKT/mTOR信号通路诱导白血病细胞HL-60凋亡的机制研究
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摘要
目的探究亚硒酸钠诱导白血病细胞HL-60凋亡的信号通路及其对信号通路的影响。方法应用不同浓度的亚硒酸钠处理白血病细胞HL-60,或者应用亚硒酸钠按照不同的时间点处理白血病细胞HL-60,使用流式细胞仪检测亚硒酸钠对HL-60细胞凋亡的影响。应用10μmol/L亚硒酸钠处理HL-60细胞,Western blot检测p-PI3K、总PI3K、p-AKT、总AKT、p-m TOR和总m TOR的表达情况。在过表达PI3K的HL-60细胞中应用10μmol/L亚硒酸钠处理白血病细胞HL-60,流式细胞仪检测HL-60细胞的凋亡情况,Western blot检测p-PI3K、总PI3K、pAKT、总AKT、p-m TOR和总m TOR的表达变化。同时以加蒸馏水处理的HL-60细胞为空白对照组。结果与空白对照组凋亡比例(0.045±0.0013)相比,10μmol/L及20μmol/L亚硒酸钠处理组可有效诱导白血病细胞HL-60凋亡[凋亡比例分别为(0.254±0.001 6)和(0.435±0.002 1)],差异均有统计学意义(P<0.05)。此外与10μmol/L的亚硒酸钠处理0 h组相比[凋亡比例(0.055±0.001 1)],10μmol/L亚硒酸钠作用24 h、48 h、72 h后,HL-60细胞的凋亡显著[凋亡比例分别为(0.179±0.0018)、(0.384±0.0023)和(0.535±0.0034)],差异均有统计学意义(P<0.05)。亚硒酸钠处理细胞能下调PI3K、AKT、m TOR的磷酸化水平,但对总的PI3K、AKT以及m TOR的表达没有影响。在过表达PI3K的细胞中,AKT、m TOR表达上调,而亚硒酸钠可逆转过表达PI3K导致的PI3K/AKT/m TOR信号通路的过度激活。过表达PI3K使细胞凋亡被抑制,而亚硒酸钠可诱导过表达PI3K的细胞凋亡增加。结论亚硒酸钠可通过抑制PI3K/AKT/m TOR信号通路的激活,诱导白血病细胞HL-60凋亡。
Objective To investigate the signal pathway for sodium selenite-induced apoptosis of leukemia cell line HL-60 and sodium selenite's effects on the signaling pathway. Methods Treating leukemia cell line HL-60 with different concentrations of sodium selenite or using sodium selenite to deal with HL-60 cells at different time points, the effect of sodium selenite on the apoptosis of HL-60 cells was detected by flow cytometry. HL-60 cells were treated with10 μmol/L sodium selenite, and the expression of p-phosphatidylinositol 3-kinase(PI3K), total(PI3K), p-AKT, total AKT, p-mammalian target of rapamycin(m TOR), and total(m TOR) were detected by Western blot. The PI3K-overexpressing HL-60 cells were treated with 10 μmol/L sodium selenite, and flow cytometry was performed to detected the apoptosis of HL-60 cells. Besides, Western blot was performed to detect the expression of p-PI3K, total PI3K, p-AKT,total AKT, p-m TOR, and total m TOR. Results Compared with the blank control group, 10 μmol/L and 20 μmol/L sodium selenite can induce the apoptosis of leukemia cells HL-60: the apoptosis ratio(0.045±0.001 3) vs(0.254±0.001 6)and(0.435±0.002 1), P<0.05. In 10 μmol/L sodium selenite treatment condition, comparing with 0 h treatment(the ratio of apoptosis), the treatment of 24 h, 48 h and 72 h significantly induced the apoptosis of HL-60 cells: the apoptosis ratio(0.055±0.001 1) vs(0.179±0.001 8),(0.384±0.002 3),(0.535±0.003 4), P<0.05. Sodium selenite could down-regulate the phosphorylation levels of PI3K, AKT and m TOR, but had no effect on the total expression of PI3K, AKT and m TOR. In PI3K-overexpressing HL-60 cells, the expression of AKT and m TOR were up-regulated, but sodium selenite could inhibit the over-activation of the PI3K/AKT/m TOR pathway resulting from the overexpression of PI3K. The overexpression of PI3K could inhibit the apoptosis, but sodium selenite could induce the increase of apoptosis of PI3K-overexpressing cells. Conclusion Sodium selenite can induce the apoptosis of leukemia cell line HL-60 by inhibiting the activation of PI3K/AKT/m TOR pathway.
Objective To investigate the signal pathway for sodium selenite-induced apoptosis of leukemia cell line HL-60 and sodium selenite's effects on the signaling pathway. Methods Treating leukemia cell line HL-60 with different concentrations of sodium selenite or using sodium selenite to deal with HL-60 cells at different time points, the effect of sodium selenite on the apoptosis of HL-60 cells was detected by flow cytometry. HL-60 cells were treated with10 μmol/L sodium selenite, and the expression of p-phosphatidylinositol 3-kinase(PI3K), total(PI3K), p-AKT, total AKT, p-mammalian target of rapamycin(m TOR), and total(m TOR) were detected by Western blot. The PI3K-overexpressing HL-60 cells were treated with 10 μmol/L sodium selenite, and flow cytometry was performed to detected the apoptosis of HL-60 cells. Besides, Western blot was performed to detect the expression of p-PI3K, total PI3K, p-AKT,total AKT, p-m TOR, and total m TOR. Results Compared with the blank control group, 10 μmol/L and 20 μmol/L sodium selenite can induce the apoptosis of leukemia cells HL-60: the apoptosis ratio(0.045±0.001 3) vs(0.254±0.001 6)and(0.435±0.002 1), P<0.05. In 10 μmol/L sodium selenite treatment condition, comparing with 0 h treatment(the ratio of apoptosis), the treatment of 24 h, 48 h and 72 h significantly induced the apoptosis of HL-60 cells: the apoptosis ratio(0.055±0.001 1) vs(0.179±0.001 8),(0.384±0.002 3),(0.535±0.003 4), P<0.05. Sodium selenite could down-regulate the phosphorylation levels of PI3K, AKT and m TOR, but had no effect on the total expression of PI3K, AKT and m TOR. In PI3K-overexpressing HL-60 cells, the expression of AKT and m TOR were up-regulated, but sodium selenite could inhibit the over-activation of the PI3K/AKT/m TOR pathway resulting from the overexpression of PI3K. The overexpression of PI3K could inhibit the apoptosis, but sodium selenite could induce the increase of apoptosis of PI3K-overexpressing cells. Conclusion Sodium selenite can induce the apoptosis of leukemia cell line HL-60 by inhibiting the activation of PI3K/AKT/m TOR pathway.
引文
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[2]贾永清,滕熔,胡慧仙.亚硒酸钠对HL-60细胞增殖、凋亡影响及作用机制探讨[J].交通医学,2011,25(2):121-125.
[3]王承艳,丁明孝.骨髓移植与造血干细胞研究[J].生物学通报,2009,44(1):6-9.
[4]Fang W,Han A,Bi X,et al.Tumor inhibition by sodium selenite is associated with activation of c-Jun NH2-terminal kinase 1 and suppression ofβ-catenin signaling[J].International Journal of Cancer,2010,127(1):32-42.
[5]Yang Y,Luo H,Hui K,et al.Selenite-induced autophagy antagonizes apoptosis in colorectal cancer cells in vitro and in vivo[J].Oncology Reports,2015,35(3):1255-1264.
[6]Luo H,Yang Y,Duan J,et al.PTEN-regulated AKT/Fox O3a/Bim signaling contributes to reactive oxygen species-mediated apoptosis in selenite-treated colorectal cancer cells[J].Cell Death&Disease,2013,4(2):e481.
[7]魏虎来.硒酸酯多糖和亚硒酸钠抗白血病效应的实验研究[J].兰州大学学报,1996,32(1):126-129.
[8]贾永清,滕熔,胡慧仙.亚硒酸钠对HL-60细胞增殖、凋亡影响及作用机制探讨[J].交通医学,2011,25(2):121-125.
[9]Elert E.Living with leukaemia[J].Nature,2013,498(7455):S2-S3.
[10]于方方,付菊芳,白燕妮,等.急性白血病患者生活质量与支持性照顾需求的相关性研究[J].护理管理杂志,2015,15(1):24-26.
[11]苗小艳,马红叶,张旭,等.N-糖基化修饰在髓性白血病耐药中的作用[J].中国微生态学杂志,2014,26(5):506-510.
[12]胡滨,陈一资.亚硒酸钠的急性、蓄积性、亚急性毒性研究[J].食品科学,2011,32(5):258-262.
[13]赵鹏,赵上,王艳,等.Fas及TRF1、TRF2在亚硒酸钠诱导胃癌SGC-7901细胞凋亡过程中的表达[J].营养学报,2014,36(2):149-153.
[14]王艳,赵上,苏衍萍,等.亚硒酸钠通过线粒体途径诱导人胃癌SGC-7901细胞凋亡的机制[J].解剖学报,2016,47(3):353-358.
[15]黄方,黄艾,马虹,等.亚硒酸钠诱导结直肠癌SW480细胞线粒体损伤及细胞凋亡[J].基础医学与临床,2013,33(10):1050-1054.
[16]段婧,罗慧,史可鉴,等.亚硒酸钠通过AMPK/m TOR通路调控白血病NB4细胞凋亡[J].基础医学与临床,2013,33(3):297-302.
[17]Fransecky L,Mochmann LH,Baldus C D.Outlook on PI3K/AKT/m TOR inhibition in acute leukemia[J].Molecular and Cellular Therapies,2015,3(1):2.
[18]Cantley LC.The phosphoinositide 3-kinase pathway[J].Science,2002,296(5573):1655.
[19]Matsuoka T,Yashiro M.The role of PI3K/Akt/m TOR signaling in gastric carcinoma[J].Cancers,2014,6(3):1441-1463.
[20]陈洪菊,屈艺,母得志.m TOR信号通路的生物学功能[J].生命的化学,2010,30(4):555-561.
[21]Ziegler ME,Hatch MM,Wu N,et al.m TORC2 Mediates Cxcl12-induced angiogenesis[J].Angiogenesis,2016,19(3):359-371.