Abstract
Resistance and side effects are common problems for anticancer drugs used in chemotherapy. Thus, continued research to discover novel and specific anticancer drugs is obligatory. Marine sponges hold great promise as a source of potent cytotoxic peptides with future applications in cancer treatments. This study aimed to purify and identify cytotoxic peptides from the protein hydrolysates of the giant barrel sponge Xestospongia testudinaria, guided by a cytotoxicity assay based on the human cervical cancer cell line (HeLa). Comparison among trypsin, chymotrypsin, papain and alcalase hydrolysates of X. testudinaria revealed papain hydrolysate (PH) to be the most active. PH was purified consecutively by membrane ultrafiltration, gel filtration chromatography, and reversed-phase high performance liquid chromatography (RP-HPLC). Following liquid chromatography-tandem mass spectrometric analysis, two peptides were identified from the most cytotoxic RP-HPLC fraction: KENPVLSLVNGMF and LLATIPKVGVFSILV. Between the two, only the synthetic peptide KENPVLSLVNGMF showed cytotoxicity toward HeLa cells in a dose-dependent manner. KENPVLSLVNGMF (EC50 0.67 mM) was 3.8-fold more cytotoxic compared with anticancer drug 5-fluorouracil (EC50 2.56 mM). Furthermore, KENPVLSLVNGMF show only marginal 5% cytotoxicity to Hek293, a non-cancerous, human embryonic kidney cell line, when tested at 0.67 mM. The half-life of the peptide was 3.2 ± 0.5 h in human serum in vitro, as revealed by RP-HPLC analyses. These results suggest that KENPVLSLVNGMF identified from X. testudinaria papain hydrolysate has potential applications as peptide lead in future development of potent and specific anticancer drugs.
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References
Adamson NJ, Reynolds EC (1996) Characterization of casein phosphopeptides prepared using alcalase: determination of enzyme specificity. Enzym Microb Technol 19:202–207. doi:10.1016/0141-0229(95)00232-4
Aleksandra G, Monika Ż, Tadeusz T (2010) Application of pancreatic enzymes in hydrolysis of egg-white proteins. Pol J Food Nutr Sci 60:57–61
Aleksandra Z, Marta P, Ewelina E, Marek S, Anna D, Józefa C, Tadeusz T (2012) Antioxidant and antimicrobial activity of lecithin free egg yolk protein preparation hydrolysates obtained with digestive enzymes. Funct Food Health Dis 2:487–500
Alemán A, Pérez-Santín E, Bordenave-Juchereau S, Arnaudin I, Gómez-Guillén MC, Montero P (2011) Squid gelatin hydrolysates with antihypertensive, anticancer and antioxidant activity. Food Res Int 44:1044–1051. doi:10.1016/j.foodres.2011.03.010
Arenas I, Villegas E, Walls O, Barrios H, Rodriguez R, Corzo G (2016) Antimicrobial activity and stability of short and long based arachnid synthetic peptides in the presence of commercial antibiotics. Molecules. doi:10.3390/molecules21020225
Beena N, Deepak K, Diwan SR (2016) Marine peptides as anticancer agents: a remedy to mankind by nature. Curr Protein Pept Sci 17:1–20. doi:10.2174/1389203717666160724200849
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 72:248–254. doi:10.1016/0003-2697(76)90527-3
Chai TT, Law YC, Wong FC, Kim SK (2017) Enzyme-assisted discovery of antioxidant peptides from edible marine invertebrates: a review. Mar Drugs. doi:10.3390/md15020042
Chen T-X, Nie H-L, Li S-B, Branford-White C, Su S-N, Zhu L-M (2009a) Comparison: adsorption of papain using immobilized dye ligands on affinity membranes. Colloids Surf B 72:25–31. doi:10.1016/j.colsurfb.2009.03.012
Chen Y-C, Chang H-S, Wang C-T, Cheng F-Y (2009b) Antioxidative activities of hydrolysates from duck egg white using enzymatic hydrolysis. Asian Australas J Anim Sci 22:1587–1593. doi:10.5713/ajas.2009.90119
Cheung RCF, Ng TB, Wong JH (2015) Marine peptides: Bioactivities and applications. Mar Drugs 13:4006–4043. doi:10.3390/md13074006
Cudic M et al (2002) Development of novel antibacterial peptides that kill resistant isolates. Peptides 23:2071–2083
Davis RA et al (2004) Microcionamides A and B, bioactive peptides from the philippine sponge Clathria (Thalysias) abietina. J Org Chem 69:4170–4176. doi:10.1021/jo040129h
De Jersey J (1970) Specificity of papain. BioChemistry 9:1761–1767. doi:10.1021/bi00810a015
Dennison SR, Whittaker M, Harris F, Phoenix DA (2006) Anticancer alpha-helical peptides and structure/function relationships underpinning their interactions with tumour cell membranes. Curr Protein Peptide Sci 7:487–499
El-Gamal A et al (2016) Cytotoxic compounds from the Saudi Red Sea sponge Xestospongia testudinaria. Mar Drugs 14:82
Fernández-Musoles R, Salom JB, Castelló-Ruiz M, Contreras MdM, Recio I, Manzanares P (2013) Bioavailability of antihypertensive lactoferricin B-derived peptides: transepithelial transport and resistance to intestinal and plasma peptidases. Int Dairy J 32:169–174. doi:10.1016/j.idairyj.2013.05.009
Hooper J, Soest RWMV (2002) Systema Porifera: a guide to the classification of sponges (volume 1 and 2). 1 edn. Springer US, New York. doi:10.1007/978-1-4615-0747-5
Hsu K-C, Li-Chan ECY, Jao C-L (2011) Antiproliferative activity of peptides prepared from enzymatic hydrolysates of tuna dark muscle on human breast cancer cell line MCF-7. Food Chem 126:617–622. doi:10.1016/j.foodchem.2010.11.066
Hung C-C, Yang Y-H, Kuo P-F, Hsu K-C (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. doi:10.1016/j.jff.2014.08.015
Jenssen H, Aspmo SI (2008) Serum stability of peptides. In: Otvos L (ed) Peptide-based drug design. Humana Press, Totowa, pp 177–186. doi:10.1007/978-1-59745-419-3_10
Jin D-X, Liu X-l, Zheng X-Q, Wang X-J, He J-F (2016) Preparation of antioxidative corn protein hydrolysates, purification and evaluation of three novel corn antioxidant peptides. Food Chem 204:427–436. doi:10.1016/j.foodchem.2016.02.119
Kim E-K, Kim Y-S, Hwang J-W, Lee JS, Moon S-H, Jeon B-T, Park P-J (2013) Purification and characterization of a novel anticancer peptide derived from Ruditapes philippinarum. Process Biochem 48:1086–1090. doi:10.1016/j.procbio.2013.05.004
Leng B, Liu X-D, Chen Q-X (2005) Inhibitory effects of anticancer peptide from Mercenaria on the BGC-823 cells and several enzymes. Fed Eur Biochem Soc Lett 579:1187–1190 doi:10.1016/j.febslet.2004.12.089
Li Y, Yu J (2015) Research progress in structure-activity relationship of bioactive peptides. J Med Food 18:147–156. doi:10.1089/jmf.2014.0028
Liao W, Zhang R, Dong C, Yu Z, Ren J (2015) Novel walnut peptide–selenium hybrids with enhanced anticancer synergism: facile synthesis and mechanistic investigation of anticancer activity. Int J Nanomed 2016:1305–1321. doi:10.2147/IJN.S92257
Ma S et al (2015) Isolation of a novel bio-peptide from walnut residual protein inducing apoptosis and autophagy on cancer cells. BioMed Cent Complement Altern Med 15:413. doi:10.1186/s12906-015-0940-9
Markman JL, Rekechenetskiy A, Holler E, Ljubimova JY (2013) Nanomedicine therapeutic approaches to overcome cancer drug resistance. Adv Drug Deliv Rev 65:1866–1879. doi:10.1016/j.addr.2013.09.019
Mehbub MF, Lei J, Franco C, Zhang W (2014) Marine sponge derived natural products between 2001 and 2010: trends and opportunities for discovery of bioactives. Mar drugs 12:4539–4577. doi:10.3390/md12084539
Mehbub MF, Perkins MV, Zhang W, Franco CMM (2016) New marine natural products from sponges (Porifera) of the order Dictyoceratida (2001 to 2012); a promising source for drug discovery, exploration and future prospects. Biotechnol Adv 34:473–491. doi:10.1016/j.biotechadv.2015.12.008
Ngo D-H, Vo T-S, Ngo D-N, Wijesekara I, Kim S-K (2012) Biological activities and potential health benefits of bioactive peptides derived from marine organisms. Int J Biol Macromol 51:378–383. doi:10.1016/j.ijbiomac.2012.06.001
Nguyen LT, Chau JK, Perry NA, de Boer L, Zaat SA, Vogel HJ (2010) Serum stabilities of short tryptophan- and arginine-rich antimicrobial peptide analogs. PLoS ONE. doi:10.1371/journal.pone.0012684
Nielsen PM, Petersen D, Dambmann C (2001) Improved method for determining food protein degree of hydrolysis. J Food Sci 66:642–646. doi:10.1111/j.1365-2621.2001.tb04614.x
Pan X, Zhao Y-Q, Hu F-Y, Chi C-F, Wang B (2016) Anticancer activity of a hexapeptide from skate (Raja porosa) cartilage protein hydrolysate in HeLa cells. Mar Drugs 14:153. doi:10.3390/md14080153
Papo N, Shai Y (2005) Host defense peptides as new weapons in cancer treatment. CMLS Cell Mol Life Sci 62:784–790. doi:10.1007/s00018-005-4560-2
Park YW, Nam MS (2015) Bioactive peptides in milk and dairy products: a review. Korean J Food Sci Anim Resour 35:831–840. doi:10.5851/kosfa.2015.35.6.831
Perdicaris S, Vlachogianni T, Valavanidis A (2013) Bioactive natural substances from marine sponges: new developments and prospects for future pharmaceuticals. Nat Prod Chem Res 1:1–8 doi:10.4172/2329-6836.1000114
Picot L et al (2006) Antiproliferative activity of fish protein hydrolysates on human breast cancer cell lines. Process Biochem 41:1217–1222. doi:10.1016/j.procbio.2005.11.024
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 55:8398–8403. doi:10.1021/jf0710635
Sbroggio MF, Montilha MS, Figueiredo VRGd, Georgetti SR, Kurozawa LE (2016) Influence of the degree of hydrolysis and type of enzyme on antioxidant activity of okara protein hydrolysates. Food Sci Technol (Campinas) 36:375
Song R, Wei R-b, Luo H-y, Yang Z-s (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. doi:10.1016/j.jff.2014.06.010
Suarez-Jimenez G-M, Burgos-Hernandez A, Ezquerra-Brauer J-M (2012) Bioactive peptides and depsipeptides with anticancer potential: Sources from marine animals. Mar Drugs 10:963
Umayaparvathi S, Meenakshi S, Vimalraj V, Arumugam M, Sivagami G, Balasubramanian T (2014) Antioxidant activity and anticancer effect of bioactive peptide from enzymatic hydrolysate of oyster (Saccostrea cucullata). Biomed Prev Nutr 4:343–353. doi:10.1016/j.bionut.2014.04.006
Werle M, Bernkop-Schnurch A (2006) Strategies to improve plasma half life time of peptide and protein drugs. Amino Acids 30:351–367. doi:10.1007/s00726-005-0289-3
Wiriyaphan C, Chitsomboon B, Roytrakul S, Yongsawadigul J (2013) Isolation and identification of antioxidative peptides from hydrolysate of threadfin bream surimi processing byproduct. J Funct Foods 5:1654–1664. doi:10.1016/j.jff.2013.07.009
World Health Organization (2017) 10 facts about cancer. http://www.who.int/features/factfiles/cancer/en/. Accessed Feb 22 2017
Yao JW, Xiao Y, Lin F (2012) Effect of various pH values, ionic strength, and temperature on papain hydrolysis of salivary film. Eur J Oral Sci 120:140–146. doi:10.1111/j.1600-0722.2012.00942.x
Ye J, Zhou F, Al-Kareef AM, Wang H (2015) Anticancer agents from marine sponges. J Asian Nat Prod Res 17:64–88. doi:10.1080/10286020.2014.970535
You L, Zhao M, Liu RH, Regenstein JM (2011) Antioxidant and antiproliferative activities of loach (Misgurnus anguillicaudatus) peptides prepared by papain digestion. J Agric Food Chem 59:7948–7953. doi:10.1021/jf2016368
Zhan KX et al (2014) Reniochalistatins A–E, cyclic peptides from the marine sponge Reniochalina stalagmitis. J Nat Prod 77:2678–2684. doi:10.1021/np5006778
Zheng L et al (2013) Targeting cellular apoptotic pathway with peptides from marine organisms. Biochim Biophys Acta 1836:42–48. doi:10.1016/j.bbcan.2013.02.006
Zhou X, Lu Y, Lin X, Yang B, Yang X, Liu Y (2011) Brominated aliphatic hydrocarbons and sterols from the sponge Xestospongia testudinaria with their bioactivities. Chem Phys Lipids 164:703–706. doi:10.1016/j.chemphyslip.2011.08.002
Acknowledgements
This study was financially supported by the Fundamental Research Grant Scheme of the Ministry of Higher Education, Malaysia, with Grant No. FRGS/1/2013/ST04/UTAR/02/1. We thank Keng-Fei Ooh and Yew-Wai Soon for their technical assistance especially in RP-HPLC operations. We thank Yew-Chye Law for his technical support and suggestions on the result interpretations. We also thank Dr Lai-Kuan Teh for her technical assistance in our peptide stability experiment. We are grateful to Perkin Elmer Malaysia for allowing us to perform RP-HPLC experiments on their PerkinElmer Flexar FX-20 UHPLC unit.
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Ethical approval was obtained from UTAR Scientific and Ethical Review Committee (U/SERC/40/2017). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or research committee and with Helsinki Declaration of 1975.
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Quah, Y., Mohd Ismail, N.I., Ooi, J.L.S. et al. Identification of Novel Cytotoxic Peptide KENPVLSLVNGMF from Marine Sponge Xestospongia testudinaria, with Characterization of Stability in Human Serum. Int J Pept Res Ther 24, 189–199 (2018). https://doi.org/10.1007/s10989-017-9604-6
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DOI: https://doi.org/10.1007/s10989-017-9604-6