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.

软珊瑚Sarcophyton glaucum中新型细胞毒性寡肽的纯化与鉴定

[1]Abdel-Lateff A, Alarif WM, Ayyad SEN, et al., 2015. New cytotoxic isoprenoid derivatives from the Red Sea soft coral Sarcophyton glaucum. Nat Prod Res, 29(1):24-30.

[2]Al-Lihaibi SS, Alarif WM, Abdel-Lateff A, et al., 2014. Three new cembranoid-type diterpenes from Red Sea soft coral Sarcophyton glaucum: isolation and antiproliferative activity against HepG2 cells. Eur J Med Chem, 81:314-322.

[3]Bradford MM, 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 72(1-2):248-254.

[4]Chai TT, Law YC, Wong FC, et al., 2017. Enzyme-assisted discovery of antioxidant peptides from edible marine invertebrates: a review. Mar Drugs, 15(2):42.

[5]Chao CH, Li WL, Huang CY, et al., 2017. Isoprenoids from the soft coral Sarcophyton glaucum. Mar Drugs, 15(7):202.

[6]Chen LL, Song LY, Li TF, et al., 2013. A new antiproliferative and antioxidant peptide isolated from Arca subcrenata. Mar Drugs, 11(6):1800-1814.

[7]Chen YC, Chang HS, Wang CT, et al., 2009. Antioxidative activities of hydrolysates from duck egg white using enzymatic hydrolysis. Asian Austral J Anim Sci, 22(11):1587-1593.

[8]Cordeiro, R., van Ofwegen, L., Williams, G., 2010. World List of Octocorallia. Sarcophyton glaucum (Quoy & Gaimard, 1833). http://www.marinespecies.org/aphia.php?p=taxdetails&id=209621 [Accessed on Sept. 25, 2017].

[9]Daliri EBM, Oh DH, Lee BH, 2017. Bioactive peptides. Foods, 6(5):32.

[10]de Lumen BO, 2005. Lunasin: a cancer-preventive soy peptide. Nutr Rev, 63(1):16-21.

[11]Dennison SR, Whittaker M, Harris F, et al., 2006. Anticancer α-helical peptides and structure/function relationships underpinning their interactions with tumour cell membranes. Curr Protein Pept Sci, 7(6):487-499.

[12]Fabricius KE, Alderslade P, 2001. Soft Corals and Sea Fans: A Comprehensive Guide to the Tropical Shallow Water Genera of the Central-West Pacific, the Indian Ocean and the Red Sea. Australian Institute of Marine Science, Townsville, Australia, p.264.

[13]Fahmy H, Zjawiony JK, Konoshima T, et al., 2006. Potent skin cancer chemopreventing activity of some novel semi-synthetic cembranoids from marine sources. Mar Drugs, 4(2):28-36.

[14]Fan XD, Bai L, Mao XL, et al., 2017. Novel peptides with anti-proliferation activity from the Porphyra haitanesis hydrolysate. Process Biochem, 60:97-107.

[15]Ferlay J, Soerjomataram I, Ervik M, et al., 2018. GLOBOCAN 2018, Cancer tomorrow: estimated number of incident cases from 2018 to 2040, all cancers, both sexes, all ages. International Agency for Research on Cancer, Lyon, France. http://gco.iarc.fr/tomorrow/graphic-line?type=0&population=900&mode=population&sex=0&cancer=39&age_group=value&apc_male=0&apc_female=0 [Accessed on Dec. 20, 2018].

[16]Gabernet G, Müller AT, Hiss JA, et al., 2016. Membranolytic anticancer peptides. Med Chem Commun, 7(12):2232-2245.

[17]He GQ, Xuan GD, Ruan H, et al., 2005. Optimization of angiotensin I-converting enzyme (ACE) inhibition by rice dregs hydrolysates using response surface methodology. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 6(6):508-513.

[18]Hegazy ME, El-Beih AA, Moustafa AY, et al., 2011. Cytotoxic cembranoids from the Red Sea soft coral Sarcophyton glaucum. Nat Prod Commun, 6(12):1809-1812.

[19]Hsu KC, Li-Chan ECY, Jao CL, 2011. Antiproliferative activity of peptides prepared from enzymatic hydrolysates of tuna dark muscle on human breast cancer cell line MCF-7. Food Chem, 126(2):617-622.

[20]Huang CY, Sung PJ, Uvarani C, et al., 2015. Glaucumolides A and B, biscembranoids with new structural type from a cultured soft coral Sarcophyton glaucum. Sci Rep, 5:15624.

[21]Huang YB, Wang XF, Wang HY, et al., 2011. Studies on mechanism of action of anticancer peptides by modulation of hydrophobicity within a defined structural framework. Mol Cancer Ther, 10(3):416-426.

[22]Hung CC, Yang YH, Kuo PF, et al., 2014. Protein hydrolysates from tuna cooking juice inhibit cell growth and induce apoptosis of human breast cancer cell line MCF-7. J Funct Foods, 11:563-570.

[23]Jumeri, Kim SM, 2011. Antioxidant and anticancer activities of enzymatic hydrolysates of solitary tunicate (Styela clava). Food Sci Biotechnol, 20(4):1075-1085.

[24]Kim EK, Kim YS, Hwang JW, et al., 2013. Purification and characterization of a novel anticancer peptide derived from Ruditapes philippinarum. Process Biochem, 48(7):1086-1090.

[25]Li Y, Yu JM, 2015. Research progress in structure-activity relationship of bioactive peptides. J Med Food, 18(2):147-156.

[26]Minkiewicz P, Dziuba J, Iwaniak A, et al., 2008. BIOPEP database and other programs for processing bioactive peptide sequences. J AOAC Int, 91(4):965-980.

[27]Ngo DH, Vo TS, Ngo DN, et al., 2012. Biological activities and potential health benefits of bioactive peptides derived from marine organisms. Int J Biol Macromol, 51(4):378-383.

[28]Nielsen PM, Petersen D, Dambmann C, 2001. Improved method for determining food protein degree of hydrolysis. J Food Sci, 66(5):642-646.

[29]Nurdiani R, Vasiljevic T, Yeager T, et al., 2017. Bioactive peptides with radical scavenging and cancer cell cytotoxic activities derived from Flathead (Platycephalus fuscus) by-products. Eur Food Res Technol, 243(4):627-637.

[30]Pan X, Zhao YQ, Hu FY, et al., 2016. Anticancer activity of a hexapeptide from skate (Raja porosa) cartilage protein hydrolysate in HeLa cells. Mar Drugs, 14(8):153.

[31]Pangestuti R, Kim SK, 2017. Bioactive peptide of marine origin for the prevention and treatment of non-communicable diseases. Mar Drugs, 15(3):67.

[32]Picot L, Bordenave S, Didelot S, et al., 2006. Antiproliferative activity of fish protein hydrolysates on human breast cancer cell lines. Process Biochem, 41(5):1217-1222.

[33]Qian ZJ, Je JY, Kim SK., 2007. Antihypertensive effect of angiotensin I converting enzyme-inhibitory peptide from hydrolysates of bigeye tuna dark muscle, Thunnus obesus. J Agric Food Chem, 55(21):8398-8403.

[34]Quah Y, Ismail NIM, Ooi JLS, et al., 2017. Identification of novel cytotoxic peptide KENPVLSLVNGMF from marine sponge Xestospongia testudinaria, with characterization of stability in human serum. Int J Pept Res Ther, 24(1):189-199.

[35]Schmidt N, Mishra A, Lai GH, et al., 2010. Arginine-rich cell-penetrating peptides. FEBS Lett, 584(9):1806-1813.

[36]Shan YP, Huang JF, Tan JJ, et al., 2012. The study of single anticancer peptides interacting with HeLa cell membranes by single molecule force spectroscopy. Nanoscale, 4(4):1283-1286.

[37]Simmons TL, Andrianasolo E, McPhail K, et al., 2005. Marine natural products as anticancer drugs. Mol Cancer Ther, 4(2):333-342.

[38]Song R, Wei RB, Luo HY, et al., 2014. Isolation and identification of an antiproliferative peptide derived from heated products of peptic hydrolysates of half-fin anchovy (Setipinna taty). J Funct Foods, 10:104-111.

[39]Sutradhar KB, Amin ML, 2014. Nanotechnology in cancer drug delivery and selective targeting. Int Sch Res Notices, 2014:939378.

[40]Tada N, Horibe T, Haramoto M, et al., 2011. A single replacement of histidine to arginine in EGFR-lytic hybrid peptide demonstrates the improved anticancer activity. Biochem Biophys Res Commun, 407(2):383-388.

[41]Wang B, Li L, Chi CF, et al., 2013. Purification and characterisation of a novel antioxidant peptide derived from blue mussel (Mytilus edulis) protein hydrolysate. Food Chem, 138(2-3):1713-1719.

[42]Wang YK, He HL, Wang GF, et al., 2010. Oyster (Crassostrea gigas) hydrolysates produced on a plant scale have antitumor activity and immunostimulating effects in BALB/c mice. Mar Drugs, 8(2):255-268.

[43]Wang ZJ, Zhang XW, 2016. Inhibitory effects of small molecular peptides from Spirulina (Arthrospira) platensis on cancer cell growth. Food Funct, 7(2):781-788.

[44]You LJ, Zhao MM, Liu RH, et al., 2011. Antioxidant and antiproliferative activities of loach (Misgurnus anguillicaudatus) peptides prepared by papain digestion. J Agric Food Chem, 59(14):7948-7953.

[45]Zheng LH, Lin XK, Wu N, et al., 2013. Targeting cellular apoptotic pathway with peptides from marine organisms. Biochim Biophys Acta, 1836(1):42-48.

[46]List of electronic supplementary materials

[47]Fig. S1 A representative SPE elution prolife of GF3

[48]Fig. S2 Peptide content of SPE fractions