细胞凋亡过程中半胱氨酸蛋白酶级联激活反应的分子成像

Apoptosis, or programmed cell death, is a controlled sequence of events leading to discharge of cells without releasing toxic materials to the surroundings. Under physiological conditions, apoptosis plays pivotal roles in regulating embryonic development, growth and differentiation and cause tumor cell death once triggered by chemotherapeutic drugs. Most anti-tumor drugs are taking effects by inducing cancer cell apoptosis. The development of pro-apoptosis drugs will be of great benefit to the patients. However, many pro-apoptosis drugs discovered by high throughput screening (HTS) method lose their therapy potential when applied to animal models or tested in the clinic due to the low tumor targeting or bad metastasis in vivo. Caspases are a family of cysteine-aspartic proteases involved in the process of apoptosis. Induction of apoptosis typically results in the activation of an initiator caspase such as caspase-8 or caspase-9. These caspases can then lead to the activation of the effector caspase, for example caspase-3. These caspases are responsible for the cleavage of the key cellular proteins, such as cytoskeletal proteins, which leads to the typical morphological changes observed in cells undergoing apoptosis. Although caspase imaging has been developed for years, the visualization of caspase cascade activation is rarely reported...Herein, we proposed a simple, versatile and robust one-step technique that enables real-time imaging of multiple intracellular caspase activities in living cells and animals without the need for complicated synthetic protocols. Conventional fluorogenic probes or recently reported activatable probes have been designed to target various proteases but are limited to extracellular molecules. Only a few have been applied to image intracellular proteases in living cells because most of these probes have limited cell-permeability. Our platform involves a straightforward peptide synthesis and a simple mixing step with a screened transfection agent. The transfection agent efficiently delivered the highly quenched fluorogenic probes, comprised of distinctive pairs of dyes and quenchers, to the initiator caspase-8 or caspase-9 and the effector caspase-3 in MDA-MB-435 cells, allowing imaging of various caspase activities during the apoptotic process induced by TNF-related apoptosis induced ligand (TRAIL) or other anti-cancer drugs. By combining multiple fluorogenic probes, this simple platform can be applied to study apoptotic processes in single cells and also monitoring therapeutic efficacy of cancer drugs in vivo.

细胞凋亡是细胞程序性死亡，它在生理和病理条件下都发挥着重要的生理功能。作为抗肿瘤药物的重要分支，促细胞凋亡类药物能通过诱导肿瘤细胞的凋亡而实现对肿瘤的治疗作用。然而，临床前高通量筛选的促细胞凋亡类药物中，某些化合物即便在体外显示了很好的功效，但是在体内的表现却差强人意。有效的在细胞水平和动物模型中评价药物功效能够缩短抗癌药物筛选进程。半胱氨酸蛋白酶通路是细胞凋亡的经典信号通路之一，不同的半胱氨酸蛋白酶之间的级联活化反应导致了凋亡的最终发生。申请人在大量蛋白酶分子影像研究基础上提出：以凋亡过程中的半胱氨酸蛋白酶为靶点，利用半胱氨酸蛋白酶的特异性可活化探针，采用先进的光学分子成像技术，在细胞水平上对凋亡过程中的半胱氨酸蛋白酶级联激活反应做可视化研究，建立细胞凋亡的分子成像平台，研究的最终目的是利用此平台在细胞水平和动物模型中监测细胞凋亡过程，从而评价抗肿瘤药物在体外和体内的功效。

光动力治疗多种疾病（包括癌症）已经在临床上得到认可。PDT是利用特定波长的光激发无毒的光敏剂产生有毒的活性氧以及单线态氧破坏肿瘤血管以及组织。与其他传统的治疗方法相比，PDT的优点是非侵害性，低毒性，副作用少。显然，对于那些产生化疗和放疗抗药性的人来说，PDT也是一种治疗途径，因为它通过其他途径杀死细胞。最有可能的一种PDT机制是产生活性氧以及单线态氧导致细胞毒性。在PDT过程中直接实时监测细胞凋亡有助于：（1）研究PDT毒性与细胞变化的关系（2）阐明PDT的机制（3）预测及评价治疗效果（4）促进光敏剂的筛选及优化。尽管有许多研究报道了PDT可引发细胞凋亡，但是鲜有研究阐明其中的细节。当下，监测细胞凋亡的金标准是利用细胞凋亡检测试剂盒来检测。虽然这种方法很简便，但是不直接不明确。以半胱氨酸和天冬氨酸定向的蛋白酶的凋亡成像引起了极大地研究兴趣，因为这些蛋白酶与细胞凋亡密切相关。有很多研究报道了活细胞中显像胱天蛋白酶的表达。其中，由特异性肽底作为linker合成的荧光共振能量转移对（FRET）是一种新型的细胞凋亡显像剂，由于它们可以提高靶-背景比，灵敏度，特异性。之前的研究报道有以下几个方面：利用肽转染剂实时监测caspase-8 和 caspase-3的激活；利用其它纳米材料实时监测caspase-8/9/3的激活；基于FRET的具有胱天蛋白酶靶向的材料用于显像细胞凋亡；基于荧光和发光蛋白的具有胱天蛋白酶靶向的材料用于显像细胞凋亡。然而这些方法监测caspase的激活较为繁琐，例如，添加外源细胞凋亡诱导剂或使用基因工程细胞。尽管一些光敏剂可以在PDT过程中实时提供caspase的激活的静态图像，但是由于光敏剂的低耐光性以及较差的稳定性限制了其灵敏度以及在体内的应用。此外，复杂的合成步骤需要光敏剂有荧光性能以及易于修饰。综上所述，设计一个简便，稳定，灵敏，高的靶-背景比的细胞凋亡显像剂是十分必要的。在自然科学基金支持下，我们合成了一种稳定性和灵敏度都很高的探针（FABP/ZnPc），同时实现光敏剂（ZnPc）递送，以及在PDT过程中实时监测细胞凋亡。ZnPc在近红外光照射下被激活并且引发细胞凋亡，ZnPc外层的FABP被胱天蛋白酶（caspase-3）酶解，产生很强的荧光信号并实时监测凋亡。纳米材料采用生物相容性很好的铁蛋白，它可以在酸性条件下分散，在中性条件在中性条件