Objective: To explore whether bortezomib and a Bcl-2 inhibitor exhibit synergistic anti-tumor effect in human acute T lymphoblastic leukemia cells.
Methods: MTT assay was used to determine the cytotoxicity of bortezomib in the absence or presence of Bcl-2 inhibitors (obatoclax, AT-101 and ABT-199) in Jurkat cells. The effects of drug treatment on the expression of Bcl-2 family proteins, LC3B, p62, ubiquitin, BiP/Grp78, p-JNK, p-p38 and CHOP proteins were examined by Western blotting. Flow cytometry was used to determine the effects of bortezomib and Bcl-2 inhibitors (obatoclax, AT-101 and ABT-199) on cell apoptosis. Quantitative real-time PCR was used to measure the mRNA expression levels of the key regulatory factors of unfolded protein reaction (UPR). A zebrafish xenograft model was used to study the anti-tumor effect of bortezomib, obatoclax and their combination in vivo.
Results: Bortezomib or Bcl-2 inhibitors alone inhibited the cell viability of Jurkat cells, but only obatoclax and bortezomib showed synergistic cytotoxicity and pro-apoptotic effect. Obatoclax, rather than AT-101 and ABT- 199, blocked autophagic flux in the cells evidenced by concomitant accumulation of LC3B-Ⅱ and p62. Both bortezomib and obatoclax alone caused accumulation of polyubiquinated proteins, and their combination showed a synergistic effect, which was consistent with their synergistic cytotoxicity. The dual blockade of proteasome and autophagy by the combination of bortezomib and obatoclax triggered unfolded protein response followed by cell apoptosis. Preventing UPS dysfunction by tauroursodeoxycholic acid (TUDCA) significantly attenuated the cytotoxicity and pro-apoptotic effect of bortezomib in combination with obatoclax. In zebrafish xenograft models, bortezomib combined with obatoclax significantly decreased tumor foci formation.
Conclusions: Bortezomib and obatoclax for dual blockade of protein degradation pathways show synergistic anti-tumor effect in human acute T lymphoblastic leukemia cells.
目的: 探讨硼替佐米联用obatoclax对人急性T淋巴白血病细胞Jurkat是否具有协同抗肿瘤作用。
方法: MTT法检测单用硼替佐米和联用Bcl-2抑制剂（obatoclax，AT-101和ABT-199）对Jurkat细胞活力的影响。采用Western blot方法检测药物对Bcl-2家族蛋白、LC3B、p62、ubiquitin、BiP/Grp78、p-JNK、p-p38和CHOP蛋白表达的影响。硼替佐米和Bcl-2抑制剂对细胞凋亡的影响。实时荧光定量PCR检测未折叠蛋白反应（UPR）关键调节因子的mRNA表达水平。斑马鱼异种移植模型研究单药以及两药联用的体内抗肿瘤作用。
结果: 单用硼替佐米和Bcl-2抑制剂均可抑制Jurkat细胞活力，但联用时仅obatoclax与硼替佐米具有协同细胞毒作用，引起细胞凋亡增多。obatoclax阻断自噬流，伴随LC3B-Ⅱ和p62的蓄积。此外，单用硼替佐米和obatoclax可诱导泛素化蛋白的蓄积，两药联用对泛素化蛋白蓄积具有协同作用，与药物联用的协同细胞毒作用实验结果一致。硼替佐米和obatoclax联用对蛋白酶体和自噬的双重阻断触发了未折叠蛋白反应，诱导细胞凋亡。内质网应激抑制剂牛磺熊去氧胆酸（TUDCA）可减弱硼替佐米和obatoclax联用的细胞毒作用，减少细胞凋亡。在斑马鱼体内联用硼替佐米和obatoclax显著地减少肿瘤灶形成。
结论: 硼替佐米与obatoclax联用双重阻断蛋白质降解途径对人急性T淋巴白血病细胞具有协同抗肿瘤作用。
Keywords: Bcl-2; autophagy; bortezomib; human acute T lymphoblastic leukemia cells; obatoclax; synergism; unfolded protein response; zebrafish xenograft model.