rsTRAIL联合阿糖胞苷诱导HL-60细胞凋亡及其机制的研究
摘要
目的:探讨人重组可溶性TRAIL(rsTRAIL)单独和联合阿糖胞苷(Ara-c)诱导HL-60细胞凋亡作用及其机制,为其应用于临床治疗急性白血病提供实验依据。
方法:在体外以人类急性白血病细胞株HL-60细胞为研究对象,分成5组进行细胞培养:对照组、Ara-c组、rsTRAIL组、同时给药组(Ara-c+rsTRAIL)、先后给药组(Ara-c→rsTRAIL)。MTT比色法测定HL-60细胞生长抑制率;Annexin V/PI染色、流式细胞术检测HL-60细胞凋亡率;流式细胞术检测不同浓度Ara-c对HL-60细胞表面DR5表达水平的影响;流式细胞术检测rsTRAIL单药组及先后给药组对HL-60细胞表面DR5表达水平和HL-60细胞内caspase8活性的影响。
结果:(1)MTT实验结果显示各组HL-60细胞生长抑制率为:对照组5.20±0.65;5mg/L Ara-c组11.53±0.51;rsTRAIL组(50、100、200μg /L):16.45±1.35、22.86±0.81、29.79±1.88;不同浓度的同时给药组:27.78±2.63、33.36±2.01、36.35±3.27;不同浓度的先后给药组:34.08±3.44、41.21±5.98、44.45±4.55。与对照组比较,50、100、200μg /L浓度的rsTRAIL能抑制HL-60细胞生长,在50-200μg /L浓度范围内生长抑制率呈现剂量依赖性(P<0.001)。同时给药组HL-60细胞生长抑制率大于单用同等浓度的rsTRAIL组和Ara-c组(P<0.001)。先后给药组细胞生长抑制率大于相应浓度的同时给药组,有统计学差异(P<0.05)。(2)流式细胞术分析显示各组HL-60细胞凋亡率:对照组13.41±1.52;Ara-c组61.67±2.79;rsTRAIL组(50、100、200 ug/L)25.78±2.21、36.76±0.62、44.50±2.78;不同浓度的同时给药组74.45±5.06、77.39±5.34、80.66±6.27;不同浓度的先后给药组85.05±3.77、86.33±4.83、87.71±4.78。与对照组比较,50、100、200ug/L浓度的rsTRAIL能诱导HL-60细胞凋亡,在50-200ug/L浓度范围内诱导凋亡率呈现剂量依赖性(P<0.001)。同时给药组HL-60细胞凋亡率大于相应浓度的单用同等浓度的rsTRAIL组和Ara-c组(P<0.05)。先后给药组HL-60细胞凋亡率大于同时给药组(P<0.05)。(3)Ara-c(2.5mg/L、5mg/L、10mg/L )作用24小时后,HL-60细胞表面DR5表达水平分别为:1.22±0.18、3.02±0.03、3.56±0.45。对照组HL-60细胞表面DR5表达水平为1.20±0.16。10mg/L Ara-C组细胞表面DR5表达水平最高,5mg/LAra-C组次之,2.5mg/L Ara-c组与对照组最低(P<0.001)。(4)流式细胞术检测HL-60细胞表面DR5表达水平结果显示:不同浓度的rsTRAIL组分别为0.61±0.05、0.91±0.02、1.29±0.04。不同浓度的先后给药组分别为0.90±0.03、1.31±0.06、3.69±0.09。先后给药组HL-60细胞表面DR5表达水平大于单用同等浓度的rsTRAIL组(P<0.001)。(5)流式细胞术检测HL-60细胞内caspase-8表达水平结果显示:不同浓度的rsTRAIL组分别为74.86±1.57、78.26±1.30、83.69±1.42。先后给药组分别为83.31±1.24、87.46±2.43、91.08±2.29。先后给药组HL-60细胞内caspase-8的活性水平大于单用同等浓度的rsTRAIL组(P<0.05)。
结论:(1)rsTRAIL能抑制HL-60细胞生长,诱导HL-60细胞凋亡,在50-200ug/L浓度范围内,诱导细胞凋亡率呈现剂量依赖性。(2)Ara-c能上调HL-60细胞表面DR5表达水平,增强rsTRAIL诱导HL-60细胞凋亡的作用。(3)Ara-c和rsTRAIL联合用药时,先加Ara-c、再加rsTRAIL诱导HL-60细胞凋亡的效果比同时给药效果好。(4)Ara-c增强rsTRAIL诱导HL-60细胞凋亡的机制可能由Ara-c上调HL-60细胞表面DR5表达水平和/或增强细胞内caspase8的活性实现的。
Objective:To investigate the combined effect of rsTRAIL with Ara-c or alone on HL-60 leukemia cell lines and its mechanism,and to provide experimental basis for treatment of acute leukemia.
Methods: Human leukemia cell line HL-60 was cultured in vitro.HL-60 cell was separated into 5 groups:control group, Ara-c group,rsTRAILgroup, Ara-c+rsTRAIL group, Ara-c followed by rsTRAIL group(Ara-c→rsTRAIL group). The cytotoxic effect were meatured by MTT assay;Cell apoptosis rate was determined by flow cytometry after Annexin V/PI staining; HL-60 cell was treated with Ara-c of different concentration for 24 hours, expression level of DR5 on surface of HL-60 cells was determined by flow cytometry. Expression level of DR5 on surface of HL-60 cells and caspase-8 activition in HL-60 cells of rsTRAIL group、Ara-c→rsTRAIL group was determined by flow cytometry.
Results:(1)The result by MTT assay: control group 5.20±0.65;Ara-c group 11.53±0.51;rsTRAIL group16.45±1.35、22.86±0.81、29.79±1.88,respectively;Ara-c+rsTRAIL group27.78±2.63、33.36±2.01、36.35±3.27,respectively;Ara-c→rsTRAIL group34.08±3.44、41.21±5.98、44.45±4.55,respectively. 50-200ug/L rsTRAIL could inhibit the proliferation of the HL-60 cell in concentration-dependent manners(P<0.001).The effects presented by cotreatment with Ara-c and rsTRAIL were significantly higher than Ara-c or rsTRAIL used alone(P<0.001). The effects presented by sequential treatment of HL-60 cells with Ara-c followed by rsTRAIL were significantly higher than cotreatment with rsTRAIL and Ara-c(P<0.05).(2)The result of percentage of apoptisis cells: control group 13.41±1.52;rsTRAIL group 25.78±2.21、36.76±0.62、44.50±2.78,respectively;Ara-c+rsTRAIL group74.45±5.06、77.39±5.34、80.66±6.27,respectively;Ara-c→rsTRAIL group 85.05±3.77、86.33±4.83、87.71±4.78,respectively. The apoptosis rate of rsTRAIL group was higher than that of control group,The apoptosis-induction of rsTRAIL was concentration-dependen(tP<0.05). The apoptosis rate of cotreatment with Ara-c and rsTRAIL was higher than that of Ara-c group and rsTRAIL group alone(P<0.001). The apoptosis rate of sequential treatment of HL-60 cells with Ara-c followed by rsTRAIL was higher than that of cotreatment with Ara-c and rsTRAIL(P<0.05)(.3)After treatment with Ara-c(2.5 mg/L、5 mg/L、10mg/L)for 24 hours,the expression level of DR5 on HL-60 was 1.22±0.18、3.02±0.03、3.56±0.45,respectively. The expression level of DR5 of control group was 1.20±0.16。The expression level of DR5 in 10mg/L Ara-c group was the highest, the expression level of DR5 in 5mg/L Ara-c group was the second, the expression level of DR5 in 2.5mg/L Ara-c group and control group was the lowes(tP<0.001)(.4)The expression level of DR5 on HL-60 of rsTRAIL group was 0.61±0.05、0.91±0.02、1.29±0.04。The expression level of DR5 on HL-60 of Ara-c→rsTRAIL group was 0.90±0.03、1.31±0.06、3.69±0.09,respectively. The expression level of DR5 was significant higher in sequential treatment of HL-60 cells with Ara-c followed by rsTRAIL than that in rsTRAIL group alone(P<0.001)(.5)The expression level of caspase-8 of rsTRAIL group was 74.86±1.57、78.26±1.30、83.69±1.42。The expression level of caspase-8 of Ara-c→rsTRAIL group was 83.31±1.24、87.46±2.43、91.08±2.29,respectively. The expression level of caspase-8 was significant higher in HL-60 with Ara-c followed by rsTRAIL than that in rsTRAIL group alone(P<0.05).
Conclusion: (1)RsTRAIL could inhibit the proliferation of HL-60 cells, rsTRAIL could induced apoptosis of HL-60 cells.The apoptosis-induction of rsTRAIL was concentration-dependent.(2)Ara-c could upregulated DR5 expression on the surface of HL-60 cells ,and enhance effect of rsTRAIL-inducing apoptosis(.3)Sequential treatment of HL-60 cells with Ara-c followed by rsTRAIL induced significantly more apoptosis than cotreatment with rsTRAIL and Ara-c(.4)Ara-c and rsTRAIL has a synergistic inhibitory effect on growth of HL-60 cells,the mechanism may correlate with the up-regulation the expression level of DR5 and/or caspase-8 of HL-60 cells.
方法:在体外以人类急性白血病细胞株HL-60细胞为研究对象,分成5组进行细胞培养:对照组、Ara-c组、rsTRAIL组、同时给药组(Ara-c+rsTRAIL)、先后给药组(Ara-c→rsTRAIL)。MTT比色法测定HL-60细胞生长抑制率;Annexin V/PI染色、流式细胞术检测HL-60细胞凋亡率;流式细胞术检测不同浓度Ara-c对HL-60细胞表面DR5表达水平的影响;流式细胞术检测rsTRAIL单药组及先后给药组对HL-60细胞表面DR5表达水平和HL-60细胞内caspase8活性的影响。
结果:(1)MTT实验结果显示各组HL-60细胞生长抑制率为:对照组5.20±0.65;5mg/L Ara-c组11.53±0.51;rsTRAIL组(50、100、200μg /L):16.45±1.35、22.86±0.81、29.79±1.88;不同浓度的同时给药组:27.78±2.63、33.36±2.01、36.35±3.27;不同浓度的先后给药组:34.08±3.44、41.21±5.98、44.45±4.55。与对照组比较,50、100、200μg /L浓度的rsTRAIL能抑制HL-60细胞生长,在50-200μg /L浓度范围内生长抑制率呈现剂量依赖性(P<0.001)。同时给药组HL-60细胞生长抑制率大于单用同等浓度的rsTRAIL组和Ara-c组(P<0.001)。先后给药组细胞生长抑制率大于相应浓度的同时给药组,有统计学差异(P<0.05)。(2)流式细胞术分析显示各组HL-60细胞凋亡率:对照组13.41±1.52;Ara-c组61.67±2.79;rsTRAIL组(50、100、200 ug/L)25.78±2.21、36.76±0.62、44.50±2.78;不同浓度的同时给药组74.45±5.06、77.39±5.34、80.66±6.27;不同浓度的先后给药组85.05±3.77、86.33±4.83、87.71±4.78。与对照组比较,50、100、200ug/L浓度的rsTRAIL能诱导HL-60细胞凋亡,在50-200ug/L浓度范围内诱导凋亡率呈现剂量依赖性(P<0.001)。同时给药组HL-60细胞凋亡率大于相应浓度的单用同等浓度的rsTRAIL组和Ara-c组(P<0.05)。先后给药组HL-60细胞凋亡率大于同时给药组(P<0.05)。(3)Ara-c(2.5mg/L、5mg/L、10mg/L )作用24小时后,HL-60细胞表面DR5表达水平分别为:1.22±0.18、3.02±0.03、3.56±0.45。对照组HL-60细胞表面DR5表达水平为1.20±0.16。10mg/L Ara-C组细胞表面DR5表达水平最高,5mg/LAra-C组次之,2.5mg/L Ara-c组与对照组最低(P<0.001)。(4)流式细胞术检测HL-60细胞表面DR5表达水平结果显示:不同浓度的rsTRAIL组分别为0.61±0.05、0.91±0.02、1.29±0.04。不同浓度的先后给药组分别为0.90±0.03、1.31±0.06、3.69±0.09。先后给药组HL-60细胞表面DR5表达水平大于单用同等浓度的rsTRAIL组(P<0.001)。(5)流式细胞术检测HL-60细胞内caspase-8表达水平结果显示:不同浓度的rsTRAIL组分别为74.86±1.57、78.26±1.30、83.69±1.42。先后给药组分别为83.31±1.24、87.46±2.43、91.08±2.29。先后给药组HL-60细胞内caspase-8的活性水平大于单用同等浓度的rsTRAIL组(P<0.05)。
结论:(1)rsTRAIL能抑制HL-60细胞生长,诱导HL-60细胞凋亡,在50-200ug/L浓度范围内,诱导细胞凋亡率呈现剂量依赖性。(2)Ara-c能上调HL-60细胞表面DR5表达水平,增强rsTRAIL诱导HL-60细胞凋亡的作用。(3)Ara-c和rsTRAIL联合用药时,先加Ara-c、再加rsTRAIL诱导HL-60细胞凋亡的效果比同时给药效果好。(4)Ara-c增强rsTRAIL诱导HL-60细胞凋亡的机制可能由Ara-c上调HL-60细胞表面DR5表达水平和/或增强细胞内caspase8的活性实现的。
Objective:To investigate the combined effect of rsTRAIL with Ara-c or alone on HL-60 leukemia cell lines and its mechanism,and to provide experimental basis for treatment of acute leukemia.
Methods: Human leukemia cell line HL-60 was cultured in vitro.HL-60 cell was separated into 5 groups:control group, Ara-c group,rsTRAILgroup, Ara-c+rsTRAIL group, Ara-c followed by rsTRAIL group(Ara-c→rsTRAIL group). The cytotoxic effect were meatured by MTT assay;Cell apoptosis rate was determined by flow cytometry after Annexin V/PI staining; HL-60 cell was treated with Ara-c of different concentration for 24 hours, expression level of DR5 on surface of HL-60 cells was determined by flow cytometry. Expression level of DR5 on surface of HL-60 cells and caspase-8 activition in HL-60 cells of rsTRAIL group、Ara-c→rsTRAIL group was determined by flow cytometry.
Results:(1)The result by MTT assay: control group 5.20±0.65;Ara-c group 11.53±0.51;rsTRAIL group16.45±1.35、22.86±0.81、29.79±1.88,respectively;Ara-c+rsTRAIL group27.78±2.63、33.36±2.01、36.35±3.27,respectively;Ara-c→rsTRAIL group34.08±3.44、41.21±5.98、44.45±4.55,respectively. 50-200ug/L rsTRAIL could inhibit the proliferation of the HL-60 cell in concentration-dependent manners(P<0.001).The effects presented by cotreatment with Ara-c and rsTRAIL were significantly higher than Ara-c or rsTRAIL used alone(P<0.001). The effects presented by sequential treatment of HL-60 cells with Ara-c followed by rsTRAIL were significantly higher than cotreatment with rsTRAIL and Ara-c(P<0.05).(2)The result of percentage of apoptisis cells: control group 13.41±1.52;rsTRAIL group 25.78±2.21、36.76±0.62、44.50±2.78,respectively;Ara-c+rsTRAIL group74.45±5.06、77.39±5.34、80.66±6.27,respectively;Ara-c→rsTRAIL group 85.05±3.77、86.33±4.83、87.71±4.78,respectively. The apoptosis rate of rsTRAIL group was higher than that of control group,The apoptosis-induction of rsTRAIL was concentration-dependen(tP<0.05). The apoptosis rate of cotreatment with Ara-c and rsTRAIL was higher than that of Ara-c group and rsTRAIL group alone(P<0.001). The apoptosis rate of sequential treatment of HL-60 cells with Ara-c followed by rsTRAIL was higher than that of cotreatment with Ara-c and rsTRAIL(P<0.05)(.3)After treatment with Ara-c(2.5 mg/L、5 mg/L、10mg/L)for 24 hours,the expression level of DR5 on HL-60 was 1.22±0.18、3.02±0.03、3.56±0.45,respectively. The expression level of DR5 of control group was 1.20±0.16。The expression level of DR5 in 10mg/L Ara-c group was the highest, the expression level of DR5 in 5mg/L Ara-c group was the second, the expression level of DR5 in 2.5mg/L Ara-c group and control group was the lowes(tP<0.001)(.4)The expression level of DR5 on HL-60 of rsTRAIL group was 0.61±0.05、0.91±0.02、1.29±0.04。The expression level of DR5 on HL-60 of Ara-c→rsTRAIL group was 0.90±0.03、1.31±0.06、3.69±0.09,respectively. The expression level of DR5 was significant higher in sequential treatment of HL-60 cells with Ara-c followed by rsTRAIL than that in rsTRAIL group alone(P<0.001)(.5)The expression level of caspase-8 of rsTRAIL group was 74.86±1.57、78.26±1.30、83.69±1.42。The expression level of caspase-8 of Ara-c→rsTRAIL group was 83.31±1.24、87.46±2.43、91.08±2.29,respectively. The expression level of caspase-8 was significant higher in HL-60 with Ara-c followed by rsTRAIL than that in rsTRAIL group alone(P<0.05).
Conclusion: (1)RsTRAIL could inhibit the proliferation of HL-60 cells, rsTRAIL could induced apoptosis of HL-60 cells.The apoptosis-induction of rsTRAIL was concentration-dependent.(2)Ara-c could upregulated DR5 expression on the surface of HL-60 cells ,and enhance effect of rsTRAIL-inducing apoptosis(.3)Sequential treatment of HL-60 cells with Ara-c followed by rsTRAIL induced significantly more apoptosis than cotreatment with rsTRAIL and Ara-c(.4)Ara-c and rsTRAIL has a synergistic inhibitory effect on growth of HL-60 cells,the mechanism may correlate with the up-regulation the expression level of DR5 and/or caspase-8 of HL-60 cells.
引文
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[1] Wiley SR,Schooley K,Smolak PJ,et a1.1dentification and characterization of a new member of the TNF family that induces apoptosis[J].Immunity,1995,3(6):673-682.
[2] Li LY, Shu Hb.Molecular mechanisms of TRAIL-induced apoptosis of cancer cells[J].Chinese Science Bulletin, 2001,9(5):707-709.
[3] 范清林,宋礼华,魏伟.TRAIL的诱导肿瘤细胞凋亡与临床[J].中国药理学通报2003,19(9):976-981
[4] Kimberley FC,Screaton GR.. Following a TRAIL: Update on a ligand and its five receptors [J] . Cell research, 2004,14(5):359-372.
[5] Waiant H. TNF—related apoptosis inducing ligang (TRAIL) and its receptors in tumor surveillance[J].Apoptosis,2002,7(5):449～59.
[6] Youngleem Kim ,Dai-Wu Seol . TRAIL, a mighty apoptosis inducer[J]. Mol. Cells, 2003,!5(3):283-293.
[7] Kim K,Fisher MA,Xu SQ el a1. Molecular determinants of response to TRAIL in killing of normal and cancer cells[J]. Clin Cancer Res,2000,6(2):335-346.
[8] 张劲崎. TRAIL的研究现状及其在血液系统肿瘤治疗中的应用[J].国外医学输血及血液学分册,2002,25(3):249-252.
[9] 祝瑾,李宁川,程宏. TRAIL诱导白血病细胞凋亡的分子机制研究[J].实用临床医药杂志,2004,8(3):36-40.
[10] 刘洪波,范学工,李宁. 肿瘤坏死因子相关凋亡诱导配体的凋亡诱导机制及其药物开发[J].中国药科大学学报,2004,35(4):381-384.
[11] 史娟,张锦春,畅晓燕等.重组可溶性TRAIL的表达与生物学活性[J].中国生物工程杂志,2003,23(6)46-48.
[12] Yagita H,Takeda K,Hayakawa Y et al. TRAIL and its receptors as targets for cancer therapy[J]. Cancer sci ,2004,95(10):777-783.
[13] Walczak H, Miller RE, Ariail K, et al. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med 1999;5:157-163
[14] Shankar S, Srivastava RK. Enhancement of therapeutic potential of TRAIL by cancer chemotherapy and irradiation: mechanisms and clinical implications[J]. Drug Resist Updat ,2004; 7: 139–56.
[15] Ashkenazi A, Pai RC, Fong S, et al. Safety and antitumor activity of recombinant soluble Apo2 lligand.[J].clin.Invest 1999,104:155-162
[16] Wen JH, Ramadevi N, Nguyen D, et al. Antileukemic drugs increase death receptor 5 levels and enhanceApo-2L–induced apoptosis of human acute leukemia cells[J]. Blood, 2000,96(12):3900-3906
[17] Lacour S, Hammann A,Wotawa A,et al. Anticancer Agents Sensitize Tumor Cells to Tumor Necrosis Factor-related Apoptosis-inducing Ligand-mediated Caspase-8 Activation and Apoptosis[J]. Cancer res,2001,61(2):1645-1651
[18] Jones DT, Ganeshaguru K, Mitchell WA . Cytotoxic drugs enhance the ex vivo sensitivity of malignant cells from a subset of acute myeloid leukaemia patients to apoptosis induction by tumour necrosis factor receptor-related apoptosis-inducing ligand[J].Br J Haematol. 2003, 121(5):713-20
[19] 赵缇,王红祥.肿瘤坏死因子相关凋亡诱导配体联合阿糖胞苷诱导急性髓细胞性白血病细胞凋亡的研究[J].临床血液学杂志,2004,3(17)105-106.
[20] 赵缇,王红祥.肿瘤坏死因子相关凋亡诱导配体联合三氧化二砷对急性髓细胞性白血病细胞凋亡的影响[J].中华血液学杂志,2005,1(26):51-52