汉防己甲素对人多发性骨髓瘤干细胞增殖、凋亡的影响及机制
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摘要
目的:研究汉防己甲素对人多发性骨髓瘤干细胞CD138~- B细胞增殖、凋亡的影响及作用机制。方法:利用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)比色法检测汉防己甲素对CD138~- B细胞增殖的影响;应用流式细胞仪检测汉防己甲素对CD138~- B细胞周期的影响以及药物诱导细胞凋亡的作用;应用蛋白免疫印迹法(Western blot)检测汉防己甲素对CD138~- B细胞中裂解的半胱氨酸天冬氨酸蛋白水解酶(cleaved-Caspase)-3,cleaved-Caspase-9蛋白表达的影响。结果:与空白组及溶剂组比较,10,20,30,40μmol·L~(-1)汉防己甲素处理6,12,24 h对CD138~- B细胞的增殖均具有显著的抑制作用(P<0.01);与空白组及溶剂组比较,汉防己甲素30,40μmol·L~(-1)时,G_0/G_1期细胞显著增多(P<0.01),S期细胞明显减少(P<0.05,P<0.01),汉防己甲素10,20,30,40μmol·L~(-1)时,细胞凋亡率显著增加(P<0.01);与空白组及溶剂组比较,20,30,40μmol·L~(-1)汉防己甲素处理后,cleaved-Caspase-3,cleaved-Caspase-9蛋白表达显著升高(P<0.01),10μmol·L~(-1)汉防己甲素处理时,cleaved-Caspase-3蛋白表达明显增高(P<0.05)。结论:汉防己甲素通过阻滞细胞周期在G_0/G_1期抑制CD138~- B细胞的增殖,通过活化细胞内与凋亡相关蛋白cleaved-Caspase-3,cleaved-Caspase-9促进细胞凋亡。
Objective: To investigate the effects and mechanism of tetrandrine on proliferation and apoptosis in multiple myeloma stem cells. Method: Methylthiazolyldiphenyl-tetrazolium bromide(MTT) assay was used to detect the survival of cancer cell. Flow cytometer was used to detect the cell circles and apoptotic induced by tetrandrine. Western blot were used to detect the activity of cleaved-Caspase-3,cleaved-Caspase-9. Result:Compared with blank group and solution group,10,20,30,40 μmol·L~(-1)tetrandrine treatment for 6,12,24 h could significantly inhibit the proliferation of CD138~- B cells(P<0.01). Compared with blank group and solution group,the cells in G_0/G_1 phase increased significantly(P<0.01),and the cells in S phase significantly decreased(P<0. 05,P<0.01),when being treated with 30,40 μmol·L~(-1)tetrandrine. The apoptotic rate increased significantly at concentrations of 10,20,30,40 μmol·L~(-1)of tetrandrine(P<0.01). Compared with blank group and solution group,the expression of cleaved-Caspase-3,cleaved-Caspase-9 protein was significantly increased after being treated with 20,30,40 μmol·L~(-1)tetrandrine(P<0.01),the expression of cleavedCaspase-3 protein was significantly increased when being treated with 10 μmol·L~(-1)tetrandrine(P<0. 05).Conclusion: Tetrandrine can arrest CD138~- B cell line in the G_0/G_1 phase,inhibit the cell survival,and enhance CD138~- B cell apoptosis by promoting the activity of cleaved-Caspase-3,cleaved-Caspase-9.
Objective: To investigate the effects and mechanism of tetrandrine on proliferation and apoptosis in multiple myeloma stem cells. Method: Methylthiazolyldiphenyl-tetrazolium bromide(MTT) assay was used to detect the survival of cancer cell. Flow cytometer was used to detect the cell circles and apoptotic induced by tetrandrine. Western blot were used to detect the activity of cleaved-Caspase-3,cleaved-Caspase-9. Result:Compared with blank group and solution group,10,20,30,40 μmol·L~(-1)tetrandrine treatment for 6,12,24 h could significantly inhibit the proliferation of CD138~- B cells(P<0.01). Compared with blank group and solution group,the cells in G_0/G_1 phase increased significantly(P<0.01),and the cells in S phase significantly decreased(P<0. 05,P<0.01),when being treated with 30,40 μmol·L~(-1)tetrandrine. The apoptotic rate increased significantly at concentrations of 10,20,30,40 μmol·L~(-1)of tetrandrine(P<0.01). Compared with blank group and solution group,the expression of cleaved-Caspase-3,cleaved-Caspase-9 protein was significantly increased after being treated with 20,30,40 μmol·L~(-1)tetrandrine(P<0.01),the expression of cleavedCaspase-3 protein was significantly increased when being treated with 10 μmol·L~(-1)tetrandrine(P<0. 05).Conclusion: Tetrandrine can arrest CD138~- B cell line in the G_0/G_1 phase,inhibit the cell survival,and enhance CD138~- B cell apoptosis by promoting the activity of cleaved-Caspase-3,cleaved-Caspase-9.
引文
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[14]陈修平,唐正海,石哲,等.肿瘤生物学研究热点与抗肿瘤中药研发策略[J].中国中药杂志,2015,40(17):3416-3422.
[15]HUANG S Y,LIN C W,LIN H H,et al.Expression of cereblon protein assessed by immunohisto chemicalstaining in myeloma cells is associated with superior response of thalidomide and lenalidomide based treatment,but not bortezomib-based treatment,in patients with multiple myeloma[J].Ann Hematol,2014,93(8):1371-1380.
[16]LI J,ZHU J,CAO B,et al.The m TOR signaling pathway is an emerging therapeutic target in multiple myeloma[J].Curr Pharm Des,2014,20(1):125-135.
[17]Tucci M,Stucci S,Savonarola A,et al.An imbalance between Beclin-1 and p62 expression promotes the proliferation of myeloma cells through autophagy regulation[J].Exp Hematol,2014,42(10):897-908.
[18]张淑瑜,于燕莉,刘世君,等.复方汉防己颗粒中防己的成分鉴别及含量测定[J].中国药事,2013,27(7):725-728.
[19]QIU W,SU M,XIE F,et al.Tetrandrine blocks autophagic flux and induces apoptosis via energetic impairment in cancer cells[J].Cell Death Dis,2014,5(3):463-473.
[20]伍义文,童健,张福伟,等.汉防己甲素对食管癌细胞凋亡相关基因Survivin、Caspase-3表达的影响[J].中药材,2013,36(6):986-988.
[2]Larocca A,Cavallo F,Bringhen S,et al.Aspirin or enoxaparin thromboprophylaxis for patients with newly diagnosed multiple myeloma treated with lenalidomide[J].Blood,2012,119(4):933-939.
[3]王曼,李树仁.基因工程在骨髓间充质干细胞治疗心肌梗死中的应用[J].中国老年学杂志,2015,35(10):269-271.
[4]Colak S,Medema J P.Cancer stem cells-important players in tumor therapy resistance[J].FEBS J,2014,281(21):4779-4791.
[5]Kim D,Park C Y,Medeiros B C,et al.CD19-CD45low/-CD38 high/CD138+plasma cells enrich for human tumorigenic myeloma cells[J].Leukemia,2012,26(12):2530-2537.
[6]DING Q,Lee Y K,Schaefer E K,et al.A TALEN genome-editing system for generating human stem cellbased disease models[J].Cell Stem Cell,2013,12(2):238-251.
[7]CHU J,HE S,DENG Y,et al.Genetic modification of T cells redirected toward CS1 enhances eradication of myeloma cells[J].Clin Cancer Res,2014,20(15):3989-4000.
[8]Soto H,Erasme D.Dclk1 distinguishes between tumor and normal stem cells in the intestine[J].Nat Genet,2013,45(1):98-103.
[9]WAN J,LIU T,MEI L,et al.Synergistic antitumour activity of sorafenib in combination with tetrandrine is mediated by reactive oxygen species(ROS)/Akt signaling[J].Br J Cancer,2013,109(2):342-350.
[10]GAO S,CUI Y L,YU C Q,et al.Tetrandrine exerts antidepressant-like effects in animal models:role of brain-derived neurotrophic factor[J].Behav Brain Res,2013,238(1):79-85.
[11]QIN R,SHEN H,CAO Y,et al.Tetrandrine induces mitochondria-mediated apoptosis in human gastric cancer BGC-823 cells[J].PLo S One,2013,8(10):e76486.
[12]YAN T,SUN A,LIU R,et al.Simultaneous determination of fangchinoline and tetrandrine in Stephania tetrandra S.Moore,by using 1-alkyl-3-methylimidazolium-based ionic liquids as the RP-HPLC mobile phase additives[J].Anal Chim Acta,2013,767(6):148-154.
[13]HUANG A C,LIAN J C,LIN M W,et al.Tetrandrine induces cell death in SAS human oral cancer cells through caspase activation-dependent apoptosis and LC3-Ⅰand LC3-Ⅱactivation-dependent autophagy[J].Int J Oncol,2013,43(2):485-494.
[14]陈修平,唐正海,石哲,等.肿瘤生物学研究热点与抗肿瘤中药研发策略[J].中国中药杂志,2015,40(17):3416-3422.
[15]HUANG S Y,LIN C W,LIN H H,et al.Expression of cereblon protein assessed by immunohisto chemicalstaining in myeloma cells is associated with superior response of thalidomide and lenalidomide based treatment,but not bortezomib-based treatment,in patients with multiple myeloma[J].Ann Hematol,2014,93(8):1371-1380.
[16]LI J,ZHU J,CAO B,et al.The m TOR signaling pathway is an emerging therapeutic target in multiple myeloma[J].Curr Pharm Des,2014,20(1):125-135.
[17]Tucci M,Stucci S,Savonarola A,et al.An imbalance between Beclin-1 and p62 expression promotes the proliferation of myeloma cells through autophagy regulation[J].Exp Hematol,2014,42(10):897-908.
[18]张淑瑜,于燕莉,刘世君,等.复方汉防己颗粒中防己的成分鉴别及含量测定[J].中国药事,2013,27(7):725-728.
[19]QIU W,SU M,XIE F,et al.Tetrandrine blocks autophagic flux and induces apoptosis via energetic impairment in cancer cells[J].Cell Death Dis,2014,5(3):463-473.
[20]伍义文,童健,张福伟,等.汉防己甲素对食管癌细胞凋亡相关基因Survivin、Caspase-3表达的影响[J].中药材,2013,36(6):986-988.