Sturzu Alexander, Sheikh Sumbla, Echner Hartmut, Deeg Martin, Nägele Thomas, Weidenmaier Christopher, Schwentner Christian, Horger Marius, Ernemann Ulrike, Heckl Stefan
Department of Neuroradiology University of Tübingen Hoppe-Seyler-Str.3 72076 Tübingen, Germany.
Med Chem. 2016;12(5):412-8. doi: 10.2174/1573406412666151112125622.
Second mitochondrial activator of caspase (Smac) is a short mitochondrial peptide. When released from the mitochondria into the cytoplasm, it binds to inhibitor of apoptotic proteins (IAPs) within the cytoplasm and prevents them from inhibiting apoptosis.
Delivery of external synthetic Smac peptide into the cytoplasm of malignant cells could greatly improve the efficiency of apoptosis-inducing chemotherapeutic agents.
In our study different conjugates based on the seven N-terminal amino acids AVPIAQK of Smac (SmacN7) were produced to obtain a cytoplasm-directed Smac variant. SmacN7 and a point mutant (AVPKAQK) were coupled either to rhodamine alone or to both rhodamine and undecylic aldehyde, which is an antagonist of the Lily-of-the-valley fragrance receptor. The fifth conjugate consisted of rhodamine coupled only to undecylic aldehyde, without SmacN7. The uptake of these five conjugates into three different human cell lines was characterized and quantified by confocal laser scanning microscopy and flow cytometry. A caspase apoptosis assay was performed for cells incubated with the five different conjugates after induction of apoptosis.
The coupling of undecylic aldehyde to SmacN7 increased the cellular uptake of the correct and mutant conjugates.
Caspase 3/7 apoptosis tests after induction of apoptosis with staurosporine or UV irradiation showed that the coupling of SmacN7 with undecylic aldehyde resulted in a greatly increased adjuvant pro-apoptotic effect compared to the separate components and a mutant SmacN7 peptide sequence in the LNCaP prostate carcinoma cells compared to the benign prostate hyperplasia (BPH) cells and the human embryonal kidney (HEK) cells.
半胱天冬酶的第二个线粒体激活剂（Smac）是一种短小的线粒体肽。当它从线粒体释放到细胞质中时，会与细胞质中的凋亡蛋白抑制剂（IAPs）结合，阻止它们抑制细胞凋亡。
将外部合成的Smac肽递送至恶性细胞的细胞质中可极大提高诱导凋亡的化疗药物的效率。
在我们的研究中，基于Smac的七个N端氨基酸AVPIAQK（SmacN7）制备了不同的缀合物，以获得一种靶向细胞质的Smac变体。SmacN7和一个点突变体（AVPKAQK）分别单独与罗丹明偶联，或与罗丹明和十一醛偶联，十一醛是铃兰香味受体的拮抗剂。第五种缀合物仅由与十一醛偶联的罗丹明组成，不含SmacN7。通过共聚焦激光扫描显微镜和流式细胞术对这五种缀合物在三种不同人类细胞系中的摄取进行了表征和定量。在用五种不同缀合物孵育的细胞诱导凋亡后进行了半胱天冬酶凋亡测定。
十一醛与SmacN7偶联增加了正确和突变缀合物的细胞摄取。
用星形孢菌素或紫外线照射诱导凋亡后的半胱天冬酶3/7凋亡试验表明，与单独的组分相比，SmacN7与十一醛偶联在LNCaP前列腺癌细胞中产生了大大增强的辅助促凋亡作用，与良性前列腺增生（BPH）细胞和人胚肾（HEK）细胞相比，SmacN7肽序列发生了突变。
