重组人可溶性 TRAIL 的制备及其联合硼替佐米诱导肿瘤细胞的凋亡作用

摘要

目的：构建重组人肿瘤坏死因子相关的凋亡诱导配体（ tumor necrosis factor－related apoptosis－indu－cing ligand，TRAIL）原核表达质粒pET－28a （＋）－TRAIL114－281，优化蛋白表达和纯化条件，制备重组人可溶性TRAIL并鉴定其活性。方法：使用CCK－8初步验证TRAIL是否具有抑制肿瘤细胞生长的生物活性；将制备的TRAIL单独或联合50 nmol／L硼替佐米应用于H460细胞（对TRAIL敏感）和K562细胞（对TRAIL抵抗）24 h，流式细胞术检测细胞凋亡率，比色法检测caspase－8、－9、－3的活化程度，Western blot分析细胞中Bax、Bcl－2和cFLIP蛋白的表达。流式细胞术检测硼替佐米处理H460细胞和K562细胞24 h后DR4和DR5的表达量变化。结果：制备了具有生物学活性且性质稳定的重组人可溶性TRAIL，且成功诱导H460和K562细胞凋亡。不同浓度TRAIL处理H460细胞后其凋亡率随着TRAIL浓度升高而显著升高（P＜0．05），但K562细胞凋亡率并未随着TRAIL浓度明显升高。联合用药组的H460和K562细胞凋亡率均显著高于单独用药组（P＜0．05），凋亡过程中caspase－8、－9、－3均被活化，药物处理组的Bcl－2和cFLIP表达量均比对照组下降，尤其联合用药组表达量下降最为显著（ P＜0．05），而Bax表达量无明显变化。硼替佐米处理H460和K562细胞后DR4和DR5表达量均上调（ P＜0．05）。结论：硼替佐米能协同TRAIL启动内源性凋亡途径诱导H460和K562细胞凋亡，其可能机制是通过上调死亡受体DR4和DR5的表达量、下调抗凋亡蛋白Bcl－2和cFLIP的表达量来实现的。%[ ABSTRACT] AIM:To construct a prokaryotic expression plasmid to produce recombinant human tumor necrosis factor-related apoptosis-inducing ligand ( TRAIL) and to verify the biological activity of TRAIL.METHODS: The pro-karyotic expression plasmid pET-28a (+)-TRAIL114-281 was constructed.Human soluble TRAIL was obtained through opti-mized inducing protein expression and purification conditions.The biological activity of TRAIL was verified by CCK-8 as-say.The apoptosis-inducing effect of TRAIL alone and/or in combination with proteasome inhibitor bortezomib ( Velcade, PS-341) on the tumor cell lines H460 ( TRAIL-sensitive) and K562 ( TRAIL-resistance) for 24 h was determined.The ap-optotic rates of the cells were analyzed by flow cytometry with Annexin V-FITC/PI staining.The activities of caspase-8,-9 and -3 in the cells were detected by colorimetric method.The protein expression of Bax, Bcl-2 and cFLIP was measured by Western blot.The expression of DR4 and DR5 in the H460 cells and K562 cells after treated with bortezomib for 24 h was detected by flow cytometry.RESULTS:The recombinant human soluble TRAIL protein with stable bioactivity was success-fully acquired, which induced apoptosis in H460 cells and K562 cells.After treatment with different concentrations of TRAIL, the apoptotic rate of H460 cells was significantly increased with the increase in the concentration of TRAIL ( P<0.05), but the apoptotic rate of K562 cells was not affected by the increasing TRAIL concentration.Apoptotic rate in com-bination group was obviously higher than that in single group ( P<0.05 ) .In the process of apoptosis, the activities of caspase-8,-9 and -3 in H460 cells and K562 cells were both increased.The expression of Bcl-2 and cFLIP in treatment groups ( especially the combination group) was decreased compared with control group.No significant change of the Bax expression level was observed.The expression of DR4 and DR5 in the H460 cells and K562 cells was significantly up-regu-lated after treated with bortezomib ( P<0.05 ) .CONCLUSION: Bortezomib combined with recombinant human soluble TRAIL synergistically induces apoptosis in tumor cell lines H460 and K562 through initiating intrinsic apoptotic pathways by up-regulating death receptors DR4 and DR5, and reducing the expression of antiapoptotic proteins Bcl-2 and cFLIP.