Role of Hsp-70 in Triptolide-Mediated Cell Death of Neuroblastoma
Role of Hsp-70 in Triptolide-Mediated Cell Death of Neuroblastoma
复制标题
DOI：
10.1016/j.jss.2010.04.047
复制
发表时间：
2010-09-01
影响因子：
2.2
通讯作者：
Saluja, Ashok K.
中科院分区：
文献类型：
--
作者：
Antonoff, Mara B.;Chugh, Rohit;Saluja, Ashok K.
背景我们最近的工作表明，雷公藤内酯醇治疗神经母细胞瘤在体外引起凋亡细胞死亡，并减少体内肿瘤的大小。雷公藤甲素治疗与Hsp-70表达减少有关，提示细胞杀伤机制涉及Hsp-70抑制。本研究的主要目的是探讨热休克蛋白-70在雷公藤内酯醇介导的神经母细胞瘤细胞死亡中的作用。用Hsp-70特异性siRNA转染神经母细胞瘤细胞。随后测量了活力、半胱天冬酶活性和磷脂酰丝氨酸外化。开发了原位、同基因小鼠肿瘤模型，随机小鼠每天接受雷公藤内酯醇或载体的注射。21 d处死小鼠。免疫组化法检测残留肿瘤组织中Hsp-70的表达，末端脱氧核苷酸转移酶缺口末端标记法（TUNEL）检测肿瘤组织中凋亡细胞。用siRNA靶向沉默Hsp-70显著降低细胞活力，增强caspase-3活性，并导致膜联蛋白-V染色增加。这些效果与雷公藤内酯醇治疗后获得的结果平行。雷公藤甲素处理的小鼠的残留肿瘤表现出最小的HSP-70免疫组化染色，而对照肿瘤染色显着。用TUNEL法检测，治疗组小鼠的肿瘤显示出明显的染色，而对照组肿瘤没有显示出凋亡的证据。使用siRNA抑制神经母细胞瘤中的Hsp-70表达导致凋亡性细胞死亡，类似于雷公藤内酯醇的作用。雷公藤甲素治疗小鼠的残留肿瘤表达Hsp-70水平降低，并表现出显着的凋亡。这些发现支持Hsp-70抑制在雷公藤内酯醇介导的神经母细胞瘤细胞死亡中起重要作用的假设（C）2010 Elsevier Inc. All rights reserved.
Background. Our recent work demonstrated that treatment of neurobastoma with triptolide causes apoptotic cell death in vitro and decreases tumor size in vivo. Triptolide therapy has been associated with reduced expression of Hsp-70, suggesting a mechanism of cell killing involving Hsp-70 inhibition. The principal objective of this study was to investigate the role of Hsp-70 in triptolide-mediated cell death in neuroblastoma.Materials and Methods. Neuroblastoma cells were transfected with Hsp-70-specific siRNA. Viability, caspase activity, and phosphatidylserine externalization were subsequently measured. An orthotopic, syngeneic murine tumor model was developed, and randomized mice received daily injections of triptolide or vehicle. At 21 d, mice were sacrificed. Immunohistochemisty was used to characterize Hsp-70 levels in residual tumors, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed to identify cells undergoing apoptosis.Results. Targeted silencing of Hsp-70 with siRNA significantly decreased cellular viability, augmented caspase-3 activity, and resulted in increased annexin-V staining. These effects parallel those findings obtained following treatment with triptolide. Residual tumors from triptolide-treated mice showed minimal staining with Hsp-70 immunohistochemistry, while control tumors stained prominently. Tumors from treated mice demonstrated marked staining with the TUNEL assay, while control tumors showed no evidence of apoptosis.Conclusions. Use of siRNA to suppress Hsp-70 expression in neuroblastoma resulted in apoptotic cell death, similar to the effects of triptolide. Residual tumors from triptolide-treated mice expressed decreased levels of Hsp-70 and demonstrated significant apoptosis. These findings support the hypothesis that Hsp-70 inhibition plays a significant role in triptolide-mediated neuroblastoma cell death (C) 2010 Elsevier Inc. All rights reserved.