Abstract
Globally, peptide-based anticancer therapies have drawn much attention. Marine organisms are a reservoir of anticancer peptides that await discovery. In this study, we aimed to identify cytotoxic oligopeptides from Sarcophyton glaucum. Peptides were purified from among the S. glaucum hydrolysates produced by alcalase, chymotrypsin, papain, and trypsin, guided by a methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay on the human cervical cancer (HeLa) cell line for cytotoxicity evaluation. Purification techniques adopted were membrane ultrafiltration, gel filtration chromatography, solid phase extraction (SPE), and reversed-phase high-performance liquid chromatography (RP-HPLC). Purified peptides were identified by de novo peptide sequencing. From papain hydrolysate, three peptide sequences were identified: AGAPGG, AERQ, and RDTQ (428.45, 502.53, and 518.53 Da, respectively). Peptides synthesized from these sequences exhibited cytotoxicity on HeLa cells with median effect concentration (EC50) values of 8.6, 4.9, and 5.6 mmol/L, respectively, up to 5.8-fold stronger than the anticancer drug 5-fluorouracil. When tested at their respective EC50, AGAPGG, AERQ, and RDTQ showed only 16%, 25%, and 11% cytotoxicity to non-cancerous Hek293 cells, respectively. In conclusion, AERQ, AGAPGG, and RDTQ are promising candidates for future development as peptide-based anticancer drugs.
概要
目的
纯化和鉴定软珊瑚Sarcophyton glaucum 蛋白水解 物中的细胞毒性肽。
创新点
首次从软珊瑚S. glaucum 中发现具有癌细胞毒性 的寡肽,开发了一种有潜力的新型抗癌药物。
方法
通过碱性蛋白酶、胰凝乳蛋白酶、木瓜蛋白酶和 胰蛋白酶产生软珊瑚S. glaucum 蛋白质水解物, 并通过MTT 法评估其对人宫颈癌HeLa 细胞的 毒性。通过膜超滤、凝胶过滤色谱、固相萃取和 反相高效液相色谱等技术进一步提纯肽;采用从 头测序法(de novo sequencing)进行肽鉴定;根 据所鉴定的序列,进一步确定合成肽的细胞毒 性。
结论
从软珊瑚S. glaucum 的木瓜蛋白酶水解物中鉴定 出三种新型肽序列:AGAPGG、AERQ 和RDTQ (分子量分别为428.45、502.53 和518.53 Da)。 此三种寡肽具有对HeLa 的细胞毒性,其半最大 效应浓度(EC50)值各为8.6、4.9 和5.6 mmol/L, 为抗癌药物5-氟尿嘧啶的3.3、5.8 和5.1 倍。当 测试对非癌细胞Hek293 的毒性时,这三种肽仅 显示16%、25%和11%的细胞毒性。因此,AERQ、 AGAPGG 和RDTQ 有很大的潜力成为肽类抗癌 药物
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Acknowledgements
We thank Ms. Nina Ann Jin HO of the Institute of Biological Sciences, Faculty of Science, University of Malaya, Malaysia, for SCUBA diving and sampling assistance in the field.
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Project supported by the Fundamental Research Grant Scheme of the Ministry of Higher Education, Malaysia (FRGS/1/2013/ST04/UTAR/02/1)
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Quah, Y., Mohd Ismail, N.I., Ooi, J.L.S. et al. Purification and identification of novel cytotoxic oligopeptides from soft coral Sarcophyton glaucum. J. Zhejiang Univ. Sci. B 20, 59–70 (2019). https://doi.org/10.1631/jzus.B1700586
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DOI: https://doi.org/10.1631/jzus.B1700586