Epinecidin-1 (epi) was identified from orange-spotted grouper (Epinephelus coioides) and exhibits diverse biological activities. The aims of this study were to investigate how the distribution of positively charged amino acid residues affects epi-mediated cytotoxicity and to examine the molecular mechanism underlying epi-induced cytotoxicity in U87MG human glioblastoma cells. MTS/PMS and trypan blue exclusion assay were used to measure cell viability. Necrotic cell death was confirmed by detecting cyclophilin A release and propidium iodide incorporation. DNA damage was evaluated by measuring phosphorylated H2AX. Intracellular reactive oxygen species (ROS) were analyzed by flow cytometry using dihydroergotamine. Mitochondrial membrane potential was detected by flow cytometry using tetramethylrhodamine, ethyl ester. Overall, we found that epi caused cytotoxicity in U87MG cells by inducing DNA damage and necrosis through mitochondrial hyperpolarization and subsequent ROS production. The proper folding of epi into an α-helical structure was essential for epi-mediated anti-glioblastoma effects. In addition, NFκB signaling was activated in U87MG cells after exposure to epi. Suppression of NFκB further enhanced epi-induced cytotoxicity, ROS generation and DNA damage, indicating that NFκB may play a protective role in epi-induced cytotoxicity. Our findings may be useful for the design and improvement of antimicrobial peptides with anti-cancer activity.

中文翻译：

---

抗菌肽epinecidin-1中带正电荷的氨基酸残基的分布对于体外胶质母细胞瘤细胞毒性及其潜在机制至关重要。

Epinecidin-1（epi）是从橙斑石斑鱼（Epinephelus coioides）中鉴定出来的，具有多种生物活性。这项研究的目的是调查带正电荷的氨基酸残基的分布如何影响Epi介导的细胞毒性，并研究U87MG人胶质母细胞瘤细胞中Epi诱导的细胞毒性的分子机制。MTS / PMS和锥虫蓝排除法用于测量细胞活力。通过检测亲环蛋白A释放和碘化丙锭掺入证实坏死细胞死亡。通过测量磷酸化的H2AX评估DNA损伤。使用二氢麦角胺通过流式细胞仪分析细胞内活性氧（ROS）。使用四甲基罗丹明，乙酯通过流式细胞术检测线粒体膜电位。全面的，我们发现Epi通过线粒体超极化和随后的ROS产生诱导DNA损伤和坏死而引起U87MG细胞的细胞毒性。Epi正确折叠成α螺旋结构对于Epi介导的抗成胶质细胞瘤作用至关重要。此外，暴露于Epi后，U87MG细胞中的NFκB信号被激活。NFκB的抑制进一步增强了Epi诱导的细胞毒性，ROS生成和DNA损伤，表明NFκB可能在Epi诱导的细胞毒性中起保护作用。我们的发现可能对设计和改进具有抗癌活性的抗菌肽有用。Epi正确折叠成α螺旋结构对于Epi介导的抗成胶质细胞瘤作用至关重要。此外，暴露于Epi后，U87MG细胞中的NFκB信号被激活。NFκB的抑制进一步增强了Epi诱导的细胞毒性，ROS生成和DNA损伤，表明NFκB可能在Epi诱导的细胞毒性中起保护作用。我们的发现可能对设计和改进具有抗癌活性的抗菌肽有用。Epi正确折叠成α螺旋结构对于Epi介导的抗成胶质细胞瘤作用至关重要。此外，暴露于epi后，U87MG细胞中的NFκB信号传导被激活。NFκB的抑制进一步增强了Epi诱导的细胞毒性，ROS生成和DNA损伤，表明NFκB可能在Epi诱导的细胞毒性中起保护作用。我们的发现可能对设计和改进具有抗癌活性的抗菌肽有用。