development of survivin targeted therapy for Adult T-cell leukemia/lymphoma

Survivin是一种凋亡抑制蛋白，在成人T细胞白血病/淋巴瘤（ATL）中高表达，与化疗耐药相关。在几乎所有的人类恶性肿瘤中都有报道生存素的过表达，但在正常组织中检测不到，这使得生存素靶向治疗成为一种有吸引力的ATL治疗策略。Survivin是一种同源二聚体，通过N端单一球状杆状病毒IAP重复（BIR）结构域与XIAP相互作用，保护XIAP不被泛素化，增加其稳定性，促进caspase-9抑制。在本研究中，我们合成了在Val 89和Lys 103之间的Survivin同源二聚体形成序列（PTD-（89- 103 aa））和在Lys 15和Met 38之间的XIAP相互作用序列（PTD-（15- 38 aa）），并在它们的氨基端添加达特蛋白的蛋白转导结构域（PTD）以使其具有细胞渗透性。各种白血病细胞系（ATL细胞系S1 T和MT 2，白血病细胞系C12和C14）的生长 ...更多信息 细胞系HL 60、NB 4、K562、Jurkat）与PTD-（15- 38 aa）孵育2小时后，而在正常细胞系、肺成纤维细胞HEL和正常人脐静脉内皮细胞HUVEC中未观察到生长抑制。用PTD-（15- 38 aa）处理S1 T细胞2小时后，通过Giemsa染色观察到显著的细胞死亡。流式细胞仪检测Annexin V和PI染色的S1 T细胞凋亡比例呈剂量依赖性增加。免疫印迹法未检测到caspase-9、caspase-3和PARP的降解。出乎意料的是，我们发现PTD-（15- 38 aa）处理引起LC 3-II表达的剂量依赖性增加和p62表达的降低。这些结果表明PTD-（15- 38 aa）诱导的S1 T细胞凋亡中有自噬的参与。在共转染Flag-XIAP和Myc-survivin的COS细胞中，采用免疫沉淀法研究PTD-（15- 38 aa）对survivin与XIAP相互作用的影响。与预期相反，PTD-（15- 38 aa）剂量依赖性地增强Survivin与XIAP的结合。这些结果表明，PTD-（15- 38 aa）强烈抑制白血病细胞，特别是ATL细胞的增殖，并且caspase活化不参与PTD-（15- 38 aa）诱导的自噬介导的凋亡。少

Survivin, an inhibitor of apoptosis protein, is highly expressed in adult T-cell leukemia/lymphoma (ATL), and associated with chemotherapy resistance. Specifically overexpression of survivin has been reported in almost all human malignancies, but not detectable in normal tissues, making survivin targeted therapy an attractive ATL therapy strategy. Survivin is functioning as a homodimer, and interacts with XIAP through N-terminal single globular baculovirus IAP repeat (BIR) domain, protecting XIAP from ubiquitination and increasing its stability that promotes caspase-9 inhibition. In this study, peptides containing the survivin sequence for homodimer formation between Val89 and Lys103 (PTD-(89-103aa)), and the sequence for interacting with XIAP between Lys15 and Met38 (PTD-(15-38aa)), adding the protein transduction domains (PTD) of TAT protein to their amino-terminals for cell permeable, were synthesized. The growth of various leukemia cell lines (ATL cell lines S1T and MT2, leukemia c … More ell lines HL60, NB4, K562, Jurkat) was strongly inhibited after 2 hr incubation with PTD-(15-38aa). However, no growth suppression was observed in the normal cell lines, lung fibroblast cells HEL and Normal Human Umbilical Vein Endothelial Cells HUVEC. Significant cell death of S1T cells was observed after 2hr treatment with PTD-(15-38aa) by Giemsa staining. The proportion of apoptosis fraction estimated by FACS with Annexin V and PI staining in S1T cells was increased in the dose dependent manner. However, Degradation of caspase-9, caspase-3 and PARP was not detected by immunoblotting. Unexpectedly, we found that PTD-(15-38aa) treatment caused a dose dependent increase in the expression of LC3-II and decrease in the expression of p62. These results indicated that autophagy involved in the PTD-(15-38aa)-induced apoptosis in S1T cells. The effect of PTD-(15-38aa) on the interacting of survivin with XIAP was investigated in COS cells co-transfected with Flag-XIAP and Myc-survivin by immunopreciptation. Contrary to expectation, PTD-(15-38aa) dose dependently enhanced the binding of survivin with XIAP. These results suggested that PTD-(15-38aa) strongly inhibited the proliferation of leukemia cells, specifically ATL cells, and caspase activation is not involved in the induction of autophagy- mediated apoptosis by PTD-(15-38aa). Less