J. Korean Soc. Appl. Biol. Chem. 54(1), 59-65 (2011) Article

Effect of Partially Purified Lipid from the coruscus on Apoptosis in Cancer Cells

Eun-Kyung Kim1, Yon-Suk Kim1, Seung-Jae Lee1, You-Jin Jeon2, Chang-Bum Ahn3, Yong-Tae Kim4, Hyun Kang1, Jae-Hyun Jeong5, Sang-Ho Moon6, Byong-Tae Jeon6*, and Pyo-Jam Park1* 1Department of Biotechnology, Konkuk University, Chungju 380-701, Republic of 2Faculty of Applied Marine Science, Cheju National University, Jeju, 690-756, Republic of Korea 3Division of Food Science and Aqualife Medicine, Chonnam National University, Yeosu 550-749, Republic of Korea 4Department of Food Science & Biotechnology, Kunsan National University, Kunsan 573-701, Republic of Korea 5Department of Food Science & Biotechnology, Chungju National University, Chungju 380-702, Republic of Korea 6Korean Nokyong Research Center, Konkuk University, Chungju 380-701, Republic of Korea Received July 20, 2010; Accepted October 7, 2010

Anticancer effects of the mussel (Mytilus coruscus) cultivated in Korea were determined. Lipid extracts of Mytilus coruscus were prepared using extractions in several organic solvents: methanol, chloroform, hexane, methanol:chloroform=1:1, and chloroform:hexane=1:1. Anticancer activities of the extracts were evaluated using apoptosis assays analyzed by flow cytometry. The hexane extract exhibited the highest anticancer activity. This extract was further separated and purified using thin layer chromatography, through which lipid compounds were isolated, and their components were analyzed; the major fatty acids were eiocosadienoic acid (EA, C20:2), eicosapentaenoic acid (EPA, C20:5), and docosahexaenoic acid (DHA, C22:6), and these components were higher than those of the cultivated in New Zealand and Italy. The partially purified lipid compounds exhibited an anticancer effect in several cancer cell lines. These results indicate that lipid extract of M. coruscus effectively inhibits in vitro tumor growth by inducing apoptosis of cancer cells. Key words: apoptosis, cancer cells, lipids extract, Mytilus coruscus, thin layer chromatography

Mytilus coruscus belongs to the family , one grayanus, possessed high immunomodulating activity of the main cultured of marine shellfish in Korea, [Ovodova et al., 1992]. Glycogen, a polysaccharide and has been used as food and medicine for thousands of extracted from Perna canaliculus, has been found to have years. As a traditional medicine, it has been frequently anti-inflammatory activity [Miller et al., 1993]. Moreover, prescribed by practitioners to regulate the functions of heparin-like substance able to bind antithrombin III liver and kidney, strengthen the immune system, for the (ATIII), isolated from the marine clams Anomalocardia treatment of male impotence, female menoxenia, and brasiliana, was found to have potent anticoagulant other disorders [Li and Ding, 2006]. activity [Cesaretti et al., 2004]. Until now, little attention had been paid to the lipid Research on fatty acid composition of the mussel has compounds in this bivalve. Few reports have investigated been undertaken by laboratories in New Zealand, Bulgaria, the structure and bioactivity of other shellfish. Mytilan, Russia, Italy, and other countries [Stefanov et al., 1992; isolated from the mantle of the mussel Crenomytilus Murphy et al., 2002; Pirini et al., 2007; Korostetski et al., 2008]. Many of the previous studies mostly concentrated *Corresponding authors on the cultivation conditions; thus, little information is Phone: +82-43-840-3588; Fax: +82-43-852-3616 available on the mussels cultivated in Korea and their E-mail: [email protected] (Pyo-Jam Park) anticancer activity. Phone: +82-43-840-3523; Fax: +82-43-851-0932 The objective of the present work was to investigate the E-mail: [email protected] (Byong-Tae Jeon) fatty acid composition and the anticancer effects of M. doi:10.3839/jksabc.2011.008 coruscus on breast, lung, and liver cancer cells. 60 Eun-Kyung Kim et al.

Materials and Methods Apoptosis analysis. The harvested cells were fixed with ethanol (containing 0.5% Tween-20) for 24 h, Materials. Silica gel 60 was obtained from Merck incubated with 50 μg/mL propidium iodide (PI) and 1 μg/ (Darmstadt, Germany). Thin layer chromatography (TLC) mL RNase A at 37oC for 30 min, and analyzed by flow silica gel HLF plates were purchased from Analtech, Inc. cytometry using a FACScan (BD, Franklin Lakes, NJ). (Newark, DE), and TLC silica gel 60F254 plates were The cells belonging to the sub-G1 population were obtained from Merck. Dulbecco's Modified Eagle considered as apoptotic cells, and the percentage of each Medium (DMEM), RPMI medium 1640, fetal bovine phase of the cell cycle was determined. serum (FBS), and penicillin-streptomycin were obtained Fatty acid analysis. Fatty acids of freeze-dried M. from Invitrogen Corporation (Carlsbad, CA). RNase A Coruscus corresponding to 20 mg dry mass were analyzed and Tween-20 were purchased from Novagen (Darmstadt, as follows. Fatty acid methyl esters (FAME), obtained by Germany) and USB (Cleveland, OH), respectively. M. transmethylation of total lipids with a 14% BF3 methanolic coruscus was purchased from a local aquafarm (Yoesu, solution according to the method of Morrison and Smith Korea). All other reagents were of the highest grade [1964], were analyzed on a Agilent 6890 gas-liquid available commercially. chromatograph equipped with a fused silica capillary Preparation of lipid extracts from M. coruscus. column SP2560 (100 m×0.25 mm) and flame-ionization Freeze-dried M. coruscus was suspended in several detector held at 260oC. The oven temperature was organic solvents such as methanol, chloroform, hexane, programmed from 140 to 240oC at 4oC/min and final methanol:chloroform, methanol:hexane for 24 h. The isotherm has been of 240oC for 20 min. The carrier gas lipid extracts were vacuum-dried and stored at −20oC was nitrogen at 1.0 mL/min. The data were processed until used. The recovery rates of each lipid extract from using Agilent Chem Station for GC System. FAME M. coruscus were 15.4 and 7.4% after the second and the mixtures were identified by a combination of different third preparative TLCs. procedures, as described by Pirini et al. [2007]. Open column chromatography using silica gel. Statistical analysis. The data are presented as means Twenty milliliters of the hexane extract (g/mL) was ±SD. The values were evaluated by paired t-test and one- loaded onto a silica gel column (30×400 mm) equilibrated way analysis of variance (ANOVA) followed by post-hoc and eluted with hexane/diethyl ether/acetic acid (80:20:2, Duncan’s multiple range tests. All analyses were performed v/v/v). Each fraction was collected at a volume of 50 mL, using an SPSS system (SPSS, Chicago, IL). and was then applied to silica gel plates (Silica gel HLF). Purification of anticancer lipid. Among the extracts Results and Discussion of the five organic solvents, hexane extract, showing the highest anticancer activity, was selected for further The regulation of cell growth is a homeostatic balance purification. Concentrated lipid samples (200 μL) were between stimulatory and inhibitory signals. Negative growth applied to silica gel plates (Silica gel HLF) and eluted control by tumor suppressor genes, differentiation factors, with hexane/diethyl ether/acetic acid (80:20:2, v/v/v) and programmed cell death (apoptosis) are the commonly according to the method described by McPhee et al. targeted mechanisms exploited for strategies in the [2007]. Briefly, lipids were visualized by spraying the treatment of malignancies and other diseases. Among plate with 20% sulfuric acid. Upon illumination with UV them, apoptosis is a highly attractive and widely studied light (254 nm), individual lipid classes were detected as area to search for more effective agents in the treatment violet spots. After the first TLC, individual classes were of human cancers. A wide variety of in vivo and in vitro scraped off, and their anticancer effects were determined. studies published in recent years have suggested that The fraction displaying the highest anticancer activity many chemotherapeutic agents could induce apoptotic from the first TLC classes was further purified using TLC cell death in different cancer cells [Hannun, 1997; Byrd et in the same manner as the first TLC method. al., 1998]. For this reason, the anticancer activities of the Cell culture. Prostate (PC3), breast (MDA-MB-231), organic solvent extracts were evaluated for their ability to lung (NCI-A549), and liver (HepG2) cancer cells were induce apoptosis using flow cytometry. Among the various cultured and maintained in DMEM. The prostate cancer organic solvent extracts of M. coruscus, the highest cells were then cultured and maintained in RPMI medium anticancer activity was observed in the hexane extract, 1640 supplemented with 100 U/mL penicillin, 100 μg/ exhibiting an apoptosis rate of 40.9% at 0.5 mg/mL in mL streptomycin, and 10% FBS and maintained at 37oC human prostate cancer cells, PC3, with no cytotoxicity under a humidified atmosphere with 5% CO2. All treatments effect; apoptosis rates of control and DMSO were 2.4 and were performed at 30% confluence. 4.9% at 0.5 mg/mL, respectively (Fig. 1). Therefore, the Anticancer effects of Mytilus coruscus 61

Fig. 1. Anticancer effects of various organic solvent extracts from M. coruscus on PC3, prostate cancer cell. 1. Methanol; 2. Chloroform; 3. Hexane; 4. Methanol/chloroform=1/1, 5. Chloroform/hexane=1/1. The cells were treated with 0.5 mg/mL of each organic solvent fraction of M. coruscus. Values are means±SD of triplicate experiments. Lower case letters a-d indicate values are significantly different at p<0.05 as analyzed by Duncan’s multiple range test.

Fig. 2. Anticancer effects of hexane extracts of M. coruscus on PC3 cells. a. The first TLC of hexane extracts from M. coruscus; b. Anticancer effects of first TLC fractions of M. coruscus on PC3 cells. Values are means±SD of triplicate experiments. Lower case letters a-d indicate values are significantly different at p<0.05 as analyzed by Duncan’s multiple range test. hexane extracts were selected for further study. In the hexane extracts, study on the mechanism in the addition, because S and G2 phase arrests were detected in pathway is ongoing. 62 Eun-Kyung Kim et al.

Fig. 3. Anticancer effects of hexane extracts of M. coruscus on PC3, prostate cancer cells. a. Second TLC of hexane extracts from M. coruscus; b. Anticancer effects of second TLC fractions of M. coruscus on PC3 cells; c. TLC fractions of each purification step of M. coruscus compared with EPA as a standard. Values are means±SD of triplicate experiments. Lower case letters a-d indicate values are significantly different at p<0.05 as analyzed by Duncan’s multiple range test. Anticancer effects of Mytilus coruscus 63

Table 1. Fatty acid components of MC-III-b, anticancer- 6/n-3 fatty acids was lower in MC-III-b (0.258) than in effective partially purified lipids of M. coruscus the hexane extract (0.401) (data not shown). Previous Fatty acid works on the ratio of n-6/n-3 fatty acids effect on cancer Content (%) Compound Molecular formula cells has shown that decreased n-6/n-3 fatty acid ratio reduces the risk of cancer [Esterbauer et al., 1991; Myristic acid C14:0 0.007 Neuhouser et al., 2007]. Therefore, these fatty acid Palmitic acid C16:0 0.019 compositions of MC-III-b, the hexane extracts of M. Palmitoleic acid C16:1 0.016 coruscus cultivated in Korea, could be assumed to Stearic acid C18:0 3.050 substantially contribute to the anticancer properties due to Heptadecenoic acid C17:0 2.404 the decreased lipid peroxidation and low n-6/n-3 fatty Oleic acid C18:1 ω-6-c 2.625 acid ratio. Elaidic acid C18:1 ω-6-t 1.588 The use of hexane extract of M. coruscus at 30 μg/mL Linoleic acid C18:2 3.636 for the treatment of various cancer cells, including breast, Linolenic acid C18:3 1.920 lung, and liver cancer cells, resulted in apoptosis rates of Eicosadienoic acid C20:2 8.543 22.7, 45.6, and 37.3%, respectively, in a dose-dependent Eicosapentaenoic acid C20:5 33.3560 manner (Fig. 4). These results showed the lipid extract of Heneicosanoic acid C21:0 1.784 M. coruscus has a potent anticancer effect. Behenic acid C22:0 3.076 Dietary fat has been observed to affect the gene Docosahexaenoic acid C22:5 10.6000 expression, in addition to its role as an important source of energy and its effects on membrane lipid composition To purify the anticancer lipid, the hexane extracts of M. [Vock et al., 2007]. In mammals, the expressions of many coruscus were subjected to TLC after open column genes have been shown to be modulated by fatty acids in chromatography (data not shown). Three fractions were a positive or a negative manner, leading to pronounced detected: MC-I, MC-II, and MC-III (Fig. 2a). The highest changes in metabolism, cell differentiation, and growth anticancer activity was observed in MC-III, exhibiting an [Black et al., 2000; Pegorier et al., 2004]. Fatty acids apoptosis rate of 66.1% at 10 μg/mL (Fig. 2b). Therefore, (FAs) are major components of biological cell membranes the MC-III fraction was selected for further study. The that play important roles in intracellular signaling and as MC-III fraction was subjected to TLC, and two individual precursors for ligands that bind to nuclear receptors. FAs fractions were detected: MC-III-a and MC-III-b (Fig. 3a). are chemically classified as saturated and unsaturated The highest anticancer activity was observed in MC-III-b, (monounsaturated and polyunsaturated), and their structures exhibiting an apoptosis rate of 77.9% at 10 μg/mL (Fig. affect their biological activities [Baylin et al., 2002]. It is 3b). Therefore, MC-III-b was selected for analysis of widely recognized that linolenic acid, EPA, DHA, and fatty acid composition. MC-III-b was further purified other PUFAs have antitumor effects [Karmali et al., 1984; using TLC (Fig. 3c), and the resulting isolate exhibited Begin et al, 1988]. PUFAs inhibit cancer-cell growth, fewer bands. MC-III-b was found to be composed of induce apoptosis, and increase the efficiency of chemo- three major polyunsaturated fatty acids (PUFAs), including therapeutic drugs [Terry et al., 2003]. eicosadienoic acid (EA) (8.5%), eicosapentaenoic acid In the present research, the mussels cultivated in Korea (EPA) (33.4%), and docosahexaenoic acid (DHA) (10.6%) were found to contain higher level of EPA than the green- (Table 1), and has a high PUFA content (over 55%). lipped mussels cultivated in New Zealand. In addition, Omega-3 fatty acids were the main components in the concentrations of PUFAs were higher than those of PUFAs. Of the major fatty acids in MC-III-b, the the mussels cultivated in Italy. On the contrary, the concentration of EPA was higher than the mussel contents of SFA were lower than those found in the cultivated in New Zealand (19.8%), a green-lipped mussel Italian mussels. Moreover, the ratio of n-6/n-3 fatty acids [Murphy et al., 2002]. Moreover, the concentration of was considerably lower in MC-III-b than in the hexane PUFAs (58.1%) was higher than the mussels cultivated in extract. These results indicate the fatty acid composition Italy (51.7%). On the contrary, the content of saturated of the mussels cultivated in Korea has a greater health fatty acid (SFA) (5.5%) was lower than that of the Italian benefit than mussels cultivated in other countries. mussels (37.7%) [Ventrella et al., 2008]. However, In summary, a purified fraction of the hexane extract of because their results were obtained using different M. coruscus, designated MC-III-b, was found to be high extraction methods from that used in the present study, in EA, EPA, and DHA, and exhibited a strong anticancer one must exercise utmost discretion in comparing our effect in vitro. MC-III-b increased apoptosis in human data with the literature values. In addition, the ratio of n- prostate, breast, lung, and liver cancer cells, but did not 64 Eun-Kyung Kim et al.

Fig. 4. Anticancer effects of MC-III-b of M. coruscus on MDA-MB-231, breast cancer cells (a), A549, lung cancer cells (b), and HepG2, liver cancer cells (c). Values are means±SD of triplicate experiments. * (at p<0.05) and ** (at p<0.01) indicate values are significantly different as analyzed by paired t-test. increase apoptosis in normal liver cells (data not shown). Acknowledgments. This work was supported by a Presumably, the reason why there is no cytotoxicity in special grant from Konkuk University in 2010. normal cells is that the extract was composed of natural products from edible shellfishes, and MC-III-b could References contain a potential cancer therapy agent. Our results suggest that MC-III-b could effectively inhibit in vitro Baylin A, Kabagambe EK, Siles X, and Campos H (2002) tumor growth by inducing apoptosis of cancer cells, but Adipose tissue biomarkers of fatty acid intake. Am J Clin the mechanism for inducing apoptosis in tumor cells is Nutr 76, 750-757. not known and needs further investigation. Begin ME, Ells G, and Horrobin DF (1988) Polyunsaturated Anticancer effects of Mytilus coruscus 65

fatty acid induced cytotoxicity against tumor cells and its 139-142. relationship to lipid peroxidation. J Natl Cancer Inst 80, Morrison WR and Smith LM (1964) Preparation of fatty acid 188-194. methylesters and dimethylacetals from lipids with boron Black PN, Faergeman NJ, and DiRusso CC (2000) Long-chain fluoride-methanol. J Lipid Res 5, 600-608. acyl-CoAdependent regulation of gene expression in Murphy KJ, Mooney BD, Mann NJ, Nichols PD, and Sinclair bacteria, yeast and mammals. J Nutr 130, 305S-309S. AJ (2002) Lipid, FA, and sterol composition of New Byrd JC, Shinn C, Waselenko JK, Fuchs EJ, Lehman TA, Zealand green lipped mussel (Perna canaliculus) and Nguyen PL, Flinn IW, Diehl LF, Sausville E, and Grever Tasmanian blue mussel (Mytilus edulis). Lipid 37, 587-595. MR (1998) Flavopiridol induces apoptosis in chronic Neuhouser ML, Barnett MJ, Kristal AR, Ambrosone CB, King lymphocytic leukemia cells via activation of caspase-3 I, Thornquist M, and Goodman G (2007) (n-6) PUFA without evidence of bcl-2 modulation or dependence on increase and dairy foods decrease prostate cancer risk in functional p53. Blood 92, 3804-3816. heavy smokers. J Nutr 137, 1821-1827. Cesaretti M, Luppi E, Maccari F, and Volpi N (2004) Isolation Ovodova RG, Glazkova VE, Mikheyskaya LV, Molchanova and characterization of a heparin with high anticoagulant VI, Isakov VV, Ovodov YS, and Molina LEF (1992) The activity from the clam Tapes phylippinarum: Evidence for structure of mytilan, a bioglycanimmunomodulator isolated the presence of a high content of antithrombin III binding from the mussel Crenomytilus grayanus. Carbohydr Res site. Glycobiology 14, 1275-1284. 223, 221-226. Esterbauer H, Schaur RJ, and Zollner H (1991) Chemistry and Pegorier JP, May CL, and Girard J (2004) Control of gene biochemistry of 4-hydroxynonenal, malonaldehyde and expression by fatty acids. J Nutr 134, 2444S-2449S. related aldehydes. Free Radic Biol Med 11, 81-128. Pirini M, Manuzzi MP, Pagliarani A, Trombetti F, Borgatti AR, Hannun YA (1997) Apoptosis and the dilemma of cancer and Ventrella V (2007) Changes in fatty acid composition chemotherapy. Blood 89, 1845-1853. of Mytilus galloprovincialis (Lmk) fed on microalgal and Karmali RA, Marsh J, and Fuchs C (1984) Effect of omega-3 wheat germ diets. Comp Biochem Physiol 147, 616-626. fatty acids on growth of a rat mammary tumor. J Natl Stefanov K, Seizova K, Brechany EY, and Christie WW (1992) Cancer Inst 73, 457-461. An unusual fatty acid composition for a fresh-water mussel, Korostetski EI, Boroda AV, and Odintsova NA (2008) Changes Unio tumidus, from Bulgaria. J Nat Prod 55, 979-981. in the lipid composition of embryonic cells of the mussel Terry PD, Rohan TE, and Wolk A (2003) Intakes of fish and Mytilus trossulus during cryopreservation. Biofizika 53, marine fatty acids and the risks of cancers of the breast and 658-665. prostate and of other hormone-related cancers: a review of Li PP and Ding XL (2006) The manufacture and nutritional the epidemiologic evidence. Am J Clin Nutr 77, 532-543. analysis of the functional natural sauce from the decoction Ventrella V, Pirini M, Pagliarani A, Trombetti F, Manuzzi MP, of Mytilus edulis. Condiment 2, 17-19. and Borgatti AR (2008) Effect of temporal and McPhee S, Hodges LD, Wright PFA, Wynne PM, Kalafatis N, geographical factors on fatty acid composition of M. Harney DW, and Macrides TA (2007) Anti-cyclooxygenase galloprovincialis from the Adriatic sea. Comp Biochem effects of lipid extracts from the New Zealand green-lipped Physiol B 149, 241-250. mussel, Perna canaliculus. Comp Biochem Physiol B 146, Vock C, Gleissner M, Klapper M, and Döring F (2007) 346-356. Identification of palmitate-regulated genes in HepG2 cells Miller TE, Dodd J, Ormrod DJ, and Geddes R (1993) Anti- by applying microarray analysis. Biochim Biophys Acta inflammatory activity of glycogen extracted from Perna 1770, 1283-1288. canaliculus (NZ green-lipped mussel). Agents Actions 38,