Preparation of recombinant human soluble TRAIL and its inducing effect on apoptosis of tumor cells by TRAIL combined with bortezomib
10.3969/j.issn.1000-4718.2015.11.003
- VernacularTitle: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
- Author:
Xueyan LI
;
Xia XU
- Publication Type:Journal Article
- Keywords:
Tumor necrosis factor-related apoptosis-inducing ligand;
Bortezomib;
Apoptosis
- From:
Chinese Journal of Pathophysiology
2015;(11):1933-1942
- CountryChina
- Language:Chinese
-
Abstract:
[ 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.
