Sahin Deniz, Taflan Sevket Onur, Yartas Gizem, Ashktorab Hassan, Smoot Duane T
Department of Molecular Biology and Genetics, Istanbul Technical University, Istanbul, Turkey. Email:
Asian Pac J Cancer Prev. 2018 Apr 25;19(4):927-932. doi: 10.22034/APJCP.2018.19.4.927.
Background: Gastric cancer is the second most common cancer among the malign cancer types. Inefficiency of traditional techniques both in diagnosis and therapy of the disease makes the development of alternative and novel techniques indispensable. As an alternative to traditional methods, tumor specific targeting small peptides can be used to increase the efficiency of the treatment and reduce the side effects related to traditional techniques. The aim of this study is screening and identification of individual peptides specifically targeted to human gastric cancer cells using a phage-displayed peptide library and designing specific peptide sequences by using experimentally-eluted peptide sequences. Methods: Here, MKN-45 human gastric cancer cells and HFE-145 human normal gastric epithelial cells were used as the target and control cells, respectively. 5 rounds of biopannning with a phage display 12-peptide library were applied following subtraction biopanning with HFE-145 control cells. The selected phage clones were established by enzyme-linked immunosorbent assay and immunofluorescence detection. We first obtain random phage clones after five biopanning rounds, determine the binding levels of each individual clone. Then, we analyze the frequencies of each amino acid in best binding clones to determine positively overexpressed amino acids for designing novel peptide sequences. Results: DE532 (VETSQYFRGTLS) phage clone was screened positive, showing specific binding on MKN-45 gastric cancer cells. DE-Obs (HNDLFPSWYHNY) peptide, which was designed by using amino acid frequencies of experimentally selected peptides in the 5th round of biopanning, showed specific binding in MKN-45 cells. Conclusion: Selection and characterization of individual clones may give us specifically binding peptides, but more importantly, data extracted from eluted phage clones may be used to design theoretical peptides with better binding properties than even experimentally selected ones. Both peptides, experimental and designed, may be potential candidates to be developed as useful diagnostic or therapeutic ligand molecules in gastric cancer research.
胃癌是恶性肿瘤类型中第二常见的癌症。传统技术在该疾病的诊断和治疗方面效率低下，使得开发替代的新技术变得不可或缺。作为传统方法的替代，肿瘤特异性靶向小肽可用于提高治疗效率并减少与传统技术相关的副作用。本研究的目的是使用噬菌体展示肽库筛选和鉴定特异性靶向人胃癌细胞的单个肽，并通过使用实验洗脱的肽序列设计特定的肽序列。方法：在此，分别使用MKN - 45人胃癌细胞和HFE - 145人正常胃上皮细胞作为靶细胞和对照细胞。在用HFE - 145对照细胞进行减法生物淘选后，应用噬菌体展示12肽库进行5轮生物淘选。通过酶联免疫吸附测定和免疫荧光检测建立所选噬菌体克隆。我们首先在五轮生物淘选后获得随机噬菌体克隆，确定每个单个克隆的结合水平。然后，我们分析最佳结合克隆中每种氨基酸的频率，以确定用于设计新肽序列的正向过表达氨基酸。结果：DE532（VETSQYFRGTLS）噬菌体克隆筛选呈阳性，显示在MKN - 45胃癌细胞上有特异性结合。通过使用第5轮生物淘选实验选择的肽的氨基酸频率设计的DE - Obs（HNDLFPSWYHNY）肽在MKN - 45细胞中显示出特异性结合。结论：单个克隆的选择和表征可能为我们提供特异性结合肽，但更重要的是，从洗脱的噬菌体克隆中提取的数据可用于设计具有比实验选择的肽更好结合特性的理论肽。实验性和设计性肽都可能是在胃癌研究中开发为有用的诊断或治疗配体分子的潜在候选物。
