神经纤毛蛋白-1 结合肽融合白喉毒素诱导非小细胞肺癌细胞系凋亡。
Neuropilin-1 Binding Peptide as Fusion to Diphtheria Toxin Induces Apoptosis in Non-small Cell Lung Cancer Cell Line.
机构信息
Department of Biology, Faculty of Basic Sciences, Islamic Azad University, Science and Research Branch, Tehran, Iran.
Venom and Biotherapeutics Molecules Laboratory, Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
出版信息
Curr Pharm Des. 2024;30(17):1317-1325. doi: 10.2174/0113816128292382240325074032.
BACKGROUND
Targeted cancer therapy can be considered as a new strategy to overcome the side effects of current cancer treatments. Neuropilin-1 (NRP-1) is a transmembrane glycoprotein that is expressed in endothelial cells and tumor vessels to stimulate angiogenesis progression. Targeted diphtheria toxin (DT)- based therapeutics are promising tools for cancer treatment. This study aimed to construct a novel NRP-1 binding peptide (as three repeats) (CRGDK) as a fusion to truncated DT (DTA) (DTA-triCRGDK) for targeted delivery of DT into NRP-1 expressing cells.
METHODS
The concept of DTA-triCRGDK was designed, synthesized and cloned into the bacterial host. Expression of DTA-triCRGDK was induced by Isopropyl ß-D-1-thiogalactopyranoside (IPTG) and purification was performed using Ni-NTA chromatography. Biological activity of DTA-triCRGDK was evaluated using MTT, apoptosis, and wound healing assays. In addition, expression levels of apoptotic Bax, Bcl2, and Casp3 genes were determined by Real-time PCR.
RESULTS
Cytotoxicity analysis showed the IC values of DTA-triCRGDK for A549 and MRC5 were 0.43 nM and 4.12 nM after 24 h, respectively. Bcl2 expression levels decreased 0.4 and 0.72 fold in A549 and MRC5, respectively. However, Bax and Casp3 expression level increased by 6.75 and 8.19 in A549 and 2.51 and 3.6 in MRC5 cells.
CONCLUSION
Taken together, DTA-triCRGDK is a promising tool for targeted therapy of NRP-1 overexpressing cancer cells.
背景
靶向癌症治疗可以被认为是克服当前癌症治疗副作用的一种新策略。神经纤毛蛋白-1（NRP-1）是一种跨膜糖蛋白，在血管内皮细胞和肿瘤血管中表达，以刺激血管生成进展。靶向白喉毒素（DT）为基础的治疗方法是癌症治疗的有前途的工具。本研究旨在构建一种新型的 NRP-1 结合肽（作为三个重复）（CRGDK）作为融合物与截断的 DT（DTA）（DTA-triCRGDK），用于将 DT 靶向递送至 NRP-1 表达细胞。
方法
设计、合成并克隆了 DTA-triCRGDK 的概念，使其进入细菌宿主。用异丙基-β-D-1-硫代半乳糖吡喃糖苷（IPTG）诱导 DTA-triCRGDK 的表达，并通过 Ni-NTA 色谱进行纯化。使用 MTT、凋亡和划痕愈合测定评估 DTA-triCRGDK 的生物学活性。此外，通过实时 PCR 测定凋亡 Bax、Bcl2 和 Casp3 基因的表达水平。
结果
细胞毒性分析表明，DTA-triCRGDK 在 A549 和 MRC5 中的 IC 值分别为 24 小时后 0.43 nM 和 4.12 nM。Bcl2 表达水平在 A549 和 MRC5 中分别降低了 0.4 和 0.72 倍。然而，Bax 和 Casp3 的表达水平在 A549 中分别增加了 6.75 和 8.19 倍，在 MRC5 中分别增加了 2.51 和 3.6 倍。
结论
总之，DTA-triCRGDK 是一种有前途的用于治疗 NRP-1 过表达癌细胞的靶向治疗工具。
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