Bfl-1蛋白C-端结构域短肽诱导细胞凋亡及自噬的作用与机制

A major focus in current cancer research is to develop small molecules or peptides as potential therapeutic agents or diagnostic tools for cancer treatment. Compared with conventional chemotherapy or radiotherapy agents, peptide drugs have the advantage of tissue-specific targeting, thus off-target effects could be minimized. Previously, we identified a novel mitochondrial targeting peptide, an amphipathic tail-anchoring peptide (ATAP) derived from C-terminal α-helix region of the human anti-apoptotic protein Bfl-1, is a potent inducer of apoptosis by targeting mitochondria permeability transition. By linking ATAP to an internalizing RGD peptide (iRGD) or folate, selective targeting for ATAP to tumor cell was achieved. In vivo xenograft studies with multiple prostate cancer cell lines showed that intravenous administration of ATAP-iRGD and ATAP-folate suppressed tumor growth. Toxicological studies revealed that repetitive intravenous administration of ATAP-iRGD did not produce significant toxicity in the SV129 mice. ATAP could distribute along the mitochondria network and triggered mitochondria-dependent cell death through release of cytochrome c. However, this cell death is also accompanied by LC3‑I to LC3‑II conversion, suggesting that autophagy represents an alternative induction of ATAP peptide. In this study, it will be important to determine the mechanisms that underlie the functional interaction ATAP and its associated proteins in regulating the mitochondria permeability transition associated with apoptotic and autophagic cell death.

肿瘤患者常见的化疗、放疗手段往往伴随着严重的副作用, 而多肽药物因高效、低毒的特点成为抗肿瘤研究的新热点。我们前期发现Bcl-2家族成员Bfl-1蛋白C-端序列中29个氨基酸组成的双亲性多肽（ATAP）可以诱发细胞凋亡。根据ATAP结构域设计的多肽药物ATAP-iRGD及纳米颗粒都可以有效抑制前列腺癌和食管癌等癌细胞在体外及裸鼠体内的生长，而且对正常组织的毒性非常低，具有用于癌症治疗的潜力。ATAP结构域可以特异性聚集于线粒体膜，破坏线粒体外膜完整性，最终诱发细胞的凋亡，这一过程也伴随着自噬的发生。ATAP结构是如何引起线粒体功能变化并诱导细胞凋亡及自噬进程的？作用机制是什么？目前还知之甚少。本项目拟检测ATAP多肽诱导肿瘤细胞凋亡及自噬的作用与机制进行深入研究，为设计以ATAP为基础的多功能新型多肽药物提供更多理论依据。

Bcl-2家族成员Bfl-1蛋白C-端序列中29个氨基酸组成的双亲性多肽（ATAP，Amphipathic tail-anchoring peptide）可以诱发Caspase依赖性的细胞凋亡。ATAP多肽结构包括一个双亲性的α-螺旋，其N-端和C-端均有线粒体定位信号。连接了iRGD的ATAP-iRGD多肽可以特异性靶向多种癌细胞，并聚集于细胞内线粒体膜，破坏线粒体外膜完整性，最终诱发细胞的凋亡和细胞自噬的发生，这一过程伴随着自噬相关蛋白LC3I/II的累积。在此过程中，ATAP在线粒体中的正确定位起到关键作用。我们的结果证明ATAP-iRGD诱导的凋亡和自噬过程为相互拮抗的关系，当ATAP-iRGD诱导的细胞凋亡过程被抑制时，自噬反应水平更高，反之亦然。Bfl-1高表达的乳腺癌细胞系MDA-435表现出对于ATAP-iRGD多肽的极端不敏感，推断Bfl-1蛋白与ATAP特异性竞争线粒体上同一位点从而降低了对ATAP的敏感性，进一步证明ATAP发挥细胞毒性作用有赖于其在细胞线粒体上的正确定位。. 由于ATAP-iRGD多肽的水溶性偏低，我们进一步探索设计新的ATAP癌细胞靶向递送方式。利用叶酸(folate)分子与癌细胞表面高表达的叶酸受体(folate receptor)的高亲和性这一特征，我们设计了三步化学合成途径，成功的连接folate分子到ATAP N端的氨基上，合成了以ATAP为基础的多功能新型化合物药物分子folate-ATAP. 共聚焦显微镜显示folate-ATAP可以进入癌细胞内并聚集在线粒体网络内，细胞活性实验表明folate-ATAP有效抑制癌细胞的生长，以上结果说明folate-ATAP具有发展为癌症治疗药物的潜力。.