Purification of a Novel Peptide Derived from a Shellfish, Crassostrea gigas, and Evaluation of Its Anticancer Property Sun Hee Cheong, †,∥ Eun-Kyung Kim, ∥,‡ Jin-Woo Hwang, † Yon-Suk Kim, † Jung-Suck Lee, § Sang-Ho Moon, ‡,∥ Byong-Tae Jeon, ‡,∥ and Pyo-Jam Park* ,†,∥ † Department of Biotechnology, Konkuk University, Chungju 380-701, Republic of Korea ‡ Division of Food Bioscience, Konkuk University, Chungju 380-701, Republic of Korea § Industry-Academic Cooperation Foundation, Jeju National University, Jeju 690-756, Republic of Korea ∥ Korea Nokyong Research Center, Konkuk University, Chungju 380-701, Republic of Korea ABSTRACT: A novel anticancer peptide was purified from Crassostrea gigas and investigated for its cytotoxic activity. To prepare the peptide, eight proteases were employed for enzymatic hydrolysis. Flavourzyme hydrolysate, which showed clearly superior cytotoxic activity on prostate cancer cells, was further purified using a membrane system and consecutive chromatographic methods. Finally, a novel anticancer peptide was obtained, and the sequence was partially purified as His-Phe- Asn-Ile-Gly-Asn-Arg-Cys-Leu-Cys at the N-terminus. The peptide purified from C. gigas effectively induced the death of prostate, breast, and lung cancer cells but not normal liver cells. This is the first report of an anticancer peptide derived from the enzymatic hydrolysates of C. gigas. KEYWORDS: Crassostrea gigas, anticancer peptide, cancer cells, enzymatic hydrolysis, flavourzyme ■ INTRODUCTION Crassostrea gigas, known as the Pacific oyster, is a widely cultivated aquatic species with an annual world production of 4.6 million metric tons in 2006. 1 There are more than 100 species of oysters worldwide. Oysters are a nutrient-rich food source that includes minerals such as calcium, phosphorus, and iron, various vitamins and amino acids, ands proteins, lipids, and glycogen. Taurine and glycogen from oysters affect many chronic diseases, hepatitis, and eye strength recovery. Further, the oyster is rich in selenium, which supports a number of cellular functions, including heavy metal detoxification. 2 C. gigas has been thoroughly researched in areas related to the food industry, and much is known about their culture conditions, cDNA cloning or gene expression during develop- ment, and environment. 2−4 Recently, it has been reported that oyster extracts have several bioactive peptides, including angiotensin-converting enzyme (ACE) inhibitory peptides and antifungal peptides. 5,6 However, the anticancer effects of C. gigas have not been evaluated. In recent years, peptides generated by the digestion of various proteins, including animal and plant sources, have been found to possess biofunctional activity. 7 These peptides are inactive within the sequences of their parent proteins and are released during gastrointestinal digestion or food processing. 8 Once such bioactive peptides are liberated, depending on their structural, compositional, and sequential properties, they may exhibit various biofunctional activities. The functional proper- ties of a protein can be improved by enzymatic hydrolysis under controlled conditions. Indeed, hydrolytic processes have been developed to convert underutilized materials into marketable and acceptable forms, which can then be widely used in food rather than as animal feed or fertilizer. Hydrolysis potentially influences the molecular size, hydrophobicity, and polar groups of hydrolysates. 9 Further, the characteristics of a hydrolysate directly affect its functional properties and potential as a food ingredient. Despite the intensive efforts and substantial advances that have occurred by focusing on improving treatments, cancer is still a leading cause of death worldwide. Chemotherapy is one of the major approaches for treating cancer by delivering a cytotoxic agent to the cancer cells. The main problem with chemotherapy is the inability to deliver the correct amount of drug directly to cancer cells without affecting normal cells. 10 Apoptosis as a form of programmed cell death is one of the major mechanisms of cell death in response to cancer therapies. 11 A growing body of evidence shows that most marine anticancer peptides that exhibit cytotoxicity may trigger apoptosis by targeting many cellular proteins, and the induced apoptotic process involves both intracellular and extracellular pathways. 12,13 The objective of this work was, therefore, to identify and investigate the effect of the cancer toxicity components hydrolysated from C. gigas. ■ MATERIALS AND METHODS Materials. C. gigas was obtained from a local market (Jeonnam, Korea). Dulbecco’s modified Eagle’s medium (DMEM), RPMI medium 1640, fetal bovine serum (FBS), and penicillin-streptomycin were obtained from Invitrogen Corp. (Carlsbad, CA). RNase A and Tween 20 were purchased from Novagen (Darmstadt, Germany) and USB (Cleveland, OH), respectively. Four enzymes (papain, pepsin, α- chymotrypsin, and trypsin) were obtained from Sigma Chemical Co. (St. Louis, MO), and four other enzymes (flavourzyme, neutrase, Received: July 24, 2013 Revised: November 3, 2013 Accepted: November 7, 2013 Published: November 7, 2013 Article pubs.acs.org/JAFC © 2013 American Chemical Society 11442 dx.doi.org/10.1021/jf4032553 | J. Agric. Food Chem. 2013, 61, 11442−11446