Distinct cellular and therapeutic effects of obatoclax in rituximab-sensitive and -resistant lymphomas

Bcl-2 proteins represent a rheostat that controls cellular viability. Obatoclax, a BH3-mimetic, has been designed to specifically target and counteract anti-apoptotic Bcl-2 proteins. We evaluated the biological effects of obatoclax on the anti-tumour activity of rituximab and chemotherapy agents. Obatoclax induced cell death of rituximab/chemotherapy-sensitive (RSCL), -resistant cell lines (RRCL) and primary tumour-cells derived from patients with B-cell lymphomas (N=39). Obatoclax also enhanced the activity of rituximab and had synergistic activity when combined with chemotherapy agents. The ability of Obatoclax to induce PARP cleavage varied between patient samples and was not observed in some RRCL. Inhibition of caspase activity did not affect obatoclax activity, suggesting the existence of caspase-independent death pathways. Autophagy was detected by LC3 conversion and/or electron microscopy in RRCL and in patient-derived tumour cells. Moreover, obatoclax activity was inhibited by Beclin-1 knockdown. In summary, obatoclax is an active Bcl-2 inhibitor that potentiates the activity of chemotherapy agents and, to a lesser degree, rituximab. Defining the molecular events triggered by obatoclax is necessary to further its clinical development and identify potential biomarkers that are predictive of response.     

硼替佐米联用obatoclax协同诱导人急性B淋巴细胞白血病细胞株Nalm-6细胞凋亡

目的 探讨硼替佐米分别与3种Bcl-2抑制剂（obatoclax、AT-101、ABT-199）联用诱导人急性B淋巴细胞株Nalm-6细胞凋亡的协同作用。方法 应用MTT法分别检测3种Bcl-2抑制剂单用或联用硼替佐米作用于Nalm-6细胞48 h后的细胞活力。通过流式细胞术和蛋白质印迹分析分别检测不同药物单独或联合作用于Nalm-6细胞后的细胞凋亡情况及Bcl-2家族蛋白、泛素、微管相关蛋白1轻链3B（LC3B）、p62、免疫球蛋白结合蛋白（Bip）、磷酸化p38、磷酸化JNK、C/EBP同源蛋白（CHOP）等蛋白的表达情况。同时利用qRT-PCR检测硼替佐米和obatoclax联用后细胞的内质网应激反应关键基因Bip、CHOP、活化转录因子（ATF）4、ATF6、肌醇需求酶1α（IRE1α）、X-盒结合蛋白1（XBP1）的表达水平。最后通过MTT和流式细胞术检测内质网应激反应抑制剂牛磺熊去氧胆酸（TUDCA）是否可以逆转两药联用诱导的细胞凋亡。结果 硼替佐米和3种Bcl-2抑制剂单用均可降低Nalm-6细胞的存活率。3组药物联用实验组中，仅有obatoclax＋硼替佐米表现出协同细胞毒性作用，其余两组并无此现象。Obatoclax通过增加LC3B-Ⅱ、p62蛋白的表达水平抑制Nalm-6细胞的自噬活性。与单药相比，硼替佐米和obatoclax联用后泛素化蛋白的表达显著上调。硼替佐米联用obatoclax可同时抑制自噬和泛素蛋白酶体活性，造成大量蛋白蓄积，进而激活内质网应激反应，最终诱导Nalm-6细胞凋亡。内质网应激抑制剂TUDCA可削弱硼替佐米和obatoclax联用诱导的细胞凋亡。结论 硼替佐米联用obatoclax可同时抑制自噬和蛋白酶体活性，并激活内质网应激反应协同诱导人急性B淋巴细胞白血病细胞凋亡。     

Obatoclax与MG-132对食管癌细胞CaES-17的协同抗肿瘤作用

目的探讨obatoclax联用MG-132对人食管癌细胞是否具有协同的抗肿瘤作用。方法MTT法检测obatoclax与MG-132 对人食管癌细胞CaES-17的细胞毒作用,根据IC50确定两药联用的浓度比（1∶2.4）,将两个药物从4 IC50的作用浓度起倍比稀释, 用CompuSyn软件计算两药的联用指数（CI）。采用Western blot方法研究药物对ubiquitin表达、PARP蛋白剪切、caspase-9蛋白 剪切、phospho-Histone H3蛋白及phospho-Aurora A/B/C蛋白表达的影响。采用流式细胞术检测细胞早期凋亡（Annexin-V阳 性率）及细胞周期的影响。结果Obatoclax与MG-132联合处理人食管癌细胞CaES-17,对细胞抑制率为50%及95%时相对应 的CI 值分别为0.296 及0.104,具有明显的协同抗肿瘤作用。单用obatoclax 或MG-132 均能诱导ubiquitin 表达增加、PARP及 caspase-9发生剪切,且联用组与单用组相比有统计学差异（P<0.05）;联用组phospho-Histone H3蛋白增加与单用组相比有统计 学差异（P<0.05）;phospho-Aurora A/B/C蛋白表达的增加也与单用obatoclax相比有统计学差异（P<0.05）,与单用MG-132相比 无明显改变（P>0.05）。同时,联用组Annexin-V阳性率及sub-G1期及G2/M期细胞百分比与单用组相比都显著增加,且差异均有 统计学意义（P<0.05）。结论Obatoclax联用MG-132对人食管癌细胞CaES-17具有协同抗肿瘤作用,且与诱导肿瘤细胞发生凋 亡及G2/M期阻滞有关。