SapC-DOPS nanovesicles induce Smac- and Bax-dependent apoptosis through mitochondrial activation in neuroblastomas.
SapC-DOPS nanovesicles induce Smac- and Bax-dependent apoptosis through mitochondrial activation in neuroblastomas.
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DOI：
10.1186/s12943-015-0336-y
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发表时间：
2015-04-08
期刊：
影响因子：
37.3
通讯作者：
Qi X
中科院分区：
文献类型：
--
作者：
Sulaiman MK;Chu Z;Blanco VM;Vallabhapurapu SD;Franco RS;Qi X
高毒性、发病率和继发性恶性肿瘤使得神经母细胞瘤的化疗无效，促使寻找新的化合物。纳米囊泡在癌症的成像和治疗中提供了巨大的希望。SapC-DOPS是由溶酶体蛋白saposin C和二油酰磷脂酰丝氨酸形成的稳定纳米囊泡，其对癌细胞上大量暴露的表面磷脂酰丝氨酸具有强亲和力。在这里，我们表明，SapC-DOPS有效地靶向和抑制神经母细胞瘤的生长，并阐明SapC-DOPS在体外神经母细胞瘤中的作用的分子机制。在静脉注射荧光标记的SapC-DOPS的异种移植小鼠中评估了神经母细胞瘤的体内靶向作用。异种移植肿瘤也被用来证明其治疗效果。使用TUNEL测定在肿瘤切片中评价体内细胞凋亡诱导。SapC-DOPS诱导细胞凋亡的潜在机制通过测量细胞活力、线粒体膜电位（Δ pM）、流式细胞术DNA片段化分析以及通过免疫印迹分析培养的神经母细胞瘤细胞的胞质溶胶或线粒体组分中的第二类来源于caspase的激活物（Smac）、Bax、细胞色素c（Cyto c）和Caspase-3来解决。SapC-DOPS显示出特异性靶向作用，并阻止了小鼠中人神经母细胞瘤异种移植物的生长。在体外培养的神经母细胞瘤细胞中，凋亡的发生是通过一系列的步骤进行的，包括：（1）Δ β M的丢失和线粒体超氧化物的形成增加;（2）Smac、Cyto c、AIF的胞浆释放;（3）线粒体Bax的易位和聚合。ShRNA介导的Smac敲除和V5肽介导的Bax抑制降低了胞质Smac和Cyto c的释放，沿着半胱天冬酶的激活并消除了细胞凋亡，表明Smac和Bax是SapC-DOPS作用的关键介质。类似地，用β-胡萝卜素稳定剂米松酸预处理减少了细胞凋亡，表明Δ β-胡萝卜素M的丧失对SapC-DOPS活性至关重要。细胞凋亡诱导并不严重依赖于活性氧（ROS）的产生和亲环素D，因为预处理与N-乙酰半胱氨酸和环孢素A，分别没有阻止Smac或Cyto C的释放。两者合计，我们的研究结果表明，SapC-DOPS的行为，通过一个由Smac和Bax的早期释放伴随着一个ESTA介导的途径。SapC-DOPS的特异性肿瘤靶向能力和抗癌功效支持其作为神经母细胞瘤治疗中的双重成像和治疗剂的潜力。本文的在线版本（doi：10.1186/s12943-015-0336-y）包含补充材料，可供授权用户使用。
High toxicity, morbidity and secondary malignancy render chemotherapy of neuroblastoma inefficient, prompting the search for novel compounds. Nanovesicles offer great promise in imaging and treatment of cancer. SapC-DOPS, a stable nanovesicle formed from the lysosomal protein saposin C and dioleoylphosphatidylserine possess strong affinity for abundantly exposed surface phosphatidylserine on cancer cells. Here, we show that SapC-DOPS effectively targets and suppresses neuroblastoma growth and elucidate the molecular mechanism of SapC-DOPS action in neuroblastoma in vitro. In vivo targeting of neuroblastoma was assessed in xenograft mice injected intravenously with fluorescently-labeled SapC-DOPS. Xenografted tumors were also used to demonstrate its therapeutic efficacy. Apoptosis induction in vivo was evaluated in tumor sections using the TUNEL assay. The mechanisms underlying the induction of apoptosis by SapC-DOPS were addressed through measurements of cell viability, mitochondrial membrane potential (ΔΨM), flow cytometric DNA fragmentation assays and by immunoblot analysis of second mitochondria-derived activator of caspases (Smac), Bax, Cytochrome c (Cyto c) and Caspase-3 in the cytosol or in mitochondrial fractions of cultured neuroblastoma cells. SapC-DOPS showed specific targeting and prevented the growth of human neuroblastoma xenografts in mice. In neuroblastoma cells in vitro, apoptosis occurred via a series of steps that included: (1) loss of ΔΨM and increased mitochondrial superoxide formation; (2) cytosolic release of Smac, Cyto c, AIF; and (3) mitochondrial translocation and polymerization of Bax. ShRNA-mediated Smac knockdown and V5 peptide-mediated Bax inhibition decreased cytosolic Smac and Cyto c release along with caspase activation and abrogated apoptosis, indicating that Smac and Bax are critical mediators of SapC-DOPS action. Similarly, pretreatment with the mitochondria-stabilizing agent bongkrekic acid decreased apoptosis indicating that loss of ΔΨM is critical for SapC-DOPS activity. Apoptosis induction was not critically dependent on reactive oxygen species (ROS) production and Cyclophilin D, since pretreatment with N-acetyl cysteine and cyclosporine A, respectively, did not prevent Smac or Cyto c release. Taken together, our results indicate that SapC-DOPS acts through a mitochondria-mediated pathway accompanied by an early release of Smac and Bax. Specific tumor-targeting capacity and anticancer efficacy of SapC-DOPS supports its potential as a dual imaging and therapeutic agent in neuroblastoma therapy. The online version of this article (doi:10.1186/s12943-015-0336-y) contains supplementary material, which is available to authorized users.
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